The CD274 (PD-L1)/PDCD1 (PD-1) immune checkpoint interaction may promote cancer progression, but the expression patterns and prognostic significance of PD-L1 and PD-1 in the colorectal cancer microenvironment are inadequately characterised.We used a custom 9-plex immunohistochemistry assay to quantify the expression patterns of PD-L1 and PD-1 in macrophages, T cells, and tumour cells in 910 colorectal cancer patients. We evaluated cancer-specific mortality according to immune cell subset densities using multivariable Cox regression models.– macrophages, PD-L1+ macrophages were more likely M1-polarised than M2-polarised and located closer to tumour cells. PD-L1+ macrophage density in the invasive margin associated with longer cancer-specific survival . T cell densities associated with longer cancer-specific survival regardless of PD-1 expression (Ptrend < 0.005 for both PD-1+ and PD-1– subsets). Higher densities of PD-1+ T cell/PD-L1+ macrophage clusters associated with longer cancer-specific survival (Ptrend < 0.005).Compared to PD-L1+ macrophages show distinct polarisation profiles (more M1-like), spatial features , and associations with favourable clinical outcome. Our comprehensive multimarker assessment could enhance the understanding of immune checkpoints in the tumour microenvironment and promote the development of improved immunotherapies.PD-L1 Colorectal cancer is the second leading cause of cancer death worldwide with over 900,000 deaths in 2020 . The assMacrophages are inflammatory cells which have shown to associate with cancer progression . They arIn this study, we used multiplex immunohistochemistry and machine learning-based image analysis to comprehensively characterise PD-L1 and PD-1 immune checkpoint expression in M1-like and M2-like macrophages, T cells, and tumour cells in a large, population-based colorectal cancer cohort of 910 patients. Our primary aim was to (i) evaluate the expression patterns and prognostic significance of PD-L1 and PD-1 in immune cells and tumour cells. As secondary aims, we (ii) clarified the associations between immune checkpoint expression and tumour characteristics, and (iii) investigated the infiltration patterns and prognostic role of M1-like and M2-like polarised macrophages. We hypothesised low expression of PD-L1 and PD-1 immune checkpoints and higher density of M1-like macrophages to associate with favourable colorectal cancer outcome.BRAF V600E mutation status with immunohistochemistry . The possible range for values was from 0  to 300  . For surT cells were further subdivided into PD-1 positive or negative based on a fixed cut-off value for their cytoplasmic PD-1 staining intensity (40 intensity units).spatstat (2.2–0) package to calculate nearest neighbour distances (NNDs), which measure the distance from a specific point  to its closest neighbour point of specific category . For visualisation, scaled intensities of macrophage polarisation markers as a function of NND from tumour cells were plotted with ggplot2 (3.3.3) package using generalised additive model smoothing [formula y ~ s(x)]. For further spatial analyses, we calculated the density of PD-1+/PD-L1+ clusters defined as PD-1+ T cell located within 20-μm distance from the closest PD-L1+ macrophage. The radius was selected in order to identify cells with capability for direct cell-cell interaction consistent with prior reports , high tumour grade , MMR deficiency (both P < 0.0001) and BRAF mutation . The findings were mainly similar for PD-1+ and PD-1− T cells  and PD-1− T cells (R = 0.50)  macrophage population, which did not reach statistical significance either in the tumour centre or the invasive margin in univariable . When macrophages were classified according to their PD-L1 expression, higher density of PD-L1+ macrophages associated with improved cancer-specific survival in both the tumour centre and the invasive margin in univariable analyses    .We first examined the prognostic role of total  Table . We furt+ T cell density in the tumour centre (Q4 vs. Q1) was 0.46 (95% CI: 0.30–0.71), while the HR for high PD-1− T cell density (Q4 vs. Q1) was 0.48 (95% CI: 0.32–0.70) and the HR for high T cell density (Q4 vs. Q1) was 0.49 (95% CI: 0.33–0.73). Full multivariable Cox regression models with all variables are shown in Supplementary Table Higher T cell density associated with better cancer-specific survival regardless of PD-1 expression in both univariable Fig. , Table 3+ T cell density in tumour groups defined by PD-L1+ macrophage densities or tumour cell PD-L1 expression. These analyses indicated that the survival associations of PD-1+ T cells did not statistically significantly differ according to the density of PD-L1+ macrophages or PD-L1 expression in tumour cells in univariable .Considering potential interactions between PD-1 and PD-L1, we next evaluated the prognostic value of PD-1Ptrend = 0.0004, HR for high (Q4 vs. Q1) 0.54, 95% CI: 0.38–0.78] (Supplementary Table Ptrend > 0.005). To evaluate the prognostic value of PD-L1 expression in differently polarised macrophage subpopulations, we calculated macrophage densities based on subpopulations defined by both PD-L1 expression and polarisation state , while the densities of PD-L1− M1-like macrophages, PD-L1+ M2-like macrophages, or PD-L1− M2-like macrophages did not significantly associate with the survival in multivariable models . These results indicate that higher densities of PD-L1+ and M1-like macrophages associate with favourable prognosis, which is further highlighted when PD-L1 expression and polarisation are examined simultaneously.In secondary analyses, we investigated the prognostic significance of M1-like and M2-like macrophage densities. Higher M1-like macrophage density associated with longer cancer-specific survival in both the tumour centre and the invasive margin in univariable analyses  and M1-like macrophages were 48% closer to tumour cells than M2-like macrophages (P < 0.0001)  or T cells  with tumour cells, as measured with mean NNDs from immune cell to the closest tumour cell, did not significantly associate with cancer-specific survival (P > 0.005)  Fig. . To furt01) Fig. . This pl+ macrophage to evaluate the possibility for PD-1−PD-L1 interactions. We found that PD-1+ T cells were located closer to both tumour cells  and PD-L1+ macrophages  than PD-1− T cells .For T cells, we measured NNDs to the closest tumour cell and to the closest PD-L1lls Fig. . To evalses Fig. . The proIn the present study, we used multiplex immunohistochemistry combined with digital image analysis and quantitative density and spatial analysis to comprehensively characterise the expression of PD-L1 (CD274) and PD-1 (PDCD1) immune checkpoints in the colorectal cancer microenvironment. This analysis, conducted in a large, population-based cohort of 910 colorectal cancer patients expands the knowledge on immune checkpoint expression patterns, their prognostic value, and associations with T cell and macrophage infiltration and may help to develop cancer therapies.+ macrophages in the invasive margin associated with longer colorectal cancer-specific survival independent of stage, grade, and MMR status. Our finding is concordant with some prior studies which have reported association between high PD-L1 expression in immune cells and better survival [+ expression in tumour cells [The expression of PD-L1 and PD-1 is often elevated in cancer and their interaction may repress the cytotoxic activity of T cells and thus promote tumour immune escape . Howeversurvival –23 and aur cells –27. DiveThe detection and phenotyping of macrophages are challenging because of their plasticity and the lack of specific or standardised immunohistochemical markers for various subpopulations . We used+ macrophages were more likely M1-polarised than M2-polarised, which could partly explain their comparable spatial arrangement and prognostic value. The associations of macrophage polarisation phenotypes with PD-L1 expression are not well-characterised, but may be affected by the cytokine environment [+ M1-like phenotype showing the strongest association with a favourable outcome.In analyses combining PD-L1 expression and polarisation status of macrophages, PD-L1ironment . In partironment , 34. Howironment , 35, 36.+ and M1-like macrophages) also showed the strongest associations with favourable clinical outcome. Shorter average distance of M1-like than M2-like macrophages from the closest tumour cell has been reported also in prior studies of gastric [Some recent studies have reported the co-localisation of immune cells with tumour cells to be strongly prognostic , 37–39.  gastric , lung [2 gastric  and panc gastric  cancers.+ and PD-1− T cells showed strong associations with favourable survival, and their HR point estimates were very close to that of the overall T cell population. This supports the strong prognostic significance of T cells in general [+ cell densities and longer survival [Our study addressed the prognostic significance of T cell subsets defined by PD-1 expression. We found that higher densities of both PD-1 general , and indsurvival , 24, 25.+ and PD-L1+) cells, as well as their co-localisation. In line with prior reports [+ macrophage and T cell densities. Furthermore, this correlation was higher for PD-1+ T cells than for PD-1− T cells. A prior study [+ T cell densities associated with favourable outcome regardless of PD-L1 expression in tumour cells or PD-L1+ macrophage densities. Furthermore, higher density of PD-1+ T cell/PD-L1+ macrophage clusters strongly associated with longer cancer-specific survival.To evaluate the possibility of PD-1/PD-L1 interactions occurring in the tumour microenvironment, we assessed the correlations between the densities of immune checkpoint expressing  may associate with better clinical outcomes among patients with a given cancer type. Those apparent paradoxical findings can be explained by interpersonal heterogeneity of cancer [Fusobacterium nucleatum, might associate with T cell count, PD-1 and PD-L1 expression, and colorectal cancer prognosis [In contrast to our hypotheses, higher densities of PD-L1f cancer . Our finf cancer , 38. Favf cancer . Furtherrognosis .To our knowledge, this is the first study examining the PD-L1/PD-1 expression along with macrophage polarisation using multimarker analysis. However, the findings of this study should be interpreted with some caution. First, the analyses were conducted using tissue microarrays, which may not fully represent the immune infiltration in the whole tumour area . To incrBRAF mutation status representing two key molecular features of colorectal cancer. Multiplex immunohistochemistry staining together with machine learning-based image analysis enabled accurate phenotyping of each cell with multimarker combination in a single batch, facilitating the consistency and reproducibility of the analysis. Flow cytometry and RNA sequencing methods are other commonly used myeloid cell phenotyping methods but, unlike multiplex immunohistochemistry, they do not provide the spatial information of the cells.This study has important strengths. Our study included a large, comprehensively analysed , 46–49 pIn conclusion, this study shows that higher density of PD-L1 expressing macrophages and their spatial proximity with PD-1 expressing T cells associate with prolonged survival of colorectal cancer patients. Our results highlight the utility of detailed multimarker analysis in understanding the role of PD-L1 and PD-1 expression in cancer immune escape and developing improved immunotherapies.Supplementary material


