The TBC1 domain family member 10B (EPI64B/TBC1D10B), a member of the RabGAP EPI64 subfamily, contains a TBC domain that confers GTPase-activating protein activity. Even though overexpression of TBC1D10B has been reported to promote tumor invasion and metastasis in gastric adenocarcinoma, the prognostic value of TBC1D10B and its correlation with DNA methylation and immune infiltration in hepatocellular carcinoma are still not known. Transcriptional expression profiles of TBC1D10B between hepatocellular carcinoma tissues and normal tissues were downloaded from The Cancer Genome Atlas and Gene Expression Omnibus. The Clinical Proteomic Tumor Analysis Consortium and the Human Protein Atlas were used to assess the TBC1D10B protein expression. The biological functions of TBC1D10B were evaluated by the Metascape database and by Gene Set Enrichment Analysis (GSEA). Receiver operating characteristic (ROC) curve analysis was used to distinguish hepatocellular carcinoma from adjacent normal tissues. The effect of TBC1D10B on survival was estimated using the Kaplan–Meier method. DNA methylation in the TBC1D10B gene was assessed using the online MEXPRESS and MethSurv tools. The association between TBC1D10B mRNA expression and immune cell infiltration was investigated by the TIMER2 web server, tumor immune estimation resource and single-sample GSEA. This study found that TBC1D10B is highly expressed in hepatocellular carcinoma and that increased TBC1D10B mRNA expression is associated with female sex, lower Body Mass Index, high level of alpha fetal protein, and worse clinical stages. The mRNA and protein levels of TBC1D10B were verified in cells. Functional annotation indicated enrichment with negative regulation of the cell cycle, extracellular matrix, and corresponding pathways in the high-TBC1D10B phenotype. The ROC curve analysis showed that, with a cutoff level of 2.912, the accuracy, sensitive, and specificity in differentiate TBC1D10B hepatocellular carcinoma from adjacent controls were 0.931, 0.920, and 0.802, respectively. Kaplan–Meier survival analysis showed that hepatocellular carcinoma patients with high TBC1D10B had a worse prognosis than those with low TBC1D10B, especially in patients with a weight below 70 kg, height above 170 cm, and histological G2 and G3. We also found that the methylation of TBC1D10B was associated with the prognosis in patients with hepatocellular carcinoma. Moreover, correlation analysis indicated that TBC1D10B mRNA expression was positively correlated with infiltration levels of most immune cells, but negatively correlated with Th17 and cytotoxic cells infiltration. Our study indicates that increased TBC1D10B expression in hepatocellular carcinoma may play a role in tumorigenesis by regulating the cell cycle and extracellular matrix. TBC1D10B may be a novel prognostic and predictive marker and immune therapeutic target in hepatocellular carcinoma patients. The development of hepatocellular carcinoma is a multiple process that involves sustained inflammatory conditions, including hepatocyte necrosis and regeneration, related to fibrotic deposition2. Although several studies have shown that surgical resection, ablation, transplantation, chemoembolization3, and the tyrosine-kinase inhibitors lenvatinib4 and sorafenib5 are treatments with survival benefits, the characteristics of multifocal development and drug-resistance preclude treatment being curative in most hepatocellular carcinoma cases7. Given that hepatocellular carcinoma is the result of epigenetic modifications and somatic genomic alterations in passenger and driver genes2, the further understanding of the molecular classifications of hepatocellular carcinoma that predict disease progression or recurrence is of paramount importance.Hepatocellular carcinoma, the third leading cause of cancer death, is the most frequent primary liver malignancy and an important medical problem. Most hepatocellular carcinoma cases occur in eastern Asia and sub-Saharan Africa, where the most prevalent risk factors are aflatoxin B1 and hepatitis B exposure9. Rab GTPase is involved in regulating intracellular vesicular transport spatially and temporally by switching between inactive and active states10, which are catalyzed by GTPase activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs), respectively11. More importantly, Rabs and their effectors are found to be overexpression or aberrant function in hepatocellular carcinoma, including Rab1A12, Rab5A13, Rab1814, Rab3115 and Rab27A16. The Tre-2/Bub2/Cdc16 (TBC) domain is a conserved catalytic domain with ~ 200 amino acids presenting in various molecules in eukaryotic organisms17 as Rab-GAPs18. The TBC1D10 subfamily members  possess a TBC domain and have been reported to exhibit GAP activities toward all Ras and certain Rabs20. Each TBC1D10 member exhibits distinct Ras/Rab specificities, as well as distinct biological functions: TBC1D10A exhibits its effects on angiogenesis21 and selective autophagy22 through binding to Rab3A and 35, respectively; TBC1D10B participate in regulating vesicle-mediated membrane traffic by binding to Rab35, 3A, and 27A25; and TBC1D10C serves as a negative modulator in T-cell activation through dual inhibition of both calcineurin and Ras pathways26. Additionally, the overexpression of EPI64 has been proven to contribute to exosome secretion in lung cancer17, and aberrant activation of the Ras pathways has been causally observed in a human hepatocellular carcinoma that is characterized by shorter survival27, indicating potential roles of TBC1D10 in tumor progression and prognosis. In light of the fact that the knockdown of TBC1D10B attenuates the viability, apoptosis, and proliferation ability of gastric adenocarcinoma cells28, we asked whether increased TBC1D10B in hepatocellular carcinoma may play a role in tumorigenesis and have prognostic value for clinical outcomes.The Rab GTPase family comprises a large number of small GTPases that exist in two conformational states: an inactive GDP-bound protein and an active GTP-bound proteinTherefore, we carried out a bioinformatics analysis to identify the significance of TBC1D10B in liver hepatocellular carcinoma (LIHC) tumorigenesis and prognosis. We found an overexpression of TBC1D10B in LIHC and explored its potential role in LIHC tumorigenesis. Then, a significant correlation was observed between TBC1D10B and several clinicopathological characteristics. Furthermore, we identified the diagnostic and prognostic values of TBC1D10B. Our study provides a novel insight into the underlying mechanisms of LIHC tumorigenesis and reveals TBC1D10B as a potential diagnostic and prognostic biomarker in LIHC.33typesofcancer using UCSC Xena platform (http://xena.ucsc.edu/), an online tool for the exploration of gene expression and clinical phenotype data. Expression data were Log2 transformed, and Wilcoxon rank sum test conducted on these tumor types; P < 0.05 were considered to indicate differential expression between tumor and normal tissues. The data analysis was conducted using R software , and the R package “ggplot2” was used to draw box plots. Then, we downloaded high-throughput RNA-sequencing data of 424 samples from the TCGA data set. The 424 samples included 50 matched normal-liver cancer samples  and 324 unmatched liver cancers. The expression of TBC1D10B was evaluated using the downloaded data, and expression levels were compared between 50 cancer samples and 50 matched adjacent normal samples in LIHC.RNA sequencing and related clinical data were downloaded from the Cancer Genome Atlas (TCGA) DESeq2”29, based on the negative binomial generated linear models in which the log-fold change is larger than 1.5 and the adjusted P-value less than 0.05. Visualizations of DEGs, such as heatmaps and volcano plots, were generated using the R package “pheatmap” and “EnhancedVolcano.” As the TCGA cohort has already obtained Ethics Committee Approval, this study did not require additional approval.In total, 374 LIHC patients were separated into high- and low-TBC1D10B expression groups based on the median TBC1D10B value. Differentially expressed gene (DEG) analyses between the two groups by a two-tailed hypothetical test were conducted using the R package “https://www.ncbi.nlm.nih.gov/geo/) for external verification of TBC1D1B expression in LIHC. GSE45267 obtained 48 primary HCC samples and 39 non-cancerous tissues, whereas GSE101685 included 8 normal tissue and 24 HCC patients with T1, T3a, and T3b. For GSE87630, there were 64 tumor tissue samples and 30 non-tumor tissue samples, whereas GSE57957 included 39 primary HCC tissue