Journal: bioRxiv

Article Title: APEX1 Protects Cardiomyocytes Against Oxidative Stress by Regulating p53 Protein Stability

doi: 10.64898/2026.03.17.712520

Figure Lengend Snippet: APEX1 interacts with p53 and regulates the protein stability of p53. (A) Co-localisation of APEX1 and p53 in AC16 cardiomyocytes. APEX1 identified with APEX1 antibody (red), p53 identified by p53 antibody (green) and nuclei were stained with DAPI (blue). (Scale bar: 50μm). (B) Reciprocal Co-IP confirms the interaction between APEX1 and p53. (C) The mRNA expression levels of APEX1 and p53 were detected by qRT-PCR after APEX1 knockdown (n=6). (D) The protein expression level of p53 was detected by Western blot after APEX1 knockdown (n=3). (E) The protein expression level of p53 was detected by Western blot after APEX1 knockdown and MG132 treatment (n=7). (F) Co-immunoprecipitation (Co-IP) analysis of APEX1 interaction with P53 in the presence of MG132. Statistical analysis was performed with Student’s t-test or one-way analysis of variance (ANOVA). Results presented as mean ± SEM. A dot represents an independent biological sample. **p <0 .01; ***p< 0.001; ns not significant.

Article Snippet: The cell lysates were incubated on ice for 30 minutes at 4°Cand then centrifuged at 14,000 × g for 15 minutes at 4°C Protein A/G Magnetic Beads (HY-K0202, MedChemExpress) were pre-incubated with an anti-p53 antibody and then added to the cell lysates for overnight incubation at 4°C.

Techniques: Staining, Co-Immunoprecipitation Assay, Expressing, Quantitative RT-PCR, Knockdown, Western Blot, Immunoprecipitation

Journal: bioRxiv

Article Title: APEX1 Protects Cardiomyocytes Against Oxidative Stress by Regulating p53 Protein Stability

doi: 10.64898/2026.03.17.712520

Figure Lengend Snippet: The role of p53 in the oxidative stress response of AC16 cells. (A) The protein level of p53 in AC16 cells were detected by Western blot (n=10). (B) CCK-8 assay was used to assess the effect of p53 overexpression on AC16 cell viability (n = 5). (C) Overexpression of p53 significantly attenuated the increase in LDH levels (n = 6). (D) Overexpression of p53 reduced ROS levels in cells subjected to oxidative stress injury (n = 4). (D) P53 overexpression upregulated GPX4 protein expression after H/R (n = 6). Statistical analysis was performed with Student’s t-test or one-way analysis of variance (ANOVA). Results presented as mean ± SEM. A dot represents an independent biological sample. *p < 0.5; ***p < 0.001.

Article Snippet: The cell lysates were incubated on ice for 30 minutes at 4°Cand then centrifuged at 14,000 × g for 15 minutes at 4°C Protein A/G Magnetic Beads (HY-K0202, MedChemExpress) were pre-incubated with an anti-p53 antibody and then added to the cell lysates for overnight incubation at 4°C.

Techniques: Western Blot, CCK-8 Assay, Over Expression, Expressing

Journal: bioRxiv

Article Title: APEX1 Protects Cardiomyocytes Against Oxidative Stress by Regulating p53 Protein Stability

doi: 10.64898/2026.03.17.712520

Figure Lengend Snippet: APEX1 regulates myocardial oxidative stress injury via p53. (A) Overexpression of p53 ameliorates the reduction in AC16 cell viability induced by APEX1 knockdown (n = 6). (B) Overexpression of p53 reverses the increase in LDH release caused by APEX1 knockdown (n = 6). (C) Overexpression of p53 alleviates the intracellular reactive oxygen species (ROS) accumulation triggered by APEX1 deficiency (n = 5). (D) Overexpression of p53 restores the downregulation of glutathione peroxidase 4 (GPX4) protein expression resulting from APEX1 knockdown (n = 3). Statistical analysis was performed with One-way analysis of variance (ANOVA). Results presented as mean ± SEM. A dot represents an independent biological sample. **p < 0.01; ***p < 0.001 .

