Figure 6

Knockdown of Hsp27 decreased the activity of NF-κB in BCSCs. (A) AS-B145 or ALDH+ AS-B244 cells were transfected with negative control siRNA (Ctrl) or Hsp27 siRNA (KD) for 24 h or 48 h and total proteins were harvested to detect the expression of IκBα, phosphor-IκBα (p-IκBα), Hsp27 and β-actin by Western blot. Inserted values indicated relative proteins expression in comparison with ctrl siRNA. (B) AS-B145 or ALDH+ AS-B244 cells were transfected with negative control siRNA (Ctrl) or Hsp27 siRNA (KD) for 48 h and nuclear (Nuc) and cytosolic (Cyto) proteins were isolated to detect NF-κB, histone H1, Hsp27 and β-actin by Western blot. Inserted values indicated relative proteins expression in comparison with ctrl siRNA. (C) AS-B145 or ALDH+ AS-B244 cells were co-transfected with NF-κB reporter vector, Renilla luciferase vector and negative control siRNA (ctrl-si)/or Hsp27 siRNA (si-Hsp27) for 48 h. Luciferase activity was determined by dual luciferase assay. Data were presented as relative luciferase activity of ctrl siRNA. *, P < 0.05. (D) AS-B145 or AS-B244 cells were treated with JSH-23 (20, 10 or 5 μM) or 0.1% DMSO for 48 h and ALDH+ population were determined by ALDEFLUOR assay. Data were presented as relative percentage of DMSO. *, P < 0.05; #, P < 0.01. ALDH, aldehyde dehydrogenase; BCSCs, breast cancer stem cells; NF-κB, nuclear factor kappa B