samples and paired normal tissue samples.In addition, we selected 4 datasets: GSE45267 (GLP570), GSE101685 (GLP570), GSE87630 (GPL6947) and GSE57957 (GPL10558) from the Gene Expression Omnibus  to present a thorough analysis of TBC1D10B protein expression and its phosphorylation from the Clinical Proteomic Tumor Analysis Consortium data set. Meanwhile, the IHC images of TBC1D10B protein expression in normal tissue and liver LIHC tissue were downloaded from the Human Protein Atlas (HPA) (http://www.proteinatlas.org/) and analyzed.To evaluate the difference in TBC1D10B protein expression levels between a primary tumor and normal tissue, we implemented UALCAN . The reverse transcriptase reaction was performed using RevertAid RT Kit (Thermo Scientific). The PCR reaction was performed using SYBR Green . Data were analyzed using the 2−ΔΔCt method. The primers sequences for TBC1D10B and GAPDH are as follows: TBC1D10B forward primer (5′-CAGGAGGCATTGCTGATGAT-3′) and TBC1D10B reverse primer (5′-GAAGGCTGGGGCTCATTT-3′); GAPDH forward primer (5′-CCATCCCTTCCCTCACCCACTG-3′) and GAPDH reverse primer (5′-TCTCCCGCTCCTTCTCCTGTTTC-3′).A total of four cell lines including human hepatoma cell lines  and the WRL68 non-tumor human embryo liver cells, which were obtained from Cell Bank of Chinese Academy of Science . Huh-7, BEL-7402 and SK-HEP-1 were cultured in DMEM  with 10% fetal bovine serum . WRL68 was cultured in RPMI-1640  containing 10% fetal bovine serum. All cells were maintained in a humidified incubator with 37 ℃, 5% CO30. Briefly, total protein was extracted from cells by using RIPA extraction buffer with protease inhibitors  according to the manufacturer’s instructions. After electrophoresis, proteins were electroeluted onto a polyvinylidenedifluoride (PVDF) memberane (Invitrogen). The membrane was blocked with Tris-buffered saline containing 0.1% Tween 20 and 5% milk for 2 h at room temperature, and then incubated with the following antibodies against TBC1D10B , β-actin  at 4℃ overnight. The membrane was incubated at room temperature for 1 h with HRP-conjugated secondary antibodies. Proteins were visualized by chemiluminescence.Western blot was performed as previously described31. The enrichment factors were larger than 1.5, and the minimum counts larger than 3. For the analysis, the P-value threshold was set to 0.01. The Gene Set Enrichment Analysis (GSEA) of DEGs between the two groups was performed using the R package “clusterProfiler”32. The gene set collection C2 (curated gene sets) was obtained from the Molecular Signature Database as the reference gene sets. We finally identified a total of 772 clusters for which a false discovery rate (FDR) of < 0.25 and a P-value of < 0.05 were considered significant. Protein–protein interaction networks were constructed based on the STRING database (https://string-db.org/) and were visualized in Cytoscape software33.Functional annotation of the identified DEGs was performed using the Metascape database and online toolggplot2.”The Wilcoxon rank sum test (continuous variables) or Pearson’s chi-square test (rank variables) was used to examine the relationship between TBC1D10B expression levels and clinicopathological characteristics. Visualizations of clinical phenotype correlations were generated using the R package “34. The R packages “survminer” and “survival” were applied to Kaplan–Meier (K-M) analysis and the clinicopathological subgroup study. All statistical analyses were carried out in R v. 3.6.3. with a P-value of ≤ 0.05.Receiver operating characteristic (ROC) analysis was used to estimate the predictive value of TBC1D10B for LIHC diagnoses. Information on LIHC patients’ clinical outcomes, including overall survival, disease-specific survival, and progression-free interval, was obtained from a published studyhttps://mexpress.be/). Next, we analyzed the prognostic value of the TBC1D10B methylation level in LIHC using MethSurv (https://biit.cs.ut.ee/methsurv/), a web tool for providing survival analysis based on TCGA data.The methylation data of TBC1D10B from multiple probes  were obtained from the MEXPRESS website . In addition, we performed GSEA using the package “GSVA” to present the infiltration enrichment of 19 common immune cells, including activated DCs (aDCs), immature DCs (iDCs), plasmacytoid DCs, T cells, T helper (Th) cells, type 1 Th cells (Th1s), type 2 Th cells (Th2s), type 17 Th cells (Th17s), T gamma delta cells, T effector memory (Tem) cells, regulatory T (Treg) cells, T central memory cells, T follicular helper (Tfh) cells, eosinophils, cytotoxic cells, mast cells, natural killer (NK) cells, NK 56− cells, and NK 56+ cells. Then, the relationship between TBC1D10B and immune infiltration was estimated by Spearman’s analysis and the Wilcoxon rank sum test.We used the “Immune-Gene” module of TMIER.2 to explore the correlation between TBC1D10B expression and immune infiltrates  in the high-TBC1D10B group. Representative DEGs were also illustrated by heatmaps , mitotic spindle checkpoint , mitotic metaphase and anaphase , mitotic G1 and G1-S transition , and mitotic prometaphase . TBC1D10B-associated DEGs were also enriched in cancer pathways , progression-free interval , and disease-specific survival .To verify the clinical benefits of TBC1D10B evaluation, we initially determined the predictive value on discriminating LIHC diagnosis by using an ROC curve. We observed that TBC1D10B showed significant sensitivity and specificity for LIHC prognosis , clinical stages I − II , clinical stages T1 and T2 , clinical N0 stage , clinical M0 stage , non-overweight , no residual tumor , prothrombin time less than 4 s , and liver fibrosis   whereas it was positively correlated with the methylation level of the cg27159695 . Interestingly, the MethSurv analysis showed that patients with high methylation of cg09439599 had significantly worse survival outcomes than patients with low methylation (P < 0.001) while patients with high methylation of cg27159695 had a better survival outcome than patients with low methylation (P < 0.05) , iDC , NK CD56dim cells , NK cells , T helper cells , Tem , Tfh , Th1 , and Th2 , but cytotoxic cells  and Th17  exhibited a significantly negative TBC1D10B expression  was associated with a poor prognosis of LIHC while TBC1D10B methylation at the non-promoter region (cg09439599) correlated with a good prognosis. Therefore, additional evidence is needed of the potential role of TBC1D10B DNA methylation in the tumorigenesis of LIHC.DNA methylation reportedly regulates cell differentiation and participates in tumorigenesis53 and neutrophils54. As dedicated professional antigen-presenting cells, dendritic cells can prime T-cells against tumor-associated antigens, which are involved in the progression of hepatocellular carcinoma55. Moreover, an increased number of both circulating and intra-tumoral CD8+ T cells in hepatocellular carcinoma predicts high postoperative recurrences and poor prognoses56. Our study has for the first time demonstrated that high TBC1D10B expression is positively correlated with CD8+ T cells, B cells, CD4+ T cells, macrophages, neutrophils, dendritic cells, and most other immune cells. These findings indicate that the dysfunction of TBC1D10B may contribute to cancer cell immune escape. Notably, we found a significantly negative correlation between TBC1D10B expression and cytotoxic cell infiltration, which are the preferred immune cells for targeting cancer57. Therefore, the roles and functions of TBC1D10B need to be further studied to provide a new strategy for tumor immunotherapy.Because LIHC is an inflammation-associated tumor, the immunosuppressive microenvironment of LIHC contributes to immune escape and tolerance through multiple routes. Substantial clinical data and experimental research have confirmed that tumor initiation, tumor progression, and malignant metastasis are related to macrophagesOur combined findings show for the first time that the mRNA expression of TBC1D10B is upregulated in LIHC and significantly associated with clinical prognosis, DNA methylation, and immune cell infiltration. In addition, we uncovered a potential mechanism for TBC1D10B activity in LIHC tumorigenesis and demonstrated its predictive value in LIHC clinical outcomes. Our research suggests that TBC1D10B may be regarded as a novel prognostic and predictive marker and therapeutic target for hepatocellular carcinoma patients, including RNA-targeting treatment, such as antisense oligonucleotides or RNA-targeting Cas9.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Figures.Supplementary Information 5.Supplementary Information 6.