O-glycan fragments generated by the in-source decay (ISD) of glycopeptides and glycoproteins containing O-glycans that occurs during intense laser irradiation, enabling direct O-glycan analysis. Furthermore, we demonstrated that it is possible to investigate the internal structure of each O-glycan fragment with pseudo-MS/MS/MS using the sodium adduct ion of the O-glycan-derived ISD fragments from an intact mucin mixture.3-Amino-4-hydroxybenzoic acid (AHB) was the first matrix identified by glycoprotein glycan analysis using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). However, compared to commonly used matrices, such as 2,5-dihydroxybenzoic acid (DHB), AHB is less efficient at glycan ionization and lacks the ability to ionize other molecular species, such as peptides, and thus is no longer used. In this study, we focused on the glycan-selective ionization ability of AHB and its low-noise properties in the low-molecular-weight region, as we expected that these properties could be enhanced by adding sodium to AHB. Sodium-doped AHB (AHB/Na) selectively imparts sodium adduct ions onto  N-glycans and O-glycans. In particular, O-glycans are widely used as clinical CAs because they are diverse near protein binding sites and significantly affect peptide conformation , [Hex + HexNAc], and [Hex] from the intact cAFGPs , respectively. Interestingly, the DAN/DHB/Na and AHB/Na matrices give higher ISD peaks corresponding to the neutral loss of [Hex + HexNAc + H2O] (m/z 1465 and 2073) rather than the neutral loss of [Hex + HexNAc] (m/z 1483 and 2091) from the cAFGPs. This indicates these two matrices promote the release of a free O-glycan moiety due to β-elimination-type cleavage at the threonine side chain during the ISD fragmentation process.Cyclic antifreeze glycopeptides cAFGPs; a 23], w, w23], wdge, DAN ,25 and Ddge, DAN  are suitnt peaks b. This rnt peaks c, DAN/DHnt peaks d, and AHnt peaks e led to m/z 388 for [HexNAc + Hex + Na]+ and an m/z 406 for [HexNAc + Hex + H2O + Na]+ were observed, respectively  derived from N-glycans occurred preferentially [m/z 18) derived from O-glycans were observed.We next focused on forming ISD fragment ions corresponding to the released core 1 disaccharide moiety observed in the low-molecular-weight region . No ISD ectively . Similarentially ,20. In cm/z 406, which was formed due to the ISD of cAFGPs using a sodium adduct ion with DHB, DAN/DHB/Na, and AHB/Na matrices, was 38.7  peak signal d. The siwas 38.7 b, 79.2  without pretreatment and subjected to MALDI-ISD analysis. Unlike the cAFGP analysis, no peptide nor glycopeptide-derived PSM fragment signals were detected with the matrixes tested. As with the cAFGPs, no O-glycan-derived fragment signals were detected with the DAN matrix  and those produced by the cleavage of glycosidic bonds (B ions), supports the attribution of O-glycan-derived ions. Furthermore, C ion fragments were preferentially observed in the larger glycan regions /DHB/Na matrix was also analyzed to demonstrate that the ISD of O-glycans on PSM generated the C ions. As previously reported, the mixture was stored at 60 °C for one hour after mixing with the samples to ensure that the BOA entirely tagged the reducing ends of the free glycans in the samples [O-glycans tagged by BOA were observed in the mixture of the BOA/DHB/Na matrix and PSM. This indicates that all the O-glycan-derived signals observed in the mixture of PSM with the AHB/Na matrix were caused by the ISD of PSM. N: N-acetyl hexosamine; H: hexose, F: fucose (deoxyhexose).The t of PSM ,29. Howe samples . HoweverO-glycan-derived ISD ions observed with the AHB/Na matrix were selected as precursor ions for pseudo-MS3 analysis using MALDI-LIFT-TOF/TOF [m/z 406, which was observed in both cAFGPs and PSM, was selected as the precursor ion. An almost identical fragment pattern of the core 1 disaccharide was observed from the precursor ion formed through ISD in each analyte  yielded signal patterns indicative of sugar composition (3 spectrum of ISD precursor ions corresponding to a B ion (m/z 1102) and C ion (m/z 1120) pair with a hexasaccharide composed of one deoxyhexose, two hexoses, and three N-acetylhexosamines. A comparison of fragment patterns in both spectra shows that B-ion fragments are preferentially produced from B-derived precursor ions, whereas fragments of both B and C ions are observed as coming from C-derived precursor ions. This fragmentation pattern derived from precursor ion pairs with the same glycan composition is expected to contain detailed glycan structural information. Since glycans with free-reducing ends are prone to fragmentation, further pretreatments, such as labeling and reduction, have commonly been used for the MS analysis of released glycans from glycoproteins [O-glycan-derived B and C ions as precursor ions have been limited. As the PSM used in this study is a mixture of multiple mucins and is expected to have structural diversity even within the same glycan composition, a comparison with mucin-type glycopeptides with well-defined glycan structures, such as cAFGPs, will lead to a more detailed structural discussion.In the pseudo-MSposition . Figure proteins ,16. Ther3), O-benzylhydroxylamine (BOA), and acetonitrile (HPLC grade) were purchased from Wako Pure Chemical Industries, Ltd. . 2,2,2-Trifluoroacetic acid (TFA) was purchased from Watanabe Chemical Industry Co., Ltd. . 3-Amino-4-hydroxybenzoic acid (AHB) was purchased from Tokyo Chemical Industry . Additionally, 700 µm and 900 µm STA µFocus plates measuring 24 × 16 c were purchased from Hudson Surface Technology .Type III mucin from porcine stomach (PSM) and 1,5-diaminonaphthalene (DAN) were purchased from Sigma-Aldrich Corp. . DHB, sodium bicarbonate , 50 mM AHB in acetonitrile/H2O , 600 mM BOA in acetonitrile/H2O , 500 mM DHB in acetonitrile/H2O , and 100 mM NaHCO3 in H2O were prepared. The 50 mM AHB was vortexed and sonicated. The DAN/DHB/Na matrix was prepared by mixing 4 μL of 50 mM DAN, 2 μL of 500 mM DHB, and 1 μL of 100 mM NaHCO3 and diluting it to 100 μL with acetonitrile/H2O . The AHB/Na matrix was prepared by mixing 20 μL of 50 mM AHB and 1 μL of 100 mM NaHCO3 and diluting it to 100 μL with acetonitrile/H2O . The DAN matrix was prepared by mixing 20 μL of 50 mM DAN and diluting it to 100 μL with acetonitrile/H2O . The DHB matrix was prepared by mixing 2 μL of 500 mM DHB and diluting it to 100 μL with acetonitrile/H2O/TFA . The BOA/DHB/Na matrix was prepared by mixing 2 μL of 600 mM BOA, 2 μL of 500 mM DHB, and 1 μL of 100 mM NaHCO3 and diluting it to 100 μL with acetonitrile/H2O .Solutions of 50 mM DAN in acetonitrile/HMatrix solutions (0.35 µL) were spotted on the 700 µm or 900 µm STA µFocus MALDI plate , and then analytes of 1 g/L (0.35 µL) were deposited on the pre-spotted plates. The plates spotted with the DAN/DHB/Na, AHB Na, DAN, and DHB matrices were dried at 25 °C. The plates spotted with the BOA/DHB/Na matrix were dried in a thermostatic chamber for 1 h at 60 °C. The morphological image of each matrix on the µFocus plate was taken by polarized camera DZK 33UX250  with polarized filter equipped light source LG-PS2 .All spectra were acquired using a Ultraflex III instrument  equipped with a 200 Hz Smartbeam Nd:YAG laser (355 nm). The ISD spectra from an intact glycopeptide and glycoprotein were acquired in the positive reflectron mode with 1000 laser shots, 80% laser power, and a random walk. The generated ions were accelerated to a kinetic energy of 25.0 kV. The TOF/TOF spectra were acquired with 2400 laser shots in the parent and fragment mode using a random walk. In the LIFT-TOF/TOF mode, the ISD fragment ions were accelerated to 8 kV in the MALDI ion source and selected within a time gate. The selected ISD ion was further accelerated to 19 kV in the LIFT cell. The metastable post-source decay (PSD) ions were analyzed without any additional fragmentation process, such as collision-induced dissociation.3 analysis of intact O-linked glycopeptides and glycoproteins without pretreatment. AHB/Na facilitated the release of free O-glycans through the β-elimination of the serine/threonine residue’s side chains; this gave O-glycan-derived ISD ion peaks with a high resolution and high S/N ratio due to the uniform morphology of the matrix, allowing for high glycan-selective ionization and suppression of total ion production during intense laser irradiation to promote ISD. The ISD products of O-linked glycans were formed as B and C ion pairs. In addition to synthetic O-linked glycopeptides (cAFGPs) with well-defined structures, we have shown that glycan patterns can be detected from mixtures, such as gastric mucins, composed of multiple O-linked glycoproteins without a pretreatment such as digestion or purification. Pseudo-MS3 analysis of the O-glycan-derived B- and C-type ISD ion pairs could provide more detailed structural information about the detected O-glycans. Matrices that selectively ionize peptide and N-glycan fragments from glycoproteins through MALDI ISD analysis have been reported. This study adds AHB/Na as a suitable matrix for the direct MALDI ISD analysis of O-glycans from glycoproteins. These ultra-fast approaches can significantly contribute to the quality control of glycoproteins in pharmaceuticals, the evaluation of biological samples, and glycomics research.In conclusion, AHB/Na allows for the glycan-selective MALDI-ISD analysis and pseudo-MS


SCN5A undergoes foetal-to-adult isoform switching around birth. Conventional hiPSC-CM cultures, which are phenotypically foetal, have thus far been unable to capture mutations in adult gene isoforms. Here, we investigated whether tri-cellular cross-talk in a three-dimensional (3D) cardiac microtissue (MT) promoted post-natal SCN5A maturation in hiPSC-CMs.Human-induced pluripotent stem cell-cardiomyocytes (hiPSC-CMs) are widely used to study arrhythmia-associated mutations in ion channels. Among these, the cardiac sodium channel SCN5A exon 6B and exon 4. Electrophysiological properties of patient hiPSC-CMs in monolayer were not altered by the exon 6B mutation compared with isogenic controls since it is not expressed; further, CRISPR/Cas9-mediated excision of the foetal exon 6A did not promote adult SCN5A expression. However, when hiPSC-CMs were matured in 3D cardiac MTs, SCN5A underwent isoform switch and the functional consequences of the mutation located in exon 6B were revealed. Up-regulation of the splicing factor muscleblind-like protein 1 (MBNL1) drove SCN5A post-natal maturation in microtissues since its overexpression in hiPSC-CMs was sufficient to promote exon 6B inclusion, whilst knocking-out MBNL1 failed to foster isoform switch.We derived patient hiPSC-CMs carrying compound mutations in the adult SCN5A isoform switch in hiPSC-CMs, (ii) adult splicing of SCN5A is driven by MBNL1 in these tissues, and (iii) this model can be used for examining post-natal cardiac arrhythmias due to mutations in the exon 6B.Our study shows that (i) the tri-cellular cardiac microtissues promote post-natal The cardiac sodium channel is essential for conducting the electrical impulse in the heart. Postnatal alternative splicing regulation causes mutual exclusive inclusion of fetal or adult exons of the corresponding gene, SCN5A. Typically, immature hiPSCCMs fall short in studying the effect of mutations located in the adult exon. We describe here that an innovative tri-cellular three-dimensional cardiac microtissue culture promotes hiPSC-CMs maturation through upregulation of MBNL1, thus revealing the effect of a pathogenic genetic variant located in the SCN5A adult exon. These results help advancing the use of hiPSC-CMs in studying adult heart disease and for developing personalized medicine applications.  