Fig. 5: SMS deletion induces Bim expression via inhibition of FOXO3a acetylation. | Nature Communications

Fig. 5: SMS deletion induces Bim expression via inhibition of FOXO3a acetylation.

From: Spermine synthase and MYC cooperate to maintain colorectal cancer cell survival by repressing Bim expression

Fig. 5

a SMS-WT or SMS-KO HCT116 and DLD-1 cell lysates were immunoprecipitated with FOXO3a antibody or IgG as control followed by western blot analysis for the indicated proteins. b Representative confocal images of SMS-WT or SMS-KO HCT116 cells stained for FOXO3a (green) and DAPI (red). c The distribution of FOXO3a in the cytosol and nucleus shown in b was analyzed using Image J. d ChIP analysis of SMS-WT or SMS-KO HCT116 cell extracts using a FOXO3a antibody or an irrelevant IgG. e Bim promoter activity was assessed in SMS-WT or SMS-KO HCT116 cells. f, g SMS-KO HCT116 cells with re-expression of SMS or vector control were assessed by confocal microscopy (f) for cellular staining of SMS (purple), FOXO3a (green), and DAPI (red), followed by quantification of FOXO3a distribution in the cytosol and nucleus (g). h HCT116 cells with stable expression of two different sets of EP300 shRNAs or control shRNA were analyzed by western blot analysis for the indicated proteins. i, j Cell lysates from HCT116 cells treated with 1 mM spermidine (SPD) or PBS for 24 h, or expressing EP300 shRNAs or control shRNA, were immunoprecipitated with FOXO3a antibody or IgG as control, followed by western blot analysis for the indicated proteins (i), or by ChIP analysis (j) of FOXO3a interaction with Bim promoter. k, l Bim promoter activity was analyzed in HCT116 cells treated with 1 mM SPD or PBS as control for 24 h (k), or in HCT116 cells expressing EP300 shRNAs or control shRNA (l). m The expression levels of Bim mRNA in HCT116 cells expressing EP300 shRNAs or control shRNA were quantified by RT-PCR analysis. n HCT116 cells expressing EP300 shRNAs or control shRNA were treated with 1 µM JQ1 or DMSO for 24 h, followed by western blot analysis for the indicated proteins. Scale bar, 25 µm. All graphic data are presented as mean values ±SEM (n = 3 independent experiments). The indicated p-values were determined by two-tailed unpaired t test. Source data are provided as a Source Data file.

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