TP53 wild-type at diagnosis. An increasing number of studies are evaluating alternative targeted agents in CLL, including MDM2–p53 binding antagonists. In this study, we report the effect of splicing modulation on key proteins in the p53 signalling pathway, an important cell death pathway in B cells. Splicing modulation by E7107 treatment reduced full-length MDM2 production due to exon skipping, generating a consequent reciprocal p53 increase in TP53WT cells. It was especially noteworthy that a novel p21WAF1 isoform with compromised cyclin-dependent kinase inhibitory activity was produced due to intron retention. E7107 synergized with the MDM2 inhibitor RG7388, via dual MDM2 inhibition; by E7107 at the transcript level and by RG7388 at the protein level, producing greater p53 stabilisation and apoptosis. This study provides evidence for a synergistic MDM2 and spliceosome inhibitor combination as a novel approach to treat CLL and potentially other haematological malignancies.Several molecular subtypes of cancer are highly dependent on splicing for cell survival. There is a general interest in the therapeutic targeting of splicing by small molecules. E7107, a first-in-class spliceosome inhibitor, showed strong growth inhibitory activities against a large variety of human cancer xenografts. Chronic lymphocytic leukaemia (CLL) is a clinically heterogeneous hematologic malignancy, with approximately 90% of cases being  In ppoptosis ,17. Signpoptosis . In addipoptosis .50 = 6 nM), selectivity, and bioavailability. Both oral and intravenous formulations have been developed [Inhibiting the p53–MDM2 interaction with synthetic small molecules has been investigated as a therapeutic strategy for the activation of wild-type p53 . One of eveloped . RG7388 eveloped ,23,24,25eveloped . It is teveloped ,28,29.Here, we hypothesized that the altered splicing of p53 pathway genes has a contributory effect on the cytotoxicity of E7107, as p53 is a master regulator of cellular death signalling pathways. The potential for synergistic combination treatment with the spliceosome inhibitor E7107 and the MDM2–p53 antagonist RG7388 was investigated in leukaemia–lymphoma cell lines and primary CLL samples.50 < 15 nM) to the spliceosome inhibitor than the other two (IC50 > 60 nM). The mean IC50 values were relatively higher for HEL and HAL-01 cell lines  . No correlation was evident between E7107 sensitivity and TP53 status  or SF3B1 status  for the primary CLL samples tested  than either the cell lines or primary CLL samples (Six out of eight cell lines were notably more sensitive (ICctively) . E7107 ss tested A. Findin samples B.TP53-dependent targeted therapy agent, with the spliceosome inhibitor E7107. Although the overall single-agent cytotoxic effect of E7107 appears to be independent of TP53 mutational status, we were interested to evaluate its mechanistic effect on key proteins in the p53 signalling pathway, an important cell death pathway in B cells, before testing its combined use with a TP53-dependent agent. The Western blots revealed a marked concentration-dependent decrease in MDM2, a critical negative regulator of p53 [TP53WT cells. In addition, a lower molecular weight p53 isoform (~45 kDa) was seen in OCI-Ly3 cells treated with E7107, likely due to alternative splicing, whereas a concomitant novel high molecular weight p21WAF1 isoform (~30 kDa) was evident in TP53WT cells  transcript with primers spanning the end of exon 3 and beginning of exon 4 revealed the presence of an additional longer transcript product of approximately 1200 bp in OCI-Ly3 cells treated with E7107, which was longer than the RT-PCR product size of 84 bp expected from the processed transcript with the chosen primers (CDKN1A (p21WAF1) gene (The RT-PCR of  1303 bp D. The co 1303 bp . The RT- primers F. SequenF1) gene , which wF1) gene C.WAF1 and the intron-retaining isoform (p21L) revealed that despite retaining the N-terminal CDK binding site, the proliferating cell nuclear antigen (PCNA) interaction site  antibodies detecting different phosphorylation sites on pRB. The MDM2 inhibitor RG7388 was used for endogenous wild-type p21 induction. The result showed that exogenous p21L expression had no impact on pRB phosphorylation and hence no CDK inhibitory activity in the transduced Nalm-6 cells, in contrast to the clear suppression of RB phosphorylation seen on induction of endogenous p21WAF1 by RG7388 , however, induced endogenous p21WAF1 expression and the p21WAF1 signal was detected in both cytoplasmic and nuclear compartments  and an increase in G2/M phase (p = 0.0045). Treatment with RG7388 alone, however, resulted in a significant reduction in the S phase (p = 0.011), with increases in the cell population in G0/G1 phase (p = 0.011). E7107 treatment further decreased the proportion of cells in S phase when it was combined with RG7388   A,C.p = 0.019) and a decrease in the G2/M phase (p = 0.014). Treatment with RG7388 alone resulted in a significant increase in G0/G1 phase (p < 0.0001). E7107 treatment further decreased the proportion of cells in S phase when it was combined with RG7388   B,D.The induction of apoptosis was evaluated by caspase 3/7 enzymatic activity, which is a sensitive and specific indicator of apoptosis ,23. Wildn = 4 CLL patient samples  was evident in OCI-Ly3 cells treated with E7107, likely due to alternative splicing. Different p53 isoforms were reported in the literature with molecular weights from 26 to 48 kDa, and some of them were shown to exhibit several biological functions, modulating p53 transcriptional activity and tumour-suppressor functions [WAF1 isoform (~30 kDa) formed due to intron retention was also evident in TP53WT cells. Intron retention has been shown as a hallmark of cancer stemness and aggressiveness in multiple studies, and splicing modulation reverses this aggressive behaviour and overcomes treatment resistance in cancers [CDKN1A and MDM2, may provide insights into the studies with splicing inhibitors in terms of drug resistance/synergies and new drug combination strategies. Sequence analysis revealed that despite retaining the N-terminal CDK binding site, the proliferating cell nuclear antigen (PCNA) interaction site and nuclear localization signal domain were partially lost in the intron-retained isoform of CDKN1A encoding the p21L protein product. The alteration in the nuclear localization signal of p21L prevents its access to the nucleus, resulting in the loss of both CDK and PCNA inhibitory activity in the cellular context. Ectopic expression of the altered p21 in Nalm-6 cells using lentiviral transduction showed that the p21L protein, with an alternative C-terminus, loses its ability to act as a cyclin-dependent kinase inhibitor due to loss of the ability to localise to the nucleus , they form greater levels of p21 aberrant splicing isoform (p21L) at the same concentration of E7107 treatment . Idasanutlin was custom synthesised with >99% purity as part of the Newcastle University/Astex Pharmaceuticals Alliance and CRUK Drug Discovery Programme at the Newcastle University Cancer Centre.All of the cell lines  were obtTP53 and SF3B1 mutations, including coding region position and amino acid changes as well as del17p status.Peripheral blood samples were obtained from CLL patients