Journal: Alcohol (Fayetteville, N.Y.)

Article Title: Profiling the Oxylipidome in Aged Mice after Chronic Ethanol Feeding: Identifying Lipid Metabolites as Drivers of Hepatocyte Stress

doi: 10.1016/j.alcohol.2022.08.012

Figure Lengend Snippet: Young and aged adult WT mice were allowed free access to ethanol (32%,d25) or pair-fed control diets. A) Liver RNA was isolated and expression of senescence markers, p16 and p21 mRNA was detected in mouse livers using qRT-PCR. B/C) Paraffin-embedded liver sections were deparaffinized followed by immunodetection of PCNA and p53 and nuclei were counterstained with hematoxylin. Frozen liver sections were stained for β-galactosidase activity. Images were acquired using a ×10 objective and positive staining was quantified using Image-J. Data are from two independent trials; individual values are reported and represented as means ± SEM, n=8 pair-fed and 12 EtOH-fed mice/group. *Values were significantly different from each other (P<0.05).

Article Snippet: Paraffin-embedded liver sections were deparaffinized and stained with antibodies against NIMP-R14 (Novus Biologicals, cat NB600–1387), PCNA (Millipore, cat# MAB424R) and p53 (Novus Biologicals, cat# NB200–103).

Techniques: Control, Isolation, Expressing, Quantitative RT-PCR, Immunodetection, Staining, Activity Assay

Journal: Antioxidants

Article Title: Saposhnikovia divaricata Inhibits Inflammation, Oxidative Stress, and Ferroptosis to Alleviate DSS-Induced Ulcerative Colitis

doi: 10.3390/antiox15020258

Figure Lengend Snippet: Network pharmacology analysis identifies p53 as a core ferroptosis-related target of FF in UC. ( A ) Venn diagram illustrating the intersection of FF compound targets with ferroptosis- and UC-related targets. ( B ) Protein–protein interaction (PPI) network of the common targets. Node size and color intensity represent the degree of connectivity, with TP53 (p53) identified as the core target. ( C ) Compound-target-pathway network diagram. The inner pink nodes represent the 38 intersecting targets linking FF, UC, and ferroptosis. ( D ) Gene Ontology (GO) enrichment analysis of the common targets, categorized into Biological Process (BP, red), Cellular Component (CC, green), and Molecular Function (MF, blue). ( E ) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis.

Article Snippet: The p53 antibody was obtained from Boster Biological Technology Co., Ltd. (Pleasanton, CA, USA, Item #BM0101).

Techniques:

Journal: Antioxidants

Article Title: Saposhnikovia divaricata Inhibits Inflammation, Oxidative Stress, and Ferroptosis to Alleviate DSS-Induced Ulcerative Colitis

doi: 10.3390/antiox15020258

Figure Lengend Snippet: FF modulates the expression of ferroptosis-related proteins in colon tissue via the p53 pathway. ( A ) Representative immunohistochemical (IHC) images of p53, SLC7A11, and GPX4 expression in colon sections (scale bar = 50 μm). ( B – D ) Quantitative analysis of the relative protein expression levels of p53 (B), SLC7A11 (C), and GPX4 (D). Data are presented as the mean ± SD ( n = 3 independent experiments). ### p < 0.001 versus the control (CON) group; * p < 0.05, ** p < 0.01, *** p < 0.001 versus the DSS model group.

Article Snippet: The p53 antibody was obtained from Boster Biological Technology Co., Ltd. (Pleasanton, CA, USA, Item #BM0101).

Techniques: Expressing, Immunohistochemical staining, Control

Journal: bioRxiv

Article Title: A small molecule inhibitor of NVL suppresses tumor growth by blocking ribosome biogenesis

doi: 10.1101/2025.07.31.667081

Figure Lengend Snippet: A. Growth curves of Cas9-expressing HCT116 in the genome wide CRISPR/Cas9 knockout screen shown in . At each plotted point, cells were treated for 24 hours with vehicle or 1 µM MM17 and allowed to recover without treatment until 80% confluency (n=1). B. Immunoblots of p53 and actin (loading control) in HCT116 mCherry mock, ZsGreen p53 KO guide 1 (p53 KO-1), and ZsGreen p53 KO guide 2 (p53 KO-2) cells. C. Immunoblots of p21 and tubulin (loading control) in HCT116 mCherry mock, and ZsGreen p21 KO cells. D, E. Growth competition assay between mCherry mock and ZsGreen mock, ZsGreen p53 KO-1, ZsGreen p53 KO-2, or ZsGreen p21 KO cells. At each plotted point, cells were treated for 24 hours with MM17 ( D ) or paclitaxel ( E ) and allowed to recover without compound until confluency. (n=3 biological replicates, mean ± sem). F. Quantitation of p53 and p21 immunoblots, related to . (n = 3 biological replicates, mean ± sem). G. Immunoblot of MDM2 and Ponceau S staining (loading control) in HCT116 p53 KO cells transduced with non-target control or four sgRNAs targeting MDM2. sgMDM2 guide 3 was selected for subsequent experiments. H. Cresyl violet staining after 1 week of antibiotics selection of HCT116 cells expressing empty vector, ectopic MDM2 WT, or MDM2 C311F transduced with mock or MDM2 guide. I. Immunoblots of MDM2 and actin (loading control) of cell lines in (H). J. Quantitation of p53 and p21 immunoblots, related to . (n = 3 biological replicates, mean ± sem). K. Viability assay of HCT116 cells treated with etoposide for 72 hours. (n = 3 biological replicates, mean ± sem). L. Quantitation of p53 and p21 immunoblots, related to . (n = 3 biological replicates, mean ± sem).

Article Snippet: The following primary antibodies were used: anti-NVL (Bethyl Laboratories #A304-863A); anti-eL36 (Proteintech #15145-1-AP); anti-eS17 (Abcam #ab128671); anti-p53 (Cell Signaling #9282); anti-p21 (Cell Signaling #2947); anti-Fibrillarin (Cell Signaling #2639); anti caspase-3 (D3R6Y) (Cell Signaling #14220); anti-FLAG m2 peroxidase HRP conjugated (Cell Signaling #a8592); anti-MDM2 (Cell Signaling #86934); anti-SNAP (NEB #P9310S); anti-alpha tubulin HRP conjugated (Cell Signaling #9099); anti-beta actin HRP conjugated (Santa Cruz Biotechnology #47778).

Techniques: Expressing, Genome Wide, CRISPR, Knock-Out, Western Blot, Control, Competitive Binding Assay, Quantitation Assay, Staining, Transduction, Selection, Plasmid Preparation, Viability Assay

Journal: bioRxiv

Article Title: A small molecule inhibitor of NVL suppresses tumor growth by blocking ribosome biogenesis

doi: 10.1101/2025.07.31.667081

Figure Lengend Snippet: A. Genome wide CRISPR/Cas9 knockout screen in HCT116 cells. The log2 fold change for each gene comparing samples treated with vehicle or 1 µM MM17 was determined by averaging the log2 fold changes of its associated sgRNAs and subtracting the average log2 fold change of non-targeting sgRNAs. The top two most enriched genes, and one of the biogenesis factors removed by Rix7 are indicated in red and labeled. B. Immunoblots of p53, p21, and tubulin (loading control) in HCT116 NVL WT and NVL R403W CRISPR cells treated with 3 µM MM17. C . Cryo-EM structure of the MDM2 299-340 –5S RNP complex (PDB ID - 8BGU; MDM2 - orange, uL5 - turquoise, uL18 - red, 5S rRNA - pink) . MDM2 C311 residue (yellow) and Zn 2+ (purple) at the MDM2-uL5 binding interface are labeled. D, E. Immunoblots of p53, p21 and actin (loading control) in HCT116 MDM2 WT and MDM2 C311F cells treated with MM17 ( D ) or etoposide (E) for 24 hours. F. Cell cycle analysis of HCT116 mock and p53 KO cells treated with 3 µM MM17. (n = 3 biological replicates, mean ± sem; two-tailed unpaired t-test, ***p<0.001, **p<0.01, ns = non-significant). G. Live cell imaging analysis of relative cell area of HCT116 mock and p53 KO cells treated with 3 µM MM17 for 72 hours. (n = 3 biological replicates, mean ± sem).

