Zebrafish have been found to be a premier model organism in biological and regeneration research. However, the comprehensive cell compositions and molecular dynamics during tissue regeneration in zebrafish remain poorly understood. Here, we utilized Microwell-seq to analyze more than 250,000 single cells covering major zebrafish cell types and constructed a systematic zebrafish cell landscape. We revealed single-cell compositions for 18 zebrafish tissue types covering both embryo and adult stages. Single-cell mapping of caudal fin regeneration revealed a unique characteristic of blastema population and key genetic regulation involved in zebrafish tissue repair. Overall, our single-cell datasets demonstrate the utility of zebrafish cell landscape resources in various fields of biological research. Danio rerio) is widely accepted as an in vivo model to study vertebrate biological processes for its similarity with human  wild-type Tübingen strain was raised and maintained in standard zebrafish units at Core Facilities, Zhejiang University School of Medicine. The adult zebrafish aged from 4 to 12 months were studied. The zebrafish research analysis conducted in this study was approved by the Ethics Committee of the Zhejiang University Laboratory Animal Center.Zebrafish  were kept in system water at 28.5°C and the system water with BMP inhibitors was replaced daily. 10 μM DMH1 (Target Mol), and 10 μM Dorsomorphin (Target Mol) were used as specific BMP inhibitors. All were dissolved in DMSO and final DMSO concentration in fish water was 0.1%. The control zebrafish were kept in fish water with 0.1% DMSO. The lengths of caudal fin were detected after 3 days of BMP inhibitors treatment.The diameter and depth of the microwells are 28 and 35 μm, respectively. First, a silicon plate containing 100,000 microwells was manufactured by Suzhou Research Materials Microtech Co. Ltd. . The silicon microwell plate was then used as a mold to make a PDMS plate with the same number of micropillars. Prior to experiments, a disposable agarose microwell plate was made by pouring 5% agarose solution onto the surface of the PDMS plate. Both the silicon and the PDMS plates are reusable. One silicon microwell plate allows almost permanent use.1). The barcoded oligonucleotides on the surfaces of the beads were synthesized by three rounds of split-pool. All the sequences used are the same as those reported previously (Magnetic beads (diameter 20–25 μm) coated with carboxyl groups were provided by Suzhou Knowledge & Benefit Sphere Tech. Co., Ltd.  was washed twice with 0.1 M MES (2-[N-morpholino]ethanesulfonic acid). The beads were then suspended in a final volume of 635 μl 0.1 M MES. EDC [1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride] (3.08 mg) was added to the beads, and 6.2 μl of beads was then placed in each well of a 96-well plate. Amino-modified oligonucleotides  were then added to each well. After vortexing the mixture and incubating it for 20 min at ambient temperature, 0.5 μl of mix (6 mg of EDC in 100 μl of 0.1 M MES) was distributed into each well. After an additional round of vortexing and incubation for 20 min at ambient temperature, an additional 0.5 μl of mix (6 mg of EDC in 100 μl of 0.1 M MES) was distributed into each well. After vortexing and incubation for 80 min at ambient temperature, the beads were collected in 1 ml of 0.1 M PBS containing 0.02% Tween-20. After centrifugation, the supernatant was carefully removed. The beads were then washed twice in 1 ml of TE (pH 8.0).2O. To remove complementary chains, the beads were placed in a 95°C water bath for 6 min and separated using a magnet, removing the supernatant quickly, 2 times. The beads could be stored in TE-TW  for 4 weeks at 4°C.In the second split-pool, the beads were washed with water and divided among the wells of another 96-well plate containing PCR mix  and 5 μM oligonucleotides. The oligonucleotides in each tube encoded a sequence with reverse complementarity to linker 1, a unique barcode and a linker 2 sequence. The PCR program was as follows: 94°C for 5 min; 5 cycles of 94°C for 15 s, 48.8°C for 4 min, and 72°C for 4 min; and a 4°C hold. The third split-pool procedure was the same as the second one. The PCR program was as follows: 94°C for 5 min, 48.8°C for 20 min, 72°C for 4 min and a 4°C hold. Beads were mixed sufficiently between denaturation (95°C) and primer annealing (48.8°C) in every cycle. The oligonucleotides used in each tube encoded a linker 2 reverse-complementary sequence, a unique barcode, a unique molecular identifier (UMI) sequence and a poly-T tail. All the oligonucleotides were synthesized by Sangon Biotech Co. Ltd. with HPLC purification. To remove the chains without the third barcoded sequence, beads were collected and suspended in 200 μl of exonuclease I mix [containing 1× exonuclease I buffer and 1 U/μl exonuclease I (NEB)], and incubated at 37°C for 15 min (beads were mixed by rotary mixer). After being washed with 200 μl of TE-TW and 200 μl of 10 mM Tris-HCl pH 8.0, beads were resuspended in 1 ml of ddHg for 5 min at 4°C and then re-suspended in 3 ml of cold DPBS. After passage through a 40-μm strainer (Biologix), the cells were washed twice, centrifuged at 300 × g for 5 min at 4°C, and re-suspended at a density of 1 × 105 cells/ml in cold DPBS containing 2 mM EDTA.Dissociation of embryonic tissues was performed similarly as previously described . In brieCell concentration should be carefully controlled during Microwell-seq. Both cell and bead concentrations were estimated using a haemocytometer. The proper cell concentration is ∼100,000/ml (with 10% of the wells occupied by single cells). The proper bead concentration is ∼1,000,000/ml (with every well occupied by single beads). An evenly distributed cell suspension was pipetted onto the microwell array, and extra cells were washed away. To eliminate cell doublets, the plate was inspected under a microscope. Cell doublets were reduced by pipetting over the region of high cell density. The bead suspension was then loaded into the microwell plate, and the plate was placed on a magnet. Excess beads were washed away slowly. Cold lysis buffer  was pipetted over the surface of the plate and removed after 12 min of incubation. The beads were then collected, transferred to an RNase-free tube, and washed once with 1 ml of 6 × SSC, once with 500 μl of 6 × SSC and once with 200 μl of 50 mM Tris-HCl pH 8.0. Finally, ∼50,000 beads were collected in a 1.5-ml tube.2 (Ambion), 2.5 mM dithiothreitol, 1 mM dNTP and 1 μM TSO primer. The beads were incubated at 42°C for 90 min with mixing on a rotary mixer and then washed with 200 μl of TE-SDS (1 × TE + 0.5% sodium dodecyl sulfate) to inactivate reverse transcriptase.In this procedure, the instructions from the Smart-seq2 protocol were followed . BrieflyThe beads were washed with 200 μl of TE-TW and 200 μl of 10 mM Tris-HCl pH 8.0, resuspended in 100 μl of exonuclease I mix containing 1× exonuclease I buffer and 1 U/μl exonuclease I (NEB), and incubated at 37°C for 60 min with mixing on a rotary mixer to remove oligonucleotides that did not capture mRNA. The beads were then pooled and washed once with TE-SDS, once with 1 ml of TE-TW and once with 200 μl of 10 mM Tris-HCl pH 8.0.The beads were distributed into 4 PCR tubes. To each tube, 12.5 μl of PCR mix [1 × HiFi HotStart Readymix (Kapa Biosystems) and 0.1 μM TSO_PCR primer] was added. The PCR program was as follows: 98°C for 3 min; 4 cycles of 98°C for 20 s, 65°C for 45 s, and 72°C for 6 min; 10–14 cycles of 98°C for 20 s, 67°C for 20 s, and 72°C for 6 min; 72°C for 10 min; and a 4°C hold. After pooling all PCR products, AMPure XP beads (Beckman Coulter) were used to purify the cDNA library.The purified cDNA library was fragmented using a customized transposase that carries two identical insertion sequences. The customized transposase was included in the TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme). The fragmentation reaction was performed according to the instructions provided by the manufacturer. We replaced the index 2 primers (N5××) in the kit with our P5 primer to specifically amplify fragments that contain the 3′ ends of transcripts. Other fragments will form self-loops, impeding their binding to PCR primers. The PCR program was as follows: 72°C for 3 min; 98°C for 30 s; 5 cycles of 98°C for 15 s, 60°C for 30 s, and 72°C for 3 min; 72°C for 5 min; and a 4°C hold. The PCR product was purified using AMPure XP beads. Then, 25 μl of PCR mix (1× HiFi HotStart Readymix and 0.1 μM 2100 primer) was added to each sample. The PCR program was as follows: 95°C for 3 min; 8 cycles of 98°C for 20 s, 60°C for 15 s, and 72°C for 15 s; 72°C for 5 min; and a 4°C hold. To eliminate primer dimers and large fragments, AMPure XP beads were then used to purify the cDNA library. The size distribution of the products was analyzed on an Agilent 2100 bio-analyzer, and a peak in the 400–700 bp range was observed. Finally, the samples were subjected to sequencing on an Illumina Hiseq system.in situ hybridization (WISH) with DIG (Digoxyigenin)-labeled was performed using standard protocols as described previously , c1qtnf5 , clu , ecrg4a , fgfbp2a . Photos were taken under a Leica M216 optics.Whole mount eviously . To resoD. rerio GRCz10 genome using STAR  of transcript counts derived from mitochondria-encoded genes were also excluded.Standard procedures for processing the Microwell-seq datasets were performed using the protocols described in the previously published paper . Reads fing STAR  and the 2(TPM/100 + 1) transformed, then the number of UMI and the percentage of mitochondrial gene content were regressed out according to published method   was usedd method . About 22(TPM/10 + 1) transformed. We selected about 3,000 highly-variable genes according to their average expression and dispersion, regressed out UMI and gene numbers, scaled each gene to unit variance with clip values exceeding a standard deviation of 10. We chose about 50 PCs for the PCA, and we computed the neighborhood graph of cells. We then used the Louvain clustering to cluster cells with resolution = 2.5 and k = 10. Finally, 63 clusters for the zebrafish landscape were produced and marker genes were calculated by Wilcoxon rank sum test.For processing of the complete zebrafish tissue dataset , we used Scanpy  in pythoTo make the gene expression profiles of cross-species cell types comparable, we downloaded the homology correspondences between zebrafish and human, mouse, axolotl provided by dmodENCODE . The genWGCNA (V1.69) was performed with default parameters . In brie4 from caudal fin regeneration and non-regeneration single-cell gene expression profiles , by comparing each regulon enrichment score to a null model using msVIPER algorithm5. Master TFs and cofactors regulated each other and their downstream target genes were visualized by Cytoscape (V3.8.2).VIPER (V1.20.0) was performed to computational inference of TF activity from gene expression profile data with default parameters . Brieflyprofiles . We then6. D. rerio pathway database for enrichment was downloaded from https://www.wikipathways.org/index.php/Download_Pathways.Signature enrichment of regeneration relevant clusters against others was performed by gene set enrichment analysis (GSEA) (V1.0). The GSEA was implemented using JAVA downloaded from the Broad InstituteAnalysis of potential receptor–ligand pairings was performed using the method CellPhoneDB (V1.0.0) . First, t-test was performed to compare the differences between two groups. All quantitative experiments were repeated at