Fig. 6

Autophagy/RELB suppress activation of TGFβ gene promoters independent of NF-κB binding elements. a WT, ΔATG5 and ΔRELB A549 cells were subjected to chromatin immunoprecipitation (ChIP) for dimethyl-K4-histone H3 (H3Me2K4). Precipitating proximal promoter DNA was quantified by qRT-PCR, expressed as a percentage of input (means, n = 3, ±S.D., *P < 0.05 or **P < 0.01 vs. WT cells, two-tailed t-test). Gene names are shown above the charts. b–e HEK239T cells were co-transfected for 30 h with a firefly luciferase reporter driven by b–d a SMAD-response element (SRE) or e an NF-κB binding consensus element (NBE), and a constitutive Renilla luciferase control plasmid, along with expression vectors for the indicated forms of epitope-tagged RELB (wild-type, WT, or R141A Y142A, AA), SMAD2, SMAD3 and/or SMAD4 or an empty vector (EV) control. Luciferase assays were then performed as described in Methods (left, firefly/Renilla ratios expressed in arbitrary units, means, n = 3, ±S.D., *P < 0.05 or *P < 0.01 vs. EV, two-tailed t-test). Immunoblotting was performed to assess transfected factor expression for each replicate (right, representative blots). Uncropped blots are available in Supplementary Fig. 10