Fig. 2: The lipid kinase activity of Vps34 is dispensable for Rev-Erbα protein expression.
From: Class 3 PI3K coactivates the circadian clock to promote rhythmic de novo purine synthesis
a,b, Immunoblot analysis (n = 3) of dexamethasone-synchronized MEFs treated with dimethyl sulfoxide (DMSO, control) or Vps34 inhibitor (SAR405). Densitometry analyses of Rev-Erbα protein levels, normalized to Actin, presented as the fold change over DMSO-treated MEF cells (right). b, Relative transcript levels (n = 8; b) of dexamethasone-synchronized MEFs treated with DMSO (control) or Vps34 inhibitors (SAR405 and PIK-III). *P < 0.05 for SAR405 versus DMSO and **P < 0.05 for PIK-III versus DMSO; two-way ANOVA with Benjamini–Hochberg correction. a,b, Data are the mean ± s.e.m. from three independent experiments. c, ChIP–qPCR of Bmal1 recruitment to the Nr1d1 and Dbp promoters in MEFs treated with SAR405 or DMSO as in a collected at 24 h post synchronization. Data are the mean ± s.e.m. fold enrichment from three independent experiments (n = 9). *P < 0.05; two-way ANOVA with Benjamini–Hochberg correction. d, Immunoblot analysis of total protein extracts (n = 4). Densitometric analyses of Rev-Erbα levels normalized to Actin (right). e, Relative transcript levels of the indicated genes in dexamethasone-synchronized control (GFP) and Vps34-depleted (CRE) Vps34f/f MEFs. d,e, Data are the mean ± s.e.m. fold change compared with GFP-treated MEFs from three independent experiments (n = 5 for GFPCT16 and n = 6 for all other groups). Rhythmicity was determined using JTK_CYCLE (Supplementary Table 1). f, ChIP–qPCR of Bmal1 recruitment to the Nr1d1 and Dbp promoters in GFP and CRE MEFs collected at 24 h post synchronization. Data are the mean ± s.e.m. from three independent experiments (n = 10 for GFP ChIP-IgG and n = 11 for all other groups). *P < 0.05; two-way ANOVA with Benjamini–Hochberg correction. Source data and unprocessed blots are provided.