Article Snippet: The following primary antibodies were used: anti-NVL (Bethyl Laboratories #A304-863A); anti-eL36 (Proteintech #15145-1-AP); anti-eS17 (Abcam #ab128671); anti-p53 (Cell Signaling #9282); anti-p21 (Cell Signaling #2947); anti-Fibrillarin (Cell Signaling #2639); anti caspase-3 (D3R6Y) (Cell Signaling #14220); anti-FLAG m2 peroxidase HRP conjugated (Cell Signaling #a8592); anti-MDM2 (Cell Signaling #86934); anti-SNAP (NEB #P9310S); anti-alpha tubulin HRP conjugated (Cell Signaling #9099); anti-beta actin HRP conjugated (Santa Cruz Biotechnology #47778).

Techniques: Genome Wide, CRISPR, Knock-Out, Labeling, Western Blot, Control, Cryo-EM Sample Prep, Residue, Binding Assay, Cell Cycle Assay, Two Tailed Test, Live Cell Imaging

Journal: bioRxiv

Article Title: A small molecule inhibitor of NVL suppresses tumor growth by blocking ribosome biogenesis

doi: 10.1101/2025.07.31.667081

Figure Lengend Snippet: A. Immunoblots and quantitation of caspase-3 and actin (loading control) in HCT116 cells treated with 1 µM MLN4924 or 3 µM MM17. (n = 3 biological replicates, mean ± sem). B. Flow cytometry-based cell cycle analysis in HCT116 cells treated with vehicle or 3 µM MM17 for 3 days. (n = 3 biological replicates, mean ± sem; two-tailed unpaired t-test, ***p<0.001, **p<0.01, *p<0.05). C. Immunoblots of p53 and actin (loading control) in HCT116 mock engineered or p53 knockout (KO) cells. D. Viability (log 2 fold change relative to vehicle) of 855 cancer cell lines treated with 10 µM MM17, grouped by their p53 mutation or copy number deletion status. Each dot represents a cell line. (two-tailed paired t-test, ****p<0.0001).

Article Snippet: The following primary antibodies were used: anti-NVL (Bethyl Laboratories #A304-863A); anti-eL36 (Proteintech #15145-1-AP); anti-eS17 (Abcam #ab128671); anti-p53 (Cell Signaling #9282); anti-p21 (Cell Signaling #2947); anti-Fibrillarin (Cell Signaling #2639); anti caspase-3 (D3R6Y) (Cell Signaling #14220); anti-FLAG m2 peroxidase HRP conjugated (Cell Signaling #a8592); anti-MDM2 (Cell Signaling #86934); anti-SNAP (NEB #P9310S); anti-alpha tubulin HRP conjugated (Cell Signaling #9099); anti-beta actin HRP conjugated (Santa Cruz Biotechnology #47778).

Techniques: Western Blot, Quantitation Assay, Control, Flow Cytometry, Cell Cycle Assay, Two Tailed Test, Knock-Out, Mutagenesis

Journal: bioRxiv

Article Title: A small molecule inhibitor of NVL suppresses tumor growth by blocking ribosome biogenesis

doi: 10.1101/2025.07.31.667081

Figure Lengend Snippet: A bioavailable analog MM927 inhibits colorectal and leukemia tumor xenograft growth in vivo without overt toxicity. A. Chemical structure of MM927 and the potency (IC50) of its impact on the viability of HCT116. B. Pharmacokinetic analysis of MM927 in mouse plasma after a 10 mg/kg intraperitoneal (IP) injection (n = 3 mice/group, mean ± sem). C, D. Representative microscopy images ( C ) and quantitation ( D ) showing immunofluorescence of p53 (magenta), Ki-67 (green), and nucleus (DAPI, blue) in NVL WT and NVL R403W CRISPR tumor xenografts implanted in either flank of the same mouse. Mice were treated with 35 mg/kg MM927 IP twice daily for 3 days, and tumors were harvested 6 hours after the last dose. (Scale bar = 10 µm; n = 5 mice/group, mean ± sem). E. Effect of MM927 (35 mg/kg IP twice daily for 21 days) on HCT116 NVL WT or NVL R403W CRISPR tumor xenograft growth (n = 10 mice/group, mean ± sem). F . Tumor volume measurements in (E) at the end of 21 days or euthanasia due to tumor diameter exceeding 2 cm (n = 10 mice per group, mean ± sem; two-tailed unpaired t-test, ** p < 0.01, ns = non-significant). G. Body weight % changes in HCT116-tumor bearing mice at the end of MM927 treatment (n = 20 mice/group, mean ± sem; two-tailed unpaired t-test, ns = non-significant). H. Levels of hemoglobin, white blood cells, platelets, liver enzymes AST and ALT, and renal clearance of blood urea nitrogen (BUN) 3 hours after the last dose (n = 10 mice per group, mean ± sem; two-tailed unpaired t-test, **p<0.01, *p<0.01, ns = non-significant). I. Representative images and quantitation of leukemic disease burden by bioluminescent imaging of luciferase expressing MOLM-13 cells. (n = 8 mice/group, mean ± sem; two-tailed unpaired t-test, *** p < 0.001). J, K . Flow cytometry-based quantitation of MOLM-13 cells (human CD45 positive) in the mouse bone marrow ( J ) and peripheral blood ( K ) (n = 8 mice/group, mean ± sem; two-tailed unpaired t-test, ****p<0.0001). L . Body weight changes at the end of MM927 treatment in mice xenografted with MOLM-13 (n = 8 mice/group, mean ± sem; two-tailed unpaired t-test, ns = non-significant).

Article Snippet: The following primary antibodies were used: anti-NVL (Bethyl Laboratories #A304-863A); anti-eL36 (Proteintech #15145-1-AP); anti-eS17 (Abcam #ab128671); anti-p53 (Cell Signaling #9282); anti-p21 (Cell Signaling #2947); anti-Fibrillarin (Cell Signaling #2639); anti caspase-3 (D3R6Y) (Cell Signaling #14220); anti-FLAG m2 peroxidase HRP conjugated (Cell Signaling #a8592); anti-MDM2 (Cell Signaling #86934); anti-SNAP (NEB #P9310S); anti-alpha tubulin HRP conjugated (Cell Signaling #9099); anti-beta actin HRP conjugated (Santa Cruz Biotechnology #47778).

Techniques: In Vivo, Clinical Proteomics, Injection, Microscopy, Quantitation Assay, Immunofluorescence, CRISPR, Two Tailed Test, Imaging, Luciferase, Expressing, Flow Cytometry

Journal: bioRxiv

Article Title: A small molecule inhibitor of NVL suppresses tumor growth by blocking ribosome biogenesis

doi: 10.1101/2025.07.31.667081

Figure Lengend Snippet: A. Polysome profiling of HCT116 NVL WT ( A ) and NVL R403W CRISPR ( B ) cells treated with 0.5 µM MM927 for 24 hours. B, C. Representative microscopy images and quantitation of existing (green) and newly synthesized (magenta) eL36-SNAP in NVL WT ( B ) and NVL R403W CRISPR ( C ) cells treated with 0.5 µM MM927 for 24 hours. (Scale bar = 10 µm; n = 3 biological replicates, mean ± sem; two-tailed unpaired t-test, **p<0.01, ns = non-significant). Data from the vehicle group is re-used from these are done in the same experiment. D. Representative microscopy images and quantitation of existing (green) and newly synthesized (magenta) eS17-SNAP in HCT116 cells treated with 0.5 µM MM927 for 24 hours. (Scale bar = 10 µm; n = 3 biological replicates, mean ± sem; two-tailed unpaired t-test, **p<0.01, ns = non-significant). Data from the vehicle group is re-used from , as these are done in the same experiment. E. Flow cytometry-based analysis of cell cycle profile in HCT116 mock and p53 KO cells treated with 0.5 µM MM927. (n = 3 biological replicates, mean ± sem; two-tailed unpaired t-test, ***p<0.001, **p<0.01, *p<0.05, ns = non-significant). F. Viability assay of HCT116 NVL R403W CRISPR cells treated with MM927 for 72 hours. (N = 3 biological replicates, mean ± sem).

