Journal: Cell death discovery

Article Title: ABCB1 confers resistance to carboplatin by accumulating stem-like cells in the G2/M phase of the cell cycle in p53 null ovarian cancer.

doi: 10.1038/s41420-025-02435-7

Figure Lengend Snippet: Fig. 5 Reversal of the resistant phenotype conferred by upregulated ABCB1 through ΔNp73 suppression. A Quantitative gene expression of ΔNp73 in nonR and carR-SKOV3 cells (p = 0.0005). B Immunoblotting analysis of ΔNp73 protein levels in nonR and carR-SKOV3 cells via immunoblotting assay. Validation of ΔNp73 silencing in carR-SKOV3 compared to non-treated and negative control (NC) transfected-groups both at protein (C) and mRNA (D) levels (non- vs NC-transfected; p = 0.351, non- vs siΔNp73-transfected; p = 0.00002). E Quantitative gene expression of ABCB1 in carR-SKOV3 (non-treated, siNC-transfected, and siΔNp73-transfected) cells (non-treated vs siNC-transfected; p = 0.468, non-treated vs siΔNp73-transfected; p = 0.00005). F Relative cell growth rates of siΔNp73-transfected carR-SKOV3 cells compared to non- transfected and NC-transfected cells (24 h: non-treated vs siNC-transfected; p = 0.017, 48 h: non-treated vs siNC-transfected; p = 0.0002). G Comparison of Ki67 expression in carR-SKOV3 (non-treated, siNC-transfected, and siΔNp73-transfected) cells (non-treated vs siNC- transfected; p = 0.013). H Cell viability assay in carR-SKOV3 (non-treated, siNC-transfected, and siΔNp73-transfected) compared to nonR- SKOV3 after induction of additional damage up to 72 h. Data represent the means ± SD from triplicate experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, NS not significant.

Article Snippet: After blocking procedure, membranes were incubated with primary antibodies against p53 (7F5), phospho-p53 (E9Y4U; Ser15), PARP1 (46D11), cleaved-PARP1 (D64E10; Asp214), BCL-XL (54H6), phospho-cdc25c (63F9; Ser216), cyclin B1 (D5C10), ΔNp73 (38C674.2; Novus, USA), and β-actin (all from Cell signaling, USA), respectively, and finally visualized by ECL solution (Thermo, USA).

Techniques: Gene Expression, Western Blot, Biomarker Discovery, Negative Control, Transfection, Comparison, Expressing, Viability Assay

Journal: PLoS ONE

Article Title: Proinflammatory Cytokines and Bile Acids Upregulate ΔNp73 Protein, an Inhibitor of p53 and p73 Tumor Suppressors

doi: 10.1371/journal.pone.0064306

Figure Lengend Snippet: Exposure of esophageal cells to bile acids at low pH leads to accumulation of the ΔNp73 protein and its translocation to the nucleus. A. Representative immunohistochemical staining for ΔNp73 in the human Barrett’s esophagus (top left panel), esophageal tumor (top right panel), normal stomach and esophagus (bottom panels). Patients with Barrett’s metaplasia express high levels of ΔNp73 in epithelial cells whereas expression of this protein is low in the normal stomach and esophagus. Esophageal tumor specimen was used as a positive control. B. Endogenous ΔNp73 protein was immunoprecipitated from cellular extracts of CP-A cells 7 hours after treatment with 100 µM BA cocktail, pH 4.0 for 30 min. Non-specific rabbit antibody was used as a negative control (N/S). Input protein levels were normalized to β-actin. C. Western blot analysis of total cell extracts from SK-GT-4 cells stably transfected with ΔNp73α plasmid. Cells were collected at the indicated time after treatment with 100 µM BA cocktail, pH 4.0 for 30 min. D. Representative immunofluorescent staining for the ΔNp73 protein using an anti-FLAG tag antibody in stably transfected SK-GT-4 cells before and after BA/A treatment. Cells were fixed 7 hours after treatment with 100 µM BA cocktail, pH4.0 for 30 min. Graph shows percentage of cells with nuclear localization of ΔNp73α protein (***p<0.001). E. Levels of ΔNp73 mRNA were not increased following BA/A treatment. Real-time PCR analysis was performed in CP-A cells at the indicated time after treatment with BA/A (100 µM, 30 min).

Article Snippet: Immunohistochemical staining of BE esophageal tissues was performed using ΔNp73-specific antibody (Imgenex) at 1∶200 dilution as previously described .

