Journal: bioRxiv

Article Title: The RNA Editome of Esophageal Squamous Cell Carcinoma Identifies an ADAR1-CDK13 Editing Feedback Loop Mediating cGAS–STING Activation in Early Tumorigenesis

doi: 10.64898/2026.01.29.701210

Figure Lengend Snippet: (a) Dot plot showing editing levels of differentially edited recoding sites in genes in ESCC and CTRL. (b) Amino acid preferences of differentially edited recoding sites in genes. (c) Heatmap showing RNA editing levels of recoding genes containing at least one differentially edited recoding site. (d) Sanger sequencing chromatograms for two differentially edited recoding sites at position 1984 in NBPF8 and position 308 in CDK13 . The orange box highlights the peak of adenosine or guanosine. Edited nucleotide was shaded in red. (e,f) Kaplan-Meier curves for survival of high RNA A-to-I editing (e) and expression (f) versus low CDK13 editing and expression cohorts in NM and M ESCC, respectively. NM: nonmetastatic, M: metastatic. P values were assessed by Cox regression. (g) Overall RNA A-to-I editing levels of CDK13 across ESCC and paired CTRL samples, and across NM and M ESCC, respectively. (h) CDK13 expression across ESCC and CTRL, and NM and M samples. (i) RNA A-to-I editing in CDK13 in two groups of high and low CDK13 expression based on the mean. (j) Correlation analysis of RNA A-to-I editing and expression of CDK13 . (k) Expression of ADARs across samples of ESCC and CTRL. Significant differences were assessed by the Wilcoxon test.

Article Snippet: The membrane was blocked with 5% non-fat milk powder in 1 × TBST for 1-2 hours at RT, followed by overnight incubation (∼16 h) at 4°C refrigerator with primary antibody against ADAR1 (Santa Cruz, sc-73408; 1:200 dilution) or CDK13 (Bethyl, A301-458A-BL; 1:2000 dilution).

Techniques: Sequencing, Expressing

Journal: bioRxiv

Article Title: The RNA Editome of Esophageal Squamous Cell Carcinoma Identifies an ADAR1-CDK13 Editing Feedback Loop Mediating cGAS–STING Activation in Early Tumorigenesis

doi: 10.64898/2026.01.29.701210

Figure Lengend Snippet: (a) Correlation of the RNA editing rates (upper panel) or RNA editing events (lower panel) in CDK13 with the expression of ADAR1, ADAR2, and ADAR3 . (b) Illustration of RNA immunoprecipitation (RIP) using KYSE30 and KYSE150 cells. Cells were collected and then equally split into three parts for agarose-conjugated antibody incubation, followed by washing and RNA extraction. (c) Immunoblotting examining ADAR1 abundance in distinct fractions of ADAR1 immunoprecipitation. GAPDH was used as loading control. Abbreviations represent CE: crude extract; SN: supernatant; IP: Immunoprecipitation. (d) Distribution of peaks (KYSE30 and KYSE150) and DRS across genomics elements. (e) Circos plot showing the hg38 genomic landscape of A-to-I editing events. From outermost to innermost, tracks 1-5 showed the editing densities (sites per million base pairs) in tumor (red bars) versus adjacent normal (blue bars) tissues for exons, introns, UTRs, ncRNAs, and intergenic regions, respectively; tracks 6-7 displayed scatter plots of peak scores for KYSE30 and KYSE150 cell lines. Peaks were annotated according to its genomic location, with red dots (exonic), blue (intronic), green (regulatory), and grey (intergenic). The inner links showed the genes correlated with ADAR in A-to-I editing, and the link to CDK13 was highlighted. (f) Distribution of RNA editing sites across genomics elements in two KYSE cells. (g) Distribution of RNA editing sites resided in different elements of protein-coding RNAs. (h) Counts of A-to-I DRS within or outside peaks. (i) Relative expression of ADAR1 and ADAR2 in siRNA ADAR1 , ADAR2 or control (Scramble) knockdown cells. Error bars represent ±S.D. of the mean from two technical replicates . (j) Sanger sequencing chromatograms for two recoding sites CDK13 Q103R ( CDK13 c.A308G ) and NBPF8 I662V ( NBPF8 c.A1984G ) were examined from cDNA of siRNA ADAR1 , ADAR2 or Scramble knockdown cells. (k) RNA secondary structure of unedited (upper panel) and edited (low panel) sequence surrounding c.A308G in CDK13 .