Article Snippet: The following primary antibodies were used: anti-NVL (Bethyl Laboratories #A304-863A); anti-eL36 (Proteintech #15145-1-AP); anti-eS17 (Abcam #ab128671); anti-p53 (Cell Signaling #9282); anti-p21 (Cell Signaling #2947); anti-Fibrillarin (Cell Signaling #2639); anti caspase-3 (D3R6Y) (Cell Signaling #14220); anti-FLAG m2 peroxidase HRP conjugated (Cell Signaling #a8592); anti-MDM2 (Cell Signaling #86934); anti-SNAP (NEB #P9310S); anti-alpha tubulin HRP conjugated (Cell Signaling #9099); anti-beta actin HRP conjugated (Santa Cruz Biotechnology #47778).

Techniques: CRISPR, Microscopy, Quantitation Assay, Synthesized, Two Tailed Test, Flow Cytometry, Viability Assay

Journal: bioRxiv

Article Title: A small molecule inhibitor of NVL suppresses tumor growth by blocking ribosome biogenesis

doi: 10.1101/2025.07.31.667081

Figure Lengend Snippet: MM927 studies in HCT116 tumor xenografts. A. Immunoblots of Flag (NVL) and tubulin (loading control) in HCT116 NVL AID/AID cells ectopically expressing empty vector, human NVL, or mouse NVL treated with Ph-IAA for 6 hours. B. Viability assay of cell lines in (A) treated with 0.03 µM Ph-IAA for 72 hours (n = 2 biological replicates, mean ± sem). C. Viability assay of HCT116 NVL AID/AID cells ectopically expressing human NVL or mouse NVL in treated with MM927 and 0.03 µM Ph-IAA for 72 hours (n = 2 biological replicates; mean ± sem). D, E. Immunoblots ( D ) and quantitation ( E ) of p53, p21, and tubulin (loading control) of HCT116 NVL WT and NVL R403W CRISPR tumor xenografts implanted in either flank of the same mouse treated with one 35 mg/kg MM927 IP injection. Right two lanes are in vitro samples of HCT116 cells treated with 10 µM MM17 for 24 hours. (n=2 to 3 biological replicates, mean ± sem). F. Individual tumor volume measurements in . Each line represents a mouse. G. Pharmacokinetic quantitation of MM927 3 hours after the last dose in the mouse plasma and tumors (n = 10 mice/group, mean ± sem).

Article Snippet: The following primary antibodies were used: anti-NVL (Bethyl Laboratories #A304-863A); anti-eL36 (Proteintech #15145-1-AP); anti-eS17 (Abcam #ab128671); anti-p53 (Cell Signaling #9282); anti-p21 (Cell Signaling #2947); anti-Fibrillarin (Cell Signaling #2639); anti caspase-3 (D3R6Y) (Cell Signaling #14220); anti-FLAG m2 peroxidase HRP conjugated (Cell Signaling #a8592); anti-MDM2 (Cell Signaling #86934); anti-SNAP (NEB #P9310S); anti-alpha tubulin HRP conjugated (Cell Signaling #9099); anti-beta actin HRP conjugated (Santa Cruz Biotechnology #47778).

Techniques: Western Blot, Control, Expressing, Plasmid Preparation, Viability Assay, Quantitation Assay, CRISPR, Injection, In Vitro, Clinical Proteomics

Journal: bioRxiv

Article Title: A small molecule inhibitor of NVL suppresses tumor growth by blocking ribosome biogenesis

doi: 10.1101/2025.07.31.667081

Figure Lengend Snippet: A. Immunoblots of p53 and actin (loading control) in MOLM-13 mock engineered or p53 knockout (KO) cells. B. Viability assay of cell lines in (A) treated with MM17 for 72 hours. (n = 3 biological replicates, mean ± sem). C. Immunoblots and quantitation of caspase-3 and actin (loading control) in MOLM-13 mock and p53 KO cells treated with 1 µM MLN4924 or 3 µM MM17. (n = 3 biological replicates, mean ± sem). D. Flow cytometry-based apoptosis analysis and quantitation in MOLM-13 mock and p53 KO cells treated with 1 µM MLN4924 or 3 µM MM17. (n = 3 biological replicates, mean ± sem). E. Schematic illustrating the proposed mechanism of cellular response to MM17. F. Immunoblots of NVL and actin (loading control) in MOLM-13 cells expressing empty vector, NVL WT, or NVL R403W. G. Growth curves of cell lines in (F). n = 3 biological replicates, mean ± sem). H. Viability assay of cell lines in (F) treated with MM927 for 72 hours. (n = 2 biological replicates, mean ± sem).

Article Snippet: The following primary antibodies were used: anti-NVL (Bethyl Laboratories #A304-863A); anti-eL36 (Proteintech #15145-1-AP); anti-eS17 (Abcam #ab128671); anti-p53 (Cell Signaling #9282); anti-p21 (Cell Signaling #2947); anti-Fibrillarin (Cell Signaling #2639); anti caspase-3 (D3R6Y) (Cell Signaling #14220); anti-FLAG m2 peroxidase HRP conjugated (Cell Signaling #a8592); anti-MDM2 (Cell Signaling #86934); anti-SNAP (NEB #P9310S); anti-alpha tubulin HRP conjugated (Cell Signaling #9099); anti-beta actin HRP conjugated (Santa Cruz Biotechnology #47778).

Techniques: Western Blot, Control, Knock-Out, Viability Assay, Quantitation Assay, Flow Cytometry, Expressing, Plasmid Preparation

Journal: bioRxiv

Article Title: An interferon-independent innate immune response to double stranded RNA in embryonic stem cells

doi: 10.64898/2026.03.24.713854

Figure Lengend Snippet: ( A ) Volcano plots of differentially expressed genes in control and dsRNA transfected mESCs. Dashed lines indicate fold change (log 2 FC > 1.0) and p value cutoffs (p < 0.05). WT, wild type. ( B ) GO enrichment analyses of the dsRNA induced top 100 genes identified in (A). WT, wild type. ( C ) Heatmap of the expression of the dsRNA induced top 100 genes in WT mESCs in control or dsRNA transfected Prkra KO, Dhx9 KO, p53 KO, and Stat1 KO mESCs (n = 3 for each group). FC, fold change; WT, wild type; KO, knockout; Ctrl, control. ( D ) RT-qPCR results showing expressional fold changes of the indicated ISG genes ( Isg15 , Cxcl10 , Oasl1 , and Ifih1 ) and p53 target genes ( Pmaip1 and Trp53inp1 ) in dsRNA transfected vs control mESCs of WT, Prkra KO, Dhx9 KO, and p53 KO. Ordinary one-way ANOVA with multiple comparisons. n.s., not significant. ****, p < 0.0001. Ctrl, control; WT, wild type; KO, knockout.

Article Snippet: The following antibodies were used for WB or IP analysis: anti-Tubulin (1:5000 for WB, 66031-1-Ig, Proteintech), anti-Actin (1:5000 for WB, 66009-1-Ig, Proteintech), anti-p53 (1:5000 for WB and 2 μg for each IP, 10442-1-AP, Proteintech), anti-p53 (1:1000 for WB, ab77813, Abcam), anti-Puromycin (1:1000 for WB, MAB3343, Sigma-Aldrich), anti-Stat1 (1:1000 for WB and 2 μg for each IP, 14994S, Cell Signaling Technology), anti-p701-Stat1 (1:1000 for WB, 9167S, Cell Signaling Technology), anti-Dhx9 (1:5000 for WB and 2 μg for each IP, 17721-1-AP, Proteintech), anti-Mdm2 (1:1000 for WB, 66511-1-Ig, Proteintech), anti-Ubiquitin (1:1000 for WB, sc-8017, Santa Cruz Biotechnology), anti-Cul4A (1:1000 for WB, 14851-1-AP, Proteintech), anti-Ddb1 (1:1000 for WB, PA5-21282, Invitrogen), anti-Ddb2 (1:1000 for WB, ab77765, Abcam), anti-Rbx1 (1:1000 for WB, 14895-1-AP, Proteintech), anti-Gapdh (1:5000 for WB, 60004-1-Ig, Proteintech), anti-J2 (2 μg for each IP, 76651L, Cell Signaling Technology), anti-Tcf4 (1:1000 for WB, 05-511, Millipore), mouse control IgG (2 μg for each IP, B900620, Proteintech), and rabbit control IgG (2 μg for each IP, B900610, Proteintech); HRP conjugated anti-mouse (1:5000 for WB, 31430, Invitrogen) or rabbit (1:5000 for WB, 31460, Invitrogen) IgG (H+L) was used as the secondary antibody for WB analysis.