Techniques: Translocation Assay, Immunohistochemical staining, Staining, Expressing, Positive Control, Immunoprecipitation, Negative Control, Western Blot, Stable Transfection, Transfection, Plasmid Preparation, FLAG-tag, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Proinflammatory Cytokines and Bile Acids Upregulate ΔNp73 Protein, an Inhibitor of p53 and p73 Tumor Suppressors

doi: 10.1371/journal.pone.0064306

Figure Lengend Snippet: A. Dynamics of c-Abl phosphorylation in BA/A-treated cells. SK-GT-4 cells were treated with 100 µM BA cocktail, pH 4.0. Extracts were collected at the indicated time and analyzed by Western blotting. Early (5–30 minutes) and late (4–24 hours) time points are shown. B. Western blot analysis for phospho-ΔNp73(Y50) in SK-GT-4 cells stably transfected with ΔNp73α. Cells were collected at indicated time after treatment with BA/A (100 µM, 30 min). C. c-Abl chemical inhibitor Imatinib downregulates the ΔNp73 protein. SK-GT-4 cells were pretreated with Imatinib (30 µM) for 1 hour, treated with BA/A (100 µM) for 30 minutes and then incubated for an additional 7 hours in the presence of the indicated inhibitor. Total cell extracts were analyzed by Western blotting. D. Inhibition of c-Abl by siRNA leads to downregulation of the ΔNp73 protein in BA/A-treated cells. SK-GT-4 cells were transfected with c-Abl-specific siRNA for 48 hours, treated with BA/A (100 µM) for 30 minutes, and then collected 7 hours after treatment. Levels of ΔNp73 were analyzed by Western blotting. E. Transfection of constitutively active c-Abl leads to upregulation of the ΔNp73 protein. SK-GT-4 cells were transfected with the c-Abl (P242E/P249E)-pcDNA3 vector for 24 hours and analyzed by Western blotting.

Article Snippet: Immunohistochemical staining of BE esophageal tissues was performed using ΔNp73-specific antibody (Imgenex) at 1∶200 dilution as previously described .

Techniques: Western Blot, Stable Transfection, Transfection, Incubation, Inhibition, Plasmid Preparation

Journal: PLoS ONE

Article Title: Proinflammatory Cytokines and Bile Acids Upregulate ΔNp73 Protein, an Inhibitor of p53 and p73 Tumor Suppressors

doi: 10.1371/journal.pone.0064306

Figure Lengend Snippet: A. Dynamics of IKKα/β phosphorylation in BA/A-treated cells. SK-GT-4 cells were treated with 100 µM BA cocktail, pH 4.0. Extracts were collected at the indicated time points and analyzed by Western blotting. Early (5–30 minutes) and late (4–24 hours) time points are shown. B. IKK kinase inhibitor Bay11–7085 downregulates the ΔNp73 protein. SK-GT-4 cells were pretreated with Bay11–7085 (10 µM) for 1 hour, treated with BA/A (100 µM) for 30 minutes and then incubated for an additional 7 hours in the presence of the indicated inhibitor. Total cell extracts were analyzed by Western blotting. C. Inhibition of IKK kinases by siRNA leads to downregulation of the ΔNp73 protein in BA/A-treated cells. SK-GT-4 cells were transfected with IKKα- or IKKβ- specific siRNA for 48 hours, treated with BA/A (100 µM) for 30 minutes, and then collected 7 hours after treatment. Levels of ΔNp73 were analyzed by Western blotting. D. Transfection of constitutively active IKKα and IKKβ mutants leads to upregulation of the ΔNp73 protein. SK-GT-4 cells were transfected with the indicated mutants for 24 hours and analyzed by Western blotting.

Article Snippet: Immunohistochemical staining of BE esophageal tissues was performed using ΔNp73-specific antibody (Imgenex) at 1∶200 dilution as previously described .

Techniques: Western Blot, Incubation, Inhibition, Transfection

Journal: PLoS ONE

Article Title: Proinflammatory Cytokines and Bile Acids Upregulate ΔNp73 Protein, an Inhibitor of p53 and p73 Tumor Suppressors

doi: 10.1371/journal.pone.0064306

Figure Lengend Snippet: A. Dynamics of p38 protein phosphorylation in BA/A-treated cells. SK-GT-4 cells were treated with 100 µM BA cocktail, pH 4.0. Extracts were collected at the indicated time and analyzed by Western blotting. Early (5–30 minutes) and late (4–24 hours) time points are shown. B. p38 MAPK kinase inhibitor SB203580 downregulates the ΔNp73 protein. SK-GT-4 cells were pretreated with SB203580 inhibitor (10 µM) for 1 hour, treated with BA/A (100 µM) for 30 minutes and then incubated for an additional 7 hours in the presence of the inhibitor. Total cell extracts were analyzed by Western blotting. C. Downregulation of p38 MAPK by siRNA leads to inhibition of the ΔNp73 protein in BA/A-treated cells. SK-GT-4 cells were transfected with p38-specific siRNA for 48 hours, treated with BA/A (100 µM) for 30 minutes, and collected 7 hours after treatment. Protein levels of ΔNp73 were analyzed by Western blotting. D. Co-transfection of p38 MAPK with MKK6 (S207E/T211E) mutant, which activates p38 kinase, leads to upregulation of the ΔNp73 protein. SK-GT-4 cells were transfected with the indicated constructs for 24 hours and then analyzed by Western blotting.