least 3 times independently.All the results were presented as mean ± SEM. Two-tailed Student’s Single-cell RNA sequencing technologies enable identification of cell identity. To comprehensively characterize different cell types in zebrafish, we performed mRNA-seq expression profiling in individual cells isolated from major tissues using Microwell-seq  Figure . We collleg1.1 and bhmt. C50 partly derived from zebrafish embryos and was identified as primitive hepatocyte with additional high level of dao.1 and acot17. Compared with C7, C9, and C50 showed a closer spatial relationship in the t-SNE map. Similar to human and mouse liver cell atlas, the heterogeneity of zebrafish hepatocyte may imply zonation features at the single-cell level , C8 expressed high level of fibroblast growth factor binding protein (fgfbp2a) (C10 in replicate 2), C7 and C13 differed mainly in higher expression of proliferation genes (C4 and C8 in replicate 2), such as hmgn2, pcna  from two independent biological replicates termed replicate 1 and replicate 2  Figure . An inden2, pcna .col1a1a and col1a2, showed significantly enhanced expression, indicating that stromal cells mainly participated in blastema formation. The result is in accordance with previous findings that connective tissues transit to a blastema state during adult axolotl and frog limb regeneration  that express high levels of c1qtnf5 and ecrg4a during hair cell regeneration  between uninjured (0 dpa) and regenerated fin (3 dpa). Upregulated markers in blastema-like cells during caudal fin regeneration were identified . Extraceneration . Besidesdization . We founneration . clu, a tem cell , exhibittwist1a, prrx1a, msx1b, fosab, etc., as well as low activity TFs such as tp63, elf3 and klf2b  directly interpret the genome, exerting control over processes that specify cell types and controlling specific pathways . To undend klf2b . Previountiation .bmp4 was only expressed in 4 clusters of blastema, bmp2a was enriched in C4 and C8, and bmp1a was highly expressed in C7 and C13  signaling pathway was found to be strongly associated with the zebrafish caudal fin regenerative process , exhibit and C13 . Besides and C13 . We foun and C13  and whol and C13 . To furt and C13 . The res and C13   can be found in the article/The animal study was reviewed and approved by the Ethics Committee of the Zhejiang University Laboratory Animal Center.GG, XH, and JP were responsible for the conception, design, and study supervision. MJ, HC, CG, and YL were responsible for the development of methodology, analysis, and experiments. YX, WE, LM, JW, and QG performed the bioinformatics analysis and constructed the website of zebrafish cell landscape. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Thyroid hormone triiodothyronine (T3) is essential for embryogenesis and is commonly used during in vitro fertilization to ensure successful implantation. However, the regulatory mechanisms of T3 during early embryogenesis are largely unknown.To study the impact of T3 on hPSCs, cell survival and growth were evaluated by measurement of cell growth curve, cloning efficiency, survival after passaging, cell apoptosis, and cell cycle status. Pluripotency was evaluated by RT-qPCR, immunostaining and FACS analysis of pluripotency markers. Metabolic status was analyzed using LC–MS/MS and Seahorse XF Cell Mito Stress Test. Global gene expression was analyzed using RNA-seq. To study the impact of T3 on lineage-specific differentiation, cells were subjected to T3 treatment during differentiation, and the outcome was evaluated using RT-qPCR, immunostaining and FACS analysis of lineage-specific markers.In this report, we use human pluripotent stem cells (hPSCs) to show that T3 is beneficial for stem cell maintenance and promotes trophoblast differentiation. T3 enhances culture consistency by improving cell survival and passaging efficiency. It also modulates cellular metabolism and promotes energy production through oxidative phosphorylation. T3 helps maintain pluripotency by promoting ERK and SMAD2 signaling and reduces FGF2 dependence in chemically defined culture. Under BMP4 induction, T3 significantly enhances trophoblast differentiation.In summary, our study reveals the impact of T3 on stem cell culture through signal transduction and metabolism and highlights its potential role in improving stem cell applications.The online version contains supplementary material available at 10.1186/s13287-022-02799-y. Human pluripotent stem cells (hPSCs), with their potential to generate all the cell types of the human body, hold great promises in regenerative medicine . In ordeOne major difference between hPSCs maintained in chemically defined media and those in feeder cell culture is the dependence on high levels of growth factors . CompariHormones play key roles in growth and development, but the impacts of hormonal control on stem cell maintenance and differentiation remain largely unexplored. As a central regulator of energy homeostasis, thyroid hormone has been shown to be essential for embryogenesis and fetal development –10. DefiThere are two main forms of thyroid hormone in the human body, triiodothyronine (T3) and thyroxine (T4). T3 is the active form of thyroid hormone, and T4 is converted to T3 to exert its effects. T3 acts mainly through its nuclear receptors TRα and TRβ to modulate transcription related to various critical processes, including metabolism, growth and development . The majIn this report, we identified T3 as one of the bioactive components present in fetal bovine serum, KOSR and AlbuMAX, and systemically studied the effect of T3 on hPSC maintenance and differentiation. T3 improves the stability of hPSC culture under suboptimal conditions, significantly decreases the dependence on FGF2 and promotes extraembryonic differentiation. Our findings show that T3 is a potent regulator of hPSC pluripotency and cell fate determination.H1 and H9 hESCs and NL-1 hiPSCs were used in this study. H1 cells were used in most of the work unless specifically described. hPSCs were cultured and maintained in E8 medium as previously described . E8 mediCGA, CGB, GCM1, GATA2 and TROP2. For mesoderm differentiation, cells were differentiated in E8 medium supplemented with 20 ng/ml BMP4 for 2 days and harvested on D2 for RT-qPCR analysis of TBXT and MIXL1 expression. For endoderm differentiation, cells were treated with 5 uM CHIR in E8 for 24 h and then changed to E8 medium supplemented with activin A (10 ng/ml) for 3 days. Expression of SOX17 and FOXA2 was analyzed by RT-qPCR. For ectoderm differentiation, cells were treated with E6 medium (E8 without FGF2 and TGFβ) with 10 μM SB431542 (Selleck) for 1 day and subsequently treated with 10 μM SB431542 and 100 nM LDN193189 (Selleck) in E6 medium for 4 days. Expression levels of PAX3 and PAX6 were examined by RT-qPCR.Lineage-specific differentiation of hESCs was conducted as previously described . BrieflyMouse embryonic stem cells (mESCs) were obtained from Deng C.’s laboratory and maintained on MEF using ESF7 medium  . Medium Human triiodothyronine (T3) ELISA Kit  and Human thyroxine (T4) ELISA kit  were used to measure the concentration of thyroid hormones. Different medium supplements were diluted in DMEM/F12 to the indicated concentrations, and ELISA was carried out following manufacturer’s instructions. Briefly, samples were added into a 96-well plate precoated with T3 or T4 antibodies, incubated at room temperature for 30 min, and the wells were washed with wash buffer twice. HRP-conjugated secondary antibodies were then added. After incubation and wash, the TMB substrate was added for color development. Stopping buffer was added 15 min later, and the absorbance at 450 nm was measured using a microplate reader. The standard curve was constructed using T3 or T4 standard solutions measured alongside the samples.g for 5 min, washed with PBS, fixed with 4% PFA for 15 min and then washed with PBS. Cells were then permeablized by 0.5% triton X-100 for 15 min and washed with FACS buffer (PBS with 0.05% triton-X100 and 1% BSA). 5 × 105 cells were incubated with primary antibodies for 1 h at room temperature, washed twice with FACS buffer and then incubated with secondary antibodies  for 1 h. Cells were washed twice with FACS buffer and analyzed by CytoFLEX S flow cytometer (Beckman Coulter). Primary antibodies used include NANOG  and CGB .Cell were dissociated by TrypLE Select  and neutralized by 2 volumes of DMEM/F12 . Cells were collected by centrifugation at 300×Cell cycle analysis was conducted using Click-iT® EdU Flow Cytometry Assay Kits (Thermo Fisher). Briefly, cells were incubated with 10 μM EdU for 30 min and then harvested by TrypLE, and then fixed and permeabilized as described above. EdU was labeled with Alexa Fluor® 647 dye, and DNA was labeled by propidium iodide. Cell cycle profile was analyzed by CytoFLEX S flow cytometer (Beckman Coulter).Cell were harvested in RNAiso Plus  and extracted as instructed by the product manual. 500 ng RNA were added in 10ul reverse transcription reaction mixture using Maxima H Minus Reverse Transcriptase kit . The resulting cDNA was diluted 20 times by distilled water, and 2 µl was used for RT-qPCR using SYBR® Premix Ex Taq kit  on a CFX384 Touch Real-Time PCR Detection System (Biorad). Gene expression was normalized to GAPDH or the control as indicated. Primers for RT-qPCR used in this study are listed in Additional file Cells were washed with PBS, fixed with 4% PFA and permeabilized by 0.5% triton-X100. Primary antibodies were applied for 2 h at room temperature. Secondary antibodies  were diluted 1:1000 and applied for 1 h at room temperature. All the antibodies were diluted in PBS with 1% BSA. Nuclei were stained with Hoechst 33342, and cell images were captured by EVOS Cell Imaging System (Thermo Fisher). Primary antibodies used include NANOG  and CGB .Cells were harvested in 2 × Laemmli buffer . Protein concentrations were determined with BCA protein assay kit . 5 μg total proteins per sample were loaded on SDS-PAGE gel and transferred to PVDF membranes. The membrane was blocked in 5% non-fat-milk in 1 × TBST at room temperature for 1 h. Primary antibodies were applied overnight at 4 °C. Peroxidase-conjugated secondary antibodies  were diluted 1:10,000 and applied for 1 h at room temperature. Signals were visualized using SuperSignal™ West Pico Plus Chemiluminescent Substrate Kit . All the antibodies were diluted in PBS with 1% BSA. Primary antibodies used include GAPDH , Phospho-p44/42 MAPK (Erk1/2) antibody , p44/42 MAPK (Erk1/2) antibody , phospho-SMAD2 antibody , Smad2 (D43B4) antibody .To set up high- and low-density culture, H1 hESCs at 70% confluence were passaged by the ratio of 1:3 for high-density culture and 1:12 for low-density culture in E8 or E8 with 500 nM T3, and cultured for 3 days until the high-density culture reached ~ 100% confluence. Cell apoptosis was analyzed by CellEvent™ Caspase-3/7 Green Detection Reagent (Thermo Fisher). Briefly, caspase 3/7 green reagent (2 μM) were added to the cell culture medium and incubated for 30 min at 37 °C. Cells were washed, observed under microscope and then harvested to be analyzed by CytoFLEX S flow cytometer (Beckman Coulter).High- and low-density H1 culture was set up as described above. Cells from high- or low-density culture were then passaged to new plates using the EDTA method  with or 4 cells/well in E8 medium with or without FGF2. Medium was changed the next day with the same treatments added, and Mito Stress Test was run the day after using Agilent Seahorse XF Cell Mito Stress Test Kit, with 500 nM T3 or 100 ng/ml FGF2 added to the assay medium in corresponding wells.Mitochondrial respiration was analyzed by Mito Stress Test on a Seahorse XF96 analyzer (Agilent) following published protocols . Prior tg for 5 min. The supernatant was extracted by nine volumes of extraction solution , vortexed and centrifuged at 10,000 × g at 4 °C for 10 min. The supernatant was used for LC–MS/MS analysis. For analysis of intracellular metabolites, cells were washed with 0.9% saline twice, and then, 500 μl/well − 80 °C precooled 80% methanol  was added. Cells were scraped off and then centrifuged at 12,500×g for 15 min. The supernatant was evaporated under nitrogen till dry and redissolved in 100 μl 50% acetonitrile for intracellular metabolic analysis.Quantification of metabolites and data processing were based on previous publications , 22. BriWaters Acquity UPLC equipped with Waters Xevo TQD detector was used for LC–MS/MS analysis. The source gas was argon, capillary voltage was 3500 V, and dissolving temperature was 500 °C. Glycolysis and PPP-related metabolites were determined by Acquity UPLC BEH HILIC column , and other metabolites were determined by Acquity UPLC BEH amide column . Column temperature was set at 40 °C.https://maayanlab.cloud/Enrichr/).hESCs were treated with different conditions and were then collected with RNAiso Plus . The RNA sequencing was carried out by Novogene and Genewiz with similar methods. Briefly, total RNA was extracted using RNAiso Plus following manufacturer’s instructions. 