Techniques: Control, Transfection, Expressing, Knock-Out, Quantitative RT-PCR

Journal: bioRxiv

Article Title: An interferon-independent innate immune response to double stranded RNA in embryonic stem cells

doi: 10.64898/2026.03.24.713854

Figure Lengend Snippet: (A) RT-qPCR results showing relative expressions of the indicated ISGs ( Isg15 , Cxcl10 , Ifih1 , and Oasl1 ) in mESCs with 0, 50, 100, and 200 ng/well (24-well plate) dsRNA transfection. Ordinary one-way ANOVA with multiple comparisons. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. (B) Western blotting results showing the translational inhibition by CHX treatment and RT-qPCR results showing relative expressions of the indicated ISGs ( Isg15 , Cxcl10 , Ifih1 , and Oasl1 ) and p53 target genes ( Pmaip1 and Trp53inp1 ) in control and dsRNA transfected mESCs with CHX treatment or not. Ctrl, control; CHX, cycloheximide; IB, immunoblotting; Ordinary one-way ANOVA with multiple comparisons. *, p < 0.05; **, p < 0.01; ****, p < 0.0001.

Article Snippet: The following antibodies were used for WB or IP analysis: anti-Tubulin (1:5000 for WB, 66031-1-Ig, Proteintech), anti-Actin (1:5000 for WB, 66009-1-Ig, Proteintech), anti-p53 (1:5000 for WB and 2 μg for each IP, 10442-1-AP, Proteintech), anti-p53 (1:1000 for WB, ab77813, Abcam), anti-Puromycin (1:1000 for WB, MAB3343, Sigma-Aldrich), anti-Stat1 (1:1000 for WB and 2 μg for each IP, 14994S, Cell Signaling Technology), anti-p701-Stat1 (1:1000 for WB, 9167S, Cell Signaling Technology), anti-Dhx9 (1:5000 for WB and 2 μg for each IP, 17721-1-AP, Proteintech), anti-Mdm2 (1:1000 for WB, 66511-1-Ig, Proteintech), anti-Ubiquitin (1:1000 for WB, sc-8017, Santa Cruz Biotechnology), anti-Cul4A (1:1000 for WB, 14851-1-AP, Proteintech), anti-Ddb1 (1:1000 for WB, PA5-21282, Invitrogen), anti-Ddb2 (1:1000 for WB, ab77765, Abcam), anti-Rbx1 (1:1000 for WB, 14895-1-AP, Proteintech), anti-Gapdh (1:5000 for WB, 60004-1-Ig, Proteintech), anti-J2 (2 μg for each IP, 76651L, Cell Signaling Technology), anti-Tcf4 (1:1000 for WB, 05-511, Millipore), mouse control IgG (2 μg for each IP, B900620, Proteintech), and rabbit control IgG (2 μg for each IP, B900610, Proteintech); HRP conjugated anti-mouse (1:5000 for WB, 31430, Invitrogen) or rabbit (1:5000 for WB, 31460, Invitrogen) IgG (H+L) was used as the secondary antibody for WB analysis.

Techniques: Quantitative RT-PCR, Transfection, Western Blot, Inhibition, Control

Journal: bioRxiv

Article Title: An interferon-independent innate immune response to double stranded RNA in embryonic stem cells

doi: 10.64898/2026.03.24.713854

Figure Lengend Snippet: (A) Schematic representation of gRNA mediated gene knockout of Dhx9 , Stat1 , p53, and Mavs genes in mESCs. WT, wild type; KO, knockout. (B) RT-qPCR results showing expressional fold changes of the indicated p53 target genes ( Pmaip1 and Trp53inp1 ) in dsRNA transfected vs control mESCs of WT or Dhx9 KO with overexpression of the indicated Dhx9 truncates shown in . Ordinary one-way ANOVA with multiple comparisons. ****, p < 0.0001. WT, wild type; KO, knockout; T, truncate; Ctrl, control.

Article Snippet: The following antibodies were used for WB or IP analysis: anti-Tubulin (1:5000 for WB, 66031-1-Ig, Proteintech), anti-Actin (1:5000 for WB, 66009-1-Ig, Proteintech), anti-p53 (1:5000 for WB and 2 μg for each IP, 10442-1-AP, Proteintech), anti-p53 (1:1000 for WB, ab77813, Abcam), anti-Puromycin (1:1000 for WB, MAB3343, Sigma-Aldrich), anti-Stat1 (1:1000 for WB and 2 μg for each IP, 14994S, Cell Signaling Technology), anti-p701-Stat1 (1:1000 for WB, 9167S, Cell Signaling Technology), anti-Dhx9 (1:5000 for WB and 2 μg for each IP, 17721-1-AP, Proteintech), anti-Mdm2 (1:1000 for WB, 66511-1-Ig, Proteintech), anti-Ubiquitin (1:1000 for WB, sc-8017, Santa Cruz Biotechnology), anti-Cul4A (1:1000 for WB, 14851-1-AP, Proteintech), anti-Ddb1 (1:1000 for WB, PA5-21282, Invitrogen), anti-Ddb2 (1:1000 for WB, ab77765, Abcam), anti-Rbx1 (1:1000 for WB, 14895-1-AP, Proteintech), anti-Gapdh (1:5000 for WB, 60004-1-Ig, Proteintech), anti-J2 (2 μg for each IP, 76651L, Cell Signaling Technology), anti-Tcf4 (1:1000 for WB, 05-511, Millipore), mouse control IgG (2 μg for each IP, B900620, Proteintech), and rabbit control IgG (2 μg for each IP, B900610, Proteintech); HRP conjugated anti-mouse (1:5000 for WB, 31430, Invitrogen) or rabbit (1:5000 for WB, 31460, Invitrogen) IgG (H+L) was used as the secondary antibody for WB analysis.

Techniques: Gene Knockout, Knock-Out, Quantitative RT-PCR, Transfection, Control, Over Expression

Journal: bioRxiv

Article Title: An interferon-independent innate immune response to double stranded RNA in embryonic stem cells

doi: 10.64898/2026.03.24.713854

Figure Lengend Snippet: ( A ) Immunoblotting images showing the expression of indicated protein in control or dsRNA transfected WT mESCs. IB, immunoblotting. ( B , C ) Co-IP assays showing the interactions of Dhx9-Mdm2 (B) or Mdm2-p53 (C) in control or dsRNA transfected WT mESCs. WCL, whole cell lysate; IP, immunoprecipitation; IB, immunoblotting. ( D , E ) In vivo ubiquitination analysis of p53 (D) or Stat1 (E) in control or dsRNA transfected WT mESCs. WCL, whole cell lysate; IP, immunoprecipitation; IB, immunoblotting. ( F , G ) In vivo ubiquitination analysis of Stat1 in control or dsRNA transfected mESCs of WT, Dhx9 KO (F), or p53 KO (G). WCL, whole cell lysate; IP, immunoprecipitation; IB, immunoblotting; WT, wild type; KO, knockout. ( H ) Immunoblotting images showing the expression of indicated protein in MG132 or Lactacystin treated WT mESCs. Ctrl, control; IB, immunoblotting. ( I ) Co-IP assays showing the interactions between Stat1 or p53 and Mdm2 or CRL ubiquitin ligase machinery. IP, immunoprecipitation; IB, immunoblotting. ( J ) Immunoblotting images showing the expression of indicated protein in Nutlin (Mdm2 inhibitor, iMdm2) or KH-4-43 (Cul4A inhibitor, iCul4A) treated WT mESCs. IB, immunoblotting. ( K ) Cut&Tag peaks (visualized by IGV) of Dhx9, p53, and Stat1 for the selected ISG genes ( Isg15 and Oasl1 ) or p53 target gene ( Pmaip1 ) in control or dsRNA transfected WT mESCs. Ctrl, control.