with informed consent, in accordance with institutional guidelines and the Declaration of Helsinki. CLL patient samples were obtained and stored under the auspices of the Newcastle Biobank (Research Ethics Committee (REC) reference 17/NE/0361). CLL diagnosis was made according to IWCLL-164 NCI 2008 criteria . Supplem5 cells/mL in 100μL of medium per well of a 96-well plate (Corning) for 24 h before treating with a range of concentrations (from 1 to 104 nM) of E7107 and/or idasanutlin for 72 h. Concentrations are indicated in the figure legends. XTT Assay Kit II  was used to measure growth inhibition compared to solvent DMSO control.Cells were seeded at 1.6 × 106 cells/mL in 100μL of medium per well of a 96-well were exposed to a range of concentrations (from 1 to 104 nM) of E7107 and/or idasanutlin for 48 h. Concentrations are indicated in the figure legends. Ex vivo cytotoxicity was assessed by XTT Assay Kit II . Results were normalised to DMSO controls and expressed as % viability.5 × 10TP53 cell lines were treated for 72 h with each agent alone and in combination simultaneously at constant 1:1 ratios at 0.25×, 0.5×, 1×, 2×, and 4× of their respective IC50 concentrations. Median-effect analysis was used to calculate combination index (CI) values [v/v) in final volume for single and combined treatments.For the combination treatment of idasanutlin and E7107, the wild-type ) values  using Ca5 cells/well were seeded in white-walled 96-well plates  and exposed to 1 nM E7107 and/or 0.2 µM idasanutlin for 24 and 48 h. Caspase 3/7 activity was assessed using Caspase-Glo 3/7 Assay . Apoptosis was also determined by examining PARP protein cleavage by Western blot.1.6 × 106 cells/mL were seeded in 1 mL per well of a 24-well plate (Corning) and exposed to idasanutlin or E7107 alone and in combination at constant 1:1 ratios at 1× of their respective IC50 concentrations for 24 h. g for 5 min and washed with 1 mL PBS, followed by further centrifugation at 500 × g for 5 min, and were then incubated in a 250 µL mixture of DNase-free RNase A (40 µg/mL), propidium iodide (24 µg/mL), and PBS at room temperature for 30 min. Samples were analysed on a FACSCaliburTM flow cytometer using CellQuest Pro software . Cyflogic Software Version 1.2.1 (CyFlo Ltd.) was used to manually gate the single cells on the FL2-A vs. FL2-W scatter plot.0.5 × 10™ Taq Green Hot Start DNA Polymerase (Invitrogen) in a GeneAmp PCR System 9700 thermal cycler  using the following primers:SF3B1 Ex14 F: ACCAACTCATGACTGTCCTTTC, R: ACAACTTACCATGTTCAATGSF3B1 Ex15-16 F: AACTTAGGTAATGTTGGGGCA, R: TCAACTGACCTGAAATGAAGAGASF3B1 Ex18 F: CCTTGGAAAAGCAGTCTAAAAGG, R: GTCAACCTTTTCTAACCACCCADNA was extracted using a QIAamp DNA Mini Kit  according to the manufacturer’s protocol. PCR reactions were run using PlatinumA PureLink™ PCR Purification Kit  was used. The purified PCR products were sent to DBS Genomics  for Sanger dideoxy sequencing. Sequencing results were analysed by visual inspection of the chromatograms and alignment with a normal reference sequence using Mutation Surveyor DNA Variant Analysis Software (SoftGenetics).™ Taq Green Hot Start DNA Polymerase (Invitrogen) in a GeneAmp PCR System 9700 thermal cycler (Applied Biosystems) using the following primers:CDKN1A transcript (spanning exons 3 to 4) F: CTGGAGACTCTCAGGGTCGA, R: CTCTTGGAGAAGATCAGCCGMDM2 transcript (spanning exons 1 to 11) F: CTGGGGAGTCTTGAGGGACC, R: CTTTCACATCTTCTTGGCTGCTotal RNA was extracted using an RNeasy Mini Kit  and cDNA was generated using a High-Capacity cDNA Reverse Transcription Kit  as described by the manufacturer. A DNase treatment step with on-column digestion was carried out using an RNase-free DNase kit (Qiagen). PCR reactions were run using PlatinumThe constitutive and altered products, purified from agarose gel, were sequenced by Sanger sequencing.6 cells/mL  were seeded in 2 mL per well of a 6-well plate (Corning) and exposed to a range of concentrations of E7107 and/or idasanutlin. Concentrations are indicated in the figure legends. Protein lysates were harvested using 2% SDS lysis buffer at 24 h, heated at 95 °C for 10 min, and sonicated. Protein concentration was measured using a Pierce™ BCA Protein Assay Kit . Primary antibodies against p53 (DO-7) , MDM2 (Ab-1) , p21WAF1 , PARP (Trevigen), SF3B1 (Sigma), Actin (Sigma), Phospho-Rb (Ser780) , Phospho-Rb (Ser807/811) , Phospho-Rb (T821) (Abcam) and secondary goat anti-mouse/rabbit horseradish peroxidase-conjugated antibodies (Dako) were used. All antibodies were diluted in 5% (w/v) nonfat milk or BSA in TBS-tween20. Proteins were visualized using enhanced chemiluminescence reagents .1 × 10L) was inserted into the Q3210 plasmid and then the lentiviral particles were produced by lentiviral packaging in HEK293T host cells. The transduced cell line was induced using 0.5 µg/mL doxycycline (Clontech Labs). Pre-B Nalm-6 isogenic cell lines expressing either endogenous SF3B1K700K or mutant SF3B1K700E were provided by H3 Biomedicine.A tetracycline-inducible (Tet-ON) lentiviral expression system, Q3210 plasmid  was used for the stable expression of the gene of interest. The aberrantly spliced transcript form of P21, P21-Long  and anti-p21 (Mouse anti-p21WAF1 (EA10), OP64, Calbiochem; 1:100 in blocking buffer). Alexa Fluor 488®-conjugated anti-mouse IgG  was used as a secondary antibody. A Leica DM6 fully automated widefield fluorescence microscope system with the LAS X software was used to capture images.Suspension cells were fixed on the slides using 100% methanol for immunofluorescence staining with DAPI  unless otherwise stated. Statistical tests were carried out using GraphPad Prism 6 software and all https://swissmodel.expasy.org/, accessed on 1 September 2021). It searches a library of experimental protein structures to determine suitable templates for a given target protein. Based on a sequence alignment between the target protein and the template structure, a 3D model for the target protein is generated [PCNA/p21 structural information for wild-type and altered p21 was identified using SWISS-MODEL workspace, which is an integrated web-based homology-modelling environment  was intron retention. It was especially noteworthy that the novel p21 isoform lost cell cycle inhibitory activity due to the disruption of motifs for nuclear localisation and CDK binding. The consequent stabilisation of p53 and compromised cyclin-dependent kinase inhibitory activity drives apoptosis and the observed synergy with response to MDM2 inhibitors. A particularly novel aspect of this synergy is the dual inhibition of MDM2; by E7107 at the transcript level and by RG7388 at the protein level, producing greater p53 stabilisation and apoptosis.Mechanistically, the notable finding was that splicing modulation with E7107 differentially affected p53 pathway gene splicing, producing exon skipping for MDM2 and consequent activation of p53, while the effect on


