Fig. 5

RELB promotes tumorigenesis and suppresses TGFβ target gene expression. a A549 ΔRELB cell clones (ΔRELB and ΔRELB-2) generated using CRISPR/Cas9 and two independent gRNA sequences were immunoblotted as shown. b A549 WT, ΔRELB and ΔRELB-2 cells were subcutaneously injected into immunocompromised mice and tumour volume was measured longitudinally (means, n = 12 flanks, ±S.E.M., *P < 0.05 or *P < 0.01 vs. WT, two-tailed t-test). c Two sequence-unrelated siRNA oligonucleotides targeting RELB were transfected in A549 cells (knockdown confirmed by immunoblotting). Expression profiling was performed vs. two sequence-unrelated non-targeting controls (Supplementary Data set 1). GSEA was performed, comparing genes differentially expressed in RELB knockdown cells with known oncogene and tumour suppressor profiles. The comparison yielding the greatest normalised enrichment score (NES), ‘TGFβ upregulated genes’, is shown here. d Fold-change heat map showing top 50 upregulated genes after RELB RNAi. Individual mean RELB siRNA values are normalised to the averaged means of the controls (siCtrls). Blue text indicates genes known to be transcriptionally upregulated downstream of TGFβ (Supplementary Table 2). e, f qRT-PCR was performed for the indicated transcripts after the following treatment regimens in A549 cells. e A549 cells were transfected with siCtrl or siRELB for 72 h. Cells were either left untreated (Ctrl) or treated with 5 ng/ml TGFβ1 for the final 16 h of transfection. f Exponentially dividing A549 WT and ΔRELB cells were compared. (means, n = 3, ±S.D., *P < 0.05 or **P < 0.01 vs. similarly treated siCtrl or WT cells, two-tailed t-test). g A549 cells were transfected with the indicated siRNA for 72 h, stained for paxillin and then imaged by widefield microscopy (scale bar = 20 µm). As a positive control, siCtrl cells were treated with 5 ng/ml TGF β1 for 8 h prior to fixation (+TGFβ). Paxillin foci were quantified on a per cell basis by image analysis (means, n = 3, ±S.E.M., *P < 0.05, two-tailed t-tests vs. siCtrl). h A549 WT or ΔRELB cells, with (+) or without (−) prior treatment with 5 ng/ml TGFβ1 for 16 h, were immunoblotted as shown (P-SMAD2 = phospho-S465/467 SMAD2, P-SMAD3 = phospho-S423/425 SMAD3). Uncropped blots are available in Supplementary Fig. 10