Article Snippet: The following antibodies were used for WB or IP analysis: anti-Tubulin (1:5000 for WB, 66031-1-Ig, Proteintech), anti-Actin (1:5000 for WB, 66009-1-Ig, Proteintech), anti-p53 (1:5000 for WB and 2 μg for each IP, 10442-1-AP, Proteintech), anti-p53 (1:1000 for WB, ab77813, Abcam), anti-Puromycin (1:1000 for WB, MAB3343, Sigma-Aldrich), anti-Stat1 (1:1000 for WB and 2 μg for each IP, 14994S, Cell Signaling Technology), anti-p701-Stat1 (1:1000 for WB, 9167S, Cell Signaling Technology), anti-Dhx9 (1:5000 for WB and 2 μg for each IP, 17721-1-AP, Proteintech), anti-Mdm2 (1:1000 for WB, 66511-1-Ig, Proteintech), anti-Ubiquitin (1:1000 for WB, sc-8017, Santa Cruz Biotechnology), anti-Cul4A (1:1000 for WB, 14851-1-AP, Proteintech), anti-Ddb1 (1:1000 for WB, PA5-21282, Invitrogen), anti-Ddb2 (1:1000 for WB, ab77765, Abcam), anti-Rbx1 (1:1000 for WB, 14895-1-AP, Proteintech), anti-Gapdh (1:5000 for WB, 60004-1-Ig, Proteintech), anti-J2 (2 μg for each IP, 76651L, Cell Signaling Technology), anti-Tcf4 (1:1000 for WB, 05-511, Millipore), mouse control IgG (2 μg for each IP, B900620, Proteintech), and rabbit control IgG (2 μg for each IP, B900610, Proteintech); HRP conjugated anti-mouse (1:5000 for WB, 31430, Invitrogen) or rabbit (1:5000 for WB, 31460, Invitrogen) IgG (H+L) was used as the secondary antibody for WB analysis.

Techniques: Western Blot, Expressing, Control, Transfection, Co-Immunoprecipitation Assay, Immunoprecipitation, In Vivo, Ubiquitin Proteomics, Knock-Out

Journal: bioRxiv

Article Title: An interferon-independent innate immune response to double stranded RNA in embryonic stem cells

doi: 10.64898/2026.03.24.713854

Figure Lengend Snippet: (A, B) RT-qPCR results showing relative expression levels of ISG15 in Nutlin (Mdm2 inhibitor, iMdm2) (A) or KH-4-43 (Cul4A inhibitor, iCul4A) (B) treated mESCs. Ctrl, control; Student’s t -test. ****, p < 0.0001. (C) Cut&Tag peaks (visualized by IGV) of Dhx9, p53, and Stat1 for the selected ISG genes ( Irf7 and Tnfaip3 ) or p53 target genes ( Trp53inp1 and Bbc3 ) in control or dsRNA transfected WT mESCs. Ctrl, control.

Article Snippet: The following antibodies were used for WB or IP analysis: anti-Tubulin (1:5000 for WB, 66031-1-Ig, Proteintech), anti-Actin (1:5000 for WB, 66009-1-Ig, Proteintech), anti-p53 (1:5000 for WB and 2 μg for each IP, 10442-1-AP, Proteintech), anti-p53 (1:1000 for WB, ab77813, Abcam), anti-Puromycin (1:1000 for WB, MAB3343, Sigma-Aldrich), anti-Stat1 (1:1000 for WB and 2 μg for each IP, 14994S, Cell Signaling Technology), anti-p701-Stat1 (1:1000 for WB, 9167S, Cell Signaling Technology), anti-Dhx9 (1:5000 for WB and 2 μg for each IP, 17721-1-AP, Proteintech), anti-Mdm2 (1:1000 for WB, 66511-1-Ig, Proteintech), anti-Ubiquitin (1:1000 for WB, sc-8017, Santa Cruz Biotechnology), anti-Cul4A (1:1000 for WB, 14851-1-AP, Proteintech), anti-Ddb1 (1:1000 for WB, PA5-21282, Invitrogen), anti-Ddb2 (1:1000 for WB, ab77765, Abcam), anti-Rbx1 (1:1000 for WB, 14895-1-AP, Proteintech), anti-Gapdh (1:5000 for WB, 60004-1-Ig, Proteintech), anti-J2 (2 μg for each IP, 76651L, Cell Signaling Technology), anti-Tcf4 (1:1000 for WB, 05-511, Millipore), mouse control IgG (2 μg for each IP, B900620, Proteintech), and rabbit control IgG (2 μg for each IP, B900610, Proteintech); HRP conjugated anti-mouse (1:5000 for WB, 31430, Invitrogen) or rabbit (1:5000 for WB, 31460, Invitrogen) IgG (H+L) was used as the secondary antibody for WB analysis.

Techniques: Quantitative RT-PCR, Expressing, Control, Transfection

Journal: bioRxiv

Article Title: An interferon-independent innate immune response to double stranded RNA in embryonic stem cells

doi: 10.64898/2026.03.24.713854

Figure Lengend Snippet: ( A-D ) Representative immunofluorescence images showing the co-staining of dsRNA (J2) and Dhx9 (A), Stat1 (B), p53 (C), or Ddb1 (D) in WT mESCs. WT, wild type. ( E-H ) Representative immunofluorescence images showing the dsRNA foci (E) and their co-staining with Stat1 (F), p53 (G), or Ddb1 (H) in Dhx9 KO mESCs. KO, knockout. ( I , J ) A model for the Dhx9-dsRNA mediated p53, Stat1 stabilization and ISG activation. In wild type mESCs, p53 and Stat1 are ubiquitinated and thus degradation by Mdm2/Cul4A containing E3 ligase complex (I). Dhx9 senses dsRNA to recruit the p53/Stat1 ubiquitination complex into the condensates while excluding their adaptor Ddb1, leading to release and stabilization of p53 and Stat1 (J).

Article Snippet: The following antibodies were used for WB or IP analysis: anti-Tubulin (1:5000 for WB, 66031-1-Ig, Proteintech), anti-Actin (1:5000 for WB, 66009-1-Ig, Proteintech), anti-p53 (1:5000 for WB and 2 μg for each IP, 10442-1-AP, Proteintech), anti-p53 (1:1000 for WB, ab77813, Abcam), anti-Puromycin (1:1000 for WB, MAB3343, Sigma-Aldrich), anti-Stat1 (1:1000 for WB and 2 μg for each IP, 14994S, Cell Signaling Technology), anti-p701-Stat1 (1:1000 for WB, 9167S, Cell Signaling Technology), anti-Dhx9 (1:5000 for WB and 2 μg for each IP, 17721-1-AP, Proteintech), anti-Mdm2 (1:1000 for WB, 66511-1-Ig, Proteintech), anti-Ubiquitin (1:1000 for WB, sc-8017, Santa Cruz Biotechnology), anti-Cul4A (1:1000 for WB, 14851-1-AP, Proteintech), anti-Ddb1 (1:1000 for WB, PA5-21282, Invitrogen), anti-Ddb2 (1:1000 for WB, ab77765, Abcam), anti-Rbx1 (1:1000 for WB, 14895-1-AP, Proteintech), anti-Gapdh (1:5000 for WB, 60004-1-Ig, Proteintech), anti-J2 (2 μg for each IP, 76651L, Cell Signaling Technology), anti-Tcf4 (1:1000 for WB, 05-511, Millipore), mouse control IgG (2 μg for each IP, B900620, Proteintech), and rabbit control IgG (2 μg for each IP, B900610, Proteintech); HRP conjugated anti-mouse (1:5000 for WB, 31430, Invitrogen) or rabbit (1:5000 for WB, 31460, Invitrogen) IgG (H+L) was used as the secondary antibody for WB analysis.

Techniques: Immunofluorescence, Staining, Knock-Out, Activation Assay, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: An interferon-independent innate immune response to double stranded RNA in embryonic stem cells

doi: 10.64898/2026.03.24.713854

Figure Lengend Snippet: (A) Immunoblotting images showing the subcellular expressions of Dhx9 protein in control or dsRNA transfected mESCs. IB, immunoblotting. (B-H) Representative immunofluorescence images showing the co-staining of dsRNA (J2) and Cul4A (B, D, and G), Mdm2 (C, E, and H), or Ddb1 (F) in WT, Dhx9 KO, or p53 KO mESCs. WT, wild type; KO, knockout. Scale bar, 5 μm.