Among the cardiac diseases that have been modelled using hiPSC-CMs are inherited cardiac arrhythmias caused by mutations in cardiac ion channels.4 The SCN5A gene encodes the pore-forming α-subunit of the cardiac sodium (Na+) voltage-gated channel Nav1.5. This channel conducts the depolarizing Na+ current (INa), which is responsible for the fast upstroke of the action potential (AP) in working cardiomyocytes; it is essential for conducting the electrical impulse in the heart. Mutations in SCN5A are associated with different cardiac diseases, including Brugada syndrome, long QT syndrome, isolated conduction defects, and dilated cardiomyopathy.5 Nav1.5 expression is developmentally regulated, with foetal - or adult isoforms generated by alternative splicing of the SCN5A exon 6 , which encodes the voltage sensor of the first channel subunit.6 The mechanism of SCN5A alternative splicing regulation and other cardiac genes during development has been studied in animal models, where the muscleblind-like RNA-binding protein 1 (Mbnl1) has been identified as key for inducing a post-natal switch.7–9 In humans, MBNL1 is sequestered in the heart of myotonic dystrophy patients causing re-expression of the foetal SCN5A exon 6A10 and the importance of MBNL proteins for late myogenic maturation has been recently demonstrated using hiPSCs.11Human-induced pluripotent stem cells (hiPSCs) are widely used to derive cardiomyocytes (hiPSC-CMs) and study cardiac diseases at the cellular and molecular level.SCN5A or in genes of Nav1.5 beta-subunits12–22 and these are in some cases notably different from those observed in commonly used heterologous expression systems,23 most likely because hiPSC-CMs carry the full complement of cardiac ion channel and accessory protein genes. Moreover, gene editing in hiPSCs allows effects of specific mutations to be distinguished from potential confounding elements resulting from line-to-line variability; patient mutations can be introduced into wild-type (WT) hiPSCs or mutations in patient hiPSC lines can be corrected.24 However, cardiomyocytes differentiated from hiPSCs have an immature  phenotype with their electrical properties, structure, and gene expression profiles resembling first or second gestational trimester foetal cardiac cells26 rather than adult cardiomyocytes. Although INa in hiPSC-CMs display half-maximal potential (V1/2) of activation and inactivation consistent with values reported for native human ventricular myocytes,27 the maximal AP upstroke velocity (Vmax) is slower than in adult cardiomyocytes.28 In addition, hiPSC-CMs mostly express the Nav1.5 foetal isoform, masking effects due to mutations located in the adult exon 6B.29 Previously, we and others addressed the issue of SCN5A in vitro maturation by culturing hiPSC-CMs for long periods to promote spontaneous maturation29 and by using a medium inducing a metabolic switch from glycolysis to fatty acid oxidation.20 We recently discovered that hiPSC-CM maturation is overall enhanced in a three-dimensional (3D) microtissue (MT) environment containing cardiac fibroblasts (CFs) and endothelial cells (ECs).30 hiPSC-CMs isolated from MTs have more hyperpolarized RMP and faster AP upstroke velocity, suggesting an electrical maturation involving Nav1.5.hiPSC-CMs can recapitulate many electrical alterations due to mutations in SCN5A adult isoform and allowed (i) evaluation of functional effects of the p.R225W mutation, located in adult exon 6B and (ii) detection of the more severe disease phenotype associated with the compound heterozygosity for p.W156X mutation,13 as observed in patients.31 Using gene-corrected isogenic hiPSCs was crucial in identifying mutation-specific effects. We also found that MBNL1 up-regulation in MTs was necessary and sufficient to induce the expression of SCN5A exon 6B.Here, we showed that maturation of hiPSC-CMs in MTs promoted expression of the 2.2.131 The proband patient , born with a severe tachycardia and conduction disorder, was treated with beta-blockers and later fitted with an ICD implant. Despite the ICD, he died from intractable ventricular arrhythmias at age 21. The parents and a sister  of the proband were asymptomatic, whereas the other sister  carrying the same mutations of the proband was affected by intractable arrhythmia and died at age of 1.The clinical characteristics of the family were previously reported.2.213 following the protocol approved by the LUMC and AMC ethical committees. Details for hiPSC culture and differentiation are reported in the A skin punch biopsy was obtained following written informed consent from the patient at the age of 18 and approval by the medical ethics committee of the Amsterdam Medical Center (AMC), in accordance with the Declaration of Helsinki. Dermal fibroblasts were reprogrammed using the Sendai virus by the Leiden University Medical Center (LUMC) hiPSC core facility, as described previously,2.3Figures 1E and 3A, Table S1) were cloned into the pSpCas9(BB)-2A-Puro (PX459) v2 plasmid encoding Cas9 nuclease and puromycin resistance (Addgene #62988).32 For the correction of c.673C > T mutation in exon 6B (p.R225W) and the excision of exon 4 of MBNL1, the Alt-R® CRISPR–Cas9 System (IDT Technologies) was used. Specific single-stranded DNA oligonucleotide (ssODN) repair template carrying the WT base and silent mutations were used for each mutation correction (see Table S1). Additional information is provided in the For the correction of c.468G>A mutation in SCN5A exon 4 (W156X) and excision of SCN5A exon 6A specific sgRNA sequences , as previously described.2.533 Cells were plated on Matrigel-coated coverslips in LI-BPEL supplemented with 50 ng/mL VEGF and 5 ng/mL bFGF, refreshed with LI-BPEL medium the next day, and analysed for electrophysiological properties 7–10 days after dissociation.Beating monolayers of hiPSC-CMs were dissociated at day 21 of differentiation using Tryple Select 0.5× (ThermoFisher Scientific) and plated onto Matrigel-coated glass coverslips at low density. Electrophysiological properties of hiPSC-CMs were analysed 10 days after dissociation. MTs were dissociated at day 21 of culture using collagenase II (Worthington Industries), as previously described.INa) was recorded with the ruptured patch-clamp technique in voltage-clamp mode. Patch-clamp solutions and protocols are reported in the Patch-clamp experiments were performed using an Axopatch 200B amplifier . Clampex 10.7 was used for data acquisition, voltage control, and analysis. APs were recorded in isolated single cells using the perforated patch-clamp technique in current-clamp mode and sodium current , or from MTs on day 21 of culture using RNeasy Micro Kit (Qiagen), according to the manufacturers’ instructions. cDNA was synthesized using iScript™ cDNA Synthesis Kit and gene expression was analysed by qPCR with IQ Syber Green Supermix (Bio-Rad), according to the manufacturer’s protocol. All qPCR reactions were performed in duplicate. Gene expression levels were normalized to the Table S2 and S3.For quantification of mRNA-splicing isoforms containing 6A or 6B exons, BioRad QX200™ Droplet Digital PCR (ddPCR) system was used following the manufacturer’s instructions. Primer and probe sequences used for qPCR and ddPCR are reported in 2.734 with parameter -m 20. Reads were aligned to the human genome  using STAR.35 BAM files were used by rMATS36 to detect differentially spliced exons and sashimi plots were made using the function rmats2sashimiplot (https://github.com/Xinglab/rmats2sashimiplot).The RNA was extracted with the RNeasy micro kit (QIAGEN) as from the manufacturer’s instructions, followed by paired-end sequencing at 150 bp, 40 million reads for each sample . Residual Illumina adapters were trimmed from the RNA sequences using cutadapt37 with parameters -t exon and -g exon_id. Transcripts per kilobase million (TPM) was calculated considering all exons counts as library size and by using the R script convertCounts. The coverage of constitutive and alternative exons for SCN5A or MBNL1 was calculated as percentage of TPM assigned to each exon bin over the TPM mean of the whole transcript.Exon counts for the SCN5A and MBNL1 canonical transcripts, ENST00000413689 and ENST00000282486, respectively, were retrieved using featureCounts38) were used for Figure S7A.RNA-seq data are available in the GEO repository with accession number GSE180290. Publicly available foetal heart (FH) and adult heart (AH) RNA-seq data  Polymerase Tailing Kit  following the manufacturer’s instructions.MBNL1 mRNA was generated by hiPSC-CMs were transfected with MBNL1 mRNA using Lipofectamine Stem Transfection Reagent (Invitrogen) according to the manufacturer’s instructions. Details are reported in the 2.9t-test or one-way ANOVA followed by Fisher LSD post hoc test. Statistical analysis was performed using OriginPro 2016 (Origin Lab), RStudio, or GraphPad Prism .Data were obtained from at least three independent differentiation- or MT formation experiments and were expressed as mean ± standard error of the mean (SEM). Data were compared using 3.3.1Figure 1A, II-3) presented with severe cardiac conduction abnormalities and ventricular arrhythmias early after birth and was genetically characterized as carrying bi-allelic mutations in the SCN5A gene.31 These were a c.468G>A nucleotide change in exon 4 of SCN5A (NM_198056.2) inherited from the father, leading to the stop codon mutation p.W156X (NP_932173.1), and a c.673C>T nucleotide change in exon 6B of SCN5A inherited from the mother. This led to the substitution of a positively charged arginine with an aromatic tryptophan (p.R225W) in the voltage-sensor segment of the first domain of the channel . When the patient turned 18, we reprogrammed dermal skin fibroblasts using the Sendai virus to generate the hiPSCW156X/R225W line. Sanger sequencing confirmed the presence of both SCN5A mutations . After several passages, hiPSCs were negative for the Sendai virus (see S1A), had a normal karyotype , expressed the pluripotency markers NANOG, SSEA4, POU5F1 , and genome-wide gene expression analysis indicated high ‘pluripotency score’ and low ‘novelty score’ with the PluriTest algorithm39 .A patient ,13 the phenotypic effects of the p.R255W Nav1.5 mutation was only studied in heterologous system31 since hiPSCs from the proband’s mother were not available. To examine the effects of this mutation specifically, we corrected the c.468G>A SCN5A mutation in the hiPSCW156X/R225W line using CRISPR–Cas9 and generated the hiPSCcorr/R225W line . One hiPSC clone was selected, in which Sanger sequencing confirmed the correction of the c.468G>A SCN5A mutation and the presence of the silent mutations in heterozygosity . No modifications were present in the in silico-predicted two intragenic Cas9 off-target sites by Sanger sequencing (data not shown). hiPSCcorr/R225W had a normal karyotype  and high pluripotency score, as confirmed by PluriTest . The original hiPSCW156X/R225W line was heterozygous for the rs6781731 SNP located in the intron downstream exon 4. The corrected hiPSCcorr/R225W was also heterozygous for this SNP, confirming that the recombination involved only the paternal allele as intended .While the p.W156X Nav1.5 mutation was previously characterized in a heterologous system3.2W156X/R225W and hiPSCcorr/R225W lines into cardiomyocytes (CMs) in monolayer . Differentiation efficiencies were comparable between the two lines and consistently yielded >90% cardiac troponin T (TNNT2)-positive cells (see S2A). The cardiac ion channel genes SCN5A, NCX1, KCND2, KCNH2, KCNQ1, and KCNJ12 were expressed at similar levels in CMs from hiPSCW156X/R225W and hiPSCcorr/R225W (see S2B).We differentiated hiPSCells see S2A. The 225W see S2B.W156X/R225W and hiPSCcorr/R225W lines. Figure 2A shows representative INa traces from hiPSCW156X/R225W- and hiPSCcorr/R225W-CMs. While the voltage dependence of INa activation and inactivation was similar between the two lines , INa density was lower in hiPSCW156X/R225W-CMs over the whole physiological voltage range , with the mean peak current density in hiPSCW156X/R225W-CMs (−31 ± 4.5 pA/pF) significantly reduced compared with hiPSCcorr/R225W-CMs (−94.2 ± 11.4 pA/pF) . Notably, the current density recorded in hiPSCcorr/R225W-CMs was comparable to that previously reported (∼ −100 pA/pF) for CMs differentiated with the same method from a WT hiPSC line (hiPSCWT/WT), whereas INa density in hiPSCW156X/R225W-CMs was similar to that recorded in hiPSC-CMs carrying only the p.W156X mutation (∼ −30 pA/pF) derived from the proband’s father .13We next examined the electrophysiological properties of CMs from hiPSCINa impacts the electrical activity of cardiomyocytes, particularly the AP upstroke velocity. We, therefore, recorded APs in hiPSCW156X/R225W- and hiPSCcorr/R225W-CMs paced at 1 Hz. To compensate for the typical depolarized RMP of foetal-like hiPSC-CMs, we used dynamic clamp to set the RMP to −86 mV.40Figure 2E shows representative AP traces recorded from hiPSCW156X/R225W- and hiPSCcorr/R225W-CMs, and their respective first derivative trace. In agreement with the differences in INa density, the maximal first derivative of AP upstroke, corresponding