Drosophila. Cells that undergo erebosis lose cytoskeleton, cell adhesion, organelles and fluorescent proteins, but accumulate Angiotensin-converting enzyme (Ance). Their nuclei become flat and occasionally difficult to detect. Erebotic cells do not have characteristic features of apoptosis, necrosis, or autophagic cell death. Inhibition of apoptosis prevents neither the gut cell turnover nor erebosis. We hypothesize that erebosis is a cell death mechanism for the enterocyte flux to mediate tissue homeostasis in the gut.Many adult tissues are composed of differentiated cells and stem cells, each working in a coordinated manner to maintain tissue homeostasis during physiological cell turnover. Old differentiated cells are believed to typically die by apoptosis. Here, we discovered a previously uncharacterized, new phenomenon, which we name erebosis based on the ancient Greek word erebos (“complete darkness”), in the gut enterocytes of adult  It has been believed that gut enterocytes continuously die through apoptosis. However, this study shows that gut enterocytes die through a novel cell death mechanism, named erebosis. Erobotic cells lack the characteristic features of apoptotic, necrotic or autophagic cell death; instead they lose their cytoskeleton, cell adhesion and organelles, and their nuclei become flat and indistinct. Drosophila gut, enterocytes, which absorb nutrition, are the major differentiated cell type  meaning deep darkness.Myo1D-Gal4, we noticed that erebotic cells (cytoplasmic GFP-negative cells) usually have nlsRFP signals  with different amounts of nuclear GFP and nuclear RFP. The first one retains nuclear RFP and faint signals of nuclear GFP. The second one has only nuclear RFP without nuclear GFP. The third one has lost both nuclear RFP and nuclear GFP. Assuming that losing fluorescence is a gradual process, we reason that, during erebosis, cells first lose cytoplasmic GFP, then nuclear GFP, followed by loss of nuclear RFP. Longer retention of nuclear RFP signals compared to nuclear GFP is likely due to the stability difference of the fluorescent proteins. For example, RFP is more resistant to low pH than GFP  (a gift from Dr. Iswar Hariharan)UAS-rpr GTrace esg-lexA::HG lexAop-Flp, ubi>>nlsGFP (a gift from Dr. Melanie Worley and Dr. Iswar Hariharan)AnceΔ110 [outcrossed to OregonR] control110 [respective control from the last outcross of AnceΔ110]AnceΔ128 [outcrossed to OregonR] control128 [respective control from the last outcross of AnceΔ128]Ance-RNAi 1 Ance-RNAi 2 [30535-3-1] (this paper)Ance-RNAi 3 [30535-2-1] (this paper)y-, w-; Ance-RNAi 2 y-, w-, control 2 [respective control from the last outcross Ance RNAi 2]y-, w-; Ance-RNAi 3 y-, w-, control 3 [respective control from the last outcross Ance-RNAi 3]Atg1 RNAi Atg2 RNAi Atg5 RNAi Atg6 RNAi Atg9 RNAi Atg12 RNAi Atg18a RNAi Drep4 RNAi Relish RNAi (a gift from Dr. Fumiaki Obata)UAS-JNK DN (a gift from Dr. Iswar Hariharan)UAS-puc (FlyORF F002209)hs-flp, tub-Gal4, UAS-nlsGFP;; tub-Gal80, FRT2A (a gift from Dr. Yuichiro Nakajima)FRT2A, FRT82B (a gift from Dr. Yuichiro Nakajima)H99, FRT2A (a gift from Dr. Yuichiro Nakajima)DroncΔ, FRT2A (a gift from Dr. Yuichiro Nakajima)mCherry RNAi attp2 attp40 Ance mRNA into VALIUM20, the TRiP protocol by Jian-Quan Ni and Norbert Perrimon at Harvard Medical School (https://fgr.hms.harvard.edu/files/fly/files/2ndgenprotocol.pdf) was followed, and the plasmids were sent to BestGene for transformation at attp2.For the design and cloning of hairpins targeting In brief, the coding CDS of Ance-RA was used for best sequence matching. Sequences with the fewest off-target effects were used to design the following oligos without 2nt shift:shRNA-Ance-30535-2.1ctagcagtCACCGACGATGTGCGCATCAAtagttatattcaagcataTTGATGCGCACATCGTCGGTGgcgshRNA-Ance-30535-2.2aattcgcCACCGACGATGTGCGCATCAAtatgcttgaatataactaTTGATGCGCACATCGTCGGTGactgshRNA-Ance-30535-3.1ctagcagtTTCCTGCAGTATCAACACCAAtagttatattcaagcataTTGGTGTTGATACTGCAGGAAgcgshRNA-Ance-30535-3.2aattcgcTTCCTGCAGTATCAACACCAAtatgcttgaatataactaTTGGTGTTGATACTGCAGGAAactgAnce/CG8827 targeting gRNAs and hs-Cas9 were supplied in DNA plasmids, together with the donor plasmid for microinjection into embryos of control strain w1118. F1 flies carrying selection marker of 3xP3 DsRed were further validated by genomic PCR and sequencing. CRISPR generates a 48-bp deletion in Ance/CG8827 deleting signal peptide (R2 to A17) of Ance/CG8827 and is replaced by cassette PBacDsRed. After confirming an insertion of the transgene, 3xP3-DsRed was excised by PiggyBac transposition.CRISPR-mediated mutagenesis was performed by WellGenetics using modified methods of the published one . In brieMidguts of adult female virgins (5 to 8 days old unless noted otherwise) were dissected in 1xPBS and fixed for 60 minutes at RT in 1xPBS with 4% paraformaldehyde , occasionally with 10% TCA on ice (junction staining). Samples were washed 3 times for >30 minutes in PBSTx (1x PBS + 0.1% Triton X-100) and then incubated with the primary antibody in 10% NGS , PBSTx overnight at 4°C. The samples were washed 3 times for >60 minutes in PBSTx before incubation with the secondary antibody in 10% NGS, PBSTx overnight at 4°C. Finally, samples were washed 5 times in PBST and mounted in Slow Fade Diamond Antifade Mountant . Fluorescent images of the whole or posterior midgut (R4) were acquired using a Zeiss LSM 880 or Zeiss LSM 900 confocal microscope at 20x and 40x magnification.The following antibodies and fluorescent dyes were used:Polyclonal rabbit-anti-Ance ,mouse-anti-Armadillo ,mouse-anti-Discs large ,mouse-anti-LaminB1 mouse-anti-KDEL rabbit-anti-GM130rabbit-anti-phospho-H3 ,rabbit-anti-cleaved-Drosophila Dcp-1 ,rabbit-anti-Fibrillarin antibody ,rabbit-anti-beta Tubulin antibody ,Alexa Fluor phalloidin ,Alexa Fluor GFP-Booster Alexa Fluor secondary antibodies ,DAPI ,Hoechst 33342 Propidium iodide Fiji and Zeiss ZEN were used for image analyses. The nuclear area was measured using maximum-intensity projections, and the nuclear height was measured on resliced images with automatic output spacing. For fluorescence signal quantifications, the mean or median intensity of a cell was measured and the background signal was subtracted.esg-expressing cells was induced in a temperature-dependent manner using the Gal80ts system. Virgin female flies were collected for up to 5 days in 18°C incubators (permissive temperature) and shifted to 30°C incubators (restrictive temperature) Flies were kept at 30°C for 7 days, dissected, and imaged as described above.The Fiji CellCounter plugin was used for cell counting of total cell numbers and/or cell subpopulations. Therefore, images were converted to maximum-intensity projections and cells were counted with reference to the respective stacked image for inclusion of the 3D tissue organization. The distance of progenitor cells to erebotic cells was measured between cell membranes. For this experiment, expression of GFP in hs-flp, tub-Gal4, UAS-nlsGFP;; tub-Gal80 FRT2A female flies were crossed to H99 FRT2A, DroncΔ FRT2A or FRT2A males to generate offspring with desired genotypes. Newly eclosed virgin female flies were kept in standard fly food for 3 to 5 days before clone induction. Then, flies were heat-shocked at 37°C for 1.5 hours in a water bath to induce clonal cells in the midgut. Intestines were dissected and stained with the anti-Ance antibody 14 days after the heat shock to examine the existence of erebotic cells in apoptosis-deficient cells.Virgin female flies were collected for 1 to 2 days and kept on standard food as described above until becoming 6 to 8 days old. Flies were fed with 100 nM propidium iodide in 5% sucrose solution