Article Snippet: The cell lysates were incubated on ice for 30 minutes at 4°Cand then centrifuged at 14,000 × g for 15 minutes at 4°C Protein A/G Magnetic Beads (HY-K0202, MedChemExpress) were pre-incubated with an anti-p53 antibody and then added to the cell lysates for overnight incubation at 4°C.

Techniques: Over Expression, Knockdown, Expressing

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: Advances in Dermatology and Allergology/Postȩpy Dermatologii i Alergologii

Article Title: Caspase-3, p53 and Bcl-2 expression in basal cell carcinoma of the eyelid

doi: 10.5114/ada.2020.98285

Figure Lengend Snippet: Immunohistochemical staining of all groups

Article Snippet: The sections were then covered with the primary antibodies diluted 1 : 500 for anti-caspase-3, 1 : 200 for anti-p53, 1 : 500 for anti-bcl-2 in TBS at 4°C overnight (Anti-caspase-3 (PA1302-1); anti-p53 (MA1078); anti-bcl-2 (MA1004) were from Boster Biological Technology Co. Ltd., USA).

Techniques: Immunohistochemical staining, Staining, Control

Journal: Biochemical and biophysical research communications

Article Title: Ribonucleotide Reductase Subunit p53R2 Regulates Mitochondria Homeostasis and Function in KB and PC-3 Cancer Cells

doi: 10.1016/j.bbrc.2011.05.114

Figure Lengend Snippet: A, q-RT-PCR analyses reveal that the steady-state p53R2 mRNA level decreased by ~90% in KB and PC-3 cells at 48 h post transfection with p53R2 siRNA. B, Western blotting analyses illustrate that the p53R2 protein amount was significantly downregulated in KB and PC-3 cells at 48 h post transfection with si-p53R2. C, Quantification of the mtDNA using real-time PCR to assess mitochondrial gene COX-1 amount relative to that of the PDE6B nuclear gene. D, Quantitative PCR analyses of mitochondrial gene ND1 amount to estimate mtDNA content relative to the β-GLOBIN nuclear gene. Data shown as the mean ± S.D. calculated from at least 3 independent experiments. * depicts p < 0.05.

Article Snippet: The rabbit p53R2 antibody was raised against a human p53R2 synthetic peptide (Acetyl-PERPEAAGLDQDERSC-amide; amino acids 4–18) conjugated to keyhole limpet hemocyanin (Rockland Immunochemicals).

Techniques: Reverse Transcription Polymerase Chain Reaction, Transfection, Western Blot, Real-time Polymerase Chain Reaction

Journal: Biochemical and biophysical research communications

Article Title: Ribonucleotide Reductase Subunit p53R2 Regulates Mitochondria Homeostasis and Function in KB and PC-3 Cancer Cells

doi: 10.1016/j.bbrc.2011.05.114

Figure Lengend Snippet: A, RR activity in cytosol extracts (CE) and mitochondrial extracts (ME) in KB and PC-3 cells is shown. Data presented as means of three separate experiments with duplicate. B, Western blot analysis of RRM1 in CE and ME. The presence of RRM1, COXIV or GAPDH was visualized using antibody against the corresponding protein. C to F, Mitochondrial (C and E) and whole cell (D and F) individual dNTP and total dNTP pools in KB (C and D) and PC-3 (E and F) cells at 48 h post transfection with p53R2 siRNA, scramble siRNA or mock cell are shown. Data are shown as the mean ± S.D. from at least 3 independent experiments.

Article Snippet: The rabbit p53R2 antibody was raised against a human p53R2 synthetic peptide (Acetyl-PERPEAAGLDQDERSC-amide; amino acids 4–18) conjugated to keyhole limpet hemocyanin (Rockland Immunochemicals).