Article Snippet: The following antibodies were used for WB or IP analysis: anti-Tubulin (1:5000 for WB, 66031-1-Ig, Proteintech), anti-Actin (1:5000 for WB, 66009-1-Ig, Proteintech), anti-p53 (1:5000 for WB and 2 μg for each IP, 10442-1-AP, Proteintech), anti-p53 (1:1000 for WB, ab77813, Abcam), anti-Puromycin (1:1000 for WB, MAB3343, Sigma-Aldrich), anti-Stat1 (1:1000 for WB and 2 μg for each IP, 14994S, Cell Signaling Technology), anti-p701-Stat1 (1:1000 for WB, 9167S, Cell Signaling Technology), anti-Dhx9 (1:5000 for WB and 2 μg for each IP, 17721-1-AP, Proteintech), anti-Mdm2 (1:1000 for WB, 66511-1-Ig, Proteintech), anti-Ubiquitin (1:1000 for WB, sc-8017, Santa Cruz Biotechnology), anti-Cul4A (1:1000 for WB, 14851-1-AP, Proteintech), anti-Ddb1 (1:1000 for WB, PA5-21282, Invitrogen), anti-Ddb2 (1:1000 for WB, ab77765, Abcam), anti-Rbx1 (1:1000 for WB, 14895-1-AP, Proteintech), anti-Gapdh (1:5000 for WB, 60004-1-Ig, Proteintech), anti-J2 (2 μg for each IP, 76651L, Cell Signaling Technology), anti-Tcf4 (1:1000 for WB, 05-511, Millipore), mouse control IgG (2 μg for each IP, B900620, Proteintech), and rabbit control IgG (2 μg for each IP, B900610, Proteintech); HRP conjugated anti-mouse (1:5000 for WB, 31430, Invitrogen) or rabbit (1:5000 for WB, 31460, Invitrogen) IgG (H+L) was used as the secondary antibody for WB analysis.

Techniques: Western Blot, Control, Transfection, Immunofluorescence, Staining, Knock-Out

Journal: bioRxiv

Article Title: An interferon-independent innate immune response to double stranded RNA in embryonic stem cells

doi: 10.64898/2026.03.24.713854

Figure Lengend Snippet: (A) dsRNA pull down assays showing the protein components of dsRNA positive foci in WT and Dhx9 KO mESCs. WCL, whole cell lysate; IP, immunoprecipitation; IB, immunoblotting. (B) Co-IP assays showing the interactions between Ddb1 and Cul4A in control or dsRNA transfected mESCs of WT, Dhx9 KO, and p53 KO. WCL, whole cell lysate; IP, immunoprecipitation; IB, immunoblotting.

Article Snippet: The following antibodies were used for WB or IP analysis: anti-Tubulin (1:5000 for WB, 66031-1-Ig, Proteintech), anti-Actin (1:5000 for WB, 66009-1-Ig, Proteintech), anti-p53 (1:5000 for WB and 2 μg for each IP, 10442-1-AP, Proteintech), anti-p53 (1:1000 for WB, ab77813, Abcam), anti-Puromycin (1:1000 for WB, MAB3343, Sigma-Aldrich), anti-Stat1 (1:1000 for WB and 2 μg for each IP, 14994S, Cell Signaling Technology), anti-p701-Stat1 (1:1000 for WB, 9167S, Cell Signaling Technology), anti-Dhx9 (1:5000 for WB and 2 μg for each IP, 17721-1-AP, Proteintech), anti-Mdm2 (1:1000 for WB, 66511-1-Ig, Proteintech), anti-Ubiquitin (1:1000 for WB, sc-8017, Santa Cruz Biotechnology), anti-Cul4A (1:1000 for WB, 14851-1-AP, Proteintech), anti-Ddb1 (1:1000 for WB, PA5-21282, Invitrogen), anti-Ddb2 (1:1000 for WB, ab77765, Abcam), anti-Rbx1 (1:1000 for WB, 14895-1-AP, Proteintech), anti-Gapdh (1:5000 for WB, 60004-1-Ig, Proteintech), anti-J2 (2 μg for each IP, 76651L, Cell Signaling Technology), anti-Tcf4 (1:1000 for WB, 05-511, Millipore), mouse control IgG (2 μg for each IP, B900620, Proteintech), and rabbit control IgG (2 μg for each IP, B900610, Proteintech); HRP conjugated anti-mouse (1:5000 for WB, 31430, Invitrogen) or rabbit (1:5000 for WB, 31460, Invitrogen) IgG (H+L) was used as the secondary antibody for WB analysis.

Techniques: Immunoprecipitation, Western Blot, Co-Immunoprecipitation Assay, Control, Transfection

Journal: bioRxiv

Article Title: An interferon-independent innate immune response to double stranded RNA in embryonic stem cells

doi: 10.64898/2026.03.24.713854

Figure Lengend Snippet: ( A ) Representative immunofluorescence images showing dsRNA (J2) and ZIKV envelope protein (ZIKV-E) in WT, Mavs KO and Dhx9 KO mESCs at 48 hpi. hpi, hours post infection; WT, wild type; KO, knockout. Scale bar, 5 μm. ( B ) RT-qPCR results showing the relative expression level of ISGs ( Isg15 , Cxcl10, Oasl1, and Ifit8 ) and p53 targets ( Pmaip1 and Trp53inp1 ) in WT or Dhx9 KO mESCs infected by ZIKV at MOI of 0.5 and 1.0 at 48 hpi. Ordinary one-way ANOVA with multiple comparisons. n.s., not significant. *, p < 0.05; ***, p < 0.001; ****, p < 0.0001. WT, wild type; KO, knockout. ( C ) RT-qPCR results showing the relative ZIKV mRNA level in WT, Dhx9 KO, and Mavs KO mESCs at the indicated time point after infection. hpi, hours post infection; WT, wild type; KO, knockout. Students’ t test. *, p < 0.05; **, p < 0.01. Significant differences between WT and Dhx9 KO were labeled in red, between WT and Mavs KO in blue, and between Dhx9 KO and Mavs KO in green. ( D ) Western blotting results showing the expression level of ZIKV envelope protein in WT, Mavs KO, and Dhx9 KO mESCs infected by ZIKV at MOI of 1.0 at 48 hpi. hpi, hours post infection; WT, wild type; KO, knockout.

Article Snippet: The following antibodies were used for WB or IP analysis: anti-Tubulin (1:5000 for WB, 66031-1-Ig, Proteintech), anti-Actin (1:5000 for WB, 66009-1-Ig, Proteintech), anti-p53 (1:5000 for WB and 2 μg for each IP, 10442-1-AP, Proteintech), anti-p53 (1:1000 for WB, ab77813, Abcam), anti-Puromycin (1:1000 for WB, MAB3343, Sigma-Aldrich), anti-Stat1 (1:1000 for WB and 2 μg for each IP, 14994S, Cell Signaling Technology), anti-p701-Stat1 (1:1000 for WB, 9167S, Cell Signaling Technology), anti-Dhx9 (1:5000 for WB and 2 μg for each IP, 17721-1-AP, Proteintech), anti-Mdm2 (1:1000 for WB, 66511-1-Ig, Proteintech), anti-Ubiquitin (1:1000 for WB, sc-8017, Santa Cruz Biotechnology), anti-Cul4A (1:1000 for WB, 14851-1-AP, Proteintech), anti-Ddb1 (1:1000 for WB, PA5-21282, Invitrogen), anti-Ddb2 (1:1000 for WB, ab77765, Abcam), anti-Rbx1 (1:1000 for WB, 14895-1-AP, Proteintech), anti-Gapdh (1:5000 for WB, 60004-1-Ig, Proteintech), anti-J2 (2 μg for each IP, 76651L, Cell Signaling Technology), anti-Tcf4 (1:1000 for WB, 05-511, Millipore), mouse control IgG (2 μg for each IP, B900620, Proteintech), and rabbit control IgG (2 μg for each IP, B900610, Proteintech); HRP conjugated anti-mouse (1:5000 for WB, 31430, Invitrogen) or rabbit (1:5000 for WB, 31460, Invitrogen) IgG (H+L) was used as the secondary antibody for WB analysis.