provided on a paper filter in a vial for 4 to 5 hours in a 25°C incubator. Midguts were quickly dissected in 1xPBS and incubated with 100 μg/ml Hoechst 33342 for 10 minutes. Samples were washed at least 2 times in drops of 1xPBS by transferring midguts carefully. After mounting the samples with space holders in 1xPBS, confocal imaging was performed immediately for a maximum time of 1 hour after mounting.Myo1D>GFP was embedded in 1% low melting agarose, was incubated in PBS with 10 μM BioTracker ATP-Red for 10 minutes, and was live imaged with the Zeiss LSM 900 confocal microscope.Biotracker ATP-Red , which was developed as a chemical ATP probe , was useMidguts were dissected from 5- to 7-day-old female flies in PBS. The dissected midguts were embedded in 2% low melting agarose and were incubated in Schneider’s medium (ThermoFisher) supplemented with 10% FBS (ThermoFisher). The R4 region was imaged with the Zeiss LSM 900 confocal microscope (40x magnification) every 5 minutes.The TUNEL assay was performed using the ApopTag Red in situ apoptosis detection kit (Millipore) according to the manufacturer’s instructions. Midguts of adult female virgins (5 to 8 days old unless noted otherwise) were dissected in 1xPBS and fixed for 60 minutes in 1xPBS with 4% paraformaldehyde at room temperature (RT). After fixation, samples were washed with PBS 0.1% Triton-X100 and incubated in equilibration buffer  for 10 seconds. Then, samples were incubated in reaction buffer  at 37°C for 1 hour. The TdT reaction mix was replaced with stop buffer  and incubated for 10 minutes at RT. Samples were then washed with PBS 0.1% Triton-X100 3 times and incubated with anti-digoxigenin antibody solution  overnight at 4°C. Samples were then washed with PBS 0.1% Triton-X100 3 times again and mounted on the slide glass.For the EdU feeding assay, a Click-iT EdU Alexa Fluor 555 Imaging Kit (Thermo Fisher Scientific C10338) was utilized. Female flies were transferred to the vials containing kimwipe soaked with 500 μM EdU and 5% sucrose and were kept for 24 hours. Midguts were dissected in 1xPBS and fixed for 60 minutes at RT in 1xPBS with 4% paraformaldehyde. After washing with PBST (0.1% Triton-X), EdU incorporation was visualized by Click reaction according to the manufacturer’s instruction. After washing with PBST for 5 minutes 3 times, guts were stained with Hoechst33342. Samples were washed with PBST for 5 minutes 3 times again and mounted on the slide glass.Virgin female flies were collected for 1 day and kept on standard food as described above until becoming 7 days old. Flies were transferred to control food  or bleomycin food ), dissected after 1 or 2 days, stained, and imaged.To raise flies under the sterile condition, eggs were collected on grape plates for 6 hours. Embryos were bleached (3%) for 2 minutes, washed, and transferred to control food  or sterile food, which was prepared by addition of penicillin and streptomycin. Virgin females and males were incubated with the food with/without the antibiotics and flipped 3 times a week until dissection at 6 to 7 days of age. Staining and imaging were performed as described above.The following GLEFA stock was generated:Myo1D-Gal4, esg-lexA-HG, lexAop-FLP, ubi>stop>nlsGFP; tub-Gal80ts.Oregon R, white1118 or UAS-p35. The progeny were kept at 18°C until 5 to 7 days old after eclosion, then the temperature was shifted to 30°C. Flies were kept at 30°C and were dissected 5 days after the temperature shift.This GLEFA stock was crossed to Guts were dissected and fixed by immersion in 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer (PB) (pH 7.4) for 30 minutes at RT. Guts were transferred to 4% paraformaldehyde in 0.1 M PB (pH 7.4) and further incubated for 3 hours at RT. After fixation, the guts were washed 3 times with 0.1 M PB (pH 7.4) for 10 minutes and permeabilized with 0.05% Triton X-100 in 0.1 M PB (pH 7.4) overnight with gentle agitation. After blocking with 1% BSA, 0.05% Triton X-100 in 0.1 M PB (pH 7.4) for 30 minutes at RT, the samples were incubated with chicken polyclonal anti-GFP antibody  for overnight at RT. The samples were washed 3 times with 0.05% Triton X-100 in 0.1 M PB (pH 7.4) for 10 minutes and incubated with biotinylated goat anti-chicken IgY antibody  for 2 hours at RT. After 3 washes with 0.05% Triton X-100 in 0.1 M PB (pH 7.4) for 10 minutes, the samples were incubated with streptavidin-nanogold AlexaFluor488  for 2 hours at RT. The samples were washed 3 times with 0.05% Triton X-100 in 0.1 M PB (pH 7.4) for 10 minutes and observed under a fluorescence stereomicroscope to confirm antibody labeling. Samples were subsequently post fixed with 1% glutaraldehyde in 0.1 M PB (pH 7.4) for 10 minutes at RT and washed 3 times with deionized water for 10 minutes at RT. Silver enhancement was performed for 4 minutes at RT using HQ Silver Enhancement Kit , according to the manufacturer’s instructions. Samples were washed 3 times with deionized water at RT and post fixed with 1% OsO4 in 0.1 M PB (pH 7.4) for 45 minutes at 4°C. After 3 washes with deionized water for 10 minutes at RT, samples were stained en bloc with 0.5% uranyl acetate for 30 minutes at RT. After 3 washes with deionized water, samples were dehydrated by graded ethanol series  for 10 minutes at each concentration, further dehydrated in 95% and 99.5% ethanol for 15 minutes at each concentration, and transferred to 100% ethanol for two 15-minute incubations. Samples were incubated in 100% acetone, transferred to a 1:1 mixture of acetone/Quetol 812 resin (Nisshin EM), and incubated overnight. After 3 transfers through Quetol 812, the R4 region was bisected with a razor to expose the region of interest, and the resin was polymerized at 60°C for >48 hours. Semithin sections (0.5 mm) were cut and stained with toluidine blue to examine the sample preparations. Ultrathin sections (50 to 70 nm) were cut and mounted on 200-mesh copper grids. The sections were stained with 0.5% uranyl acetate for 3 minutes at RT and washed with deionized water. The sections were further stained with Sato’s lead solution  for 3 miVirgin female flies were collected for 2 to 3 days and kept on standard food as described above until becoming 5 to 7 days old. In order to analyze the intestinal function, single flies were flipped to transparent cuvettes  with blue-dyed food containing 5% sucrose, 10% yeast, and 0.5% bromophenol blue for 24 hours as previously described . DefecatStatistical tests used were indicated in the figure captions. Sample sizes were determined empirically based on the observed effects.S1 Fig Control stocks control110 (A) and control128 (B) show Ance staining in an enterocyte subpopulation.  