Journal: Biomedicines

Article Title: Valemetostat–SAHA-Driven Acetylation of p53 via SET/TAF-Iβ Displacement and p300 Activation Modulates Cell Cycle Regulators in Pancreatic Cancer Cells

doi: 10.3390/biomedicines13092279

Figure Lengend Snippet: p53 acetylation and p21 expression are upregulated by SAHA/DS treatment in pancreatic cancer cells. PaCa44 pancreatic cancer cells were exposed for 48 h to SAHA (2.5 µM), DS (5 µM), or SAHA/DS combination (2.5 µM/5 µM M) or left untreated (CT). ( A ) Acetylation of p53 was evaluated by immunoprecipitation with anti-acetyl-lysine antibody and blotting with p53. Beads are shown as loading control. ( B ) Acetylation of p53 (ac p53) and total p53 expression was also evaluated by Western blotting by using anti-373/382 acetyl p53 and anti-p53 antibody. β-Actin was used as loading control. ( C ) Acetyl-histone H3 (ac H3) expression level was assessed by Western blotting in PaCa44 cells. ( D ) p53 and p21 expression as evaluated in PaCa44, PT45, and Panc-1 undergoing treatment by SAHA (2.5 µM), DS (5 µM), or SAHA/DS combination. β-Actin or GAPDH were used as controls. ( E ) Ac p53 and p21 time-course expression as evaluated by Western blotting analysis in PaCa44 cell line ( F ) p21 expression level as evaluated by Western blotting analysis in PaCa44, PT45, and Panc-1 following treatment by SAHA/DS in presence or in absence of pifithrin-α. β-Actin was used as loading control. The histograms indicate the mean plus SD of the densitometric analysis of three experiments and expressed as the ratio between molecules of interest and loading controls; p -value * < 0.05; ** < 0.01; *** < 0.001; and **** < 0.0001, as calculated by ANOVA test.

Article Snippet: First, 2 × 10 5 cells/well were seeded into 6-well plates in complete medium and treated for 48 h with the HDAC inhibitor Vorinostat (SAHA) (2.5 μM) (MedChemExpress, New York, NJ, 08852, USA, cat. HY-10221/CS-0589), the EZH2 methyltransferase inhibitor Valemetostat (DS-3201) (5 μM) (Selleckchem, Cologne, Germany, cat. S8926), or a combination of both.

Techniques: Expressing, Immunoprecipitation, Control, Western Blot

Journal: Biomedicines

Article Title: Valemetostat–SAHA-Driven Acetylation of p53 via SET/TAF-Iβ Displacement and p300 Activation Modulates Cell Cycle Regulators in Pancreatic Cancer Cells

doi: 10.3390/biomedicines13092279

Figure Lengend Snippet: p21 is upregulated at transcriptional level by SAHA/DS combination. ( A ) p21 mRNA expression in cells treated by SAHA, DS, or SAHA/DS as evaluated by q-RT PCR. ( B ) CHIP assay performed to investigate binding of p53 to p21 promoter in cells treated by SAHA, DS, or combination of both by using DOI antibody. ( C ) Acetylation of p53 and p21 expression level in Paca44 cells following transfection with p53 K382R vector (+) or with empty vector (−). GAPDH was used as loading control. Histograms indicate the mean plus SD of the densitometric analysis out of three experiments and expressed as ratio between acetyl p53/p53, p53/GAPDH and p21/GAPDH. p -value ** < 0.01; and **** < 0.0001, as calculated by ANOVA test.

Article Snippet: First, 2 × 10 5 cells/well were seeded into 6-well plates in complete medium and treated for 48 h with the HDAC inhibitor Vorinostat (SAHA) (2.5 μM) (MedChemExpress, New York, NJ, 08852, USA, cat. HY-10221/CS-0589), the EZH2 methyltransferase inhibitor Valemetostat (DS-3201) (5 μM) (Selleckchem, Cologne, Germany, cat. S8926), or a combination of both.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Transfection, Plasmid Preparation, Control

Journal: Biomedicines

Article Title: Valemetostat–SAHA-Driven Acetylation of p53 via SET/TAF-Iβ Displacement and p300 Activation Modulates Cell Cycle Regulators in Pancreatic Cancer Cells

doi: 10.3390/biomedicines13092279

Figure Lengend Snippet: p53 acetylation in response to SAHA/DS treatment correlates with changes in its interaction with p300 acetyltransferase and the cochaperone SET/TAF-Iβ. ( A ) Acetylation of p300 was investigated by immunoprecipitation with anti-acetyl-lysine antibody and blotting with an anti-p300 antibody in PaCa44 cancer cells untreated (CT) or undergoing SAHA or SAHA/DS treatment. Beads are shown as loading control. ( B ) Acetyl-p53 (ac p53) as evaluated by Western blotting in PaCa44 and PT45 cells pre-treated (+) or not (−) by p300 inhibitor (p300 inh) and then treated with SAHA/DS or left untreated (CT). β-Actin and GAPDH represent loading controls. ( C ) p21 expression level as evaluated in PaCa44 cancer cells pre-treated (+) or not (−) with p300 inhibitor and exposed to SAHA, SAHA/DS, and DS or untreated control (CT). GAPDH was used as loading control. ( D ) Interaction of mutp53 with p300 and SET/TAF1β investigated by immunoprecipitation in PaCa44 cells treated with SAHA, SAHA/DS, and DS. P53 is shown as loading control. Histograms represent mean plus SD of densitometric analysis from three experiments and expressed as ratio between ac p300/beads and p300/β-Actin ( A ), ac p53/β-Actin and ac p53/GAPDH ( B ), p21/GAPDH ( C ), and p300/p53 and SET/TAF1/p53 ( D ). p -value * < 0.05; ** < 0.01; *** < 0.001; and **** < 0.0001 as calculated by ANOVA test.