Techniques: Immunofluorescence, Infection, Knock-Out, Quantitative RT-PCR, Expressing, Labeling, Western Blot

Journal: bioRxiv

Article Title: An interferon-independent innate immune response to double stranded RNA in embryonic stem cells

doi: 10.64898/2026.03.24.713854

Figure Lengend Snippet: (A) RT-qPCR results showing the relative expression level of Pnpt1 and the indicated ISGs ( Isg15 , Cxcl10 , Oasl1 and Ifih1 ) and p53 target genes ( Pmaip1 and Trp53inp1 ) in WT or Dhx9 KO mESCs with Pnpt1 knockdown by two independent shRNAs. Ordinary one-way ANOVA with multiple comparisons. n.s., not significant; ****, p < 0.0001. WT, wild type; KO, knockout. (B) RT-qPCR results showing the relative expression level of the indicated ISGs ( Isg15 , Cxcl10 , Oasl1 and Ifih1 ) and p53 target genes ( Pmaip1 and Trp53inp1 ) in WT or Dhx9 KO mESCs treated with BAY1217389 at the concentration of 10 and 50 mM. Ordinary one-way ANOVA with multiple comparisons. n.s., not significant; ****, p < 0.0001. WT, wild type; KO, knockout.

Article Snippet: The following antibodies were used for WB or IP analysis: anti-Tubulin (1:5000 for WB, 66031-1-Ig, Proteintech), anti-Actin (1:5000 for WB, 66009-1-Ig, Proteintech), anti-p53 (1:5000 for WB and 2 μg for each IP, 10442-1-AP, Proteintech), anti-p53 (1:1000 for WB, ab77813, Abcam), anti-Puromycin (1:1000 for WB, MAB3343, Sigma-Aldrich), anti-Stat1 (1:1000 for WB and 2 μg for each IP, 14994S, Cell Signaling Technology), anti-p701-Stat1 (1:1000 for WB, 9167S, Cell Signaling Technology), anti-Dhx9 (1:5000 for WB and 2 μg for each IP, 17721-1-AP, Proteintech), anti-Mdm2 (1:1000 for WB, 66511-1-Ig, Proteintech), anti-Ubiquitin (1:1000 for WB, sc-8017, Santa Cruz Biotechnology), anti-Cul4A (1:1000 for WB, 14851-1-AP, Proteintech), anti-Ddb1 (1:1000 for WB, PA5-21282, Invitrogen), anti-Ddb2 (1:1000 for WB, ab77765, Abcam), anti-Rbx1 (1:1000 for WB, 14895-1-AP, Proteintech), anti-Gapdh (1:5000 for WB, 60004-1-Ig, Proteintech), anti-J2 (2 μg for each IP, 76651L, Cell Signaling Technology), anti-Tcf4 (1:1000 for WB, 05-511, Millipore), mouse control IgG (2 μg for each IP, B900620, Proteintech), and rabbit control IgG (2 μg for each IP, B900610, Proteintech); HRP conjugated anti-mouse (1:5000 for WB, 31430, Invitrogen) or rabbit (1:5000 for WB, 31460, Invitrogen) IgG (H+L) was used as the secondary antibody for WB analysis.

Techniques: Quantitative RT-PCR, Expressing, Knockdown, Knock-Out, Concentration Assay

Journal: bioRxiv

Article Title: An interferon-independent innate immune response to double stranded RNA in embryonic stem cells

doi: 10.64898/2026.03.24.713854

Figure Lengend Snippet: ( A ) Volcano plots of differentially expressed genes in control and dsRNA transfected hESCs. Dashed lines indicate fold change (log 2 FC > 1.0) and p value cutoffs (p < 0.05). ( B ) Heatmap of the expression of the dsRNA induced top 100 genes and p53 target genes in control (n = 3) or dsRNA (n = 3) transfected hESCs. FC, fold change. ( C ) GO enrichment analyses of the dsRNA induced top 100 genes identified in (A and B). ( D ) RT-qPCR results showing relative expression levels of the indicated ISGs ( ISG15 , CXCL10 , OASL1 , and IFIH1 ) and p53 target genes ( PMAIP1 and TRP53INP1 ) in control and dsRNA transfected hESCs. Student’s t -test. **, p < 0.01; ****, p < 0.0001. Ctrl, control. ( E ) Representative immunofluorescence images showing the co-staining of dsRNA (J2) and DHX9 in hESCs. ( F ) Immunoblotting images showing the expression of indicated proteins in Nutlin (MDM2 inhibitor, iMDM2) or KH-4-43 (CUL4A inhibitor, iCUL4A) treated hESCs. IB, immunoblotting. ( G ) RT-qPCR results showing relative expression levels of ISG15 in Nutlin (MDM2 inhibitor, iMDM2) or KH-4-43 (CUL4A inhibitor, iCUL4A) treated hESCs. Student’s t -test. ****, p < 0.0001. Ctrl, control.

Article Snippet: The following antibodies were used for WB or IP analysis: anti-Tubulin (1:5000 for WB, 66031-1-Ig, Proteintech), anti-Actin (1:5000 for WB, 66009-1-Ig, Proteintech), anti-p53 (1:5000 for WB and 2 μg for each IP, 10442-1-AP, Proteintech), anti-p53 (1:1000 for WB, ab77813, Abcam), anti-Puromycin (1:1000 for WB, MAB3343, Sigma-Aldrich), anti-Stat1 (1:1000 for WB and 2 μg for each IP, 14994S, Cell Signaling Technology), anti-p701-Stat1 (1:1000 for WB, 9167S, Cell Signaling Technology), anti-Dhx9 (1:5000 for WB and 2 μg for each IP, 17721-1-AP, Proteintech), anti-Mdm2 (1:1000 for WB, 66511-1-Ig, Proteintech), anti-Ubiquitin (1:1000 for WB, sc-8017, Santa Cruz Biotechnology), anti-Cul4A (1:1000 for WB, 14851-1-AP, Proteintech), anti-Ddb1 (1:1000 for WB, PA5-21282, Invitrogen), anti-Ddb2 (1:1000 for WB, ab77765, Abcam), anti-Rbx1 (1:1000 for WB, 14895-1-AP, Proteintech), anti-Gapdh (1:5000 for WB, 60004-1-Ig, Proteintech), anti-J2 (2 μg for each IP, 76651L, Cell Signaling Technology), anti-Tcf4 (1:1000 for WB, 05-511, Millipore), mouse control IgG (2 μg for each IP, B900620, Proteintech), and rabbit control IgG (2 μg for each IP, B900610, Proteintech); HRP conjugated anti-mouse (1:5000 for WB, 31430, Invitrogen) or rabbit (1:5000 for WB, 31460, Invitrogen) IgG (H+L) was used as the secondary antibody for WB analysis.

Techniques: Control, Transfection, Expressing, Quantitative RT-PCR, Immunofluorescence, Staining, Western Blot

Journal: bioRxiv

Article Title: An interferon-independent innate immune response to double stranded RNA in embryonic stem cells

doi: 10.64898/2026.03.24.713854

Figure Lengend Snippet: (A, B) Representative immunofluorescence images showing the co-staining of dsRNA (J2) and STAT1 (A) and TP53 (B) in hESCs. Scale bar, 5 μm. (C, D) RT-qPCR results showing relative expression levels of the indicated ISGs ( CXCL10 , IFIH1 , and OASL ) and TP53 target genes ( PMAIP1 and TRP53INP1 ) in Nutlin (MDM2 inhibitor, iMDM2) (C) or KH-4-43 (CUL4A inhibitor, iCUL4A) (D) treated hESCs. Student’s t -test. ****, p < 0.0001. Ctrl, control. (E) RT-qPCR results showing relative expression levels of the indicated ISGs ( Isg15 , Cxcl10 , Ifih1 , and Oasl1 ) and p53 target genes ( Pmaip1 and Trp53inp1 ) in N2a cells with sgRNAs against Mavs or Dhx9 transfection. Ordinary one-way ANOVA with multiple comparisons. ****, p < 0.0001.

