Journal: Oncogenesis

Article Title: Non canonical BRCA1 promotes cell survival via modulating PARP13-mediated SEC61G mRNA decay

doi: 10.1038/s41389-025-00578-x

Figure Lengend Snippet: A A2780 cells were transfected with myc-BRCA1 and flag-PARP13 as indicated, followed by IP analysis of BRCA1/PARP13 interaction with or without IR (5 Gy) treatment. B Pharmacological inhibition of PIKK kinases. Cells were pretreated with DNA-PKi (NU7441, 10 μM), ATRi (VE821, 2 μM), or ATMi (KU55933, 10 μM) for 4 h prior to IR (5 Gy) treatment. BRCA1/PARP13 interaction was analyzed by IP at 2 h after IR treatment. C Subcellular fragmentation analysis of BRCA1/PARP13 interaction in A2780 cells. D Western blot analysis of γH2AX levels in WT or PARP13-KO A2780 cells after treatment with IR (5 Gy), etoposide (Eto,10 μM for 12 h), or camptothecin (CPT, 5 μM for 12 h). E Immunofluorescence (IF) staining of Rad51/γH2AX foci formation in WT and PARP13-KO cells following CPT-induced DNA damage, n = 200. F Colony formation analysis of WT and PARP13-KO A2780 cells exposed to IR (2 Gy), Eto (200 nM), or CPT (5 nM). Colonies were quantified after 7-10 days of culture. Data are presented as mean ± SD, n = 3. ∗∗∗ p < 0.001 by one-way ANOVA with Tukey’s test ( E ) or two-way ANOVA with Tukey’s test ( F ). ns no significance.

Article Snippet: Etoposide (#T0132) and CPT (#T1123) were obtained from TargetMol (Boston, MA).

Techniques: Transfection, Inhibition, Western Blot, Immunofluorescence, Staining

Journal: International Journal of Molecular Sciences

Article Title: HDAC Inhibitors Enhance the Chemosensitivity of Osteosarcoma Cells to Etoposide by Suppressing the Hippo/YAP Signaling Pathway

doi: 10.3390/ijms26188935

Figure Lengend Snippet: The inhibitory effects of HDACis (TSA and NAM), VP16 and their combination therapy in U2OS and SJSA-1 osteosarcoma cell lines. ( A – C ) Cytotoxicity of U2OS, SJSA-1 and O-ASCs cells following treatment with various concentrations of TSA (0.0625, 0.125, 0.25, 0.5, and 1 µM), NAM (0.625, 1.25, 2.5, 5, and 10 mM), and/or VP16 (2.5, 5, 10, 20, and 40 µM) for 72 h. The dotted lines represented a half inhibitory effect. ( D – F ) The fraction affected (Fa)-combination index (CI) plots illustrated that HDACis show a clear synergistic interaction with VP16 in U2OS, SJSA-1 and O-ASCs cells for 72 h of co-treatment, as evidenced by the majority of CI values being less than 1. Com, indicates VP16/TSA/NAM combination. ( G , H ) Viability of U2OS and SJSA-1 cells after exposure to TSA (1 μM) alone, NAM (5 mM) alone, VP16 (40 μM) alone, and their dual or three drug combinations for 72h. ( I , J ) Viability of U2OS and SJSA-1 cells subjected to VP16 treatment for 48 h, and accompany with sequential exposure to TSA/NAM for 30 and 48 h. Data are presented as the mean ± SD from three independent experiments. Statistical significance was calculated using one-way ANOVA followed by Tukey’s multiple comparisons test (ns: not significant, * p < 0.05, ** p < 0.01, and *** p < 0.001). The symbol “#” denotes comparisons between any individual drug and the combination (Com) group, with ## p < 0.01, ### p < 0.001.

Article Snippet: Next day, the cells were exposed to different varying concentrations of TSA (T6270, TargetMol, Boston, MA, USA), NAM (72340, Sigma-Aldrich, St. Louis, MO, USA) and/or VP16 (T0132, TargetMol, Boston, MA, USA) for 72 h. Meanwhile, the doses were selected based on previously published studies that demonstrated efficacy on similar cell types [ , , ].

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: HDAC Inhibitors Enhance the Chemosensitivity of Osteosarcoma Cells to Etoposide by Suppressing the Hippo/YAP Signaling Pathway

doi: 10.3390/ijms26188935

Figure Lengend Snippet: HDACis potentiated the anti-tumor efficacy of VP16 in osteosarcoma cell lines. ( A ) The cell morphology of U2OS and SJSA-1 osteosarcoma cells following 24 h of exposure to HDACis (TSA 1 μM and NAM 5 mM) and/or VP16 (40 μM) using microscopy. Scale bar: 170 μm. ( B – E ) The crystal violet staining images alongside quantitative outcomes from cell growth assays conducted on U2OS and SJSA-1 cells. The combination of HDACis (TSA 1 μM and NAM 5 mM) with VP16 (40 μM) effectively inhibit the cell proliferation. All data are presented as mean ± SD from three replicates. Statistical significance was determined using two-way ANOVA followed by Sidak’s multiple comparisons test, and indicated by *** p < 0.001.

Article Snippet: Next day, the cells were exposed to different varying concentrations of TSA (T6270, TargetMol, Boston, MA, USA), NAM (72340, Sigma-Aldrich, St. Louis, MO, USA) and/or VP16 (T0132, TargetMol, Boston, MA, USA) for 72 h. Meanwhile, the doses were selected based on previously published studies that demonstrated efficacy on similar cell types [ , , ].

Techniques: Microscopy, Staining

Journal: International Journal of Molecular Sciences

Article Title: HDAC Inhibitors Enhance the Chemosensitivity of Osteosarcoma Cells to Etoposide by Suppressing the Hippo/YAP Signaling Pathway

doi: 10.3390/ijms26188935

Figure Lengend Snippet: Effect of HDACis and VP16 alone or in combination on apoptosis of osteosarcoma cells. ( A , C ) A representative images showed apoptosis in U2OS and SJSA-1 cells after 24 h of treatment with HDACis (TSA 1 μM and NAM 5 mM) and/or VP16 (40 μM). The color scale ranges from blue (low density) to red (high density), indicating the local density of cells. ( B , D ) The apoptosis proportion of U2OS and SJSA-1 cells based on ( A , C ). ( E ) Western blot assay determined the expression changes in apoptosis related protein including PARP, cleaved-caspase 3, Bcl-2, with beta-actin serving as a reference protein. ( F ) Quantification analysis of the Western blot band intensities, derived from the data in ( E ), showing the relative expression of cleaved-PARP, cleaved-Caspase 3, and Bcl 2 proteins normalized to beta-actin, based on the data presented in ( E ). All data are shown as mean ± SD and are derived from three independent replicates. P values were calculated using one-way ANOVA followed by Tukey’s multiple comparisons test, and indicated by * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Next day, the cells were exposed to different varying concentrations of TSA (T6270, TargetMol, Boston, MA, USA), NAM (72340, Sigma-Aldrich, St. Louis, MO, USA) and/or VP16 (T0132, TargetMol, Boston, MA, USA) for 72 h. Meanwhile, the doses were selected based on previously published studies that demonstrated efficacy on similar cell types [ , , ].

Techniques: Western Blot, Expressing, Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: HDAC Inhibitors Enhance the Chemosensitivity of Osteosarcoma Cells to Etoposide by Suppressing the Hippo/YAP Signaling Pathway

doi: 10.3390/ijms26188935

Figure Lengend Snippet: Comparison of the antitumor efficacy of the VP16/TSA/NAM combination therapy versus doxorubicin. ( A , B ) The cell viability of U2OS and SJSA-1 cells were assessed for 48 h exposure to combination treatment (TSA 1 μM, NAM 5 mM and VP16 40 μM) or Dox (2 μM). ( C ) The cell morphology of U2OS and SJSA-1 cells after 48 h of exposure to either the combination treatment (TSA 1 μM, NAM 5 mM and VP16 40 μM) or Dox (2 μM), as observed under a microscope. Scale bar: 170 μm. ( D , E ) Cell proliferation analysis of U2OS and SJSA-1 cells treated with the combination treatment (TSA 1 μM, NAM 5 mM and VP16 40 μM) or Dox (2 μM) after 48 h. Data are shown as means ± SD with three replicates. ns: not significant. Statistical significance was calculated using one-way ANOVA followed by Tukey’s multiple comparisons test. ** p < 0.01, *** p < 0.001 indicated the statistics significance.

Article Snippet: Next day, the cells were exposed to different varying concentrations of TSA (T6270, TargetMol, Boston, MA, USA), NAM (72340, Sigma-Aldrich, St. Louis, MO, USA) and/or VP16 (T0132, TargetMol, Boston, MA, USA) for 72 h. Meanwhile, the doses were selected based on previously published studies that demonstrated efficacy on similar cell types [ , , ].

Techniques: Comparison, Microscopy

Journal: International Journal of Molecular Sciences

Article Title: HDAC Inhibitors Enhance the Chemosensitivity of Osteosarcoma Cells to Etoposide by Suppressing the Hippo/YAP Signaling Pathway

doi: 10.3390/ijms26188935

Figure Lengend Snippet: Differentially expressed genes and pathways after combination treatment with HDACis and VP16 in U2OS osteosarcoma cells. ( A ) Heatmap presentation of 4611 differentially expressed genes influenced by the treatments in U2OS cells. ( B ) KEGG analysis pertaining to the differentially expressed genes. Red box highlighted the Top 1 significantly enriched pathway. ( C ) GSEA focusing on the Hippo signaling pathway in cells subjected to combination treatment compared to control cells. ( D ) Differential gene clustering heatmap representing the Hippo signaling pathway in U2OS cells subjected to treatment with HDACis and/or VP16.

Article Snippet: Next day, the cells were exposed to different varying concentrations of TSA (T6270, TargetMol, Boston, MA, USA), NAM (72340, Sigma-Aldrich, St. Louis, MO, USA) and/or VP16 (T0132, TargetMol, Boston, MA, USA) for 72 h. Meanwhile, the doses were selected based on previously published studies that demonstrated efficacy on similar cell types [ , , ].

Techniques: Control

Journal: International Journal of Molecular Sciences

Article Title: HDAC Inhibitors Enhance the Chemosensitivity of Osteosarcoma Cells to Etoposide by Suppressing the Hippo/YAP Signaling Pathway

doi: 10.3390/ijms26188935

Figure Lengend Snippet: HDACis augmented the sensitivity of osteosarcoma cell to VP16 by suppressing the Hippo pathway. ( A ) RT-qPCR results of U2OS cells treated with HDACis (TSA 1 μM and NAM 5 mM) and/or VP-16 (40 μM) after 24 h of treatments. ( B ) Western blotting and quantification representative protein expression of YAP1 in U2OS and SJSA-1 cells subjected to HDACis and/or VP-16 treatments for 24 h, with beta-actin serving as a reference protein. ( C ) Flowcytometry analysis of osteosarcoma cells after treatment with VP16/TSA/NAM and/or YAP inhibitor (Verteporfin 1 μM) for 24 h. VP, Verteporfin. The color scale, ranging from blue to red, represents the local density of cells, with blue indicating low density and red indicating high density. All data are presented as mean ± SD, with three replicates. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test. ns: not significant. * p < 0.05, ** p < 0.01, *** p < 0.001 indicated the statistics significance.

Article Snippet: Next day, the cells were exposed to different varying concentrations of TSA (T6270, TargetMol, Boston, MA, USA), NAM (72340, Sigma-Aldrich, St. Louis, MO, USA) and/or VP16 (T0132, TargetMol, Boston, MA, USA) for 72 h. Meanwhile, the doses were selected based on previously published studies that demonstrated efficacy on similar cell types [ , , ].

Techniques: Quantitative RT-PCR, Western Blot, Expressing