1 µg total RNA was used for library preparation. mRNA was extracted using Poly(A) mRNA Magnetic Isolation Module. The sequencing libraries were prepared by KAPA method. Sequencing was performed on the Illumina HiSeq/Novaseq platform, in a 2 × 150 bp paired-end (PE) configuration. The gene reads were aligned to reference genome by Hisat2 (v2.0.1). Reference genome sequences and gene model annotation files were downloaded from ENSEMBL; Hisat2 (v2.0.1) was used to index reference genome sequence; clean data were then aligned to reference genome via software Hisat2 (v2.0.1). The reference genome is homo_GRCh38, and the GTF reference annotation file is in version Homo_sapiens.GRCh38.102. The reads were converted into transcripts by reference transcript annotation. Gene expression levels are calculated from the read numbers that align to exons. The exon-overlapping reads were quantitated using Htseq software following the intersection-non-empty model. Gene read counts were normalized using Transcripts Per Million (TPM). Log2  values were used to generate heatmaps using R package gplots (3.6.1). Differentially expressed genes (DEG) were picked using R package EdgeR based on p value < 0.01, fold change > 1.5 or < − 1.5. The heatmap cluster was established using the Euclidean distances between selected genes in each sample. Gene set enrichment analysis was carried out using Enrichr  Statistical analysis was carried out by two-tailed Student’s t test using Excel. Given the important roles of thyroid hormone in growth and development and its reported high binding ratio to albumin , we exam3) worked synergistically to reduce the percentage of caspase positive cells  leads to medium acidosis and cell death due to the lactic acid released during glycolysis in hESCs . An incrCell dissociation is a necessary step in stem cell passaging, but is also an aggressive procedure which often results in a high cell death rate. Multiple techniques, including the use of ROCK inhibitors during passaging, have been developed, aiming to improve hESC survival after dissociation , 25. HigWe further examined effect of T3 on hESCs under regular maintenance conditions. When T3 was added to E8 medium, a normal hESC growth rate was maintained during a 96-h time period Fig. A. Cell cNANOG and POU5F1 was not altered significantly after treatment with T3 in E8 medium for five passages . It implied that ROS production could be affected by thyroid hormone. We showed that ROS level was indeed suppressed by T3 at high cell density  and TGFβ 1.74 ng/ml) to maintain pluripotency. When FGF2 and TGFβ are removed, hESCs will spontaneously differentiate, and the expression of pluripotency markers will decrease  for 6 days  was elevated during trophoblast differentiation, but its expression was significantly suppressed by T3  or high density (>90% confluence) with or without 500 nM T3. Medium pH was measured using a pH meter after 24 hours. n = 3 biological repeats. B. Immunostaining showing effects of T3 and NaHCO3 on caspase 3/7 activity in high-density H1 culture. H1 cells were cultured in E8 with or without NaHCO3 (20mM) and T3 (500nM) for 3 days until 100% confluence. Caspase 3/7 activity were detected using caspase 3/7 green detection reagent. Scale bar, 100 μm. C. FACS analysis of caspase 3/7 activity in high-density H1 culture. H1 cells were cultured in E8 with or without NaHCO3 (20mM) and T3 (500nM) for 3 days until 100% confluence. Caspase 3/7 activity were detected using caspase 3/7 green detection reagent. D-F. Impact of T3 on hESC survival after passaging from high- or low-density culture. H1 cells were pretreated with or without 500 nM T3 at high or low density, then passaged to a new plate with or without ROCK inhibitor Y27632 and counted on the next day. G. Impact of transient application of T3 during hESC passaging from high-density culture. H1 cells were cultured in E8 medium until high density without pretreatment, then passaged to a new plate in the presence or absence of T3 (500nM) and counted on the next dayAdditional file 2: Figure S2. T3 is compatible with long-term hESC maintenance in E8 medium. Related to Figure 2. A. Effect of T3 on pluripotency in H9 hESCs. H9 cells were maintained in E8 medium with or without T3 (500nM) for five passages, and the expression of pluripotency markers NANOG and POU5F1 were examined by real time PCR. GAPDH was used as internal control and gene expression was normalized to the level in E8 culture. B. Morphology of H9 hESCs cultured with or without T3 for five passages. Scale bar, 200µm. C. Effect of T3 on pluripotency in NL-1 hiPSC. NL-1 cells were maintained in E8 medium with or without T3 (500nM) for five passages, and the expression of pluripotency markers NANOG and POU5F1 were examined by real time PCR. GAPDH was used as internal control and gene expression was normalized to the level in E8 culture. D. Morphology of NL-1 hiPSCs cultured with or without T3 for five passages. Scale bar, 200µm. E. T3 enhances hESC mitochondrial spare respiration capacityAdditional file 3: Figure S3. T3 promotes pluripotency under suboptimal conditions. Related to Figure 4. A. Western blot analysis of Smad2 (Ser245/250/255) phosphorylation. H1 cells were cultured in E8 medium with or without T3 for 4 days with passaging on day 3, and then changed to E8 medium with or without TGFβ for another 24 hours before collection for analysis. GAPDH was used as loading control. Data are representative of three independent experiments. B. Effect of T3 on pluripotency in the absence of TGFβ. H1 cells were cultured in E8 medium without TGFβ for 5 passages in the presence or absence of T3, and the mRNA levels of pluripotency marker NANOG and POU5F1 were analyzed by qPCR. GAPDH was used as internal control, and gene expression was normalized to the level in E8 culture. Data are representative of three independent experiments. C. T3 enhances hESC mitochondrial spare respiration capacity in the medium without FGF2Additional file 4: Figure S4. T3 promotes trophoblast differentiation in hESCs. Related to Figure 5. A. Effect of T3 on BMP4-induced trophoblast differentiation in mESCs. mESC were differentiated toward trophoblast linage under BMP4 treatment in the absence or presence of 500 nM T3 for 6 days. The expression of Gata2, Gata3 and Hand1 were analyzed by real time PCR. B-C. Effect of T3 on BMP4-induced trophoblast differentiation in H9 hESCs (B) and NL-1 hiPSCs (C). H9 and NL-1 cells were differentiated toward trophoblast linage under BMP4 treatment in the absence or presence of 500 nM T3 for 6 days. The expression of CGA, CGB, GCM1 and GATA2 were analyzed by real time PCRAdditional file 5. Table S1. Primers used for real time PCR.Additional file 6. Table S2. LC-MS/MS analysis of intracellular levels of metabolites.Additional file 7. Table S3. LC-MS/MS analysis of amino acid consumption in culture medium.Additional file 8. Table S4. GO term of the RNA-seq analysis.


CDKN2A, encoding the tumor suppressor p16, are responsible for a large proportion of familial melanoma cases and also increase risk of pancreatic cancer. We identified four families through pancreatic cancer probands that were affected by both cancers. These families bore a germline missense variant of CDKN2A (47T>G), encoding a p16-L16R mutant protein associated with high cancer occurrence. Here, we investigated the biological significance of this variant. When transfected into p16-null pancreatic cancer cells, p16-L16R was expressed at lower levels than wild-type (WT) p16. In addition, p16-L16R was unable to bind CDK4 or CDK6 compared with WT p16, as shown by coimmunoprecipitation assays and also was impaired in its ability to inhibit the cell cycle, as demonstrated by flow cytometry analyses. In silico molecular modeling predicted that the L16R mutation prevents normal protein folding, consistent with the observed reduction in expression/stability and diminished function of this mutant protein. We isolated normal dermal fibroblasts from members of the families expressing WT or L16R proteins to investigate the impact of endogenous p16-L16R mutant protein on cell growth. In culture, p16-L16R fibroblasts grew at a faster rate, and most survived until later passages than p16-WT fibroblasts. Further, western blotting demonstrated that p16 protein was detected at lower levels in p16-L16R than in p16-WT fibroblasts. Together, these results suggest that the presence of a CDKN2A (47T>G) mutant allele contributes to an increased risk of pancreatic cancer as a result of reduced p16 protein levels and diminished p16 tumor suppressor function.Germline mutations in  CDKN2A affecting the encoded p16INK4A protein (hereafter referred to as p16) are a major risk factor for familial melanoma, with a high proportion (40–60%) of families in which multiple individuals develop melanoma-carrying germline mutations in CDKN2A (CDKN2A mutations are associated with higher risk of pancreatic adenocarcinoma (PDAC), with some evidence for increased cancers at other sites , that encodes the variant protein p16-L16R. Although CDKN2A encodes two distinct tumor suppressor proteins by alternative splicing, p16, and p14ARF, the 47T>G nucleotide alteration only affects the sequence of p16. The L16 residue of p16 is evolutionarily conserved throughout eutherian mammals, marsupials, and birds /p16-L16R, confirmed by a commercial genetic testing laboratory (CDKN2A (47T>G) mutation. There were 30 individuals who were found not to carry the mutation (age range 30–71); among these noncarriers there were five reported cancers ; none reported having multiple cancers. The remaining noncarriers reported no cancer history. In contrast, among 40 kindred members who were mutation carriers (age range 31–73), more cancers were reported: two carriers had pancreatic cancer only, one carrier had pancreatic cancer and melanoma, and one carrier had pancreatic cancer and colorectal cancer. Thirteen carriers had melanoma only, and three carriers reported other cancers . Among these carriers with cancer, four reported having additional cancers. The segregation of the mutation in these kindreds is consistent with autosomal dominant inheritance of the cancer phenotypes. These findings demonstrate a higher occurrence of cancer in 16-L16R carriers (50%) than noncarriers (16.7%) within these kindreds. Similarly, a kindred with the p16-L16R mutation was previously reported in which six out ten carriers presented with melanoma . This conformational change is not surprising as Leu16 is buried in WT-p16. Replacement of Leu16 by a bulky, positively charged residue (Arg16) would therefore be expected to disrupt the protein structure. Indeed, we observed clear opening of p16 helix α1 that harbors Arg16 after 100 ns of simulation (C). This dramatic change in conformation most likely disrupts the interaction of p16 with its binding partners. We note that helix α1 contributes to intermolecular contacts in the X-ray structure of p16 in complex with CDK4  simulations of WT and L16R mutant using the nuclear magnetic resonance (NMR) spectroscopy solution structure of p16 as a starting model . While tmulation C. This dith CDK4 . FurtherG12V and an shRNA against p16 (A and D). In contrast, transfection with p16-L16R elicited only a small decrease in the proportion of cells in the sum of G2 and S phases compared with controls, and the decrease in G2+S was significantly less than that observed with p16-WT (A and D). The transfection efficiency for p16 in Panc1 cells was shown to be ∼17% for WT and 18% for L16R p16 as estimated by immunofluorescent staining . These data indicate that the p16-L16R variant has reduced protein levels and is deficient in suppressing G1-S phase progression compared with the p16-WT under the same transfection conditions.To assess the functional consequences of the L16R mutation, p16-null Panc1 and MiaPaca2 pancreatic cancer cells , and an inst p16  , indicating that decreased p16-L16R protein levels cannot be explained by reduced p16-L16R mRNA expression. Transfection of untagged WT and L16R p16 in Panc1 cells and detection using a p16 antibody also showed significantly lower levels of L16R than WT p16 (F), indicating that reduced expression of L16R is not influenced by the presence of the FLAG epitope tag. Since individual codons can have an impact on protein translational efficiency and protein folding (G). p16-L16R with codons CGG and AGA exhibited mRNA levels that were not significantly lower than that of WT p16 (E). AGG-L16R p16 had significantly lower mRNA levels than p16-WT and AGA-L16R p16, suggesting that there might be a codon bias effect against the AGG codon. . Together, these data suggest that the decreased protein level observed for p16-L16R is likely determined by a posttranscriptional mechanism.p16-FLAG mRNA expression was not significantly different between WT and mutant p16 transfections in Panc1 cells at 24 and 30 h C, indican WT p16 F, indica folding , 24, we  folding G. p16-L1f WT p16 E. AGG-L1versus different amounts of p16-WT DNA and performing flow cytometry 48 h after transfection. The L16R mutant exhibited significantly less cell cycle suppression than the WT transfected at DNA ratios of 1, ½, or 1/4 compared with p16-L16R and was similar in cell cycle suppression effects to the WT expressed using 1/6 of the DNA compared with p16-L16R (A). However, western blotting of cells from the same transfection experiments showed that p16-L16R protein expression was higher than that of the 1/4 ratio p16-WT transfection (B). These results indicate that the L16R mutant is less functional than the WT p16 even when protein levels are equivalent.The functionality of the L16R mutant protein in cell cycle suppression was further evaluated by transfecting p16-L16R p16-L16R A. Howevesfection B. These C), indicating that the p16-L16R has a reduced affinity for CDK4/6. These results are consistent with the diminished ability of p16-L16R to inhibit cell cycle progression (A and 3A).The L16R mutant was evaluated by its ability to coimmunoprecipitate endogenous CDK4 and CDK6. Panc1 cells were transfected with p16-L16R or with a reduced DNA amount of p16-WT to yield a similar level of protein. After 48 h, cell lysates were immunoprecipitated using an anti-FLAG antibody and subsequently analyzed by western blotting for FLAG-p16, CDK4, and CDK6. The L16R mutant showed almost no co-immunoprecipitation with CDK4/6, whereas the p16-WT co-immunoprecipitated appreciable levels of CDK4 and CDK6 C, indicagression A and 3A.versus L16R p16, p16-WT protein expression was tested after the knockdown of CDK4 or CDK6 to determine if WT-p16 stability is affected by loss of binding to CDK4/6. Cells were treated for 24 h with siRNA against CDK4, CDK6, or a nontargeting control. Cells were then transfected with p16-WT for 48 h and analyzed by western blotting. Although CDK4 and CDK6 protein levels were decreased ∼90% by their respective siRNAs, there was no change in WT-p16 expression with knockdown of either CDK4 or CDK6  and untagged p16-L16R (B) protein levels were increased by treatment with the proteasome inhibitor MG132, but did not reach p16-WT levels. p16-L16R-FLAG protein levels were increased up to p16-WT-FLAG levels upon treatment with another proteasome inhibitor, bortezomib, when expressed in Panc1 cells and RPM1-7951 melanoma cells, a cell line homozygous for p16-L16R (C and D). These results suggest that ectopically expressed p16-L16R protein is more rapidly degraded by the proteasome that the WT protein and is stabilized by bortezomib.To gain further insight into the mechanisms underlying the increased stability of WT  or CDK6 . Thus, t16R-FLAG A and untp16-L16R B proteinp16-L16R , C and DA). However, only the L16R mutant and one variant (Δ86–92) containing a seven amino acid deletion showed reduced cell cycle suppression compared with the WT protein . Thus, under our experimental conditions, we were unable to identify any loss of function for most of these variants which might explain their association with cancer risk. It is possible that differences in function of these variants were not ascertainable using assays based on p16 overexpression, or that these variants affect other noncanonical functions of p16 , which we have not assessed /p16-L16R allele, and HSFs from those identified as noncarriers were p16-WT.The above studies demonstrate that p16-L16R has reduced protein expression and diminished ability to suppress cell cycling compared with p16-WT when expressed ectopically in p16-null Panc1 cells. We next wanted to determine if any loss of p16-L16R function could be detected when this protein is expressed endogenously, as occurs in human carriers. Thus, normal human skin fibroblasts (HSFs) were isolated from punch biopsies taken from p16-L16R family members with L16R (n = 8) and WT (n = 7) p16 genotypes and used as a surrogate for cells that might be at risk of transformation as a result of p16-L16R carriage. Genomic DNA sequencing verified that all HSFs from previously identified L16R individuals were heterozygous for 5 cells/10 cm dish), cultured for a week, trypsinized, counted, and then replated as above. This process was repeated until HSFs stopped replicating. Average growth rates from passages 12 to 15 were significantly higher for p16-L16R than p16-WT cells,  (A and B). Growth rates of individually derived fibroblasts from weeks 12 to 30 are shown in Fig. S5A. Notably, several L16R fibroblasts from patients who had been diagnosed with two or more cancers  exhibited the highest growth rates . When averaged separately, the four HSFs from individuals who had one or more cancers  had significantly higher growth rates (31.5%/day) than WT HSFs . Growth rates of p16-L16R HSFs from donors with no cancer (20%/day) were higher than average WT values, but not significantly so. Beginning at passage 16, some fibroblasts ceased to proliferate. However, a trend toward higher growth rates for p16-L16R samples versus p16-WT fibroblasts could be seen at almost all passages . We also evaluated cell proliferation rates of selected WT and L16R HSFs by FACS analysis. Individual WT HSFs exhibited a smaller percentage of cells in G2 and S phases than L16R fibroblasts . When pooled, L16R HSFs were significantly higher in percent S phase and G2 + S phases than WT HSFs , supporting our findings of faster cell proliferation rates in p16 L16R versus WT HSFs as measured by cell counting (A and B).To evaluate the impact of p16-L16R expression on growth, passage 12 fibroblasts from individual donors were plated at equal numbers (6 × 10ctively) , A and BA). The average terminal passage was 29.9 from among p16-L16R carrier fibroblasts and 25 for p16 WT HSFs (A). Together, these results indicate that on average p16-L16R-expressing fibroblasts had greater growth rates and tended to survive to later passages than p16-WT cells. Although samples sizes are small, the data suggest that p16-L16R expression confers a greater proliferative capacity on cells. Proliferation rates of individual fibroblasts were inversely correlated with patient age (r = −0.59. p = 0.04) when samples with multiple cancers and the greatest growth rates were excluded (B), consistent with a relationship between fibroblast growth rate and donor age reported in previous studies (p = 0.04) was observed (B).By passage 30, 5/7 p16-WT cells had ceased to proliferate, but only 2/8 p16-L16R cells had stopped growing A. The av WT HSFs A. Togethexcluded B, consisobserved B.A). By passage 7, p16 was detected in some samples of both genotypes. In HSFs (passage 11) cultured for 1 week in normal media, p16 protein levels were decreased in L16R versus WT-p16 fibroblasts, but differences between groups were not statistically significant (B). Challenging cells with nutrient depletion is reported to increase p16 protein expression in fibroblasts (C and D). Thus, the occurrence of heterozygous p16-L16R in HSFs can lead to an overall decrease in endogenous p16 protein expression, consistent with our findings using ectopically overexpressed p16 WT and L16R in multiple cell types. Since we had observed that the proteasome inhibitor, bortezomib, protected overexpressed p16-L16R from degradation (C and D), we treated HSFs endogenously expressing WT or heterozygous L16R p16 with bortezomib and evaluated the effects by western blotting. However, bortezomib had no impact on endogenous protein levels of WT or L16R p16 in HSFs (A) and also had no impact on p16 protein levels of p16-WT (501MEL and HEK293) or p16-L16R (RPMI-7951) cells (B). Thus, the decreased levels of endogenous p16 detected in p16-L16R heterozygous HSFs do not appear to be due to bortezomib-sensitive proteasomal degradation.We also investigated the impact of carrying the p16-L16R allele on p16 protein expression in HSFs. At low passages, little endogenous p16 protein was detectable by western blotting in WT or L16R fibroblasts A. By pasnificant B. Challeroblasts , 32. Thuroblasts , C and Dradation , C and D in HSFs A and als1) cells B. Thus, CDKN2A(47T>G)/p16-L16R variant in four multigeneration kindreds of Mayo Clinic patients that contained members diagnosed with familial pancreatic cancer and melanoma. Here, we demonstrated that when expressed in p16-null pancreatic cancer cells, p16-L16R had little ability to suppress the cell cycle and exhibited decreased binding to CDK4 and CDK6 compared with p16-WT. These findings provide evidence that p16-L16R is a bona fide deleterious mutation, consistent with the association of this variant with increased occurrence of melanoma and pancreatic cancer. In addition, we found that p16-L16R exhibited lower protein levels compared