Ance protein is not detectable in 2 Ance mutant stocks AnceΔ110 (C) and AnceΔ128 (D). (E) Secreted Ance protein is present outside of cells (yellow arrowhead marks cellular F-actin) on the apical/luminal side of the midgut epithelium . The dotted rectangle is magnified at the left bottom. (F) Quantification of the number of Ance+ enterocytes in the R4 region over time. (G) Ance+ enterocytes are present in the R4 region of a 60-day-old fly. (H) This picture is the male midgut, which shows a similar expression pattern of Ance to the female one. The rest of pictures in the paper are all female guts. (I) Some Ance+ enterocytes show weak or absent genome staining (arrowhead) by Hoechst 33342. (J) Nuclear LaminB1 is absent on the nucleus of Ance+ cells depicted in the single-plane image and the orthogonal projection (arrowhead). (K) The orthogonal view of Phalloidin staining shows a drastic reduction of F-actin in Ance+ enterocytes (arrowhead). (L-N) Labeling of cell adhesion components by Dlg (septate junctions), β-catenin/Arm (adherens junctions) or cad::GFP shows reduced signals in Ance+ enterocytes in the single-plane image and the orthogonal projection (arrowhead). Statistical significance was determined by using one-way ANOVA with Dunnett’s multiple comparisons (F). (PDF)Click here for additional data file.S2 Fig(A-C) Ance+ cells show an inverse relationship with GFP fluorescence driven by 3 different Gal4-lines, actin-Gal4 (A), tubulin-Gal4 (B), and the mainly posterior midgut-specific Npc1b-Gal4 (C). (D) In addition to GFP, Ance+ entrocytes show decreased RFP signals. (E) Quantification of GFP and RFP signals in normal enterocytes and erebotic cells reveals a significant reduction of both fluorescent proteins in erebotic cells. Statistical significance was determined by using a two-tailed unpaired t test (E). (PDF)Click here for additional data file.S3 Fig Immunostaining of Ance marks erebotic cells, which are negative for Myo1D-driven GFP fluorescence and can be observed in control (A) as well as with inhibition of apoptosis by ectopic p35 expression in the midgut (B). (C) Quantification of the percentage of erebotic cells in control and p35 expression, shown in A and B.  Ance+ cells are present in the midgut of dpfK1 (Darkk11502) mutant flies at the age of 2 days (G) and 20 days (H). (F-H) Induction of homozygous clones by MARCM for H99 or the Dronc null mutation shows the presence of Ance protein in clone cells that are weakly labeled by nlsGFP (arrowheads), indicating that apoptosis-deficient mutant clones undergo erebosis. Statistical significance was determined by using a two-tailed unpaired t test (C). (PDF)Click here for additional data file.S4 Fig(A) The infection-inducible cellular shedding reporter CB, which reflects upd2 induction, does not drive expression of nls-mCherry in erebotic cells labeled by Ance immunostaining. (B-E) Manipulation of JNK or IMD pathway components such as expression of JNKDN (C) or puc (D) and knockdown of relish (E) does not suppress the presence of erebotic cells. (F) Quantification of blocking JNK or IMD pathways as shown in B-E. (G-J) Similar to control conditions (G-H), erebotic cells can be observed in flies raised under the sterile condition in females (I) and males (J). Statistical significance was determined by using one-way ANOVA with Dunnett’s multiple comparison (F). (PDF)Click here for additional data file.S5 Fig(A-H) Erebotic cells are present upon knockdown of Atg genes, indicating that autophagy is not required for erebosis. (I) Quantification of the percentage of erebotic cells upon enterocyte specific Atg gene knockdown indicates that blocking autophagy does not suppress erebosis. Statistical significance was determined by using one-way ANOVA with Dunnett’s multiple comparison (I). (PDF)Click here for additional data file.S6 Fig Enterocyte-specific knockdown of the caspase-activated DNAse Drep4 does not prevent nick formation in DNA of erebotic cells shown by the presence of TUNEL staining in GFP−/Ance+ cells.  Erebotic cells are present upon enterocyte-specific expression of Drep4 RNAi. (E) Quantification of the percentage of erebotic cells shows that the Drep4 knockdown does not induce reduction of erebosis. (F) Tubulin is reduced in erebotic cells. (G) In addition to nuclear LaminB1, erebotic cells decrease the nucleolus component Fibrillarin. Statistical significance was determined by using one-way ANOVA with Dunnett’s multiple comparison (E). (PDF)Click here for additional data file.S7 FigA-C) Enterocyte-specific knockdown of Ance using 2 different RNAi lines  does reduce the abundance of Ance protein compared to control (A) but does not eliminate the presence of erebotic cells indicated by absence of GFP fluorescence. (D) Quantification of the Ance signal intensity in erebotic enterocytes with Ance RNAis. (E) Quantification of the percentage of erebotic enterocytes with Ance RNAis. (F) Quantification of the mitotic cell numbers in whole midguts identified by phospho-H3 immunostaining in 2 Ance mutants and their respective control lines that were either kept on control food or food supplemented with bleomycin.  Overexpression of Ance specifically in enterocytes does not affect the presence of erebotic cells. (I) Quantification of the percentage of erebotic cells upon overexpression of Ance shows no change in the frequency of erebosis. (J)hh-Gal4–driven Ance expression increases the amount of Ance protein in the posterior wing disc, indicating that the UAS-Ance construct is functional. (K) Representative images of fecal deposits on a glass cuvette after defecation assay performance using 2 different RNAi lines for enterocyte-specific Ance knockdown and their respective control flies. (L) Quantification of fecal deposits within 24 hours. (M) Quantification of the percentage of concentrated fecal deposits within 24 hours. Ance knockdown reduces the amounts of concentrated fecal deposits. Ance-Gal4 drives GFP expression in both erebotic cells and cells surrounding them in the R4. Although cells in late erebosis with high Ance have very little GFP signals (yellow arrowhead), cells in intermediate erebosis with a moderate amount of Ance have clear GFP signals (white arrowhead). (P) The Ance signal peptide deletion mutant, which lacks the signal peptide (R2 to A17), does not demonstrate specific patterns of Ance immunostaining, suggesting that secretion of Ance might be important for it to accumulate in erebotic cells. Statistical significance was determined by using a two-tailed paired t test  or one-way ANOVA with Dunnett’s multiple comparison . ((PDF)Click here for additional data file.S8 Fig Quantification of the mitotic cell numbers in whole midguts identified by phospho-H3 immunostaining of 3 different control conditions (2 TRiP control lines and mCherry RNAi) and 2 constructs that inhibit apoptosis expressed in enterocytes . Flies were kept either on control food (A) or on food supplemented with bleomycin (B) for 1 day. (C) Quantification of EdU-incorporated progenitor in posterior midguts. Note that we excluded Edu-positive endoreplicating enterocytes from counting. (D) A schematic of GLEFA system, which enables both gene manipulation in enterocytes and lineage tracing from progenitor cells. (E) Lineage tracing by GLEFA demonstrates that p35 expression in enterocytes does not affect proliferation of progenitor cells. (F) Quantification of the mitotic cell numbers in whole midguts identified by phospho-H3 immunostaining in dpfK1 (Darkk11502) mutant and heterozygous control. Statistical significance was determined by using a two-tailed unpaired t test  or one-way ANOVA with Dunnett’s multiple comparison (E). (PDF)Click here for additional data file.S1 Movie(MP4)Click here for additional data file.S1 DataThe numerical values used for all main and supporting figures are provided.(XLSX)Click here for additional data file.