to the maximal upstroke velocity (Vmax), was significantly lower in hiPSCW156X/R225W- compared with hiPSCcorr/R225W-CMs . As observed for INa, Vmax values from hiPSCcorr/R225W-CMs were similar to those previously reported for WT hiPSC-CMs using the same dynamic clamp technique.41 Other AP parameters, namely RMP, AP amplitude (APA), and AP duration (APD), were not different between the two lines (see S2C).Altered ines see S2C.INa and Vmax, we measured exon 6A and 6B expression in CMs from hiPSCW156X/R225W and hiPSCcorr/R225W lines. Using digital droplet PCR (ddPCR) we quantified exon 6A- and exon 6B-containing transcripts (see S2D and E). Figure 2G illustrates representative ddPCR results for hiPSCW156X/R225W- and hiPSCcorr/R225W-CMs. Exon 6B-containing SCN5A transcripts accounted for approximately 5% and were much lower than exon 6A-containing transcripts . In FH and AH tissues, exon 6B-containing transcripts accounted for 50% and 94% of all transcripts, respectively, in line with previous reports,42 and thus were expressed at higher levels than in hiPSC-CMs .To check whether both mutations contributed to ipts see S2D and EW156X/R225W and hiPSCcorr/R225W-CMs expressed the foetal SCN5A isoform almost exclusively and INa density and Vmax reduction in the compound mutant was due to the p.W156X Nav1.5 mutation. The effect of p.R225W mutation could not be properly determined.These results indicated that hiPSC3.3SCN5A exon 6B expression and thus reveal the effect of the p.R225W Nav1.5 mutation, we used CRISPR–Cas9 to excise this exon from both SCN5A alleles in the hiPSCW156X/R225W line. As illustrated in Figure 3A, we designed 2 gRNAs targeted to the flanking regions of exon 6A and identified a clone in which exon 6A had been excised in both alleles. The actual excision spanned a larger intronic region than intended by the strategy design . In the hiPSC lines with complete exon 6A excision (referred to as 6A-KO hiPSCW156X/R225W), we corrected the p.W156X mutation in exon 4 (generating the 6A-KO hiPSCcorr/R225W line), using CRISPR–Cas9 with the same strategy as earlier . The excised hiPSC lines differentiated into CMs with similar efficiencies and beating properties as the parental hiPSC line (data not shown). As expected, exon 6A was not expressed at all in excised CMs at 21 or 60 days of culture, whereas expression of exon 6B was detected (see S3B). To examine SCN5A transcripts expressed by CMs from the excised hiPSC lines, we amplified by PCR a region between exon 3 and exon 8 of SCN5A . Here, we only used the 6A-KO hiPSCcorr/R225W line, since in the 6A-KO hiPSCW156X/R225W line, the stop codon p.W156X mutation in exon 4 leads to nonsense-mediated mRNA decay13 and could confound the results. Gel electrophoresis of PCR products revealed three distinct bands . Sanger sequencing demonstrated these corresponded to three different transcript species: the highest band represented the adult isoform of SCN5A transcript including exon 6B; the middle band, a transcript in which exon 6B was skipped, and in the lowest band, both exons 5 and 6B were skipped .To investigate whether the removal of exon 6A might force cted see S3B. To eSCN5A isoform was increased compared with the non-excised line, we performed ddPCR analysis, normalizing the expression of exon 6B with that of the TATA-binding protein (TBP). Exon 6B expression was similar in the excised hiPSC-CMs and in the parental non-excised hiPSC-CMs . Since exon 6B-containing transcripts were likely the only functional transcript species produced in the exon 6A-excised lines, we investigated whether this would unmask the functional phenotype of the p.R225W Nav1.5 with electrophysiology. We attempted to record INa in 6A-KO hiPSCW156X/R225W- and 6A-KO hiPSCcorr/R225W-CMs but INa was virtually absent in all (n = 9) 6A-KO hiPSCW156X/R225W-CMs , while 50% of the 6A-KO hiPSCcorr/R225W cells (n = 10) showed very small INa  with mean peak current density of −12.9 ± 5.5 pA/pF. Analysis of SCN5A transcript expression by RNA-seq showed that upon excision of exon 6A, not only was there spurious mRNA produced but also the intronic region between exon 5 and exon 6B was aberrantly included at a similar level to the exons .To investigate whether the overall expression of the adult SCN5A exon 6A impairs the splicing mechanism, leading to abnormally spliced transcripts of unknown significance and the inclusion of intronic regions in the transcript population. Importantly, 6B-containing transcript levels were not sufficient to generate detectable INa for comparison of the two mutant lines.Taken together, these results demonstrated that excision of 3.433 could promote SCN5A exon 6B expression and thus reveal the effect of the p.R225W mutation. The cardiac MTs were generated using defined ratios of the three cell types as shown in Figure 4A and dissociated into single cells after 21 days of culture to analyse INa by patch clamp . Since INa had not been characterized in hiPSC-CMs derived in this way before, we used hiPSC-CMs from hiPSCWT/WT as a process control.43 In addition, we corrected the p.R225W mutation in hiPSCcorr/R225W, generating the isogenic control hiPSCcorr/corr (see S4A). Correction of the mutation in hiPSCcorr/corr was confirmed by Sanger sequencing (see S4B). The line had a normal karyotype and differentiated efficiently into cardiomyocytes (see S4C and D). Four groups of MTs were analysed, containing CMs from hiPSCW156X/R225W, hiPSCcorr/R225W, hiPSCcorr/corr . INa current density showed instead a genotype-specific reduction, with INa significantly lower along the physiological voltage range in hiPSCW156X/R225W-CMs compared with all the other lines, and significantly lower in hiPSCcorr/R225W- compared with hiPSCcorr/corr and hiPSCWT/WT-CMs .We next tested whether hiPSC-CM maturation in cardiac MTs containing hiPSC-derived CFs and cardiac ECs,corr see S4A. Corrcing see S4B. The ytes see S4C and Dorr/corr , and hiPINa on the AP. Figure 4F shows representative AP traces and the derivative of the AP upstroke recorded in hiPSC-CMs dissociated from MTs and paced at 1 Hz. Vmax was significantly smaller in hiPSCW156X/R225W-CMs compared with CMs from the other lines , showing that the compound mutations have an additive effect on INa density, thus impacting on CM electrical activity. Other AP parameters such as spontaneous resting membrane potential, APA and APD were instead similar in the different lines (see S5).Finally, we examined the effect of the altered ines see S5.3.5W156X/R225W- and hiPSCcorr/R225W-2D CMs and 3D MTs after 21 days of culture. Gene ontology (GO) analysis showed that genes related to GO terms of heart development and contraction had a similar expression in the two hiPSC lines, both in 2D CMs and in MTs (see S6A), indicating that the cells had reached similar levels of differentiation and maturation. Our single-cell (sc) RNA-seq datasets from MTs and hiPSC-CMs previously published from the same WT line used here confirmed that SCN5A is only expressed in the hiPSC-CM population .30 Analysis by ddPCR showed that MTs from hiPSCW156X/R225W, hiPSCcorr/R225W, and hiPSCWT/WT expressed similar fractions of SCN5A exon 6B , which accounted for around 27% of total SCN5A transcripts . Notably, the fraction of exon 6B-containing transcripts was 5–6 times higher in MTs from hiPSCW156X/R225W and hiPSCcorr/R225W compared with their respective hiPSC-CMs derived in monolayer . RNA-seq analysis confirmed that exon 6B expression was increased in MTs compared with monolayer hiPSC-CMs in both lines . Overall SCN5A expression was also higher in MTs compared with monolayer hiPSC-CMs in both lines , in agreement with scRNA-seq in hiPSCWT/WT (see S6C) and confirmed by qPCR . The total expression of exon 6 (6A+6B) was similar to upstream and downstream exons; however, the fraction of exon 6B in MTs was higher than in monolayer hiPSC-CMs in both lines . Exon 6B fraction was nonetheless lower in MTs compared with FH and AH (see S7A). Interestingly, SCN5A expression was not increased in MTs containing 6A-KO hiPSC-CMs and the expression of exons downstream of exon 6B was drastically reduced, confirming that 6A excision impaired splicing in the entire gene (see S7B).To obtain a more general overview of gene expression, we performed bulk RNA sequencing in hiPSC MTs see S6A, indiT/WT see S6C and cqPCR see S6D, leftd AH see S7A. Integene see S7B.3.69 We, therefore, investigated whether MBNL1 is involved in hiPSC-CM alternative splicing.The RNA-binding protein Mbnl1 has been previously shown to promote exon 6B- and inhibit exon 6A expression in the mouse heart.MBNL1 was up-regulated in MTs assembled using hiPSCW156X/R225W- and hiPSCcorr/R225W-CMs compared with 2D hiPSC-CMs cultures . Although we found MBNL2 was also up-regulated in MTs (see S6D), scRNA-seq from our dataset previously published showed that MBNL1 but not MBNL2 was up-regulated in the CM population of MTs compared with monolayer hiPSC-CMs (see S6C), suggesting a major role of MBNL1 specifically in CMs. In addition, MTs displayed skipping of MBNL1 exon 5, which is promoted by MBNL1 itself in adult CMs44  and thus suggesting that MBNL1 is functional in MTs.RNA-seq analysis showed that  MTs see S6D, scRN-CMs see S6C, suggMBNL1 in hiPSC-CMs. First, we transiently transfected hiPSC-CMs with an mRNA construct encoding MBNL1 and eGFP. Transfection efficiency reached around 50%, as shown by FACS analysis of eGFP+ cells  and overexpression of MBNL1 in transfected hiPSC-CMs compared with untransfected cells was confirmed by qPCR . ddPCR showed that the exon 6B fraction was increased in MBNL1-transfected hiPSC-CMs to levels comparable with those in MTs . We then knocked out MBNL1 in hiPSCs (MBNL1-KO hiPSCs) (see S8). Cardiac in vitro differentiation was not affected in MBNL1-KO hiPSCs, but MBNL1 expression in 2D hiPSC-CMs was negligible and did not differ between unexcised and MBNL1-KO lines . When included in MTs, MBNL1-KO hiPSC-CMs did not up-regulate MBNL1, differently from the unexcised line . Moreover, exon 6B fraction in MBNL1-KO MTs was comparable to the 2D cultures by ddPCR analysis, and significantly lower than in MTs composed by unexcised hiPSC-CMs .To test whether MBNL1 is necessary and sufficient to promote exon 6B inclusion, we overexpressed or knocked out SCs) see S8. CardiSCN5A exon 6B and its regulator MBNL1. This was sufficient to reveal the functional defects caused by the p.R225W mutation on INa and to dissect the different contributions of p.W156X and p.R225W Nav1.5 mutations to the disease in the patient.Taken together these results demonstrated that 3D cardiac MT constructs composed of hiPSC-cardiac ECs and -CFs promoted alternative splicing maturation in hiPSC-CMs such that they displayed increased expression of 4.45 Alternative splicing is an important mechanism in the post-natal transition and applies to many cardiac genes,46 including the cardiac sodium channel Nav1.5.29 The switch between the foetal SCN5A isoform containing exon 6A and the adult SCN5A isoform containing exon 6B is completed postnatally in mice and humans.47 Immature hiPSC-CMs predominantly express the foetal SCN5A transcript. Although this does not preclude evaluating the functional effects of SCN5A mutations using hiPSC-CMs in general, it does represent a hurdle for those mutations located in exon 6B which is expressed only postnatally. Here, we studied a patient who developed severe cardiac conduction defects early after birth and carried compound mutations in Nav1.5 (p.W156X and p.R225W), one of which (p.R225W) located in the adult exon 6B. Clinical characterization of the family indicated that the cardiac disorder was present only in individuals with compound heterozygosity for the two mutations, with individuals carrying only one of the two Nav1.5 mutations being asymptomatic.31 This suggests an additive effect of the two mutations. To dissect the contribution of each mutation, we generated a hiPSC line from the patient and an isogenic corrected line carrying only the mutation in SCN5A exon 6B (hiPSCcorr/R225W). However, when we recorded INa in hiPSCcorr/R225W-CMs, current density was similar to that measured in WT hiPSC-CMs, whereas