with the WT protein, as demonstrated by ectopic expression in cells. HSFs with heterozygous p16-L16R also showed higher proliferation and lower overall levels of p16 protein, consistent with the findings in pancreatic cancer cells. Thus, through its inability to suppress the cell cycle and decreased protein expression, the presence of p16-L16R leaves carriers at higher risk of melanoma and pancreatic cancer.We identified the in silico molecular modeling studies showed that p16-L16R is likely less stable than p16-WT. Although p16-L16R protein was protected from degradation by the proteasomal inhibitor, bortezomib, when overexpressed in cell lines, bortezomib had no protective effect on endogenously expressed p16 WT or L16R. Thus, the lower levels of endogenous p16-L16R in HSFs do not appear to involve rapid proteasomal degradation. We also demonstrated that p16-WT is not protected from degradation by binding to CDK4/6, ruling out the possibility that p16-L16R is degraded more quickly because it is not bound to CKD4 and CDK6. Interestingly, we note that several other p16 variants of unknown significance identified as associated with pancreatic cancer . p16-L16R patients without cancer (20% growth per day) showed a trend (p = 0.13 in two-tailed t-tests) toward higher growth rates than WT cells, supporting the idea that p16-L16R increases growth rates, even in subjects without cancer. Additional studies using larger samples sizes are needed to distinguish between direct p16-L16R effects on carrier HSFs and systemic effects due to previous cancers.To our knowledge, our studies are the first to investigate the impact of endogenous germline p16 mutants on cell proliferation using fibroblasts from normal skin biopsies of individuals in familial melanoma/pancreatic cancer families. Earlier studies of fibroblasts from cancer patients of unknown germline mutation status and investigations of p53 mutant HSFs have shown that cells from cancer patients possess altered growth characteristics compared with normal fibroblasts , 34. OneCDKN2A mutations develop melanoma, pancreatic cancer, or other malignancies and why penetrance of these mutations is less than 100% . Written informed consent was obtained from all participants. All participants provided a lymphocyte DNA sample, which was tested for the presence/absence of the p16-L16R variant by Sanger DNA sequencing and confirmed by commercial genetic testing . SubjectSkin biopsies were taken from volunteers from among the kindred families, including those with WT or L16R p16 status and those with or without a history of cancer. Skin punch biopsies (4 mm) were performed on a sun-unexposed area of the upper arm using sterile technique and a topical anesthetic/lidocaine and/or injectable methyl paraben-free lidocaine (placed in the skin using a needle) to minimize patient discomfort, followed by closing with 1 to 2 sutures. Samples of punch biopsies were placed in a sterile 15 ml conical test tube with biopsy transport media [Roswell Park Memorial Institute Medium (RPMI) with 1% antibiotic-antimycotic]. Sample tubes were labeled and sealed with Parafilm and stored at 4 °C for up to 3 days until processing.2 incubator for several days in growth media . After 1 week, cells were washed in PBS and passed using trypsin. Cell culture in growth media was continued until all cultured cells were fibroblasts (keratinocyte growth is inhibited under these conditions). After 2 to 3 passages, samples of HSFs were cryopreserved for reference or later use. Actively growing HSFs were used for cell biological, biochemical, and molecular studies. To verify the p16 status as WT versus L16R for each HSF, genomic DNA was isolated from HSFs using the DNeasy Blood & Tissue Kit (QIAGEN cat: 69506). PCR was then conducted using the Terra PCR Direct Red Dy Premix Kit (Takara cat: 639286) using the following primers: CAACCTGGGGCGACTTC (sense), CTGCAAACTTCGTCCTCCAG (antisense). PCR products were run on 1% Agarose gels, and ∼483 bp fragments containing a single band were then extracted using the Gel/PCR DNA Fragment Extraction Kit . Gel extracts were submitted to GENEWIZ, and Sanger sequencing was performed using the above listed antisense primer.Biopsy dissection and initial fibroblast culture were conducted under sterile conditions by the Mayo Clinic Biochemical Genetics Laboratory. Skin samples were dissected into 12 to 15 small pieces using sterile scalpel and forceps. Pieces of sample were transferred to a cell culture flask and incubated at 37 °C in a CO2 at 37 °C. Panc1 were cultured in DMEM with 10% heat-inactivated fetal bovine serum , RPMI-7951 cells in EMEM/10% FBS, and HSFs in DMEM/10% FBS plus 1:100 Penicillin-Streptomycin (Thermo). Cell lines in normal growth media (8 × 105 in 10 cm culture dishes) were transiently transfected utilizing XtremeGene HP (MilliporeSigma) using a ratio of 1 μg plasmid DNA/3 μl XtremeGene HP/ml of growth medium. CDK4 and CDK6 depletion was performed using Dharmafect1 reagent (Dharmacon) according to manufacturer's instructions with siCDK4 and siCDK6 ONTARGETplus SMARTpools, siRNAs (Dharmacon), and Allstar Neg. control siRNA .Panc1 and MiaPaca2 pancreatic adenocarcinoma cells and RPMI-7951 melanoma cells were obtained from American Type Culture Collection. HPNE cells   were a gPlasmid vector pQCXIH (Addgene plasmid 37104) containing WT p16 was purchased from Addgene and cloned into p3XFLAG-CMV-14 (Sigma-Aldrich) using the following primers and restriction sites: (EcoRI) ATAAGAGAATTCATGGAGCCGGCGGCGGGGAGC (sense) and (XbaI), CTAGCTTCTAGAATCGGGGATGTCTGAGGGACCTTCCGCG (antisense), and into the pIRES2-EGFP bicistronic vector (Clontech) using (Not1) ATAAGAATGCGGCCGCATGGAGCCGGCGGCGGGGAGCAG (sense) and (EcoR1) ATAAGAGAATTCTCAATCGGGGATGTCTGAGGGACCTTCCGCG (antisense). Mutant L16R was prepared using the Q5 Site-Directed Mutagenesis Kit from New England Biolabs. The following primers were used: sense GCTGACTGGCGGGCCACGGCC and antisense CGAAGGCTCCATGCTGCTCCC. Other p16 variants were prepared similarly.2, supplemented with 10 μg/ml leupeptin, 10 μg/ml aprotinin, 1 μg/ml pepstatin, 1 mM phenylmethyl sulphonyl fluoride, and complete protease inhibitor cocktail ]. For HSFs, 1 to 2 confluent 10 cm dishes were washed three times with ice-cold PBS. Cells were then were scraped in 80 μl lysis buffer. For transfected cells, 2 days after transfection, cells were washed three times with ice-cold PBS and then lysed in 150 μl lysis buffer. Protein lysates were dispersed by passage three times through a 25-gauge needle, then centrifuged at 17,000g, and the supernatant collected. Protein concentrations of cleared lysates were determined using the BCA protein reagent (Pierce). Lysates were then diluted in Laemmli buffer with 20 mg/ml DTT and run on 12% or 4 to 20% Bio-Rad Min-Protean TGX or Criterion TGX unstained precast gels. Gels were transferred to PVDF membranes in Towbin transfer buffer with 20% MeOH. The transferred membranes were blocked in 3% BSA in PBS with 0.3% Tween-20 (PBS-T) for 1 h. Membranes were then incubated at room temperature for 2 h with primary antibodies in PBS-T with 3% BSA. The following primary antibodies were used: M2-FLAG , vinculin  and α-tubulin  , and mouse anti-p16 . After washing, blots were incubated for 1 h with horseradish peroxidase secondary antibodies (MilliporeSigma) in PBS-T with 3% BSA. After further washes, immunoreactive signals on blots were detected with Thermo SuperSignal West Pico and West Dura Chemiluminescent Substrate kits. Signals were captured on X-Ray film and converted to digital images by scanning or scanned using a Bio-Rad ChemiDoc MP system. Blot signals were quantified using Image J 1.49v.All cells were lysed in lysis buffer .PANC1 cells cultured on 25 mm coverslips in 35 mm culture dishes were transfected for 48 h with WT or L16R p16 in the pCMV-FLAG vector. Cells were then fixed in 3.2% formaldehyde for 40 min and permeabilized in 0.1% Triton-X-100 at 4 °C for 5 min. Subsequent steps were performed at room temperature. Samples were blocked for 1 h in blocking buffer , 2 day after transfection, cells were washed with cold PBS, trypsinized, and centrifuged at 1500g for 5 min. Cell pellets were fixed by adding 430 μl of cold PBS and then adding 1 ml of 100% EtOH, dropwise. The cells were then kept at −20 °C overnight. The next day, cells were washed with cold PBS and centrifuged at 1500g for 5 min, followed by resuspension in 300 μl of PBS with 0.9 ng/μl RNAse and 0.5 ng/μl propidium iodide. FACS was performed using BD FACSCanto II Flow Cytometry System, using BDX Diva software. For each measurement, 40,000 (Panc1 and MiaPaca2) or 20,000 (HPNE) events were acquired. Analysis of cell cycle was performed using the Modfit cell cycle program (Verity Software House) to determine the fractions of cells in the G1, S, and G2/M phases from the cell cycle distribution.Panc1, MiaPaca2, and HPNE cells were transfected as above with p16-FLAG (WT or L16R) or pCMV vector only, plus EGFP-N1 at a ratio of 8:1 (p16:EGFP). After 1 day, cells were trypsinized and replated at 2 × 105 cells/10 cm dish) and cultured for 1 week. Cells were trypsinized, counted, and replated as above once per week at 6 × 105 cells/dish until they ceased proliferating. Proliferation rate (% increase in cell number per day) was calculated by the formula: 100 × (number of cells recovered/the number of cells plated the week before)/7 days per week.Passage 12 fibroblasts from individual donors expressing either WT or heterozygous p16-L16R were trypsinized, counted using a manual hemocytometer, centrifuged, and resuspended in fresh DMEM/10% FBS with 1:100 pen-strep. Cells were plated in fresh media at equal numbers . The High-Capacity cDNA Reverse Transcription kit (Applied Biosystems) was used to reverse transcribe 2 μg of RNA. A portion of the total cDNA was amplified by real-time PCR. Samples were prepared with PerfeCTa SYBR Green FastMix (Quanta BioSciences Inc) and the following primers were used to detect p16-FLAG: AATGTCGTAATAACCCCGCCCCGTTGACGC (sense); CGAAGGCTCCATGCTGCTCCC (antisense); and the housekeeping genes TBP: GGTTTGCTGCGGTAATCATGA (sense), CTCCTGTGCACACCATTTTCC (antisense); HPRT: TGGAAAAGCAAAATACAAAGCCTAAGATGA (sense), ATCCGCCCAAAGGGAACTGATAGTC (antisense). Amplification was performed using the C1000 Thermal Cycler (Bio-Rad) with BioRad CFX Manager software. p16 RNA levels were calculated following the 2ΔCt method using the corresponding levels for HPRT and TBP in the same sample.3) with a solute-wall minimum distance of 1.0 nm. Charges were neutralized with Na+ and Cl− atoms with 100 mM NaCl included to approach physiological conditions. There were 20,808 atoms in the WT system and 20,809 atoms for the simulation of L16R.For the MD simulations, we used the NMR solution structure of human p16 (PDB code 2A5E)  as startThe systems were subjected to steepest decent energy minimization with a maximum force of 200 kJ/mol/nm. The temperature and volume of each system were then equilibrated by running 200 ps of constant volume and constant temperature (NVT) equilibration at 298 K with a velocity-rescaling thermostat. This was followed by equilibration for 1 ns to a 1.0 bar constant pressure (NPT) bath using the Berendsen weak coupling method . The aboAll data calculations and statistical analyses were performed using Microsoft Excel version 16.16. 26. Results with multiple replicates were expressed as mean ± standard error (SE).All the data described are located within this article.This article contains The authors declare that they have no competing interests with the contents of this article.


