Fig. 4.

cirSMARCA5 blocks the transcription of SMARCA5 and promotes the generation of a truncated SMARCA5 protein (ΔSMARCA5). a Schematics of luciferase reporter constructs containing the SMARCA5 exon sequence as indicated (upper). The SMARCA5 exon 15-16 sequence plays an important negative role in mediating the effect of circSMARCA5 overexpression on luciferase activity (lower). b Schematics of fluorescence reporter constructs containing the SMARCA5 exon sequence as indicated (upper). MCF-7 cells were transiently transfected with these fluorescence reporters along with or without circSMARCA5 co-overexpression. After transfection for 48 hours, the reporter transcription activities were measured by flow cytometry assay. c circSMARCA5 overexpression downregulated the protein levels of SMARCA5 while upregulating truncated SMARCA5 (ΔSMARCA5) protein levels. MCF-7 cells stably overexpressing circSMARCA5 or control cells were treated with DMSO or MG132. Western blot analysis was performed using an antibody targeting the N-terminus of SMARCA5 to evaluate the expression of SMARCA5 and ΔSMARCA5. GAPDH was used as an internal control. d The ΔSMARCA5 protein was identified by mass spectrometry, and detected SMARCA5 peptides were showed in the map. The red-labeled portion is the amino acid sequence of the translated defective transcript. e MCF-7 cells expressing Flag-SMARCA5 and Flag-ΔSMARCA5 were treated with cycloheximide (CHX, 50 μg/ml). The cell lysates were subsequently harvested at sequential time points (0, 0.5, 1, 2, 4 or 8 h) after treatment, and then the cell lysates were immunoblotted with anti-Flag or anti-Actin antibody