Fig. 5 | Oncogene

Fig. 5

From: HDAC7 regulates histone 3 lysine 27 acetylation and transcriptional activity at super-enhancer-associated genes in breast cancer stem cells

Fig. 5

Long-term HDAC1/3 double knockdown maintains HDAC7 downregulation while HDAC7 knockdown represses SCTFs in stem-like BrCa cells. a Long-term HDAC7 knockdown (5.8- to 11.2-fold) with siH7 resulted in decreased HDAC1 (1.7-fold), VDR (1.9-fold), and c-MYC (2.7-fold) mRNAs in CSC-like BPLER2 (black bars) but not in nsTC-like HMLER2 (gray bars) cells. In contrast, HDAC1 knockdown with siH1 in either cell line (55.6- and 34.5-fold) did not significantly change c-MYC or HDAC7 mRNA levels while increasing EZH2 and VDR transcripts (3.3- and 1.8-fold) in BPLER2 cells after 6 days and two rounds of transient siRNA transfection. Transcript levels were normalized to ACTB and B2M and expressed relative to values in nontargeting (NT) siRNAs control. Results of real-time quantitative RT-PCR from three independent repeats were plotted in a bar chart using logarithmic scale. Error bars represent standard deviation. b Long-term transient transfections with siH1, siH7, and NT siRNAs as control resulted in stable HDAC1 and HDAC7 knockdown in BPLER2 and HMLER2 cell populations. In HDAC7-depleted BPLER2 cells, there was significant reduction of c-MYC and CD44, alterations in VDR isoforms and p27Kip1 upregulation. HMLER2 cells revealed only minimal or no change in c-MYC, CD44, VDR, EZH2, or p27Kip1 protein levels after second run of transfection with siH1 and siH7, which is consistent with a lack of MS-275 effect on these cells. c Long-term HDAC1 and HDAC3 double knockdown by combined suboptimal siH1 and siH3 concentrations resulted in reduced HDAC7 protein levels in CSC-like BPLER cells, which is also accompanied by a decrease in c-MYC and CD44 protein levels and change in VDR isoforms expression when compared with siH1/NT and siH3/NT siRNA combinations. Immunoblots of whole cell lysates were probed with the indicated antibodies, ACTB was used as loading control

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