Article Snippet: The membrane was blocked with 5% non-fat milk powder in 1 × TBST for 1-2 hours at RT, followed by overnight incubation (∼16 h) at 4°C refrigerator with primary antibody against ADAR1 (Santa Cruz, sc-73408; 1:200 dilution) or CDK13 (Bethyl, A301-458A-BL; 1:2000 dilution).

Techniques: Expressing, RNA Immunoprecipitation, Incubation, RNA Extraction, Western Blot, Immunoprecipitation, Control, Knockdown, Sequencing

Journal: bioRxiv

Article Title: The RNA Editome of Esophageal Squamous Cell Carcinoma Identifies an ADAR1-CDK13 Editing Feedback Loop Mediating cGAS–STING Activation in Early Tumorigenesis

doi: 10.64898/2026.01.29.701210

Figure Lengend Snippet: (a) Relative CDK13 expression in two stable shRNA knockdown and control KYSE30 cell lines (left) and Western blot of CDK13 in the same cell lines (right); asterisks indicate CDK13. (b) Volcano plot showing differential gene expression upon CDK13 knockdown in KYSE30 cells, with ADAR family members highlighted. (c) Expression of representative ISGs in ESCC versus control (top) and in primary versus metastatic tumors (bottom). (d) Regression analysis of six ISGs with CDK13 RNA A-to-I editing and CDK13 expression. (e) Schematic illustration of ADAR1/CDK13 regulation in ESCC prognosis. Data are mean ± S.D.; significance was assessed by Student’s t-test. Asterisks denote significance (*p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: The membrane was blocked with 5% non-fat milk powder in 1 × TBST for 1-2 hours at RT, followed by overnight incubation (∼16 h) at 4°C refrigerator with primary antibody against ADAR1 (Santa Cruz, sc-73408; 1:200 dilution) or CDK13 (Bethyl, A301-458A-BL; 1:2000 dilution).

Techniques: Expressing, shRNA, Knockdown, Control, Western Blot, Gene Expression

Journal: bioRxiv

Article Title: The RNA Editome of Esophageal Squamous Cell Carcinoma Identifies an ADAR1-CDK13 Editing Feedback Loop Mediating cGAS–STING Activation in Early Tumorigenesis

doi: 10.64898/2026.01.29.701210

Figure Lengend Snippet: (a) Reduced CDK13 signal in stable shRNA knockdown KYSE30 cells compared with controls. rRNA-depleted reads were separated by strand and normalized to CPM. (b) Volcano plot of differentially expressed genes (DEG2) in CDK13 knockdown versus control cells. DEG2 were defined as log2FC > 0.5 or < −0.5 with FDR < 0.05. Blue and red dots indicate down- and up-regulated DEG2, respectively. (c) Volcano plot of differentially phosphorylated sites (DPS). DPS were defined as log2FC > 1.5 or < −1.5 with p < 0.05. Blue and red dots represent hypo- and hyper-phosphorylated sites, respectively. (d) DEG2 is significantly enriched in immune response, cytoskeleton, and interferon signaling pathways. (e) Heatmap showing fold changes (logFC) of genes related to cGAS – STING activation, cytoskeleton regulation, STING trafficking, and interferon-stimulated gene (ISG) readout upon CDK13 knockdown in KYSE30 cells. (f) Venn diagram of DEG2 and hypo-phosphorylated sites, and the top 10 GO terms enriched among non-DEG2 DPS ranked by FDR. FDR was controlled at 5%. Blue and orange bars indicate GO terms enriched in proteins containing up- and down-phosphorylated sites, respectively. (g) Dot plot of Reactome pathways enriched for down-regulated DPS. (h) Potential CDK13 targets involved in ADAR1-dependent RNA editing. Twenty-two proteins containing 28 DPS were identified with the canonical proline residue directly following the phosphorylated amino acid . (i) Sequence conservation surrounding phosphorylated residues (±7 amino acids). Colors indicate amino acid chemistry, and bits represent residue conservation. (j) Venn diagram integrating four datasets: ADAR1-binding genes (from overlapping peaks), RNA editing-correlated genes (DEG1 and editing-correlated), DEG2, and down-regulated DPS.

