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

Journal: Oncotarget

Article Title: Tumor suppressor p53 regulates insulin receptor ( INSR ) gene expression via direct binding to the INSR promoter

doi: 10.18632/oncotarget.27645

Figure Lengend Snippet: ( A ) Total (100 μg protein), cytoplasmic (40 μg) and nuclear (40 μg) fractions of IGF1R-KD, INSR-KD and control MCF7 cells were electrophoresed through SDS-PAGE gels, blotted onto nitrocellulose membranes and incubated with antibodies against total and phospho-p53. Lamin B was used as a nuclear marker. ( B ) Relative phosphorylation of p53 in total and subcellular fractions was calculated by normalizing phospho- to total p53 levels. Data represent two independent experiments (mean ± SEM; * p < 0.01 versus respective fraction in control cells). ( C ) Effect of IGF1R-KD or INSR-KD on p53 sumoylation. Total and subcellular fractions of IGF1R-KD, INSR-KD and control MCF7 cells were immunoprecipitated with anti-p53, electrophoresed and immunoblotted with a Sumo-1 antibody. IgG was used as a control for the co-IP experiment.

Article Snippet: Protein A-peroxidase conjugated was used for p53 detection (Rockland Immunochemicals Inc, Limerick, PA, USA).

Techniques: Control, SDS Page, Incubation, Marker, Phospho-proteomics, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: Oncotarget

Article Title: Tumor suppressor p53 regulates insulin receptor ( INSR ) gene expression via direct binding to the INSR promoter

doi: 10.18632/oncotarget.27645

Figure Lengend Snippet: IGF1R-KD, INSR-KD and control MCF7 cells were transiently co-transfected with the pGL3(-877/-2) LUC INSR promoter luciferase reporter along with a wild-type or mutant (MUT143, 248 or 273) p53-encoding expression vector. Cells were harvested after 48 h and luciferase activity was measured. The activity of the INSR promoter is expressed as luciferase values normalized to total protein. A value of 100% was assigned to the promoter activity generated by the INSR promoter construct in control cells. The inset presents the basal INSR promoter activity levels at a large scale. * p < 0.01 versus empty vector-transfected cells. Experiments were performed in triplicates.

Article Snippet: Protein A-peroxidase conjugated was used for p53 detection (Rockland Immunochemicals Inc, Limerick, PA, USA).

Techniques: Control, Transfection, Luciferase, Mutagenesis, Expressing, Plasmid Preparation, Activity Assay, Generated, Construct

Journal: Oncotarget

Article Title: Tumor suppressor p53 regulates insulin receptor ( INSR ) gene expression via direct binding to the INSR promoter

doi: 10.18632/oncotarget.27645

Figure Lengend Snippet: ( A ) IGF1R-KD, INSR-KD and control MCF7 cells were co-transfected with an INSR promoter luciferase reporter along with a wild-type p53 expression vector, as described in the legend to Figure 3. After 48 h cells were harvested and the expression of the endogenous INSR was assessed by Western blots. HSC70 was measured as a loading marker. Panels ( B ) and ( C ) show scanning densitometric analyses of INSR and p53 protein levels, respectively, normalized to HSC70. * p < 0.01 versus empty vector-transfected cells.

Article Snippet: Protein A-peroxidase conjugated was used for p53 detection (Rockland Immunochemicals Inc, Limerick, PA, USA).

Techniques: Control, Transfection, Luciferase, Expressing, Plasmid Preparation, Western Blot, Marker

Journal: Oncotarget

Article Title: Tumor suppressor p53 regulates insulin receptor ( INSR ) gene expression via direct binding to the INSR promoter

doi: 10.18632/oncotarget.27645

Figure Lengend Snippet: ( A ) IGF1R-KD, INSR-KD and control MCF7 cells were co-transfected with the pGL3(-877/-2) LUC INSR promoter luciferase reporter along with an INSR-A expression vector (INSR-GFP) (or empty pEGFP vector) and a wild-type p53 expression vector (or empty pCMV). After 48 h cells were harvested and INSR promoter activity was measured as described in the legend to . The left inset presents the basal INSR promoter activity levels at a large scale. Total, cytoplasmic and nuclear fractions of IGF1R-KD cells were immunoprecipitated with an INSR antibody, electrophoresed and immunoblotted with anti-INSR and anti-p53 (right inset). IgG was used as a control for the co-IP experiment. * p < 0.01 versus control cells. § p < 0.01 versus empty pCMV-transfected cells. ( B ) Cells were transfected with the INSR promoter reporter along with an IGF1R expression vector (or empty pEGFP) and a p53 expression vector (or empty pCMV). Cells were processed as described above. Total, cytoplasmic and nuclear fractions of INSR-KD cells were immunoprecipitated with an IGF1R antibody, electrophoresed and immunoblotted with anti-IGF1R and anti-p53 (inset).