Tripidium arundinaceum dehydration-responsive element-binding transcription factor (TaDREB2B) behind the drought-responsible RD29A promoter into a commercial sugarcane cultivar FN95-1702 and subsequently conducted a series of drought tolerance experiments and investigation of agronomic and quality traits. Physiological analysis indicated that Prd29A: TaDREB2B transgenic sugarcane significantly confers drought tolerance in both the greenhouses and the field by enhancing water retention capacity and reducing membrane damage without compromising growth. These transgenic plants exhibit obvious improvements in yield performance and various physiological traits under the limited-irrigation condition in the field, such as increasing 41.9% yield and 44.4% the number of ratooning sugarcane seedlings. Moreover, Prd29A: TaDREB2B transgenic plants do not penalize major quality traits, including sucrose content, gravity purity, Brix, etc. Collectively, our results demonstrated that the Prd29A-TaDREB2B promoter-transgene combination will be a useful biotechnological tool for the increase of drought tolerance and the minimum of yield losses in sugarcane.Sugarcane is one of the most crucial sugar crops globally that supplies the main raw material for sugar and ethanol production, but drought stress causes a severe decline in sugarcane yield worldwide. Enhancing sugarcane drought resistance and reducing yield and quality losses is an ongoing challenge in sugarcane genetic improvement. Here, we introduced a  Saccharum spp. hybrid) acts as one of the important commercial crops that provides at least 60% of world sugar production  from Arabidopsis is a drought-responsive gene, several studies showed that its promoter had been used for moderate stress-inducible transgene expression in plants (green fluorescent protein (GFP) gene controlled by RD29A promoter (Prd29A) was introduced into sugarcane callus and showed that significantly induced by PEG , empty vector (EV) and Prd29A:TaDREB2B transgenic sugarcane lines generated from cv. FN95-1702 were planted in a sugarcane field in Fuzhou, Fujian province, China. For asexually propagated sugarcane, all materials used in this study come from the first generation of transgenic sugarcane. The stems with sugarcane buds were grown in the fertile soil under well-watered conditions for growth under 16 h/8 h light/dark illumination, 27°C, and 60%-70% air humidity condition. For drought treatment, soil water content and membrane permeability were monitored and represent different levels of water deficit  in the four-leaf stage sugarcane seedlings . The amino acid sequence alignments were generated by DNAMAN software. The phylogenetic tree was constructed using the Clustal X, MEGA 7 software, and iTOL website (https://itol.embl.de/).TaDREB2B gene from T. arundinaceum was inserted in Prd29A-hyg digested with PstI and SphI restriction enzyme  using ClonExpress Ultra One Step Cloning Kit  according to our previous method (Prd29A-TaDREB2B-hyg expression vector was used to transform sugarcane (s method . The genugarcane .2O. Reverse transcription-quantitative PCR (RT-qPCR) was performed on a 7500 Real-Time PCR system  in a 15 μL reaction volume , TaDREB2B .The total RNA of sugarcane leaves was extracted using RNA Extraction Kit  for gene expression, and 3 μg of total RNA was used to generate first-strand cDNA using SuperScript III reverse transcriptase  and was diluted 50-100 times with ddHn volume . The houTo determine MDA content, the crushed sugarcane leaf sample of 0.1 g was suspended with 1 mLof 0.1% trichloroacetic acid. After centrifugation at 12000 g for 2 min, 0.5 mL supernatant and 1mL of 10% trichloroacetic acid containing 0.67% thiobarbituric acid were mixed, subsequentlyboiled for 0.5 h, and rapidly cooled with ice. The OD values were measured and recorded at 450,532, and 600 nm, respectively .2O, 0.05 mL of anthrone reagent and 0.5 mL concentrated sulfuric acid, then boiled at 100°C for 1-2 min. After cooling with ice water, the OD values were measured at 630 nm  = (Xi−Xmin)/(Xmax−Xmin) when the indicator was positively correlated with drought tolerance, and the formula was U(Xi) = 1 − (Xi − Xmin)/(Xmax − Xmin) when the indicator was negatively correlated (i= ΣU(pm × Xi)/n was the overall evaluation value, in which Xi is the measured value of an index of each sugarcane line. Xmin and Xmax represent the minimum and maximum values of the index, respectively. pm is the weight coefficient of the m-th principal component and n is the number of indicators.The membership function method evaluated the drought tolerance of WT and rrelated . Xi= ΣU under the following accession numbers: AtORA47 (AT1G74930), AtDREB26 (AT1G21910), AtDEAR5 (AT4G06746), AtDEAR1 (AT3G50260), AtDEAR3 (AT2G23340), AtDEAR4 (AT4G36900), AtDDF1 (AT1G12610), AtCBF4 (AT5G51990), AtCBF2 (AT4G25470), AtCBF1 (AT4G25490), AtCBF3 (AT4G25480), AtHARDY (AT2G36450), AtERF38 (AT2G35700), AtTINY (AT5G25810), AtTINY2 (AT5G11590), AtTINY3 (AT4G32800), AtESE2 (AT2G25820), AtABI4 (AT2G40220), AtDREB2B (AT3G11020), AtDREB2C (AT2G40340), AtWIND4 (AT5G65130), AtWIND3 (AT1G36060), AtWIND1 (AT1G78080), AtRAP2.4 (AT1G22190), AtCRF4 (AT4G27950), AtRAP2.6L (AT5G13330), AtERF71 (AT2G47520), AtERF72 (AT3G16770), AtERF96 (AT5G43410), AtERF4 (AT3G15210), AtERF10 (AT1G03800). Other gene sequence information can be obtained from National Library of Medicine (https://www.ncbi.nlm.nih.gov/) under the following accession numbers: OsDREB2A (XP_025878770.1), OsDREB2B (NP_001389444.1), SsDREB2B (Sspon.001C0004630), ZmDREB2A (PWZ09406.1).The TaDREB2B gene from T. arundinaceum through BLAST analysis using the amino acid sequence of AtDREB2B. The TaDREB2B was cloned from T. arundinaceum, which contains a 978-bp open reading frame and encodes a protein with 325 amino acid residues. Phylogenetic tree analysis showed that TaDREB2B belongs to the DREB-A2 subfamily, and its orthologs are AtDREB2B , AtDREB2A, AtDREB2C, ZmDREB2A (Zea mays L.), SsDREB2B (Saccharum spontaneum L.)  and introduced into sugarcane callus  were subjected to drought (ceasing water for about 15 days), and the seedlings phenotypes were subsequently recorded. No significant growth difference was observed between all sugarcane seedlings at the four-leaf stage under normal growing conditions , and four transgenic unctions Table 1.Prd29A:TaDREB2B transgenic sugarcane line (T4) to evaluate drought-related utility in the field. We performed three analyses: (1) drought resistance experiments of the Prd29A:TaDREB2B sugarcane at the seedling stage in the greenhouse and during vigorous growth period in the field; (2) determination of key physiological characteristics of the Prd29A:TaDREB2B sugarcane in pots cultivation under rainproof shelter; (3) yield comparisons and examination of agronomic traits in the field with controlled irrigation conditions. We first performed a drought tolerance analysis for the WT and T4 line in the greenhouse. After stopping water for 7 d (mild drought), the WT seedlings displayed distinct growth retardation, but T4 line plants were still thriving. After stopping water for 15 d (severe drought), WT showed evident drought symptoms, such as leaf rolling, while the degree of leaf rolling in the T4 line was notably very slight than WT  was then introduced into a commercial sugarcane cultivar FN95-1702. The expression of TaDREB2B controlled by the RD29A promoter can be distinctly induced in a mild drought and slightly increased in severe drought stress , Guangxi Natural Science Foundation (2021JJB130425), Scientific Research and Development Fund of College of Agriculture, Guangxi University (EE101711).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. 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