Article Snippet: The membrane was blocked with 5% non-fat milk powder in 1 × TBST for 1-2 hours at RT, followed by overnight incubation (∼16 h) at 4°C refrigerator with primary antibody against ADAR1 (Santa Cruz, sc-73408; 1:200 dilution) or CDK13 (Bethyl, A301-458A-BL; 1:2000 dilution).

Techniques: shRNA, Knockdown, Control, Protein-Protein interactions, Activation Assay, Residue, Sequencing, Binding Assay

Journal: Nature Immunology

Article Title: A multi-kinase inhibitor screen identifies inhibitors preserving stem-cell-like chimeric antigen receptor T cells

doi: 10.1038/s41590-024-02042-1

Figure Lengend Snippet: a , Schematics of the MIB–MS in Jurkat cells incubated with the KIs UNC10225263A, UNC10225761A or UNC10225387B at 0.75 µM, 2.0 µM or DMSO. b , Kinases showing dose-dependent inhibition by UNC10225263A, UNC10225387B and UNC10225761A ( n = 3 replicates for UNC10225263A and UNC10225387B and n = 2 replicates for DMSO and UNC10225761A). c , Jurkat cells pre-incubated with DMSO or UNC10225263A, UNC10225761A or UNC10225387B KI (2 µM) for 6 h and then activated with 10 µg ml −1 of agonistic CD3 mAb and crosslinking secondary antibody at 37 °C for 5 min. Phosphorylation of TCR signaling molecules was measured by phospho-flow (top) and immunoblot (bottom); β-actin was used as a loading control. d , e , CAR.CD19 T cells co-transduced with lentiviruses encoding shRNA-targeting specific kinases and the percentage of CD45RA + CCR7 + cells in CD4 or CD8 CAR.CD19 T cells determined by FACS. In d , shRNA targeted CLK3, STK17B, MAP3K7, AURKA, MINK1, CDK12 or ITK, whereas, in e , it targeted ADCK1, ADCK3, MAP3K4, CDK13 or TRIM28 ( n = 8 independent T cell donors for d and n = 6 for e ). The data represent two series of separated experiments, shNC indicates small hairpin negative control and was used as negative control. The blue bars indicate kinases with knockdown that caused a significant increase in T SCM cell-like CAR T cells. f , CAR.CD19 T cells co-transduced with a combination of lentiviruses encoding shRNA-targeting ADCK3 + ITK + MAP3K4 (C1) or ADCK3 + ITK + CDK13 (C2). The percentage of CD45RA + CCR7 + cells in CD4 or CD8 CAR.CD19 T cells was determined by FACS. CAR T cells co-transduced with the shNC and cultured in DMSO or KIs were used as controls ( n = 8 independent T cell donors). g – i , ShRNA (C1) ( g ), shRNA (C2) ( h ), shNC + DMSO and shNC + KIs CAR.CD19 ( i ) T cells stimulated with CD19 + Daudi tumor cells at 1:2 E:T ratio for 3 d. Relative fold expansion to shNC + DMSO group ( h ) and percentage of the CD45RA + CCR7 + cells ( i ) of CAR.CD19 T cells were measured by FACS ( n = 5 in h and n = 4 independent T cell donors in i ). The P values were determined using two-sided, paired Student’s t -test. Data are shown as individual values and the mean ± s.d. except in b where error bars are not shown.

