1. Effects of MAX reconstitution and of BRG1 depletion in lung cancer cells.
Effects of MAX reconstitution and of BRG1 depletion in lung cancer cells. A, top, schematic depiction of the experimental design using different lentiviral constructs expressing human MAX, and the shRNA targeting BRG1 (shBRG1). Control (∅) and scramble RNAs are also shown. The shBRG1 used were validated in our previous study (16). Bottom, Western blot analyses, from total lysates, depicting MAX and BRG1 in the indicated cells. Tubulin is shown as a loading control. B, left, cell proliferation measured using MTT assays. Lines, the number of viable cells relative to the total number of cells at 0 hours. Error bars, SD. ***, P < Right, images of the MTT assays. C, schematic representation of the 5′-UTR–MAX construct. The putative glucocorticoid receptor–binding region is highlighted in green. D, Western blot analysis, from total lysates, depicting the ectopic expression of MAX from the 5′-UTR–MAX construct and from that lacking the 5′-UTR (MAX) in cells cultured in hormone-free (HF) medium or at the indicated glucocorticoid (GC) concentrations. E, Western blot analysis, from total lysates, showing the levels of ectopic expression of MAX and coexpression of MAX–shBRG1 and 5′-UTR–MAX/shBRG1 in the indicated cell lines. F, reduction of levels of endogenous MAX, after depletion of BRG1, in one MYC-amplified lung cancer cell line (H460) and in the neuroblastoma-derived SHSY-5Y cells, treated with 2 μmol/L of glucocorticoid. In the latter, two shBRG1s (#1 and #4) have been used. G, ChIP of BRG1 in the indicated cells after inducing BRG1 expression with doxycycline, followed by qPCR to determine DNA enrichment in the MAX promoter, relative to the input. The bars represent the data for the BRG1 ChIP in H1299tr-BRG1wt and the H1299tr-BRG1mt cells, as indicated. Error bars, SDs of three replicates. Under the graph there is a schematic representation of the region screened and the position (in bp) of each amplicon (vertical lines) relative to the ATG (+1). TSS, transcription start site. The bottom corresponds to the 2% agarose gel of the qPCR of the top, shown for comparison.
Octavio A. Romero et al. Cancer Discovery 2014;4:
©2014 by American Association for Cancer Research