Article Snippet: Protein A-peroxidase conjugated was used for p53 detection (Rockland Immunochemicals Inc, Limerick, PA, USA).

Techniques: Control, Transfection, Luciferase, Expressing, Plasmid Preparation, Activity Assay, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: Oncotarget

Article Title: Tumor suppressor p53 regulates insulin receptor ( INSR ) gene expression via direct binding to the INSR promoter

doi: 10.18632/oncotarget.27645

Figure Lengend Snippet: ( A ) A genomic fragment extending from nt -540 to -18 of the INS R promoter was labeled using a 5′-biotinylated antisense primer. The labeled fragment was bound to streptavidin magnetic beads, incubated with nuclear extracts of IGF1R-KD, INSR-KD or control cells, and eluted with a high-salt buffer. Western blots were performed on nuclear extracts (NE) and eluted material (E) using a p53 antibody. DNA affinity chromatography was also employed to assess the binding of endogenous INSR protein ( B ) and endogenous IGF1R protein ( C ) to the INSR promoter region.

Article Snippet: Protein A-peroxidase conjugated was used for p53 detection (Rockland Immunochemicals Inc, Limerick, PA, USA).

Techniques: Labeling, Magnetic Beads, Incubation, Control, Western Blot, Affinity Chromatography, Binding Assay

Journal: Oncotarget

Article Title: Tumor suppressor p53 regulates insulin receptor ( INSR ) gene expression via direct binding to the INSR promoter

doi: 10.18632/oncotarget.27645

Figure Lengend Snippet: IGF1R-KD, INSR-KD and control MCF7 cells were transiently co-transfected with the pGL3(-877/-2) LUC INSR promoter luciferase reporter along an Sp1 expression vector (or empty pEGFP plasmid) and a wild-type p53 vector (or empty pCMV). Cells were harvested after 48 h and promoter activity was measured. * p < 0.01 versus control cells. § p < 0.01 versus empty pCMV-transfected cells. The left inset presents the basal INSR promoter activity levels in all three cell lines at a large scale. The right inset presents the endogenous levels of Sp1 and p53 proteins in control and KD cells.

Article Snippet: Protein A-peroxidase conjugated was used for p53 detection (Rockland Immunochemicals Inc, Limerick, PA, USA).

Techniques: Control, Transfection, Luciferase, Expressing, Plasmid Preparation, Activity Assay

Journal: Oncotarget

Article Title: Tumor suppressor p53 regulates insulin receptor ( INSR ) gene expression via direct binding to the INSR promoter

doi: 10.18632/oncotarget.27645

Figure Lengend Snippet: IGF1R-KD, INSR-KD and control MCF7 cells were plated in 96-well plates in quadruplicate and, after 24 h, were transfected with p53-WT or empty vector (pCMV). Proliferation was measured after an additional 72 h using an XTT assay. A value of 100% was given to the cell number of control, untreated cells at the end of the incubation period. * p < 0.01 versus respective pCMV-transfected cells.

Article Snippet: Protein A-peroxidase conjugated was used for p53 detection (Rockland Immunochemicals Inc, Limerick, PA, USA).

Techniques: Control, Transfection, Plasmid Preparation, XTT Assay, Incubation

Journal: Oncotarget

Article Title: Tumor suppressor p53 regulates insulin receptor ( INSR ) gene expression via direct binding to the INSR promoter

doi: 10.18632/oncotarget.27645

Figure Lengend Snippet: IGF1R-KD, INSR-KD and control MCF7 cells were seeded in triplicate onto 6-well plates and, after 24 h, were transfected with p53-WT (or empty pCMV vector). After an additional 24 h, the cells were tripsinized, counted and plated again (in 6-well plates in triplicate, 10 5 cells/well) for 72 h. Cells were then permeabilized with Triton X-100, stained with propidium iodide and analyzed using a FacsCalibur system. * p < 0.01 versus respective pCMV-transfected cells.

Article Snippet: Protein A-peroxidase conjugated was used for p53 detection (Rockland Immunochemicals Inc, Limerick, PA, USA).