Article Snippet: The following antibodies were used: anti-CD3ζ (Santa Cruz, cat. no. sc166275), anti-actin (Santa Cruz, cat. no. sc47778), anti-MINK1 (Thermo Fisher Scientific, cat. no. PA5-51000), anti-AURKA (Cell Signaling Technology, cat. no. 14475s), anti-CLK3 (Cell Signaling Technology, cat. no. 3256), anti-STK17B (Proteintech, cat. no. 26600-1-AP), anti-MAP3K7 (Cell Signaling Technology, cat. no. 4505), anti-CDK12 (Cell Signaling Technology, cat. no. 11973), anti-TAOK2 (Thermo Fisher Scientific, cat. no. 21188-1-AP), anti-CDK13 (Invitrogen, cat. no. PA5-27702), anti-ADCK3 (Abcam, cat. no. ab230897), anti-ADCK1 (Sigma-Aldrich, cat. no. HPA051012), anti-KAP1 (TRIM28) (Abcam, cat. no. ab10484), anti-ZAP70 (Cell Signaling Technology, cat. no. 3165S), anti-phospho-Zap70 (Tyr319)/Syk (Tyr352) (Cell Signaling Technology, cat. no. 2701L), anti-LAT (Cell Signaling Technology, cat. no. 45533S), anti-phospho-LAT (Tyr171) (Cell Signaling Technology, cat. no. 3581), anti-p38 (Cell Signaling Technology, cat. no. 8690), anti-phospho-p38 Thr180/Tyr182 (Cell Signaling Technology, cat. no. 4511), anti-FOXO1 (Cell Signaling Technology, cat. no. 2880), goat anti-rabbit IgG-horseradish peroxidase (HRP) (Invitrogen, cat. no. 656120), rabbit anti-mouse IgG-HRP (Invitrogen, cat. no. 31430).

Techniques: Incubation, Inhibition, Western Blot, Control, Transduction, shRNA, Negative Control, Knockdown, Cell Culture

Journal: Nature Immunology

Article Title: A multi-kinase inhibitor screen identifies inhibitors preserving stem-cell-like chimeric antigen receptor T cells

doi: 10.1038/s41590-024-02042-1

Figure Lengend Snippet: ( a ) Western blots showing p38, LAT and ZAP70 phosphorylation in CAR.CD19-T cells stimulated with 1 µg/ml agonistic CD3 mAb or 1 µg/ml anti-idiotype CAR Ab in the presence of the KIs; β-actin was used as loading control. ( b, c ) Specific kinases were knockdown in CAR.CD19-T cells by using lentiviruses encoding specific shRNAs. Messenger RNA ( b ) and protein ( c ) expression of each targeted kinase in CAR.CD19-T cells after shRNA-based knocking down. ( d ) Comparison of TCF1 expression in CD4 or CD8 CAR.CD19-T cells obtained after combination of shRNA targeting ADCK3 + ITK + MAP3K4 (C1) or targeting ADCK3 + ITK + CDK13 (C2) and CAR-T cells generated in the presence of DMSO or KIs expressing the control shRNA; n = 3 independent T cell donors. Data are shown as individual values and mean ± SD; p values were determined by two-side paired student’s t test.

Article Snippet: The following antibodies were used: anti-CD3ζ (Santa Cruz, cat. no. sc166275), anti-actin (Santa Cruz, cat. no. sc47778), anti-MINK1 (Thermo Fisher Scientific, cat. no. PA5-51000), anti-AURKA (Cell Signaling Technology, cat. no. 14475s), anti-CLK3 (Cell Signaling Technology, cat. no. 3256), anti-STK17B (Proteintech, cat. no. 26600-1-AP), anti-MAP3K7 (Cell Signaling Technology, cat. no. 4505), anti-CDK12 (Cell Signaling Technology, cat. no. 11973), anti-TAOK2 (Thermo Fisher Scientific, cat. no. 21188-1-AP), anti-CDK13 (Invitrogen, cat. no. PA5-27702), anti-ADCK3 (Abcam, cat. no. ab230897), anti-ADCK1 (Sigma-Aldrich, cat. no. HPA051012), anti-KAP1 (TRIM28) (Abcam, cat. no. ab10484), anti-ZAP70 (Cell Signaling Technology, cat. no. 3165S), anti-phospho-Zap70 (Tyr319)/Syk (Tyr352) (Cell Signaling Technology, cat. no. 2701L), anti-LAT (Cell Signaling Technology, cat. no. 45533S), anti-phospho-LAT (Tyr171) (Cell Signaling Technology, cat. no. 3581), anti-p38 (Cell Signaling Technology, cat. no. 8690), anti-phospho-p38 Thr180/Tyr182 (Cell Signaling Technology, cat. no. 4511), anti-FOXO1 (Cell Signaling Technology, cat. no. 2880), goat anti-rabbit IgG-horseradish peroxidase (HRP) (Invitrogen, cat. no. 656120), rabbit anti-mouse IgG-HRP (Invitrogen, cat. no. 31430).

Techniques: Western Blot, Control, Knockdown, Expressing, shRNA, Comparison, Generated