Techniques: Activity Assay, Western Blot, Transfection

Journal: Biochemical and biophysical research communications

Article Title: Ribonucleotide Reductase Subunit p53R2 Regulates Mitochondria Homeostasis and Function in KB and PC-3 Cancer Cells

doi: 10.1016/j.bbrc.2011.05.114

Figure Lengend Snippet: A, Histogram of JC-1 staining of KB and PC-3 cells transfected with scramble or p53R2 siRNA. The JC-1 red fluorescence intensity (RFI) depicts ΔΨmt. R1 cells: RFI <300 (injured mitochondria) and R2 cells: RFI >300 (intact mitochondria). The data shown here is one representative out of 2 to 3 independent experiments. B, Relative ATP levels in KB and PC-3 cells with or without p53R2 siRNA. The level of ATP in cells transfected with scramble siRNA is designated at 100%. Bar represents mean ± S.D. of at least 3 independent experiments, * indicates p < 0.05 (p53R2 vs scramble siRNA). C, Inhibition of mitochondrial cytochrome c oxidase activity by p53R2 siRNA at 48 h post transfection. Data are presented as the mean ± SD from at least 3 independent experiments, * indicates p < 0.05 (p53R2 vs scramble siRNA).

Article Snippet: The rabbit p53R2 antibody was raised against a human p53R2 synthetic peptide (Acetyl-PERPEAAGLDQDERSC-amide; amino acids 4–18) conjugated to keyhole limpet hemocyanin (Rockland Immunochemicals).

Techniques: Staining, Transfection, Fluorescence, Inhibition, Activity Assay

Journal: Cell Death and Differentiation

Article Title: The anti-cancer agent APR-246 can activate several programmed cell death processes to kill malignant cells

doi: 10.1038/s41418-023-01122-3

Figure Lengend Snippet: a The Eμ-Myc lymphoma cell lines indicated, either parental with wt TRP53 or their TRP53 deleted derivatives, were treated for 16 h or 36 h with 15 μM APR-246 (a relatively low dose). The cells were then stained with DAPI plus A647 conjugated Annexin V and examined by flow cytometric analysis. b Western blot analysis of the indicated Eμ-Myc lymphoma cell lines, either parental with wt TRP53 or their TRP53 deleted derivatives, that had been treated for 16, 18 or 20 h with 15 μM APR-246 (a relatively low dose), showing the expression of PUMA, BIM and β-ACTIN (loading control). c Western blot analysis showing the expression of BAX and BAK in the indicated parental Eμ-Myc lymphoma cell lines expressing wt TRP53 and their absence in their BAX/BAK double knockout derivatives. Probing for β-ACTIN was used as a loading control. d Western blot analysis showing the expression of PUMA and BIM in the indicated parental Eμ-Myc lymphoma cell lines expressing wt TRP53 and their PUMA/BIM/NOXA triple knockout derivatives. Probing for β-ACTIN was used as a loading control. e NGS sequencing showing the mutations in the Noxa gene in the PUMA/BIM/NOXA triple knockout derivatives of the indicated Eμ-Myc lymphoma cell line. f The indicated Eμ-Myc lymphoma cell lines, parental as well as their BAX/BAK double knockout and PUMA/BIM/NOXA triple knockout derivatives, were treated for 48 h with the indicated concentrations of APR-246. Cell viability was measured by staining cells with PI followed by flow cytometric analysis. N = 3 independent experiments for each cell line and cell variant. Data are presented as mean ± S.D.

Article Snippet: The primary antibodies used in this study included ones against human TP53 (DO-1, Santa Cruz), mouse TRP53 (CM5, Leica), mouse PUMA (ProSci), mouse BIM (Enzo), mouse NINJ1 (ThermoFisher), mouse BAX (5B7, Sigma-Aldrich), mouse BAK (TC102, Merck) and β-ACTIN (AC40, Sigma-Aldrich), the latter used as a loading control.

Techniques: Staining, Western Blot, Expressing, Control, Double Knockout, Triple Knockout, Sequencing, Variant Assay