Mouse aortic smooth muscle cells were transfected with pre-designed Dharmacon siRNA smart pools targeting Brg1 or Brm, as well as a control siRNA, at final concentration of 50nM. 36 h after transfection, mRNA was harvested and the levels of Brg1, Brm and smooth muscle marker genes were measured by quantitative real time RT-PCR as indicated in each panel. Data presented are the mean±SEM of 6 samples from 2 independent experiments and expression levels were normalized to an hprt internal control and are expressed relative to levels in siRNA control samples (set to 1). * Indicates statistical significance as determined by a student T test (P<0.05).

SW13 cells grown in 6-well plates were co-transfected with expression plasmids (1µg) encoding either wild type Brg1 (WT) and Brm, DN-Brg1 (DN), CT-truncated Brg1 or Brm (ΔCT) or bromodomain mutants (BD) of Brg1 and Brm, together with myocardin expression plasmid (1µg, except in the absence of the Brg/Brm plasmid where 2µg were used), as indicated at the top of the panel (-, empty expression plasmid). 36 hours after transfection protein expression was analyzed by Western blotting with the indicated antibodies. Exogenous Brg1 and Brm were detected with anti-Flag antibodies (Flag) and exogenous smooth muscle myocardin was detected with an omni-epitope tag antibody (Myo(Omni)). 30µg of protein were loaded in each lane. The blots shown are representative of data obtained from 2 separate experiments. The Brm WT sample shown in panel ‘A’ is the same sample as shown in the first WT lane of panel ‘B’. Similarly the WT Brg1 samples shown in panel ‘B’ are the same samples as those shown in panel ‘A’.

A. COS cells were transduced with HA-tagged myocardin and HA-tagged SRF adenovirus. After 24 hours, nuclear protein was harvested and proteins were immunoprecipitated using Brg1, SRF or control IgG antibodies, as indicated. Immunoprecipitated proteins were identified by Western blotting using antibodies against Brg1 or the HA-epitope tags on myocardin and SRF as indicated at the right of the blot. B. A10 SMCs were transduced with omni-tagged myocardin for 24 hours. Nuclear protein was harvested and subsequently immunoprecipitated using myocardin or control IgG antibody. C. SRF, myocardin, and Brg1 or Brm (D) were transcribed and translated in vitro, the expressed proteins were then incubated together (SRF+Brg1/Brm or myocardin +Brg1/Brm) as indicated at the top of the blots. Protein mixtures were immunoprecipitated with myocardin, SRF or IgG control antibodies, as indicated below the blots. The precipitated proteins were analyzed by Western blotting, using antibodies indicated at the right of the blots. On all blots ‘input’ lanes represent 10% of the inputs that were mixed together and used for immunoprecipitates.

A. 10T1/2 cells were transduced with DN-Brg1 or control YFP adenovirus together with myocardin (solid bars) or YFP (open bars) adenovirus. 48 hrs following transduction total RNA was harvested and analyzed by qRT-PCR. Transcript levels was firstly normalized to hprt internal loading control and then normalized to their respective YFP control group. RQ=2^−ΔΔCt and ΔΔC_t = (C_{t experimental} - C_{t hprt})- (C_{t control} –C_{t hprt}). Data presented are the mean±SEM of 6 samples obtained from 2 independent experiments. B. Primary mouse aortic smooth muscle cells were transduced by DN-Brg1 or YFP control adenovirus with or without myocardin adenovirus as described in “A” (transduction efficiency of these cells was approximately 50–60%). 36–48 hours after transduction, cells were lysed, mRNA harvested and transcript levels analyzed as described above. Data presented are the mean±SEM of 3 samples obtained from one experiment. Similar results were obtained in a replicate experiment.

A–B. B22 cells (an NIH3T3 cells line that inducibly express DN-Brg1 in response to tetracycline withdrawal13) grown in the presence (control) or absence (induced DN-Brg1) of tetracycline were transduced with myocardin or YFP control adenovirus. After 30 hrs, cells were fixed and harvested for chromatin immunoprecipitation assays. Chromatin was precipitated using an antibody against SRF or using IgG negative control. The precipitated genomic DNA was purified and the presence of the promoters of SRF-dependent genes measured by real time PCR using gene specific primers8. A. The increase in SRF binding in samples transduced with myocardin is indicated relative to those transduced with YFP. These data were calculated and normalized to input levels as follows: Relative SRF binding=2^−ΔΔCt, with ΔΔC_t= (C_{t myocardin}-C_{t input})-(C_tYFP-C_{t input}). B. The relative inhibition of myocardin induced SRF binding by DN-Brg1 is shown. This was calculated as follows: Relative SRF binding= 2^−ΔΔCt, with ΔΔC_t= (C_{t DN-Brg1+Myocardin} -C_{t input})-(C_{t YFP+Myocardin} -C_{t input}). Data shown in panels ‘A’ and ‘B’ are the mean±SEM of 7 samples obtained from 3 independent experiments. A one-way t-test was performed and the asterisks indicate the results that are statistically different from 1 (P<0.05). C–D. Primary colon smooth muscle cells were prepared from 4 week old mice. The cells were transduced by DN-Brg1 or YFP control adenovirus. After 36 hours, cells were fixed and harvested for chromatin immunoprecipitation assays as above. C. The relative Brg1 binding to several SRF dependent genes in control primary smooth muscle cells is shown. The Brg1 binding to promoters were normalized to IgG control. The relative Brg1 binding was calculated as RQ=2^−ΔCt, with ΔC_t= C_{t Brg1}-C_{t IgG}. D. The relative inhibition of SRF binding by DN-Brg1 is shown. The inhibition of SRF binding by DN-Brg1 is calculated as, RQ=2^−ΔΔCt, with ΔΔC_t= (C_{t DN-Brg1}-C_{t input})-(C_{t YFP}-C_{t input}). Data presented are the mean ±SEM of 4 samples.