the compound mutant showed a reduction of INa density similar to that previously observed in hiPSC-CMs with only p.W156X mutation.13 The lack of effect of p.R225W contrasts with the strong reduction in current density shown in Xenopus laevis oocytes overexpressing the same p.R225W Nav1.5 mutation31; this could be partly due to differences between heterologous expression systems and human cells. Interestingly, the single mutation effect in patients was negligible, suggesting that there might be differences between in vitro systems and the patient phenotype. Analysis of SCN5A exon expression revealed that only a small fraction of exon 6B-containing transcripts was expressed in hiPSC-CMs, in line with previous reports in independent hiPSC lines.29 This explained the functional results and confirmed that the immature hiPSC-CM phenotype precludes analysis of mutations located in the adult SCN5A isoform.The mammalian heart undergoes extensive functional modification after birth, regulated by changes in gene expression at both transcriptional and post-transcriptional level.Scn5a exon 6B resulted in selective re-expression of foetal Nav1.5 in mice,48 our complementary approach of SCN5A exon 6A excision in hiPSCs did not lead to selective expression of the adult Nav1.5. Indeed, 6A-KO hiPSC-CMs expressed similar (low) amounts of SCN5A exon 6B compared with the original non-excised lines and presented negligible INa. This demonstrates that the exon 6B expression levels in standard monolayer hiPSC-CM cultures are not sufficient to generate INa and that INa measured in non-excised hiPSC-CMs is mainly conducted by the foetal channel. The extremely low (or negligible) INa in 6A-KO hiPSC-CMs is likely a consequence of very few functional channels formed, because 6A excision interferes with the splicing machinery, as shown by the inclusion of intronic regions and exclusion of exon 5. This indicates that some regulatory sequences important for correct splicing might be present either within the intronic region removed by the excision, or in exon 6B itself, driving its own exclusion during splicing. Further studies with consecutive removal of different sequences within the 6A region are needed to elucidate the exact mechanisms underlying the regulation of SCN5A splicing. However, the switch between exon 6A and exon 6B is likely to need not only dedicated regulatory sequences, but also specific factors which may be lacking in immature CMs.Remarkably, while genetic excision of 50 We previously showed that hiPSC-CMs in our cardiac tri-cell-type MTs undergo maturation at the functional, structural, metabolic, and gene expression levels30; however, we did not examine whether it involved alternative splicing transition. Both ddPCR and RNA-seq revealed that in cardiac MTs there is a significant increase in the fraction of SCN5A exon 6B- vs. exon 6A-containing transcripts. A similar switch between SCN5A exon 6A and exon 6B was also previously promoted by culturing hiPSC-CMs in monolayer for extended periods.29 Of note, cardiac MT maturation is less time-consuming than prolonged monolayer culture (40 days vs. more than 60), and the number and ratio of cells in MTs remain unchanged during the 21 days of culture required for maturation.30 In our experience, long-term culture of hiPSC-CMs was accompanied by some cell loss, the proliferation of non-cardiomyocyte cells and the risk of detachment of the beating monolayer from the dish. Importantly, cardiac MTs showed higher expression of the exon 6B fraction  compared with long-term monolayer hiPSC-CM culture .29 Nevertheless, levels of exon 6B expression comparable to adult human heart were not reached, suggesting that splicing regulation may still differ between in vitro and in vivo conditions. It remains to be investigated whether the effects of exon 6B mutation are revealed in other 3D or monolayer maturation systems or media.We and others developed different systems to improve the maturation of hiPSC-CMs using more physiological environments involving 3D engineering and/or co-culture with other (cardiac) cell types.30 we could analyse the electrical properties of single CMs. This revealed the contribution of p.R225W Nav1.5 mutation to the disease phenotype, uncovering the gene-dosage relationship and linking the presence of one or two mutated SCN5A alleles with a mild (∼45%) or severe (∼70%) INa reduction, respectively. Reduced INa density but no differences in the channel kinetics of activation or inactivation were measured in the p.R225W hiPSC-CMs compared with the corrected or WT hiPSC-CMs, much as has been observed for another mutation (p.I230T) in the same exon.29 A positive shift in the voltage dependence of INa activation was instead measured in the double mutant hiPSC-CMs, similar to the effects of the homozygous p.I230T Nav1.5 mutation.29 This shift can be attributed to the p.R225W mutation carried on the maternal—and only functional—allele, since the p.W156X mutation likely generates a non-functional protein, due to the stop codon in the paternal allele. In hiPSCcorr/R225W, this change was instead masked by the corrected  allele. A shift in the voltage dependence of p.R225W Nav1.5 was also observed in transfected X. laevis oocytes31; however, this was more pronounced and involved both activation and inactivation, underscoring the fact that ion channel properties are influenced by species-specific cellular context (for example ancillary proteins/subunits).The possibility to derive all cardiac MT cell components from hiPSCs allowed us to include either mutant, corrected isogenic, or WT hiPSC-CMs while the other cell types derived from the same (WT) stock, avoiding possible confounding factors. Since hiPSC-CM maturation achieved in MTs is maintained after dissociation,Mbnl1-knockout mice expressed only the foetal isoform of SCN5A in the heart.10 Here, we showed that MBNL1 expression is low in 2D hiPSC-CM monotypic cultures, while its expression increases significantly when CMs are included in MTs. Interestingly, only MBNL1 was specifically up-regulated in the CM population, and not MBNL2, another member of the same family. MBNL1 is also characterized by different developmentally regulated splicing isoforms and it is known to regulate its own splicing.51 In particular, exon 5 is included in the embryonic isoform and is gradually excluded postnatally.52 In MTs, we observed reduced expression of MBNL1 exon 5 compared with monolayer hiPSC-CMs, suggesting a functional change in MBNL1. Overall this indicates that MBNL1 is up-regulated and alternatively spliced in mature hiPSC-CMs and it might be specifically required to induce the switch in SCN5A isoform. Indeed, transient overexpression of MBNL1 increased SCN5A exon 6B fraction in hiPSC-CMs and lack of MBNL1 prevented splicing isoform switch in MTs. This confirms a key role for this factor in regulating SCN5A splicing also in human cells. Since we performed transient expression of MBNL1 in hiPSC-CMs, it remains to be elucidated whether the sole expression of the adult MBNL1 is sufficient to promote functional maturation of Nav1.5. Moreover, further studies are needed to understand how MBNL1 overexpression and knockout influence the expression of other target genes beside SCN5A. Indeed, Mbnl1 was shown to regulate the post-natal isoform switch of many other genes during mouse heart development7–9 and to repress human stem cell pluripotency while promoting differentiation.54 The expression of adult MBNL1 in hiPSC-CMs within the MT opens the possibility to study other developmentally/postnatally regulated genes using hiPSC-derived cells. The alternative splicing of the region of the voltage-sensing S4 segment is conserved also in voltage-gated sodium channels expressed in the brain, generating different isoforms underlying functional differences between foetal and adult tissues.55 It is tempting to speculate that promoting hiPSC maturation in 3D organ-specific tissues might facilitate the study of adult disease phenotypes in vitro in other organs.The comparison between immature hiPSC-CMs and more mature CMs derived from MTs allowed us to investigate the mechanism underlying the switch between foetal and adult SCN5A isoforms. Mbnl1 was previously identified as necessary for exon 6B expression in mice, as the SCN5A isoform through MBNL1 regulation and thus reveal mutation contributions, allowing dissection of ionic current changes that cause adult arrhythmic disease phenotypes in humans.In conclusion, our data demonstrate that a post-natal maturation of hiPSC-CMs is required to express the adult Cardiovascular Research online.M.B., C.L.M., and G.C.: conceptualization; G.C., G.K, A.O.V, D.O., and H.M.: formal analysis; C.L.M., M.B., and G.C.: funding acquisition; G.C., G.K., D.W.-v.O., L.Y., and A.O.V.: investigation; D.W.-v.O., M.B., and G.C.: methodology; M.B.: project administration; A.A.M.W., C.R.B., and C.C.V.: resources; M.B., A.O.V., R.P.D., and V.V.O.: supervision; and G.C., M.B., and C.L.M.: writing—original draft.cvac059_Supplementary_DataClick here for additional data file.


Caenorhabditiselegans is an excellent genetic model system with a large arsenal of forward and reverse genetic techniques. However, not all approaches are easily ported to relatedCaenorhabditisspecies (which are useful for gene conservation and gene pathway evolution studies). For CRISPR/Cas9 genetic editing, an easily screenable and dominant co-transformation marker is required – a secondary mutation that won’t impact the phenotype of a desired mutation but is capable of being screened for in heterozygous mutants. We describe here the adaptation of a dominant dumpy/roller CRISPR/Cas9-induced mutation in theC. tropicalisdpy-10orthologue. Developing genomic editing tools in the non-model system Caenorhabditis tropicalis.Drosophila melanogasterandCaenorhabditis eleganshave a wealth of genetic and molecular tools at their disposal for transgenesis, cell or gene product tagging, or the targeted alteration of gene expression and function, most species do not. In these cases, there are limited options available for developing gene editing pipelines, due to the lack of robust techniques and controls.The availability of genome sequences from a wide variety of species has allowed the field of evolutionary developmental biology to thrive. However, while the well-established model metazoans such asCaenorhabditis tropicalisis one such example. A relative ofC. elegans, it is of particular interest for the study of traits such as population genetic diversity, selfish elements, and the evolution of self-fertility from strictly outcrossing ancestorsC. elegans,C. tropicalisis easy and inexpensive to work with and maintain in the lab, possesses a transparent body with easily identifiable internal structures, is self-fertile with a short generation cycle of approximately 3.5 days, and has a sequenced genomeThe androdioecious nematodeC. tropicalis. Here we report the adaptation of a tool fromC. eleganswhere we use CRISPR/Cas9 to alter theC. tropicalisorthologue ofdpy-10into a dominant marker, to improve the identification of successful gene editing of unlinked recessive targets.However, neither genetic mutations nor transgenic lines have yet been reported forDpy-10 as a co-transformation marker for CRISPR/Cas9 use in Caenorhabditis.C. tropicalis, we required a gene that can be co-transformed in a manner that produces a dominant phenotype, so that heterozygous F1 mutants can be easily screened for amongst their wildtype siblingsC. elegansandC. briggsae, isdpy-10Injections of CRISPR/Cas9 reagents in the gonadal arms generally lead to genetic edits in haploid oocytes, which are then fertilized by non-edited haploid sperm to produce heterozygous, diploid zygotesCaenorhabditisworms,dpy-10can be used as a dominant co-transformation marker for CRISPR/Cas9, as the dumpy and/or roller phenotype does not affect their viability or fertilitydpy-10C. elegansgene can produce a dominant mutation, where the heterozygotes roll along the long axis of their body - a roller phenotype - and where the homozygotes appear shorter and fatter than roller or wildtype worms - a dumpy phenotype . We wanted to test whether theC. tropicalis dpy-10orthologue could similarly be edited as a dominant co-transformation marker.For hermaphroditicIdentification of the C. tropicalis dpy-10 orthologue.Ctr-dpy-10orthologue, and reciprocal BLASTP identifiedC. elegansandC. briggsaeDPY-10 as the strongest matchesDPY-10orthologues across multipleCaenorhabditisspecies andPristionchus pacificusC. elegansproteinC. elegansCaenorhabditisandPristionchusspecies  as a repair templateC. elegansHDR CRISPR/Cas9 protocol in related nematode speciesBased on the success of theC. tropicalisJU1373young adult hermaphrodites using the CRISPR/Cas9 editing mix, from 3 of the injected P0 worms, 12 out of 67 total adult F1 offspring presented a roller phenotype, which indicates an editing efficiency similar to the previously reported 16-18% positive HDR CRISPR/Cas9 rates inC. eleganszygotesCtr-dpy-10mutation (Ctr-dpy-10CRISPR edit (designateded73), which had the predicted effect of substituting the arginine codon CGT to the cysteine codon TGC, producing a dominant dumpy phenotype  at the University of Minnesota. C. tropicalishermaphrodites were cultured on NGM agar plates streaked withE. coliOP50, as described forC. elegansC. tropicalis DP485ed73)dpy-10, which acted as the “Search Set”, to identify the specific region to targetJU1373-3.0.1GCA_000186765.1 genome scaffold assembly was submitted to CRISPOR, after which the identifiedCtr-dpy-10region was analyzed. The guide sequence was selected based on three criteria: high “MIT Specificity Score”, high “CFD Spec. Score”, and PAM sequence being within 5 bp of the intended edit site. CRISPOR was used to help design and evaluate the on/off target potential of guide sequences forC. tropicalis CRISPR/Cas9PredictedCtr-dpy-10primers (below) were used to amplify the predicted CRISPR-edited region for sequencing.Ctr-dpy-10Guide Sequence:CGTACAGCTCGTGGTGCATACtr-dpy-10ssODN:TTCAATACGGAAAAATGAAACTTTCTGGAAATCGTACAGCATGCGGTGCATATGGCAGTGGAGCTTCTCATGGTTTTAGACCAACTGCTCtr-dpy-10 Forward Primer:AACTATTCGCGTCAGATGATCtr-dpy-10 Reverse Primer:CTCCATAAGCAGTTGGTCTAIdentification of the C. tropicalis dpy-10 orthologue.DPY-10predicted protein sequences forC. elegansandC. briggsae, collected from WormBase, were used as the “Query Sequence” for TBLASTN, and a Whole-genome Shotgun Contigs (wgs) database forCaenorhabditis tropicaliswas used as the “Search Set”. No algorithm parameters were altered. TheC. tropicalis JU1373accession set AEKS01003488.1: Contig629.1541 was identified as the container for theCtr-dpy-10orthologue and was chosen based on possessing the following elements: highest “Max Score”, “Total Score”, and “Query Cover”; and lowest “E-value”C. tropicalis NIC203 accession set was used to determine that theCtr-dpy-10gene is syntenic with theC. elegansandC. briggsaedpy-10 orthologues.C. elegansorC. briggsae was used as the “Search Set”. No algorithm parameters were altered.A reciprocal best hit strategy was also implemented: the CTR-DPY-10 predicted protein sequence, collected from WormBase-Compara, was used as the “Query Sequence” for BLASTP, and a “Non-Redundant Protein Sequences (nr)” database forHomology-directed repair CRISPR/Cas9 in C. tropicalis.C. tropicalis hermaphrodites, worm recovery, and handling. Editing efficiency was calculated as the percentage of injected worms which produced F1 rollers and the proportion of F1 progeny which displayed the roller phenotype.Please refer to Ghanta et al. 2021 for CRISPR reagent preparation, injection of young adultC. tropicalishermaphrodites were separated onto individual plates and the F1 offspring were screened over 3-5 days for any presenting a roller phenotype. No F1 dumpy worms were observed. These F1 rollers were then separated onto individual plates and their offspring were screened over 3 days to identify F2 dumpy worms. These F2 dumpy worms were likewise separated onto individual plates and allowed to undergo clonal expansion.Individual injected P0PCR amplification of Ctr-dpy-10.2and 1 mg/mL Proteinase K). Tubes containing lysis solution and worms were spun down and cooled in liquid nitrogen for 10 minutes before being heated to 65°C for 2 hours, followed by 95°C for 30 min. The resultingC. tropicalisgenomic DNA solution can be diluted 100-fold and used as a PCR template.Lysis protocol was adapted from Dr. Ian Chin-Sang. Worms were washed from a single NGM plate and resuspended in 100 µL of lysis solution ; 1 x 10’ 72°C.Ctr-dpy-10PCR products were purified using a QIAquick® PCR Purification Kit, and purified DNA was quantified using a NanoDrop® ND-1000 UV-Vis spectrophotometer. Sanger sequencing was performed by the MBSU at the University of Alberta.The


Oncogenesis 10.1038/oncsis.2016.28, published online 11 April 2016Retraction to: Figure 3a (panel β-actin) appears to overlap with Figure 2e (panel 0h vs GAPDH (36 kDa)) of .Figure 3a panel p21) appears to overlap with Figure 6b (panel APC (160 kDa)) of [ appears The Editor-in-Chief has retracted this article because several images in this article appear to overlap with those of a previously published article  by diffeIn addition, the authors have been unable to provide documents confirming that ethics approval was obtained for the study.D-Q Dou agrees with this retraction. C Liu, L-H Dai, L-Q Ma and Y-X Sun have not responded to correspondence from the Publisher about this retraction.


The P301L mutation in tau protein is a prevalent pathogenic mutation associated with neurodegenerative frontotemporal dementia, FTD. The mechanism by which P301L triggers or facilitates neurodegeneration at the molecular level remains unclear. In this work, we examined the effect of the P301L mutation on the biochemical and biological characteristics of pathologically relevant hyperphosphorylated tau. Hyperphosphorylated P301L tau forms cytotoxic aggregates more efficiently than hyperphosphorylated wildtype tau or unphosphorylated P301L tau in vitro. Mechanistic studies establish that hyperphosphorylated P301L tau exacerbates endoplasmic reticulum (ER) stress-associated gene upregulation in a neuroblastoma cell line when compared to wildtype hyperphosphorylated tau treatment. Furthermore, the microtubule cytoskeleton is severely disrupted following hyperphosphorylated P301L tau treatment. A hyperphosphorylated tau aggregation inhibitor, apomorphine, also inhibits the harmful effects caused by P301L hyperphosphorylated tau. In short, the P301L single mutation within the core repeat domain of tau renders the underlying hyperphosphorylated tau more potent in eliciting ER stress and cytoskeleton damage. However, the P301L mutation alone, without hyperphosphorylation, is not sufficient to cause these phenotypes. Understanding the conditions and mechanisms whereby selective mutations aggravate the pathogenic activities of tau can provide pivotal clues on novel strategies for drug development for frontotemporal dementia and other related neurodegenerative tauopathies, including Alzheimer’s disease. Tauopathies are neurodegenerative diseases manifested by tau inclusions in neurons, glia, or astrocytes as their shared hallmark biomarker. The brains of patients with tauopathies, such as Alzheimer’s disease, frontotemporal lobar degeneration (FTLD), Pick’s disease, corticobasal degeneration, or progressive supranuclear palsy, show protein aggregates of the microtubule-associated protein tau  . AlthougTau is a microtubule-associated protein , encodedTauopathies can be classified into primary or secondary tauopathies. In secondary tauopathies such as AD, tau dysregulation is thought to be an effect of other pathological proteins, most notably the aggregation of amyloid-β (Aβ) peptides in the senile plaques of AD patients . While rFTLD-tau is caused by mutations mapped to tau, thereby demonstrating that tau anomalies can drive neurodegeneration without Aβ. One of the most prevalent FTLD mutations is P301L (proline-to-leucine mutation) ,20. MechPathologically, AD is associated with the accumulation of misfolded proteins, metabolic derangements, elevated oxidative stress, and neuroinflammation in the brain . ParticuE. coli with a desired post-translational modification. Hyperphosphorylated tau produced by PIMAX bears GSK-3β phosphorylation marks that recapitulate many of those found in AD, which render the underlying tau prone to aggregate into a cytotoxic species without any artificial inducer such as heparin or arachidonic acid [We previously reported the synthesis and characterization of recombinant hyperphosphorylated tau by the PIMAX approach (Protein Interaction Modules-Assisted Function X) . PIMAX inic acid . In thisOne of the challenges of studying tau pathology is the prominent post-translational modifications, most notably hyperphosphorylation , that arA hallmark character of pathological tau is its ability to aggregate and cause cell death . Tau, wh50 of p-tau sometimes varies slightly, depending on the batch of protein or the passage number of cells. Two µM of p-tau gave consistent results in our cell-killing experiments. After 20 h of incubation, both P301L and WT p-tau caused a striking reduction in relative cell viability, whereas the unphosphorylated P301L tau was benign under the same test conditions (We next examined whether the P301L mutation would affect the ability of tau to cause cell death. Neuroblastoma (SH-SY5Y) cells, a model widely used for studying neurodegenerative disease, were treated with 2 µM of P301L p-tau, p-tau, and P301L tau. The LDnditions C. NoticeThe seeding activity of tau is a well-known feature of pathological tau . We suspMature paired helical filaments and straight filaments that make up neurofibrillary tangles are commonly thought to be of more pathological importance than amorphous aggregate structures . HoweverUpon inspecting different preps of the wildtype and the P301L p-tau, we frequently noticed a ~40-kDa band associated specifically with the mutant form of p-tau A. For coIf the hypothesis of P301L-specific proteolytic digestion by a bacterial endopeptidase was true, one could then further extrapolate and posit that this mutation caused a conformational alteration that consequently exposed such a cleavage site for the observed proteolysis. To explore this scenario further, we used a tau conformation antibody, MC1 . MC1 reaIn eukaryotic cells, a number of biochemical and pathophysiological challenges or the presence of aberrant proteins can interrupt the protein folding process in the endoplasmic reticulum (ER), the organelle where protein folding and assembly take place, and therefore cause the accumulation of unfolded or misfolded proteins, a condition called “ER stress”, that leads to the activation of the ER stress response or unfolded protein response (UPR) to restore protein homeostasis ,50.To understand the mechanisms by which P301L p-tau inflicts cellular damage and death, we treated the SH-SY5Y cells with a sub-lethal dose (0.5 µM) of p-tau, P301L p-tau, or P301L tau for 24 h. Quantitative real-time PCR (qPCR) analyses with the total RNAs isolated from the treated cells were performed to determine the expression profile of the genes encoding ER stress markers and major components of the ER stress-associated pro-apoptosis pathway. In response to the p-tau or P301L p-tau challenge, expression levels of the ER chaperone BiP/GRP78, a master regulator of the ER stress response , in the To see whether the highly pathogenic P301L p-tau can be controlled pharmacologically, we examined the potential of apomorphine to counter P301L p-tau aggregation and cytotoxicity in vitro. Apomorphine, a dopamine agonist for the treatment of Parkinson’s disease symptoms , was fouTo further assess whether apomorphine diminished P301L p-tau toxicity in ER stress elicitation, SH-SY5Y cells were treated with 0.5 µM P301L p-tau in the presence or absence of 5 µM apomorphine for 20 h, followed by qPCR analysis. P301L p-tau-induced expression of the transcripts encoding the ER chaperone BiP/GRP78 as well as the ER stress-associated pro-apoptotic factors ATF4, CHOP, TRB3, and GADD34 was significantly lessened by apomorphine B–F. ThisThe microtubule cytoskeleton is a dynamic structure in cells