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: 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