Techniques: Control, Transfection, Plasmid Preparation, Staining

Journal: Cancer Science

Article Title: Telomere dysfunction and inactivation of the p16 INK4a /Rb pathway in pyothorax‐associated lymphoma

doi: 10.1111/j.1349-7006.2007.00482.x

Figure Lengend Snippet: (a) Retinoblastoma (Rb), p16INK4a, and p53 protein expression in pyothorax‐associated lymphoma (PAL) cell lines. Western blotting analysis revealed that p16INK4a and Rb expression was found in none and three (OPL‐1, OPL‐4 and Deglis) of eight PAL cell lines, respectively. p53 protein was not expressed in OPL‐5, weakly expressed in OPL‐3, and expressed but small in size in OPL‐2. IB‐4 was used as control for p53 and p16INK4a proteins while Jurkat was used for Rb protein. Anti‐actin labeling showed equivalence of loading of cell lysates. 1: OPL‐1, 2: OPL‐2, 3: OPL‐3, 4: OPL‐4, 5: OPL‐5, 7: OPL‐7, De: Deglis, Pa: Pal‐1, C: control. (b) Three phospho‐Rb antibodies, which detect the targeted sites, revealed that Rb proteins expressed in three PAL cell lines (OPL‐1, OPL‐4 and Deglis) were heavily phosphorylated. Rb control proteins with and without phosphorylation in vitro by cdc2/cyclinB were provided in lanes (P) and (N), respectively. 1: OPL‐1, 4: OPL‐4, De: Deglis. (c) Methylation status of the p16 gene promoter in PAL cell lines and clinical cases. A 384 bp product containing 34 CpG sites was amplified. 16 CpG sites reported previously correspond to sites 4–19.( 20 , 21 ) Transcription start sites are shown by arrow heads. (▪) Methylated CpG, (□) unmethylated CpG. Transcription start site are shown by arrow heads.

Article Snippet: Blots were blocked in PBS containing 0.05% Tween 20 and 1% non‐fat dry milk, and incubated with each antibody at room temperature for 1 h. Antibodies used were antip16 INK4a (clone 16P04; Laboratory vision, Fremont, CA, USA), anti‐Rb (clone IF8; Laboratory vision), antiphospho‐Rb (Ser‐795), phospho‐Rb (Ser‐780), phospho‐Rb (Ser‐807/811) (cell signaling, MA), anti‐p53 (DO‐7; DAKO), anti‐hTERT (Rockland, Gilbertsville, PA, USA), and anti‐actin (Sigma‐Aldrich, Steinheim, Germany), respectively.

Techniques: Expressing, Western Blot, Control, Labeling, Phospho-proteomics, In Vitro, Methylation, Amplification

Journal: Medical Oncology (Northwood, London, England)

Article Title: A natural nephroprotective adjuvant for cancer chemotherapy: Rosmarinic acid disrupts IL-17 A-Ferroptosis coupling in Ifosfamide-induced renal injury

doi: 10.1007/s12032-026-03280-z

Figure Lengend Snippet: Changes in KIM-1/TIM-1, Caspase-3, and Nephrin protein expression levels in renal tissues across different experimental groups. In the Control and RA groups, KIM-1/TIM-1 and Caspase-3 staining was minimal, showing only faint immunohistochemical reactivity, whereas Nephrin expression exhibited a strong positive reaction (arrowhead). Conversely, the IFA group displayed markedly increased KIM-1/TIM-1 and Caspase-3 expression with intense positive staining (arrowhead), while Nephrin expression was significantly diminished. In the IFA+RA group, a reduction in KIM-1/TIM-1 and Caspase-3 expression and a partial increase in Nephrin staining were observed compared to the IFA group, with generally moderate positive reactions detected. IHC, DAB chromogen, hematoxylin counterstain; ×400 magnification. Note: Data represent mean ± SD. *p<0.05 compared to control groups, #p<0.05 compared to the IFA group.

Article Snippet: The following primary antibodies were applied: T-cell immunoglobulin and mucin domain 1 (TIM-1) / Kidney injury molecule-1 (KIM-1) (Cat. No. PA1632; Boster Biological Technology, Pleasanton, CA, USA), Nephrin (Cat. No. PA5-20330; Thermo Fisher Scientific, Waltham, MA, USA), and Caspase-3 (Cat. No. SC-56053; Santa Cruz Biotechnology, Dallas, TX, USA), each diluted at a ratio of 1:100 in antibody diluent.

Techniques: Expressing, Control, Staining, Immunohistochemical staining