Article Snippet: First, 2 × 10 5 cells/well were seeded into 6-well plates in complete medium and treated for 48 h with the HDAC inhibitor Vorinostat (SAHA) (2.5 μM) (MedChemExpress, New York, NJ, 08852, USA, cat. HY-10221/CS-0589), the EZH2 methyltransferase inhibitor Valemetostat (DS-3201) (5 μM) (Selleckchem, Cologne, Germany, cat. S8926), or a combination of both.

Techniques: Immunoprecipitation, Control, Western Blot, Expressing

Journal: Biomedicines

Article Title: Valemetostat–SAHA-Driven Acetylation of p53 via SET/TAF-Iβ Displacement and p300 Activation Modulates Cell Cycle Regulators in Pancreatic Cancer Cells

doi: 10.3390/biomedicines13092279

Figure Lengend Snippet: SAHA/DS, particularly with 5-AZA supplementation, reduces pancreatic cancer cell survival, downregulates CHK1, and increases DNA damage in pancreatic cancer cells. ( A ) Cell survival as evaluated by Trypan blue exclusion assay in PaCa44 and PT45 cell lines untreated (CT) or treated with SAHA, SAHA/DS, or DS. The histograms indicate the percentage of cell viability relative to the control; data are shown as the mean plus SD from more than three experiments. p -value * < 0.05; ** < 0.01 and **** < 0.0001 as calculated by ANOVA test. In addition, the synergistic cytotoxicity induced by combination treatment, as evaluated by the Bliss independence model, is reported (Bliss index > 1). ( B ) Cleaved Caspase 3 (cl Casp3) expression level as investigated by Western blotting in PaCa44 and PT45 cells undergoing above-reported treatments. Lamin b was used as loading control. Histograms are the mean plus SD of the densitometric analysis calculated in three experiments and expressing the ratio between cleaved Capase3 and lamin b; p -value * < 0.05 and **** < 0.0001 as calculated by ANOVA test. ( C ) Representative pictures of PaCa44 cell colonies following staining with crystal violet and histograms representing the quantitative analyses of colony formation shown as mean ± SD of percent on untreated cells (CT). ( D ) γH2AX and CHK1 expression as evaluated by Western blotting analysis in PaCa44 cells untreated (CT) or treated by SAHA, SAHA/DS, and DS. GAPDH represented the loading control. ( E ) FACS profiles of Paca44 cells treated as reported above. The numbers indicate the percentage of subG1 events. One experiment out of three is shown. ( F ) p21, CHK1, and γH2AX expression as evaluated by Western blotting in PaCa44 cells pre-treated (+) or not (−) with pifithrin-α and exposed to SAHA/DS or left untreated (CT). GAPDH was the loading control. ( G ) p21, CHK1, and γH2AX expression as evaluated by Western blotting in PaCa44 cells untreated (CT) or treated by SAHA/DS in the presence or absence of 5-AZA. GAPDH was the loading control. ( H ) Cell survival as evaluated by Trypan blue exclusion assay in PaCa44 cell lines treated by SAHA/DS in the presence or absence of 5-AZA or left untreated. Histograms represent the mean plus SD of the densitometric analysis derived from three experiments and expressed as the ratio between ( B ) cl Casp3/lamin b, ( D ) γH2AX/GAPDH and CHK1/GAPDH, ( F ) p21/GAPDH, CHK1/GAPDH, and γH2AX/GAPDH, and ( G ) p21/GAPDH and γH2AX/GAPDH. p -value * < 0.05; ** < 0.01; *** < 0.001; and **** < 0.0001 as calculated by ANOVA test.

Article Snippet: First, 2 × 10 5 cells/well were seeded into 6-well plates in complete medium and treated for 48 h with the HDAC inhibitor Vorinostat (SAHA) (2.5 μM) (MedChemExpress, New York, NJ, 08852, USA, cat. HY-10221/CS-0589), the EZH2 methyltransferase inhibitor Valemetostat (DS-3201) (5 μM) (Selleckchem, Cologne, Germany, cat. S8926), or a combination of both.

Techniques: Trypan Blue Exclusion Assay, Control, Expressing, Western Blot, Staining, Derivative Assay

Journal: Biomedicines

Article Title: Valemetostat–SAHA-Driven Acetylation of p53 via SET/TAF-Iβ Displacement and p300 Activation Modulates Cell Cycle Regulators in Pancreatic Cancer Cells

doi: 10.3390/biomedicines13092279

Figure Lengend Snippet: Diagram representing the impact of SAHA/DS treatment on mutp53, p21, and DNA damage in pancreatic cancer cells.

Article Snippet: First, 2 × 10 5 cells/well were seeded into 6-well plates in complete medium and treated for 48 h with the HDAC inhibitor Vorinostat (SAHA) (2.5 μM) (MedChemExpress, New York, NJ, 08852, USA, cat. HY-10221/CS-0589), the EZH2 methyltransferase inhibitor Valemetostat (DS-3201) (5 μM) (Selleckchem, Cologne, Germany, cat. S8926), or a combination of both.

Techniques: