Figure 2
Knockdown of Hsp27 decreased BCSCs of breast cancer cells. (A) AS-B145 cells were transfected with Hsp27 siRNA (si-Hsp27) or negative control siRNA (ctrl-siRNA) for 48 h and detected a ALDH+ population by ALDEFLUOR assay. The percentage of ALDH+ cells were calculated from samples without DEAB treatment (w/o DEAB) after gating with a cutoff line which was set according to DEAB treated (w/DEAB) cells. The quantitative results were presented as relative percentage of ctrl siRNA transfected cells. *, P < 0.05. (B) Primary (1st) mammosphere formation capacity of AS-B145 or AS-B244 cells was also analyzed after knockdown of Hsp27 for 48 h. For secondary (2nd) mammosphere cultivation, 1st spheres were collected, digested with accutase and subjected to mammosphere cultivation at 48 h post-transfected with si-Hsp27 or negative control siRNA (ctrl-siRNA). Data were collected at Day 7 post starting the cultivation and presented as relative percentage of ctrl siRNA. Bar, 100 μm. #, P < .01; *, P < 0.05. (C) The tumorigenicity of AS-B145 sphere cells after knockdown of Hsp27 (si-Hsp27) or negative control siRNA (ctrl-siRNA) was determined by xenograftment assay in NOD/SCID mice. The indicated numbers of cells were injected into mammary fat pads for 44 days and the tumor formation was monitored weekly. The cancer stem cell (CSC) frequency was calculated with ELDA software [15]. *, P < 0.05. ALDH, aldehyde dehydrogenase; BCSCs, breast cancer stem cells.