that is intimately linked, functionally and physically, to many cellular functions and organelles, including ER . DisruptDeposition of hyperphosphorylated tau in the brain is a telling pathological trait shared by neurodegenerative tauopathies, including primary tauopathies such as tau-positive frontotemporal lobar dementia and Pick’s disease and secondary tauopathies such as Alzheimer’s disease and chronic traumatic encephalopathy . The spaP301L and P301S are among the most prevalent FTLD-associated mutations and have been used in multiple animal models . HoweverThe mass spectrometry phospho-mapping results D show thS/TXXXS/TP, where the first S/T(underlined) is the site of phosphorylation that can be stimulated by a priming phosphorylation event at the +4 position (superscript-P) [262STENLK) belongs to neither, suggesting the possibility that the P301L mutation, in addition to exposing a protease site that generates the 40-kDa species, positions Ser262 in a way that makes it phosphorylatable by GSK-3β. As to the function of S262 phosphorylation, it was reported that a S262D phospho-mimicking mutation promoted GSK-3β-mediated tau phosphorylation at other sites [Besides the potential conformational alteration discussed above, Ser262 phosphorylation is yet another peculiar characteristic of P301L p-tau. S262 is uniquely phosphorylated in the P301L mutant within the PIMAX context . From mucript-P)  that faver sites . HoweverThioflavin S-based aggregation reactions and transmission electron microscopy  support https://doi.org/10.1074/mcp.O114.044412. Recombinant Protein Expression and Purification refers to the previous publication: https://doi.org/10.1007/s12035-020-02034-w. 150-mesh copper square grids were purchased from VWR. The DA9 and MC1 antibodies were generous gifts from Dr. Peter Davies. Goat-anti-Mouse IgG+IgM (10 nm gold) was a generous gift from Dr. Alicia Withrow. Cell Counting Kit-8 (CCK-8) was purchased from GlpBio. Fluorescein diacetate (FDA), propidium iodide (PI), and Thioflavin S were purchased from Sigma-Aldrich . Neurobasal medium, B27 and N2 supplements, Penicillin-Streptomycin-Glutamine and the filamentous actin (F-actin) probe, Alexa Fluor 488 phalloidin (A12379), was purchased from ThemoFisher Scientific .Plasmid and expression protocol for 1N4R p-tau refer to the previous publication: The protein band of interest was excised from the SDS-PAGE gel and washed with 25 mM ammonium bicarbonate, followed by acetonitrile. Then, the sample was reduced with 10 mM dithiothreitol at 60 °C, followed by alkylation with 50 mM iodoacetamide at RT. Then, the protein was digested with sequencing-grade trypsin  at 37 °C for 4h and then quenched with formic acid, and the supernatant was analyzed directly without further processing. Half of each digested sample was analyzed by nano LC-MS/MS with a Waters M-Class HPLC system interfaced to a ThermoFisher Fusion Lumos mass spectrometer. Peptides were loaded on a trapping column and eluted over a 75 μm analytical column at 350 nL/min; both columns were packed with XSelect CSH 5μm C18 resin (Waters). The mass spectrometer was operated in data-dependent mode, with the Orbitrap operating at 60,000 FWHM and 15,000 FWHM for MS and MS/MS, respectively. The instrument was run with a 3s cycle for MS and MS/MS. Data were analyzed using Mascot software.The SDS-PAGE gel was stained with 0.05% Coomassie blue R-250 (CBR) in 10% acetic acid and 25% isopropanol for 30 min at RT. The gel was then sequentially de-stained with 0.005% CBR in 10% acetic acid and 10% isopropanol, 0.002% CBR in 10% acetic acid, and finally 10% acetic acid for 30 min (with shaking).A solution of 6 µM tau protein with or without indicated compounds was mixed in 20 mM Tris pH 7.4, 1 mM DTT, and 20 µM ThS to a 10 µL final volume and placed into a 384-well black plate. Each plate was covered with transparent adhesive film and placed into a preheated 37-degree plate reader. ThS fluorescence was acquired every 10 min at 440/490 nm for 16 h.For the cytotoxicity assay, 2000 SH-SY5Y cells were seeded on a 96-well plate in 100 µL DMEM/F12 media. 10 µL of protein solution with or without compounds was added to each well and placed back into the incubator for 20 h. Relative cell viability was determined using either cell counting kit 8 (CCK8) or FDA/PI staining and calculated by FDA positive cells/(FDA positive cells + PI positive cells).5 cells/mL density in 100 µL DMEM for 1 h at 37 °C with 5% CO2 for attachment. The medium was changed to Neurobasal medium ; 25% of the medium was changed with fresh Neurobasal medium every three days.E16 pregnant mice (C57BL/6) were ordered from Charles River Laboratories. The cerebral cortex was dissected from embryos and then passed through a 70 μm nylon mesh filter. Primary neurons were seeded on a 96-well plate at 5 × 10A 100 µM final concentration of thioflavin S was added and stained the primary neurons for 10 min at 37 °C. The cell medium was changed to fresh Neurobasal medium. Neurons were imaged by a microscope with a 20× objective.First, 10 µM p-tau was incubated with or without compounds in 20 mM Tris buffer (pH 7.4) at 37 °C for an indicated time. The sample was 10-fold diluted and incubated with 2.5% glutaraldehyde for 5 min. Then, 20 µL of the sample was fixed on a 150-mesh copper square grid and negatively stained by 1% uranyl acetate for 10 s. The sample was imaged by JOEL 1400 Flash TEM. For immunogold staining, 20 µL of the sample was fixed on the grid with 2.5% paraformaldehyde for 5 min and blocked with 1% BSA at room temperature for 1 h. Then, the grid was incubated with primary antibody (DA9 antibody 1:1000) at 4 degrees overnight, followed by secondary 10 nm gold antibody (1:50) at room temperature for 2 h. The grid was then incubated with 2.5% glutaraldehyde for 15 min and stained with 1% uranyl acetate for 10 s. Then, the sample was imaged by JOEL 1400 Flash TEM.SDS-PAGE was transferred to the PVDF membrane. The membrane was blocked in 5% nonfat milk in TBST  for 1 h, and then incubated with the DA9  for overnight at 4 °C. After washing three times in TBST, the membrane was incubated with HRP goat anti-mouse secondary antibody  for 1 h at room temperature. The membrane was washed again 3 times in TBST and developed with the Lumi-Light Western blotting substrate for 5 min.Two microliters of the samples were gently applied to a Nitrocellulose membrane. The membrane was blocked in 5% nonfat milk in TBST  for 1 h, and then incubated with the MC-1 monoclonal antibody (at 1:1000 dilution) or DA9  for overnight at 4 °C. After washing three times in TBST, the membrane was incubated with HRP goat anti-mouse secondary antibody  for 1 h at room temperature. The membrane was washed again 3 times in TBST and developed with the Lumi-Light Western blotting substrate for 5 min.RNA was extracted from SH-SY5Y cells with TRIzol reagent (Thermo Fisher Scientific), and 500 ng of total RNA was reverse transcribed into cDNA with the High-Capacity cDNA Reverse Transcription Kit . mRNA levels were measured using RT-qPCR analysis using the SYBR green PCR Master Mix (Applied Biosystems). PCR was carried out using Applied Biosystems 7500 Real-Time PCR Systems. GAPDH, a housekeeping gene, was used as an internal control.The primer sequences used in this study:Hu BiP-F: CCTGGGTGGCGGAACCTTCGATGTGHu BiP-R: CTGGACGGGCTTCATAGTAGACCGGHu CHOP-F: GCCTTTCTCCTTTGGGACACTGTCCAGCHu CHOP-R: CTCGGCGAGTCGCCTCTACTTCCChu ATF4-F: TCAAACCTCATGGGTTCTCCAhu ATF4-R: CACAGCCAGCCATTCGGHu TRB3-F: TACCTGCAAGGTGTACCCCHu TRB3-R: GGTCCGAGTGAAAAAGGCGTAHu GADD34-F: ATGATGGCATGTATGGTGAGCHu GADD34-R: AACCTTGCAGTGTCCTTATCAGhu GAPDH-F: TGAAGGTCGGAGTCAACGGhu GAPDH-R: AGAGTTAAAAGCAGCCCTGGTGFor cytoskeleton staining, cells were incubated with Alexa Fluor 488 phalloidin (Thermo Fisher), a high-affinity filamentous actin (F-actin) probe conjugated to green-fluorescent Alexa Fluor 488 dye (Thermo Fisher) for 1 h. The stained cells were mounted with DAPI-containing ProLong Antifade Mountant (Thermo Fisher) and observed using a Cytation Confocal Imaging Reader (Biotek/Agilent). To quantitatively analyze fluorescent intensities, cells of interest and regions of interest (ROI) were selected and measured for mean fluorescence intensity using ImageJ software. Corrected Total Cell Fluorescence (CTCF) was calculated based on: CTCF = Integrated Density − (Area of Selected Cell × Mean Fluorescence of Background readings).n = 3. For comparisons between more than 2 groups , a one-way ANOVA was applied with Prism 9 (sample size n = 3). For comparisons between two groups, the Student’s t-test was applied for statistical analysis. Non-significant values are not marked to limit clutter in the data graphs. For the QPCR analysis and cytoskeleton staining quantification, biological replicates are displayed. For other experiments and figures, representative figures with technical replicates are displayed.Experiments were carried out with a minimum sample replicate size of


The Ku heterodimer, composed of subunits Ku70 and Ku80, is known for its essential role in repairing double-stranded DNA breaks via non-homologous end joining (NHEJ). We previously identified Ku70 S155 as a novel phosphorylation site within the von Willebrand A-like (vWA) domain of Ku70 and documented an altered DNA damage response in cells expressing a Ku70 S155D phosphomimetic mutant. Here, we conducted proximity-dependent biotin identification (BioID2) screening using wild-type Ku70, Ku70 S155D mutant, and Ku70 with a phosphoablative substitution (S155A) to identify Ku70 S155D-specific candidate proteins that may rely on this phosphorylation event. Using the BioID2 screen with multiple filtering approaches, we compared the protein interactor candidate lists for Ku70 S155D and S155A. TRIP12 was exclusive to the Ku70 S155D list, considered a high confidence interactor based on SAINTexpress analysis, and appeared in all three biological replicates of the Ku70 S155D-BioID2 mass spectrometry results. Using proximity ligation assays (PLA), we demonstrated a significantly increased association between Ku70 S155D-HA and TRIP12 compared to wild-type Ku70-HA cells. In addition, we were able to demonstrate a robust PLA signal between endogenous Ku70 and TRIP12 in the presence of double-stranded DNA breaks. Finally, co-immunoprecipitation analyses showed an enhanced interaction between TRIP12 and Ku70 upon treatment with ionizing radiation, suggesting a direct or indirect association in response to DNA damage. Altogether, these results suggest an association between Ku70 phospho-S155 and TRIP12. In response to DNA damage, a network of signaling pathways collectively referred to as the DNA damage response (DDR) is initiated. In the case of double-stranded breaks, once such damage has been detected, the activation of the Ataxia-telangiectasia mutated (ATM) kinase leads to the phosphorylation of numerous targets, including CHK2, 53BP1, BRCA1, p53, and histone H2A.X at serine 139 , which accumulates at DSBs [Previous work in our laboratory has implicated the Ku complex, well-established for its role as a double-stranded break (DSB) sensor and scaffold protein, in the DDR. Within the Ku70 von Willebrand A (vWA) domain, work in our laboratory previously identified serine 155 (S155) as a novel phosphorylation site that was phosphorylated in response to severe ionizing radiation (IR) ,5. To st−/− mouse embryonic fibroblast (MEF) cells expressing each mutant displayed contrasting effects in terms of cell survival. Ku70 S155D-expressing cells displayed decreased cell survival, whereas Ku70 S155A-expressing cells displayed prolonged survival despite having normal DNA repair activity [After IR treatment, Ku70activity ,5. Notabactivity ,5. TheseAltogether, these findings led to the working hypothesis that the phosphorylation of Ku70 S155 may function to promote and/or sustain a DDR at DSBs. The working model proposed that Ku70 is localized to DSBs and upon a cellular assessment of the DNA damage, if the damage was deemed overwhelming for repair, Ku70 S155 was then phosphorylated [The DDR pathway is intimately connected to the DNA repair pathway as crosstalk between the two pathways coordinates the cellular response to damage ,6. WhileAs previously hypothesized, we postulate here that the phosphorylation of Ku70 S155 initiates a key protein–protein interaction with Ku70 that leads to the observed phenotypes. Consequently, it is expected that the protein interaction(s) will be absent in Ku70 S155A cells, which cannot be phosphorylated. To our knowledge, this is the first instance of BioID2 being used to identify phospho-specific protein interactor candidates.TM 293 T-RExTM pcDNA.5/FRT/TO inducible Tet-ON expression system was used to create stable, inducible cell lines, whereby the Ku70-BioID2 fusion protein  or the Ku70 NLS-BioID2 fusion protein was stably integrated in the T-REx 293 cell line and under the control of a doxycycline-inducible promoter  , a compuTo determine the final lists of Ku70 S155-specific candidate proteins, the Ku70 S155D-specific candidates identified using both approaches described above were compared, and 17 proteins were found to be shared D. For KuTM T-REx HeLa expression system: wild-type Ku70-HA and Ku70 S155D-HA , which introduces DSBs. Previously, Fell et al. (2016) were able to demonstrate that Ku70 was phosphorylated at S155 in response to severe IR treatment, and they hypothesized that Ku70 S155 was being recruited and then subsequently phosphorylated at DSBs [p-value = 0.0612); however, there were significantly more foci on average in the IR 1 h condition compared to both no IR  and the IR 0 h  conditions , or IR 1 hr  B. We evanditions C. Finally, to determine complex formation between Ku and TRIP12 in response to DNA damage, we performed co-immunoprecipitation experiments. Ku was found to weakly co-immunoprecipitate with TRIP12 in non-treated HeLa cells, and this was greatly enhanced upon treatment of cells with IR . This anAltogether, these data demonstrate that TRIP12 and Ku interact in response to DNA damage, providing support to the idea that DNA damage-dependent Ku70 S155 phosphorylation may facilitate TRIP12 association.The Ku70 S155 residue, first identified in our laboratory as a novel phosphorylation site, was previously implicated in the DDR ,5. ExactBoth the BioID and BioID2 techniques have traditionally been used with full-length, wild-type bait proteins ,11,13,15Phosphorylation is a well-studied post-translational modification, documented to promote changes in cellular localization and translocation, signal transduction, or temporary protein interactors . Using aProtein interactions can be highly diverse. Some of the most well studied protein interactions are constitutive and strong, capable of being co-immunoprecipitated with a bait protein of interest . Such inIn this study, we chose to employ the BioID2 screening technique, which is capable of identifying, theoretically, all types of protein interactions . BioID2 was deemed a suitable and more encompassing technique as we wanted to identify additional Ku70 phospho-S155-specific factors that could have been missed in the previous Ku70 S155D pull-down [Thyroid hormone receptor-interacting protein 12 or TRIP12, is an E3 ubiquitin ligase previously implicated in the DDR for its regulation of other proteins involved directly or indirectly at DSBs ,24,25. TTo determine a context for when TRIP12 and Ku70 phospho-S155 could be interacting, we tested for a PLA association between TRIP12 and Ku70 in the presence of DSBs. Previously, Fell et al. (2016) were able to demonstrate that Ku70 S155 is phosphorylated 30 min after IR treatment, compared to untreated cells. Our PLA results with cells fixed immediately after IR (0 h IR) and cells fixed 1 h after IR (1 h IR) seem to suggest that endogenous Ku70 and TRIP12 only significantly co-localize with one another 1 h after IR treatment C. These Based on the previous model and our current preliminary results, Ku70 S155 is phosphorylated in response to DNA damage, and the co-localization between Ku70 and TRIP12 could be dependent on this phosphorylation. Since TRIP12 has previously been involved in modulating the DDR through limiting the activity of RNF168 at DNA breaks , the intWhile we have been able to establish a proximity-based association between TRIP12 and Ku70 S155D and an indirect/direct TRIP12 association with Ku70 in response to DNA damage, the context of this association needs further investigation. One possibility is that TRIP12 co-localization with Ku at DSBs promotes either activation or inhibition of its E3 activity. Alternatively, phosphorylation of Ku70 S155 could be a signal triggering its ubiquitination via TRIP12. Although we have yet to establish whether TRIP12 acts on Ku through its E3 ligase activity or whether Ku functions to modulate TRIP12 activity in response to DNA damage, there are other functional contexts that may explain this proximity-based interaction. For example, although TRIP12 is currently best known for its role as an E3 ubiquitin ligase acting on specific substrates at DSBs, TRIP12 has also recently been implicated in mitotic cell cycle progression and chromosome stability from experiments using TRIP12-depleted HeLaS3 cells , offerinTo our knowledge, this research was the first to use the BioID2 technique to screen for phospho-specific interactor candidates. In summary, we established inducible expression systems to conduct both the BioID2 screening and the validation testing. We were able to validate TRIP12 as a novel Ku70 S155D-specific interactor and provided a potential context for a TRIP12-Ku70 phospho-S155 interaction at DSBs.Previously, site-directed mutagenesis was used to introduce substitution mutations S155A and S155D within wild-type Ku70 (XRCC6) cDNA . Wild-tyTM T-RexTM expression system plasmid, pcDNA.5/FRT/TO  using a blunt–blunt restriction enzyme cloning strategy. The NLS-BioID2-HA and Ku70-BioID2-HA constructs were excised from the BioID2 plasmid by digestion with HpaI and PmeI and gel-purified prior to ligation with the EcoRV-digested pcDNA.5/FRT/TO plasmid. All cloning was verified by sequencing analysis, conducted by the DNA Sequencing Facility at Robarts Research Institute, London, ON, Canada.All four BioID2 cloning experiments were subsequently used to sub-clone the NLS-BioID2-HA or Ku70-BioID2-HA fusion constructs  into the Flp-InFor the validation testing, doxycycline-inducible Ku70-HA, Ku70 S155D-HA, and Ku70 S155A-HA stable cell lines were created. Wild-type Ku70-HA was created and then subsequently subcloned into the EcoRV site of the pcDNA.5/FRT/TO plasmid. The Ku70 S155D and A mutations were subcloned into wild-type Ku70-HA pcDNA.5/FRT/TO using BamHI and EcoRV restriction enzyme sites. The N-terminal portion of Ku70 containing the S155D or S155A mutation was PCR-amplified using the previously cloned Ku70 S155D/A-BioID2 plasmids (described earlier) as a template with primers: Ku70 Fwd BamHI (TTAATAGGATCCATGTCAGGGTGGGAGTCATATTACAAAACC) and Ku70 Rev EcoRV (TTAATAGATATCTCTGTAGAACAAGGATATGTCAAAGCCCCC), which introduced BamHI and EcoRV restriction sites to the ends of the PCR product.TM T-RexTM 293 doxycycline-inducible cells (ThermoFisher Scientific), and Flp-InTM T-RexTM HeLa doxycycline-inducible cells . All cell lines were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM)  supplemented with 10% fetal bovine serum (FBS) (Wisent Bioproducts) at 37 °C in 5% CO2. For biotinylation, cells were incubated for 24 h with media supplemented with 50 μM biotin at 37 °C in 5% CO2. Prior to doxycycline-inducible stable cell line creation, Flp-InTM T-Rex 293 and T-Rex HeLa cell lines were maintained in 15 μg/mL blasticidin (Wisent Bioproducts) and 100 μg/mL zeocin , as necessitated by the manufacturer to maintain integration of the FRT site and Tet repressor protein, respectively. After generation of the stable doxycycline-inducible cell lines, both T-RexTM 293 and T-RexTM HeLa cells were maintained in 15 μg/mL blasticidin and 150 μg/mL hygromycin (Wisent Bioproducts). To induce expression, 1 μg/mL doxycycline was added to the media for 24 h, or less for time course experiments.The following human cell lines were used in this study: HeLa , Flp-InTM T-RexTM 293 and T-Rex HeLa cell lines were used to create doxycycline-inducible stable cell lines expressing NLS-BioID-HA and Ku70-BioID2-HA  or wild-type Ku70-HA, Ku70 S155D-HA, and Ku70 S155A-HA, respectively. Stable cell lines were created by following manufacturer’s directions (ThermoFisher Scientific). Briefly, cloned pcDNA.5/FRT/TO plasmid constructs were co-transfected with pOG44 plasmid containing the Flp recombinase enzyme at a 1:9 ratio, respectively, before selection with 150 μg/mL hygromycin. Co-transfections were conducted using jetPRIME transfection reagent , following manufacturer instructions. Following successful integration events, monoclonal cells could be observed growing after approximately 10 days and were pooled and screened for expression by indirect immunofluorescence and Western blotting.Doxycycline-inducible Flp-In2 for 1 h prior to fixing and processing for PLA (below). For the co-immunoprecipitation analyses, cells were seeded onto 15 cm plates, irradiated with 40 Gy of IR, and incubated for 1 h followed by collection and extract preparation.For immunofluorescence analyses, HeLa cells were seeded onto 24-well coverslips and the next day cells were irradiated with 10 Gy IR in an X-ray cell irradiator cabinet . Coverslips were either immediately fixed afterward and processed for PLA or incubated at 37 °C in 5% CO® ChemiDocTM XRS system (Bio-Rad) with Image Lab (v6.0.1).Whole cell extracts (WCE) were prepared either using WCE buffer or RIPA lysis buffer, as previously described . ProteinTM Protein G magnetic beads (ThermoFisher Scientific) and washed five times in wash buffer  before being analyzed by Western blotting.For the Ku70 co-immunoprecipitation, wild-type HeLa cells untreated or treated with IR were collected to produce 500 µg of WCE that was diluted to 0.01% NP-40 and pre-cleared for 30 min, followed by a 2 h incubation at 4 °C with an anti-TRIP12 antibody . Immunoprecipitated proteins were bound using PierceIndirect immunofluorescence was conducted as described previously . BrieflyBioID2 was conducted exactly as previously described . SamplesSamples were quantified and processed as previously described  before sData acquisition and parameters were the same as previously described . To reithttps://reprint-apms.org; accessed on 14 March 2021). Proteins with a SAINTexpress score ≥0.6 were incorporated into a network figure, highlighting the high confidence interactors scoring ≥0.6 and ≥0.95. Protein network mapping information was visualized using Cytoscape (v3.6.1).Scaffold v4.8.7  was used to validate the MS/MS-based peptide and protein identifications, based on the Peptide Prophet algorithm  with Scap < 0.05, using Bonferroni correction for multiple testing) with a fold-change ≥5 are depicted in GO enrichment analysis by biological process was conducted for the 193 Ku70 S155D BioID2 candidate proteins with a SAINTexpress score of ≥0.6. A selection of significantly enriched biological processes . Binary DAPI images were used to highlight each nucleus (size set to 0.003-infinity) as a ROI. Similarly, binary images outlining the PLA foci (size set to 0.0003-infinity) were also acquired and merged with the DAPI outline.For each of the T-REX HeLa stable cell lines, a minimum of 600 cells were randomly counted in total from four biological replicates of PLA . Of those 600, only cells with one or more nuclear foci (cells positive for foci) were counted to determine the average number of nuclear foci per cell for each cell line, and these values were plotted as bars.Similarly, for the HeLa cells PLA, a minimum of 600 cells were also counted for all conditions  from three biological PLA replicates, each including exactly four technical replicates . Of those cells, only cells with one or more nuclear foci were counted to determine the average number of nuclear foci per cell for each cell line.p ≤ 0.0001.For both PLA analyses, the bar indicates mean with 95% confidence intervals. Statistics were performed on GraphPad Prism 8.0 using a one-way ANOVA with a Sidak’s multiple comparisons test, where **** indicates