Human Vg9/Vδ2 T cells (γδ T cells) are immune surveillance cells both in innate and adaptive immunity and are a possible target for anticancer therapies, which caninduce immune responses in a variety of cancers. Small non-peptide antigens such as zoledronate can do activation and expansion of T cells in vitro. It is evident that for adoptivecancer therapies, large numbers of functional cells are needed into cancer patients. Hence, optimization of methods needs to be carried out for the efficient expansion of these T cells.Standardization of peripheral blood mononuclear cells (PBMCs) isolation was devised. Cytokines (interleukin 2 (IL-2) and interleukin 15 (IL-15)) and zoledronate were also standardizedfor different concentrations. It was found that an increased number of PBMCs were recovered when washing was done at 1100 revolution per minute (rpm). Significantly high expansion foldwas (2524 ± 787 expansion fold) achieved when stimulation of PBMCs was done with 1 µM of zoledronate and both cytokines IL-2 and IL-15 supported the expansion and survivalof cells at the concentrations of 100 IU/ml and 10 ng/ml respectively. 14-day cultures showed highly pure (91.6 ± 5.1%) and live (96.5 ± 2.5%) expanded γδ Tcells. This study aimed to standardize an easy to manipulate technique for the expansion of γδ T cells, giving a higher yield. Cancer is a disease distinguished by abnormal growth of cells, which leads to morbidity and mortality worldwide, with approximately 18.1 million new cases and 9.6 million cancerdeaths according to the International Agency for Research on Cancer in 2018 . ConsequRPMI-1640 medium, LymphoprepTM, 0.9% saline (NaCl), Falcon tube (50 ml), Millipore filter (0.22 µM), syringe, 10% Foetus calf serum (FCS), L-glutamine , Pen/strep, MEM Sodium pyruvate (100mM), non-essential amino acids . IL-2 (100 IU/ml) were from GIBCO Life Technologies (USA). Interleukin-2and 15 were purchased from Miltenyi Biotec (Germany). Monoclonal antibodies (mAbs) specific for CD3, CD4, CD8, TCR-Vγ9 were from Beckman Coulter (USA). Live/Dead fixable aqua deadcell stain kit was from Invitrogen-Life Technologies (USA). Immunoglobulin from human and zoledronic acid injection (4 mg) were purchased from Sigma Aldrich and Cipla respectively.Venous blood samples were collected from healthy individuals  with their full consent. This study was performed in accordance with the ethical standards asgiven in the Helsinki Declaration of 1975 and revised in 2000. Ethical approval for this study was received from the institution. The following procedure was adopted for venous bloodcollection; a proper sized needle was attached to the syringe. For best visualization of the draw site was positioned and tourniquet ideally applied 3-4 inches above the puncture site.The participants were instructed to make a fist and hold it without pumping their hand. The venipuncture site was prepared using a Chlorhexadine wipe followed by air-drying. The needlewas inserted into the skin, at an angle of 15-30 degree to the skin, into the vein. All the standard safety measures were followed while drawing blood.An unopened RPMI-1650 (500 ml) was opened under a laminar flow in a sterile environment and 70 ml of medium was removed. 50 ml (10%) FCS (heat inactivated), 5 ml L-glutamine , 5 ml Pen/strep, 5 ml MEM Sodium Pyruvate, and 5 ml MEM non-essential amino acids were added to this. To avoid contamination, all solutions were passed through Millipore filters(0.22 µM) before being added to RPMI medium.A volume of 5 ml fresh blood was drawn using the method stated above. A 50 ml sterile falcon tube was taken to which 5 ml heparin/EDTA was added. Blood from the syringe was transferredto this tube and tilting the tube sideways facilitated mixing with EDTA. Five ml lymphoprepTM was added to a sterile 15 ml falcon tube and brought to room temperature. Blood was addedto the lymphoprepTM, by tilting the tube and placing the tip of the pipette on the surface of the upper layer while pouring gently. Two layers separated out: blood in the upper layerand lymphoprepTM at the bottom. The tube was centrifuged at 1680 rpm for 22 min at 18°C with acceleration set at 2 and deceleration at 0. After centrifugation, four layers were visible;the uppermost was plasma, the second hazy thin layer contained PBMCs, third layer was lymphoprepTM and bottom most layer contained RBCs. Plasma was removed by pipetting and only a fewml (2-3 ml) was left in the tube so as not to disturb any leftover PBMCs. 10 ml sterile and cold saline (0.9% NaCl) was taken in a sterile 15 ml falcon tube. The remaining plasma andthe hazy layer on top of the lymphoprepTM contained PBMCs. Approximately 3-4 ml solution was collected and added to a 15 ml tube with 10 ml normal saline and centrifuged at 1100 rpm for10 min at 4°C. After centrifugation, the supernatant was removed, first by pipetting and later using 3 ml dropper so that the supernatant could be decanted carefully, and the pelletremained undisturbed. 1 ml saline was added to the pellet and mixed well. The tip of the pipette remained inside the solution to avoid any bubbles while mixing. A further 9 ml salinewas added, and the solution centrifuged at 1100 rpm for 10 min at 4°C. After centrifugation, the supernatant was removed. 1 ml saline was added again to the pellet, mixed well andan additional 9 mL saline was also added. To estimate the number of PBMCs per mL at this stage, 20 µL of the sample was removed and the cells counted with the help of a Neubauerchamber, under a microscope . PBMC numbers usually ranged between 1-2 million cells/mL bloods. The solution was centrifuged again at 1100 rpm for 10min at 18°C. After centrifugation, the supernatant was removed, and the pellet retained. 1 ml complete RPMI (pre-warmed) medium was added to the pellet and mixed well. The remainingvolume required for an experiment was made up with complete RPMI medium. For the experiment described below 9 ml RPMI medium was added to the cells to make up the volume up to 10 ml,which contained 1 million/ml PBMCs.γδT cell in isolated PBMCs were stimulated using 1µM zoledronate and were cultured in complete RPMI 1640 medium at a density of 106 cells/ml in 24-well plate andkept in CO2 (5%) incubator at 37°C. The level of water in the incubator was checked often to prevent decrease in levels. Cytokines IL-2 (100 IU/ml) and IL-15 (20 ng/ml) were addedto the culture at day 3, 6, 8 and 11 and cells were split into fresh medium. At day 14, the percent γδT cells in culture were evaluated by flow cytometry together with theviability. Expansion fold was also determined.Approximately 50,000-500,000 PBMCs or expanded were plated in a total volume of up to 250 µl. These were centrifuge at 1100 rpm for 3 min at 4°C in a centrifuge. The pelletsof cells were then washed with 200 µl phosphate buffer saline (PBS). Three microliters of Live/Dead fixable aqua dead cell stain were added and incubated for 15 min in the darkfollowed by the addition of 200 µl PBS contains 2% FCS (FACS buffer). Post incubation, cells were centrifuged (1100 rpm for 3 mins) and the supernatant discarded. Cells were blockedwith 100 µl of 1:1000 diluted human IgG for 15 min on ice. After incubation cells were centrifuged (1100 rpm for 3 mins) and throw the supernatant. Then stained cells with 40µl cocktails of antibodies and isotype by further incubating for 20 min on ice in the dark. Approximately 160 µl FACS buffer was added after incubation and then the mixturewas centrifuged (1100 rpm for 3 mins). Finally, cells were resuspended in 100 µl FACS buffer and are ready for analysis at flowcytometer .Proliferated γδ T cell numbers depend on the starting number of cells. For this reason, the PBMC washing procedure during isolation from human blood was standardized torecover as many cells as possible. For the optimization process, PBMCs were centrifuged at different rpm  .SignifiSimilarly, different IL-15 concentrations (1-100 ng/ml) were used in γδ T cell cultures and plates. Different concentrations of IL-15 were added at day 3, 5, 8 and 11.Significantly high percent of live cells (P < 0.001) and γδ T cells (P < 0.001) were observed at 10 ng/ml and 100 ng/ml comparedBoth IL-2 and IL-15 were standardized to determine optimal concentrations to be used for γδ T proliferation. Different concentrations of IL-2 (1-200 IU/ml) were addedto γδ T cell culture plates at day 3, 5, 8 and 11, respectively. Significant differences were observed in live cells, when expanded with different concentrations of IL-2.A significantly high percent of live cells was achieved with both IL-2 100 and 200 IU/ml when compared with 1 IU/ml (p < 0.001) and 10 IU/ml (p < 0.01). Furthermore, high percentof γδ T cells (P < 0.01) were observed at 100 IU/ml of IL-2 as compared to 1 and 10 IU/ml . MoreoveDifferent concentrations of zoledronate , used as stimulant for γδ T cells, were analyzed for cell expansion in presence of both cytokines, IL-2 andIL-15. The concentration of 1 uM zoledronate led to increased percent of live expanded cells with significantly high percent of γδ T cells when compared to the 0.1 µM(p < 0.001). Furthermore, there was a significant high (p < 0.01) percent of γδ T cells were observed at 10 µM concentrations of zoledronate in 14 day cultures.In vitro proliferation of 1 µM zoledronate stimulated γδ T cells for 14 days was carried out using different cytokine(s); first condition IL-2 (100 IU/ml) alone;second condition IL-15 (10 ng/ml) alone; and third condition with both IL-2 (100 IU/ml) and IL-15 (10 ng/ml) together . PBMCs wThese expanded γδ T cells were also analyzed for activation and costimulatory cell surface molecules .SignifiFrom these findings, we propose an efficient and reproducible methodology , throughγδ T cells are unique unconventional T cells, which are distinguishable from αβ T cells. γδ T cells fulfil numerous important functions in immunity,including cytokine production in response to microbial challenges, mobilization of other types of immune cells and tumor cell killing (at least in vitro) ,12,13. It is well established that human γδ T cells have anti-cancer activity and can be safely infused into patients with different cancers. For potential cancer immunotherapy,γδ T cells are needed in large numbers, as several doses of these cells are often required for treatment. Also, it is not feasible to draw large amounts of blood from terminallyill cancer patients, which is the usual procedure for dendritic cell-based immunotherapies. Thus, a standardized method for the expansion of γδ T cells is needed with detailedmethodology for their better yield. We have established in this study a detailed method for the expansion of γδ T cells, in which significantly high yields of pure γδ T cells were recovered with high percentage of live cells after 14 days of culture. Furthermore, these expanded γδ T cells have the potential to be use as personalizedcancer therapeutics.