Article Snippet: The following antibodies were used for WB or IP analysis: anti-Tubulin (1:5000 for WB, 66031-1-Ig, Proteintech), anti-Actin (1:5000 for WB, 66009-1-Ig, Proteintech), anti-p53 (1:5000 for WB and 2 μg for each IP, 10442-1-AP, Proteintech), anti-p53 (1:1000 for WB, ab77813, Abcam), anti-Puromycin (1:1000 for WB, MAB3343, Sigma-Aldrich), anti-Stat1 (1:1000 for WB and 2 μg for each IP, 14994S, Cell Signaling Technology), anti-p701-Stat1 (1:1000 for WB, 9167S, Cell Signaling Technology), anti-Dhx9 (1:5000 for WB and 2 μg for each IP, 17721-1-AP, Proteintech), anti-Mdm2 (1:1000 for WB, 66511-1-Ig, Proteintech), anti-Ubiquitin (1:1000 for WB, sc-8017, Santa Cruz Biotechnology), anti-Cul4A (1:1000 for WB, 14851-1-AP, Proteintech), anti-Ddb1 (1:1000 for WB, PA5-21282, Invitrogen), anti-Ddb2 (1:1000 for WB, ab77765, Abcam), anti-Rbx1 (1:1000 for WB, 14895-1-AP, Proteintech), anti-Gapdh (1:5000 for WB, 60004-1-Ig, Proteintech), anti-J2 (2 μg for each IP, 76651L, Cell Signaling Technology), anti-Tcf4 (1:1000 for WB, 05-511, Millipore), mouse control IgG (2 μg for each IP, B900620, Proteintech), and rabbit control IgG (2 μg for each IP, B900610, Proteintech); HRP conjugated anti-mouse (1:5000 for WB, 31430, Invitrogen) or rabbit (1:5000 for WB, 31460, Invitrogen) IgG (H+L) was used as the secondary antibody for WB analysis.

Techniques: Immunofluorescence, Staining, Quantitative RT-PCR, Expressing, Control, Transfection

Journal: bioRxiv

Article Title: An interferon-independent innate immune response to double stranded RNA in embryonic stem cells

doi: 10.64898/2026.03.24.713854

Figure Lengend Snippet: ( A ) Volcano plots of differentially expressed genes in control and dsRNA injected zebrafish embryos at 6 hpf. Dashed lines indicate fold change (log 2 FC > 1.0). Ctrl, control. hpf, hour post fertilization. ( B ) Heatmap of the expression of the dsRNA induced ISGs and other genes in control and dsRNA injected zebrafish embryos of WT or tp53 mutant at 6 hpf (n = 3 for each group). Known p53 target genes were labeled with asterisks (*). WT, wild type; MZ tp53 , maternal and zygotic tp53 mutant; Ctrl, control. ( C ) RT-qPCR results showing expressional fold changes of the indicated ISG genes ( isg15 , cxcl12b , casp8 , and ifit8 ) and p53 target genes ( phlda3 and cdkn1a ) in dsRNA injected vs control zebrafish embryos of WT, dhx9 KO, stat1a KO and tp53 mutant. Ordinary one-way ANOVA with multiple comparisons. n.s., not significant. ****, p < 0.0001. Ctrl, control; WT, wild type; MZ tp53 , maternal and zygotic tp53 mutant; MZ dhx9 , maternal and zygotic dhx9 knockout; MZ stat1a , maternal and zygotic stat1a knockout. ( D ) Immunoblotting images showing the expression level of p53 protein in control or dsRNA injected zebrafish embryos of WT and dhx9 KO at 6 hpf. IB, immunoblotting; Ctrl, control; WT, wild type; MZ dhx9 , maternal and zygotic dhx9 knockout. ( E ) Relative SVCV dose in WT and MZ tp53 zebrafish embryos at the indicated time points after SVCV injection. WT, wild type; MZ tp53 , maternal and zygotic tp53 mutant. Students’s t test. **, p < 0.01.

Article Snippet: The following antibodies were used for WB or IP analysis: anti-Tubulin (1:5000 for WB, 66031-1-Ig, Proteintech), anti-Actin (1:5000 for WB, 66009-1-Ig, Proteintech), anti-p53 (1:5000 for WB and 2 μg for each IP, 10442-1-AP, Proteintech), anti-p53 (1:1000 for WB, ab77813, Abcam), anti-Puromycin (1:1000 for WB, MAB3343, Sigma-Aldrich), anti-Stat1 (1:1000 for WB and 2 μg for each IP, 14994S, Cell Signaling Technology), anti-p701-Stat1 (1:1000 for WB, 9167S, Cell Signaling Technology), anti-Dhx9 (1:5000 for WB and 2 μg for each IP, 17721-1-AP, Proteintech), anti-Mdm2 (1:1000 for WB, 66511-1-Ig, Proteintech), anti-Ubiquitin (1:1000 for WB, sc-8017, Santa Cruz Biotechnology), anti-Cul4A (1:1000 for WB, 14851-1-AP, Proteintech), anti-Ddb1 (1:1000 for WB, PA5-21282, Invitrogen), anti-Ddb2 (1:1000 for WB, ab77765, Abcam), anti-Rbx1 (1:1000 for WB, 14895-1-AP, Proteintech), anti-Gapdh (1:5000 for WB, 60004-1-Ig, Proteintech), anti-J2 (2 μg for each IP, 76651L, Cell Signaling Technology), anti-Tcf4 (1:1000 for WB, 05-511, Millipore), mouse control IgG (2 μg for each IP, B900620, Proteintech), and rabbit control IgG (2 μg for each IP, B900610, Proteintech); HRP conjugated anti-mouse (1:5000 for WB, 31430, Invitrogen) or rabbit (1:5000 for WB, 31460, Invitrogen) IgG (H+L) was used as the secondary antibody for WB analysis.

Techniques: Control, Injection, Expressing, Mutagenesis, Labeling, Quantitative RT-PCR, Knock-Out, Western Blot

Journal: bioRxiv

Article Title: An interferon-independent innate immune response to double stranded RNA in embryonic stem cells

doi: 10.64898/2026.03.24.713854

Figure Lengend Snippet: (A) RT-qPCR results showing relative expression levels of the indicated IFN ligands ( ifng1, ifnphi2, ifnphi3, ifnphi4, and ifnphi5 ) in control or dsRNA injected zebrafish embryos at 6 hpf. Student’s t -test; n.s., not significant; *, p < 0.05. (B-D) Schematic image showing the experiment strategy (B). WMISH showing the expression of sox17 and isg15 in zebrafish embryos at 6 hpf of control, sqt mRNA/dsRNA injection, or sqt mRNA/dsRNA injection with CHX treatment (C). Scale bar, 200 μm. RT-qPCR results showing relative expression levels of the indicated ISGs ( isg15, casp8, cxcl12b, and ifit8 ) and tp53 target genes ( cdkn1a and pdlha3 ) in control or dsRNA injected zebrafish embryos with CHX treatment or not at 6 hpf. Ordinary one-way ANOVA with multiple comparisons. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. CHX, cycloheximide.

Article Snippet: The following antibodies were used for WB or IP analysis: anti-Tubulin (1:5000 for WB, 66031-1-Ig, Proteintech), anti-Actin (1:5000 for WB, 66009-1-Ig, Proteintech), anti-p53 (1:5000 for WB and 2 μg for each IP, 10442-1-AP, Proteintech), anti-p53 (1:1000 for WB, ab77813, Abcam), anti-Puromycin (1:1000 for WB, MAB3343, Sigma-Aldrich), anti-Stat1 (1:1000 for WB and 2 μg for each IP, 14994S, Cell Signaling Technology), anti-p701-Stat1 (1:1000 for WB, 9167S, Cell Signaling Technology), anti-Dhx9 (1:5000 for WB and 2 μg for each IP, 17721-1-AP, Proteintech), anti-Mdm2 (1:1000 for WB, 66511-1-Ig, Proteintech), anti-Ubiquitin (1:1000 for WB, sc-8017, Santa Cruz Biotechnology), anti-Cul4A (1:1000 for WB, 14851-1-AP, Proteintech), anti-Ddb1 (1:1000 for WB, PA5-21282, Invitrogen), anti-Ddb2 (1:1000 for WB, ab77765, Abcam), anti-Rbx1 (1:1000 for WB, 14895-1-AP, Proteintech), anti-Gapdh (1:5000 for WB, 60004-1-Ig, Proteintech), anti-J2 (2 μg for each IP, 76651L, Cell Signaling Technology), anti-Tcf4 (1:1000 for WB, 05-511, Millipore), mouse control IgG (2 μg for each IP, B900620, Proteintech), and rabbit control IgG (2 μg for each IP, B900610, Proteintech); HRP conjugated anti-mouse (1:5000 for WB, 31430, Invitrogen) or rabbit (1:5000 for WB, 31460, Invitrogen) IgG (H+L) was used as the secondary antibody for WB analysis.

Techniques: Quantitative RT-PCR, Expressing, Control, Injection