Fig. 5

HDACs epigenetically regulate autophagy. a RT-qPCR of autophagy genes in SAHA-treated (20 μM for 24 h) HeLa (n = 5 biologically independent samples). b EGFP-LC3 expression in HeLa treated with DMSO or SAHA (20μM for 24 h) under fed or starved conditions (EBSS). Scale bar, 20 μm. c Quantification of EGFP-LC3 (n = 12 images). d Top, representative image of anti-LC3 immunoblot of HeLa treated with DMSO (D) or SAHA (S) (20 μM for 24 h) under fed or starved conditions (EBSS) with or without Bafilomycin A1 (BAF) to assess the autophagic flux; short and long exposure. Bottom, Coomassie stained immunoblot used as loading control e Quantification of LC3II/LC3I ratio (n = 5 independent experiments) normalized to the loading control and relative to DMSO FED values; D = DMSO, S = SAHA, starved = EBSS treatment. f HDAC2 occupancy of autophagy gene promoters analyzed using ChIP-seq datasets from the ENCODE/Haib project. Genes on the left of the vertical black line are occupied by HDAC2. p = 4.656e-08, odds ratio = 6.0, Fisher’s exact test. g ChIP analyses of the MAP1LC3B promoter using anti-HDAC2 antibody was performed in HeLa treated with SAHA (20μM for 24 h) or DMSO (n = 6 biologically independent samples); IgG control (n = 3 biologically independent samples). h ChIP analysis of the promoter of MAP1LC3B using acetyl histone H3 Lys 14 antibody (Acetyl-H3K14) in HeLa treated with SAHA (20 μM for 24 h) or DMSO (n = 6 biologically independent samples); IgG control (n = 3 biologically independent samples). i, j RT-qPCR of (i) Gabarapl2, Map1lc3b and (j) Pik3c3 and Sqstm1 was performed after SAHA treatment (20μM for 24 h) in WT MEFs, in MEFs with CRISPR-mediated knockout of Tfeb and Tfe3 (dKO) and in MEFs dKO silenced for Mitf (dKO + shMitf) (n ≥ 6 biologically independent samples). shC refers to cells transduced with shRNA control lentivirus. Statistical analysis was performed comparing the normalized expression of the autophagy genes in MEFs WT + shC versus dKO + shC and in MEFs WT + shC versus dKO + shMitf. Boxes represent the mean value and bar inside the box represents median value; upper bar represents maximum of distribution; lower bar represents minimum of distribution (95% confidence level). Graphs are presented as mean ± SD. Statistical analysis was performed using the Student t-test. *p < 0.05, **p < 0.01, ****p < 0.0001