Journal: Cell Death & Disease

Article Title: p53 β-hydroxybutyrylation attenuates p53 activity

doi: 10.1038/s41419-019-1463-y

Figure Lengend Snippet: a p53 kbhb in the presence or absence of BHB in cells. Western blot analysis of p53. IP was performed using BHB-K antibodies in cells transfected with p53 or both p53 and CBP and then treated with 10 mM BHB. b CBP mutants lacked the ability to exert kbhb activity on p53. Western blot analysis of p53 performed using antibodies against BHB-K, Ac-K and specific acetylated sites on p53 in IP obtained from cells transfected with p53, p53 plus wild-type CBP, or p53 plus mutant CBP and then treated with 10 mM BHB, 1 μM TSA and 5 mM nicotinamide for 24 h. c CBP β-hydroxybutyrylates p53 in vitro. Purified p53 was incubated with CBP or CBP mutants and β-hydroxybutyrate-CoA (BHB-CoA). Western blot analysis of the reaction products performed with BHB-K antibodies (top two panels), with anti-p53 (p53 labeled panel) or with anti-HA antibody (bottom panel to detect CBP). d Silencing CBP reduced the β-hydroxybutyration level of p53 in U2OS. Cells was transfected twice with 120 pmol of siRNA oligos specially targeting CBP and p300 or negative control, followed by treating with 10 mM BHB after 48 h. e p300 β-hydroxybutyrylates p53 in vitro. Purified p53 was incubated with p300 and BHB-CoA. Western blot analysis of the reaction products performed with BHB-K antibodies (top two panels), with anti-p53 (p53 labeled panel) or with anti-Flag antibody (bottom panel to detect p300)

Article Snippet: Plasmids Flag-p53, Flag-p300, CBP-HA and CBP-HA mutants were from Dr. Gu’s lab , . siRNA targeting CBP and p300 (1#: 5′-GAGGUCGUUUACAUAAATT-3′; 2#: 5′-UUUAUGUAAACGCGACCUCTT-3′) and a negative control (5′-UUCUCCGAACGUGUCACGUTT-3′) were synthesized by GenePharma (China).

Techniques: Western Blot, Transfection, Activity Assay, Mutagenesis, In Vitro, Purification, Incubation, Labeling, Negative Control

Journal: Cancers

Article Title: Stabilization of Hypoxia-Inducible Factors and BNIP3 Promoter Methylation Contribute to Acquired Sorafenib Resistance in Human Hepatocarcinoma Cells

doi: 10.3390/cancers11121984

Figure Lengend Snippet: Analysis of Bcl-2 interacting protein 3 (BNIP3) expression under hypoxic conditions and role of epigenetic modulations on chemoresistance-associated BNIP3 downregulation: ( a ) BNIP3 protein levels. Lanes 0 h show normoxic basal protein levels. a p < 0.05 vs. normoxic non-treated HepG2 cells, b p < 0.05 and c p < 0.05 vs. hypoxic non-treated and sorafenib-treated HepG2 cells, respectively, at each time point; ( b ) Confocal images of BNIP3 immunofluorescence staining (green) show BNIP3 expression after incubation under hypoxia for 24 h. DAPI staining (blue) denotes cell nucleus. Magnification: 63X, scale bar: 10 µm. a p < 0.05 and b p < 0.05 vs. non-treated and sorafenib-treated HepG2 cells, respectively; ( c ) BNIP3 mRNA levels 24 h under hypoxia were measured by qRT-PCR. a p < 0.05 and b p < 0.05 vs. non-treated and sorafenib-treated HepG2 cells, respectively; ( d ) Effect of inhibiting histone deacetylases (HDACs) on BNIP3 expression by trichostatin-A (TSA) addition. HepG2S1 and HepG2S3 cells were incubated under hypoxia during the last 24 h of every TSA treatment. First lane shows standard BNIP3 protein levels from HepG2 cells after 24 h incubation under hypoxia. Densitometry reading of each band (relative to its correspondent PCNA band) is shown under the immunoblots; ( e ) Methylation status of the BNIP3 promoter was examined performing methylation-specific PCR (MSP) after 24 h of hypoxia. Specific BNIP3 primers were used to amplify either unmethylated (U) or methylated (M) DNA; ( f ) Effect of 5-aza-2′-deoxycytidine (5-Aza) treatment on the methylation status of the BNIP3 promoter. Cells were incubated with the demethylation agent for 48 h before exposure to hypoxia plus 5-Aza for further 24 h; Impact of 5-Aza treatment during 72 h (last 24 h also under hypoxia) on ( g ) BNIP3 mRNA and ( h ) protein levels. HepG2 lanes show basal mRNA and protein levels after 24 h under hypoxia, respectively; ( i ) Effect of demethylation by 5-Aza alone or in conjunction with BNIP3 silencing on the BNIP3 expression and the resistant cell viability under hypoxia. a p < 0.05 significant differences between 5-Aza-treated and non-treated cells, b p < 0.05 significant differences between 5-Aza-treated BNIP3 siRNA and 5-Aza-treated control siRNA cells. Densitometry reading of each band (relative to its correspondent PCNA band) from ( h ) and ( i ) is shown under the immunoblot. Data from ( a – c ) are expressed as mean values of arbitrary units (a.u.) ± SD of three independent experiments. Data from ( i ) are expressed as a percentage of mean values ± SD. Full-length immunoblots are presented in .

Article Snippet: Besides, we inhibited histone deacetylation using 10, 50, and 100 nM of the HDACs inhibitor TSA (AdooQ ® Bioscience, Irvine, CA, USA) and methylation with 10 and 100 μM of the DNMT inhibitor 5-Aza (MedChemExpress, Sollentuna, Sweden).

Techniques: Expressing, Immunofluorescence, Staining, Incubation, Quantitative RT-PCR, Western Blot, Methylation, Control