Saccharomyces cerevisiae and Kluyveromyces lactis to have very similar DNA binding domains and to be necessary for derepression of a variety of genes under non-fermentative growth conditions via binding to the carbon source responsive elements (CSREs). The methylotrophic yeast Komagataella phaffii (syn Pichia pastoris) has two transcription factors (TFs), which are putative homologs of Cat8 based on sequence similarity, termed Cat8-1 and Cat8-2. It is yet unclear in which cellular processes they are involved and if one of them is actually the homolog of Sip4. To study the roles of the Cat8 homologs in K. phaffii, overexpression or deletion strains were generated for the two TFs. The ability of these mutant strains to grow on different carbon sources was tested, and transcript levels of selected genes from the carbon metabolism were quantified. Our experiments showed that the TFs are required for the growth of K. phaffii on C2 carbon sources, but not on glucose, glycerol or methanol. K. phaffii deleted for Cat8-1 showed impaired growth on acetate, while both Cat8-1 and Cat8-2 are involved in the growth of K. phaffii on ethanol. Correspondingly, both TFs are participating in the activation of ADH2, ALD4 and ACS1, three genes encoding enzymes important for the assimilation of ethanol. Different from S. cerevisiae and K. lactis, Cat8-1 is not regulating the transcription of the putative Sip4-family member Cat8-2 in K. phaffii. Furthermore, Cat8-1 is necessary for the activation of genes from the glyoxylate cycle, whereas Cat8-2 is necessary for the activation of genes from the carnitine shuttle. Neither Cat8-1 nor Cat8-2 are required for the activation of gluconeogenesis genes. Finally, the CAT8-2 gene is repressed by the Mig1-2 transcription factor on glucose and autorepressed by the Cat8-2 protein on all tested carbon sources. Our study identified the involvement of K. phaffii Cat8-1 and Cat8-2 in C2-metabolism, and highlighted similarities and differences to their homologs in other yeast species.The transcription factors Cat8 and Sip4 were described in The online version contains supplementary material available at 10.1007/s00294-021-01165-4. Komagataella phaffii (Pichia pastoris) adapts to different growth conditions through various mechanisms, including reprogramming of gene expression and protein synthesis  found in their promoters, such as the carbon source responsive elements (CSREs)  binding domain  and Sip4 (Snf1 interacting protein) possess a highly similar N-terminal zinc cluster (Zn(II)S. cerevisiae, the expression and activities of CAT8 (ScCat8) and SIP4 (ScSip4) were shown to be regulated by glucose, in a process mediated by the Snf1 kinase  Mig1 binds to the promoter of genes such as CAT8, which represses their expression. On derepressing conditions, the Snf1 kinase phosphorylates Mig1, which leads to its inactivation and the consequent induction of CAT8 transcription  and Sip4 (KlSip4) are present but the regulatory networks are different than in S. cerevisiae. The Klcat8 knock-out mutants can grow on glycerol but not on C2 carbon sources such as acetate and ethanol, which shows that unlike in S. cerevisiae, the gluconeogenesis encoding genes are not regulated by Cat8 nor Sip4 in K. lactis  by the Snf1 kinase  knock-out mutants have a similar phenotype to the wild-type in terms of gluconeogenesis, glyoxylate shunt, and ethanol utilization pathway, and CaCat8 does not seem to regulate the gluconeogenic gene PCK1 nor ICL1  (Ramirez and Lorenz Ogataea (Hansenula) polymorpha exhibit a growth defect on glycerol, ethanol, and xylose, and have a higher ethanol production from xylose fermentation  and CAT8-2 (PP7435_Chr4-0434) are found. The CAT8-2 gene was shown to be induced on limiting glucose (about 39-fold up-regulated compared to excess glucose) and on methanol (about sevenfold up-regulated compared to excess glucose), indicating that it is subject to glucose repression. No up- or down-regulation of CAT8-1 was observed in the same conditions  were used for cloning experiments.All the dy Table  were derAspergillus nidulans  and a maximum E-value of 9e−30.Eight individual protein-BLAST searches were conducted on the ‘Non-redundant protein sequences (nr)’ database of NCBI using the following 6 functionally characterized protein sequences: CBF88979.1, Cat8 of All individual BLAST search results were combined and protein ID duplicates were removed (361 sequences). Then, all sequences containing invalid protein characters (B J O U X Z) were removed from the selection (352 sequences), remaining sequences were filtered for a minimum length of 600 amino acids (275 sequences) and sequences in the selection of 99% sequence identity or higher were represented in the selection by only one sequence of that cluster to reduce sequence redundancy (157 sequences). Together with all characterized Cat8 and Sip4 sequences this resulted in a selection of 161 sequences.The selection was aligned with MAFFT G-INS-I (Katoh and Standley K. phaffii genes (1000 bps upstream of the start codon) were analyzed for the occurrence of the CSRE by MatInspector (matrix F$CSRE). Comparative analysis of TFBS in S. cerevisiae, K. lactis and K. phaffii promoters was done by Yeastract+  when needed.LB media consisted of 10 g/L soy peptone, 5 g/L yeast extract, 5 g/L NaCl. 20 g/L agar–agar was added to prepare LB agar plates.4)2HPO4, 0.8 g (NH4)2SO4, 0.49 g MgSO4 · 7H2O, 2.64 g KCl, 0.0535 g CaCl2 · 2H2O, 22 g citric acid monohydrate, 1.47 mL PTM, 2 mL biotin (0.2 g/L), 20 mL NH4OH (25%) with additional carbon source according to the purpose. For limited glucose condition 25% m2p kit Polysaccharide and 0.078% enzyme was used .ASMv6 medium contained per liter 6.3 g .PTM0 stock solution contained per liter 0.08 g NaI, 6.0 g CuSO4)2SO4, 0.2 g/L biotin, 0.8 mg/L Ca-pantothenate, 0.004 mg/L folic acid, 4 mg/L inositol, 0.8 mg/L niacin, 0.4 mg/L p-aminobenzoic acid, 0.8 mg/L pyridoxine HCl, 0.4 mg/L riboflavin, 1 mg/L H3BO3, 0.08 mg/L CuSO4, 0.2 mg/L KI, 0.4 mg/L FeCl3, 0.8 mg/L MnSO4 · H2O, 0.4 mg/L Na2MoO4 · 2H2O, 0.8 mg/L ZnSO4, 2 g/L KH2PO4, 1 g/L MgSO4, 200 mg/L NaCl, 200 mg/L CaCl2 with additional carbon source according to the purpose. For the limiting glucose condition 25% m2p kit Polysaccharide and 0.078% enzyme was used .YNB without thiamine consisted of 10 g/L  consisted of 1.8 g/L NaGenomic DNA was extracted from overnight cultures using the Wizard genomic DNA purification kit  according to the protocol of the manufacturer. All PCRs were performed using the Q5 polymerase  following the recommendations of the manufacturer or using the OneTaq 2× master mix with GC buffer . The PCRs were used to amplify DNA for cloning and for verifying positive transformants.K. phaffii CBS7435 chromosomal regions of the selected transcription factors are: PP7435_Chr2-0516 for CAT8-1 and PP7435_Chr4-0434 for CAT8-2. The sequences of these genes were retrieved from http://pichiagenome-ext.boku.ac.at.The PiCS vector series , was used for the overexpression of selected TFs .For overexpression cassettes, the CDS of the corresponding genes were amplified from the Komagataella phaffii knock‐out strains were constructed by using CRISPR/Cas9-based homology-directed genome editing  and downstream (3′) of the target gene with an approximate 1000 bp length. The primers used for amplification of these homologous fragments and elimination of internal BsaI sites are given in Supplementary table S2. The flanking upstream and downstream homologous regions of the target gene were assembled with each other in a plasmid by GGA.GAP promoter and the RPS25Att terminator into a plasmid containing the humanized Cas9 CDS under the control of PLAT1 or PPFK300 and the ScCYCtt terminator by GGA. For the integration of the knock-out cassettes, the plasmids carrying the fused homologous regions were used as templates to amplify the fragments by PCR. 3–5 µg of amplified homologous DNA and 0.5–1 µg of circular CRISPR/Cas9 plasmid DNA were simultaneously transformed into K. phaffii CBS7435 by electroporation  sequence identified in 50–200 bp upstream of the CDS of the selected TFs [see Gassler et al.  for moreK. phaffii transformation by electroporation . PCR-amplified homologous regions of knock-out cassettes were directly purified without linearizing. K. phaffii transformation was performed by electroporation  by using 0.5–1 µg of each linearized overexpression plasmid or 3–5 µg of purified knock-out fragments and 0.5–1 µg of circular CRISPR/Cas9 plasmid. Transformed cells were then regenerated by incubation at 30 °C for 1.5–3 h in YPD medium (280 rpm) and then plated on YPD plates including the appropriate antibiotics concentration . After 48–72 h at 30 °C, randomly selected transformants were streaked on selective YPD plates and incubated 48 h at 30 °C.Prior to K. phaffii CBS7435 (carrying a single native copy of the TF gene of interest). The amplifications were carried out using 4.5 µL of genomic DNA solution at a concentration of 1.777 ng/µL with 0.25 µL of both forward and reverse primers  and 5 µL of 2× qPCR S’Green BlueMix . Amplifications were done in a Rotor-Gene Q instrument . The GCN in the K. phaffii mutant strains were calculated relative to the corresponding wild-type control using the threshold cycle (ΔΔCT) method. All signals were normalized to ACT1 (PP7435_Chr3-0993). The primers used for qRT-PCR analysis are provided in Supplementary table S3.Gene copy number (GCN) was determined by quantitative real-time PCR (qRT-PCR). Genomic DNA was extracted from overnight cultures using the Wizard genomic DNA purification kit . The GCN was determined by the relative quantification of the TF of interest sequence compared to wild-type 600 0.01 in 100 μL of YNB without thiamine containing either 2% glucose, 2% glycerol, 2% ethanol or 1% methanol in a 96-well sterile microtiter plate. Growth on acetate was assessed in 100 μL of YNB or YP containing 1% or 2% acetate. The plate was incubated in a TECAN Sunrise plate reader at 30 °C for 24–48 h  with constant shaking . The absorbance at 600 nm in each well was measured every 15 min.The wild-type, overexpression and deletion strains were inoculated at OD600 of the media without cells) was subtracted from the raw OD values to obtain the corrected ODs. The corrected OD600 of the replicates for a given strain were then averaged. The average corrected OD600 was plotted against the time to obtain the growth curves. For the calculation of the specific growth rates, the average corrected OD600 was divided by the average initial OD600 and the natural logarithm was applied. The growth rate is given by the slope of the log-transformed ODs, the maximal growth rate being identified as the maximum value of the slope  and stored at −70 °C until further use. Cells were mechanically disrupted using 500 µL of glass beads in a ribolyzer (5.5 m/s for 40 s), and the total RNA extraction was performed according to the TRI reagent protocol. RNA concentrations and purity were analyzed with a Nanodrop spectrophotometer. DNAse treatment of isolated RNA samples was performed with a DNA-free kit (Invitrogen) and cDNA was synthesized using oligo(dT)23 primers  and the Biozym cDNA synthesis kit according to directions of the manufacturer . The Real-time PCR reactions were performed on a Rotor-Gene Q instrument  using Blue S’Green qPCR Mix  according to the manufacturer’s instructions. Changes in transcript levels in the K. phaffii mutant strains were calculated relative to the corresponding wild-type control using the threshold cycle (ΔΔCT) method. All signals were normalized to the expression of the actin gene ACT1 (PP7435_Chr3-0993). The primers used for qRT-PCR analysis are provided in Supplementary table S4.The wild-type, overexpression and deletion strains were grown on ASMv6 medium with limiting glucose to ODCAT8-1 and CAT8-2 with a 3xFLAG tag before their STOP codons under the control of their native promoters into K. phaffii cat8-1Δ (for Cat8-1-3xFLAG) and cat8-2Δ (for Cat8-2-3xFLAG) deletion strains. Again no clones were obtained. Since the single deletion strains of CAT8-1 and CAT8-2 were viable, it seemed that introducing a FLAG-tagged copy of Cat8-1 and Cat8-2 was detrimental to the cells. Therefore tagging of Cat8-1 and Cat8-2 with the HA tag was tested, and was proven to be a successful strategy yielding viable transformants.Initially, the tagging of Cat8-1 and Cat8-2 was tested with a 3xFLAG tag at the native locus using the CRISPR/Cas9-based homology-directed genome editing  before the STOP codons of the two genes. The PCR fragments were assembled by GGA in a vector carrying the AOX1 terminator homologous regions (for integration into the K. phaffii genome) and the KanMX marker cassette. 0.5-1 µg of linearized plasmids were transformed into K. phaffii cat8-1Δ (for Cat8-1-HA) and cat8-2Δ (for Cat8-2-HA) deletion strains. Transformants were controlled by colony PCR and qRT-PCR (for gene copy number determination). The selected clones were also grown on YNB liquid medium supplemented with 1% ethanol to make sure that the insertion of the tagged version of the proteins in the deletion strains restored their growth on this carbon source.To construct the HA-tagged Cat8-1 and Cat8-2 strains, promoter regions (upstream 1000 bp regions), coding sequences, and terminator regions of CAT8-1 and CAT8-2 were amplified from K. phaffii CBS7435 genomic DNA by PCR (Primers in Supplementary table S5). The respective promoters, the eGFP coding sequence and the ScCYCtt were assembled by GGA in a vector containing AOX1 terminator homologous regions (for integration into K. phaffii genome) and the KanMX marker cassette. 0.5–1 µg of the linearized plasmids were transformed into K. phaffii CBS7435 (WT), cat8-1Δ, cat8-2Δ, and cat8-1Δcat8-2Δ deletion strains. Transformants were controlled by colony PCR and qRT-PCR for gene copy number determination.Promoter regions (upstream 1000 bp regions) of cat8-1Δ, cat8-2Δ, and cat8-1Δcat8-2Δ deletion strains carrying the eGFP coding sequence under the control of the respective promoters were grown on ASMv6 medium with limiting glucose (polysaccharide solution) at 25 °C to OD600 7.0–8.0, washed twice in PBS and inoculated at OD600 3.5–4.5 in ASMv6 medium containing either 2% glucose, 2% glycerol, 2% ethanol or 1% methanol and grown for 5 h at 25 °C, to have an induction on glucose, glycerol, ethanol, and methanol, respectively. After induction, cells were analyzed by flow cytometry.The wild-type and positive transformants of the wild-type, 2PO4 0.24 g/L, Na2HPO4⋅2 H2O 1.8 g/L, KCl 0.2 g/L, NaCl 8 g/L) to OD600 0.2. The forward and side scatter of 10,000 cells for each sample as well as their green fluorescence  were then measured on a CytoFLEX flow cytometer (Beckman Coulter). The Kaluza analysis software (Beckman Coulter) was used to analyze the data. GFP-positive cells were gated using the WT_PCAT8-1_eGFP and WT_PCAT8-2_eGFP strains as reference.Cells were diluted in PBS , K. lactis , O. polymorpha (Cat8 942 aa) and A. nidulans (FacB 867 aa) as well as K. phaffii Cat8-1 (1036 aa) and Cat8-2 (887 aa) were aligned, and their phylogenic tree was calculated , the Sip4 clade of Saccharomycetaceae and Saccharomycodaceae (containing the characterized Sip4 proteins from S. cerevisiae and K. lactis), the Cat8 clade of Saccharomycetaceae and Saccharomycodaceae (containing the characterized Cat8 proteins from S. cerevisiae and K. lactis), the Cat8 clade of Phaffomycetaceae and Pichiaceae (containing K. phaffii Cat8-1 and O. polymorpha Cat8), and finally the Cat8 clade of Debaryomycetaceae and Metschnikowiaceae. In addition, a small clade of 8 sequences of 5 different yeast genus and one Aspergillus Cat8 is also observed , and they reached a lower OD600 at the end of the culture . In contrast, complementation of cat8-2Δ or the double knock-out cat8-1Δ cat8-2Δ with S. cerevisiae SIP4 (under control of the CAT8-2 promoter) did not rescue growth on ethanol (not shown), indicating that ScSip4 was neither able to complement Cat8-1 nor Cat8-2 under the analyzed conditions. Therefore, it was concluded that Cat8-1 and Cat8-2 are two essential transcription factors for the growth of K. phaffii on ethanol.For the knock-out strains cat8-1Δ and cat8-2Δ, and the cat8-1Δcat8-2Δ double knock-out was also assessed on acetate, another C2 carbon source. As it has been reported that K. phaffii deleted for another TF, Mxr1, only showed impaired growth on complex medium, but not YNB  expression on ethanol and methanol, respectively  or involved K. phaffii methanol metabolism .To identify potential target genes of Cat8-1 and Cat8-2, mRNA levels of 13 genes were analyzed in the overexpression and knock-out mutants of The samples for the transcript level analysis were obtained by growing the mutant strains and the wild-type in liquid cultures on minimal medium with limiting glucose to avoid glucose repression in the pre-culture and then shifting the cultures to 2% glucose, 2% glycerol, 1% methanol or 2% ethanol for 5 h to have an induction on either glucose, glycerol, methanol, and ethanol, respectively. Cells were then harvested and RNA was extracted for qRT-PCR Fig. A.Fig. 4ICRC1 and the AOX1 promoter possesses one CSRE, while the ICL1 and PEX5 promoters have two CSREs, the MLS1, MXR1, and ALD4 promoter three, the YAT2 and DAS1 promoters four, the ADH2 and FBP1 promoters five and finally the PCK1 promoter eight predicted CSRE sites.The promoters of the genes selected for the transcript level analysis were screened in silico by MatInspector  for the presence of the CSRE motif representing the binding site of Cat8 and Sip4. All of the promoters of the selected genes were shown to possess at least one predicted CSRE motif, most of them having more than two of these motifs. In particular, the On glucose and glycerol, no significant difference in transcript levels of the selected genes was observed in the different Cat8 mutant strains compared to the wild-type (data not shown).FBP1 and PCK1  were similar to that of the wild-type in the different mutant strains, showing that Cat8-1 and Cat8-2 are not involved in the regulation of these two genes , ACS1  and ALD4  were all decreased  and MLS1 , was decreased in cat8-1Δ and in the cat8-1Δcat8-2Δ double knock-out mutant but not in cat8-2Δ when compared to the wild-type. Therefore, only Cat8-1 seems to be necessary for the activation of these genes from the glyoxylate shunt in K. phaffii. For two genes from the carnitine shuttle, YAT2  and CRC1 , the expression was decreased in cat8-2Δ and in the cat8-1Δcat8-2Δ double knock-out mutant but not in cat8-1Δ when compared to the wild-type. Therefore, Cat8-2 seems to be necessary for the activation of these genes from the carnitine shuttle. Additionally, the expression of three genes important for methanol assimilation, AOX1 , DAS1  and PEX5  was assessed in the TF mutant strains on ethanol, but no specific regulation pattern was observed (Supplementary figure S2): due to the very weak expression of these genes on ethanol, the small differences observed in expression were not considered significant. Additionally the expression levels of MXR1 (PP7435_Chr4-0490) were investigated. MXR1 expression on ethanol was approximately 60% of the expression on methanol, and was found to be approximately two-fold higher in all cat8 deletion strains on ethanol (Supplementary figure S2).On ethanol, the expression of the two gluconeogenic genes ICL1 and MLS1, the two genes of the glyoxylate shunt, and the dependence of ACS1 and ALD4 on both TFs . In order to analyze a possible regulation of the CAT8-1 and CAT8-2 genes by the Cat8-1 and Cat8-2 proteins in K. phaffii, eGFP reporter strains were generated. The eGFP gene was expressed under the control of the CAT8-1 and CAT8-2 promoters, respectively. These constructs were transformed into the different knock-out mutants  and the wild-type. The obtained strains were cultivated on a minimal medium with limiting glucose and then shifted to either 2% glucose, 2% glycerol, 1% methanol or 2% ethanol for induction. After 5 h induction, the eGFP fluorescence was measured by flow cytometry . K. phaffii possesses two gene homologs of this transcription factor, termed MIG1-1 (PP7435_Chr4-0661) and MIG1-2 (PP7435_Chr1-1325). To investigate whether Mig1-1 and Mig1-2 are involved in the transcriptional regulation of CAT8-1 and CAT8-2 in K. phaffii, their transcript levels were analysed in knock-out and overexpression strains of the two Mig transcription factors . Finally, the expression of CAT8-2 is also lower than in the wild-type on ethanol in the MIG1-2_OE mutant .The ces Fig. . Therefoose Fig. A and cleant Fig. B. Indeedant Fig. C and notant Fig. D. CAT8-2S. cerevisiae: Cat8 was shown to be phosphorylated by the yeast homolog of AMPK, the Snf1 kinase complex , and Ssn3 (homolog of ScSsn3 encoded by PP7435_Chr1-1091). The generation of the SNF1-2 and SSN3 knock-out mutants was successful, but unfortunately, no transformants deleted for SNF1 could be obtained.Cat8 and Sip4 are both activated through phosphorylation in snf1-2Δ and ssn3Δ deletion strains, as well as the K. phaffii wild-type, was assessed in liquid cultures with YNB containing either 2% glucose, 2% glycerol, 1% ethanol or 1% methanol (Supplementary figure S5). No difference was observed between the specific growth rates of the snf1-2Δ knock-out mutant and the ones of the wild-type on the four-carbon sources tested. However, the specific growth rate of the ssn3Δ deletion strain is lower than that of the wild-type on glucose (0.26 h−1 for ssn3Δ and 0.31 h−1 for the wild-type), but not on glycerol, ethanol nor methanol (Supplementary figure S5). These results suggest that the two kinases Snf1-2 and Ssn3 are not necessary for activation of Cat8-1 and Cat8-2 on ethanol.The growth of the K. phaffii, which were identified as homologs of the transcription factor Cat8 from S. cerevisiae based on sequence similarity  to acetaldehyde, which is subsequently converted into acetate by aldehyde dehydrogenase (ALD). Acetate is then transformed into acetyl-Coenzyme A by acetyl-CoA synthetase (ACS) in the cytoplasm. Then, acetyl-CoA must be transferred to the mitochondria for the production of energy , Cat8-2 seems to be necessary for the activation of the carnitine shuttle (by regulating YAT2 and CRC1). In contrast, the gluconeogenic genes FBP1 and PCK1 are not regulated by neither Cat8-1 nor Cat8-2  on glucose and to a lesser extent also on glycerol  the expression of Sip4 is activated by Cat8 in S. cerevisiae and K. lactis but not in K. phaffii, and (3) the dissimilar importance of Cat8 and Sip4 in the different yeasts: although the same set of genes are targeted by Cat8/Sip4 and Cat8-1/Cat8-2 (except for the gluconeogenesis genes), the specific TF regulating the individual genes varies in the different species.Altogether, we can observe three major differences in the regulation of genes for ethanol utilization by Cat8/Cat8-1 and Sip4/Cat8-2 in the three yeast species fii Fig. : (1) CatS. cerevisiae, Cat8 and Sip4 are involved in the regulation of genes of the gluconeogenesis, therefore knocking out the genes encoding these two TFs results in a growth defect on glycerol  are knocked-out, there is no impairment of the growth on glycerol. In addition, transcript level analysis in K. phaffii showed that CAT8-1 and CAT8-2 deletions did not have a major influence on FBP1 and PCK1 expression on any of the tested carbon sources  was not studied so far but could be involved in the regulation of gluconeogenesis in this yeast.In S. cerevisiae and K. lactis, Cat8 has an important role in activating Sip4 expression on ethanol. Despite the fact that both CAT8-1 and CAT8-2 promoters carry three and two CSRE motifs, respectively (Supplementary File 2), this phenomenon is not observed in K. phaffii, suggesting that rewiring of the regulatory network of the carbon metabolism has happened somewhere during evolution.In both S. cerevisiae, Cat8 is the main activator for the growth of gluconeogenic carbon sources. In fact, the SIP4 deletion mutants do not exhibit any growth defect on any carbon source tested so far  due to its function as activator of methanol utilization genes such as AOX1 , however, we do not know if this is due to direct binding of the TFs to the CSRE present in the MXR1 promoter region or not. Additionally, there seems to be some regulatory effect of the overexpression of one transcription factor on the expression of the other on methanol, but the increased MXR1 levels were not reflected by higher levels of Mxr1 target genes such as AOX1, DAS1 or PEX5 . This fits the recent observation that integration of at least one ScCat8-TFBS into the AOX1 promoter was needed in order to make the promoter responsive to ethanol , hence these two kinases do not seem to play a major role in the regulation of ethanol utilization in K. phaffii, and are also probably not involved in the activation of Cat8-1 and Cat8-2 on this carbon source. Further investigations regarding the post-translational modifications of Cat8-1 and Cat8-2 would be necessary in order to elucidate if they are indeed activated via phosphorylation in K. phaffii, as observed for S. cerevisiae.No difference was seen in the growth of the K. phaffii, and their differential involvement in regulating the carnitine shuttle and the glyoxylate shunt, respectively. Phylogenetic analysis places Cat8-2 among the Sip4-like proteins. However, different to S. cerevisiae and K. lactis, CAT8-2 expression is not regulated by the Cat8-1 TF, but autoregulated by its own gene product. Furthermore, CAT8-2 is repressed in the presence of Mig1-2 on glucose. Despite their transcriptional induction on both ethanol and methanol, no direct impact on methanol utilization genes could be observed in the Cat8 mutant strains. Interestingly, however, the CSRE motif, which was shown to be recognized by Cat8 and Sip4 in S. cerevisiae and K. lactis, was predicted by MatInspector to be present in around 90% of the total promoters in K. phaffii . Contrary to what is described for cat8Δ and sip4Δ in S. cerevisiae, knocking-out CAT8-1 and CAT8-2 also influences the ability of K. phaffii to tolerate osmotic stress and cell wall damaging agents (Supplementary figure S6). These results suggest that these two TFs might have additional targets and a broader role in K. phaffii, both of which remain to be investigated.The present study elucidated the requirement of the two Cat8 homologs Cat8-1 and Cat8-2 for activation of the ethanol assimilation pathway in Supplementary file1 (PDF 1172 KB)Supplementary file2List of all K. phaffii CBS7435 genes with CSREs predicted by Matinspector 1000 bps upstream of their start codon (sheet Matinspector) and enrichment of gene ontology (GO) “biological functions” within theses genes  (XLSX 222 KB)Below is the link to the electronic supplementary material.