Article Snippet: Immunohistochemical staining of BE esophageal tissues was performed using ΔNp73-specific antibody (Imgenex) at 1∶200 dilution as previously described .

Techniques: Western Blot, Incubation, Inhibition, Transfection, Cotransfection, Mutagenesis, Construct

Journal: PLoS ONE

Article Title: Proinflammatory Cytokines and Bile Acids Upregulate ΔNp73 Protein, an Inhibitor of p53 and p73 Tumor Suppressors

doi: 10.1371/journal.pone.0064306

Figure Lengend Snippet: A. Dynamics of Aurora A phosphorylation in BA/A-treated cells. SK-GT-4 cells were treated with 100 µM BA cockail, pH 4.0. Extracts were collected at the indicated time points and analyzed by Western blotting. Early (5–30 minutes) and late (4–24 hours) time points are shown. B. Chemical inhibitor of Aurora A MLN8237 does not affect the ΔNp73 protein. SK-GT-4 cells were pretreated with MLN8237 (0.5 µM) for 1 hour, treated with BA/A (100 µM) for 30 minutes and then incubated for an additional 7 hours in the presence of the indicated inhibitor. Total cell extracts were analyzed by Western blotting. C. Aurora A kinase inhibitor MLN8237 efficiently blocked autophoshorylation of Aurora A kinase.

Article Snippet: Immunohistochemical staining of BE esophageal tissues was performed using ΔNp73-specific antibody (Imgenex) at 1∶200 dilution as previously described .

Techniques: Western Blot, Incubation

Journal: PLoS ONE

Article Title: Proinflammatory Cytokines and Bile Acids Upregulate ΔNp73 Protein, an Inhibitor of p53 and p73 Tumor Suppressors

doi: 10.1371/journal.pone.0064306

Figure Lengend Snippet: A. SK-GT-4 cells were treated with either BA/A (100 µM, 30 min) or cytokines (IL-1β, TNFα) or their combinations and then analyzed at the indicated time points. A combined treatment with BA/A and cytokines further enhances induction of ΔNp73 protein. B. Western blot analysis of ΔNp73 in SK-GT-4 cells treated as in A in the presence of siRNAs against p38 or IKKβ kinases. Bottom panel: downregulation of p38 and IKKβ kinases by siRNA is shown. C. Real-time PCR analysis of ΔNp73 mRNA was performed 8 hours after treatment with either IL-1β (20 ng/ml) or TNFα (20 ng/ml) in CP-A cells. D. BAR-T1 cells were treated with BA/A (100 µM) and TNFα (20 ng/ml). Cellular extracts were collected from treated and untreated cells. The endogenous ΔNp73 protein was immunoprecipitated using the ΔNp73 (N-16) antibody. The ΔNp73-p73 binding was analyzed by Western blotting with p73 antibody (Bethyl), which specifically recognizes p73 protein and does not cross-react with ΔNp73. Non-specific goat antibody was used as a negative control (N/S). Input protein levels were normalized to β-actin. Bottom panel: Relative binding of ΔNp73 to p73 was measured by densitometry; p73/IgG ratio is shown (**p<0.01). ΔNp73-p73 binding in untreated cells was arbitrarily set at 1. E. Overexpression of ΔNp73α significantly increases (**p<0.01) the survival of SK-GT-4 cells exposed to BA/A. SK-GT-4 cells, stably transfected with either ΔNp73α (ΔNp73) or empty (Vector) plasmid, were treated with BA/A (100 µM) for 20 min and cell survival was analyzed using MTT assay 24 hours after BA/A treatment.

Article Snippet: Immunohistochemical staining of BE esophageal tissues was performed using ΔNp73-specific antibody (Imgenex) at 1∶200 dilution as previously described .

Techniques: Western Blot, Real-time Polymerase Chain Reaction, Immunoprecipitation, Binding Assay, Negative Control, Over Expression, Stable Transfection, Transfection, Plasmid Preparation, MTT Assay