Fig. 2. ALDH1 activity mediates AURKA-induced self-renewal capacity.

a Immunoblot assay showing expression of TGβR1, TGβR2, SMAD3 (total and phosphorylated), and AURKA (total and phosphorylated) proteins in MDA-MB 231 and SUM149-PT cells. Densitometry analysis showing the fold change of protein levels in MDA-MB 231 and SUM149-PT cells normalized to α-Tubulin. b SUM149-PT cells were treated with 10 ng/ml TGF-β1, lenti-shRNAs, or alisertib. After 48 h incubation, ALDH1 activity was detected with Aldefluor kit and measured by FACS analysis on 10,000 events. ALDH1 inhibitor DEAB was used as the control for each sample. c Graph showing the average of ALDH1^high cells from three independent experiments (±s.d.). d SUM149-PT cells were cultured under non-adherent conditions for 24 days (three serial passages) to form tertiary mammospheres (MPS). In total, 1000 SUM149-PT cells derived from tertiary MPS were then infected with scrambled or AURKA lenti-ShRNAs and were incubated for 8 days to monitor MPS growth. Graph showing the average of ALDH1^High cells from three independent experiments (±s.d.). e FACS sorting analysis was performed on SUM149-PT secondary MPS to separate ALDH1^high from ALDH1^low cells. 10,000 ALDH1^high and ALDH1^low cells were then cultured for 8 days under non-adherent conditions to form tertiary MPS. Cancer cells were labeled with 5 μM Cell Tracker Red CMTPX (Thermo Fisher Scientific, #C34552) for 1 h and the MPS area was measured using the NIH Image-J software. Graph showing the average of ALDH1^High and ALDH1^low MPS area from three independent experiments (+/− s.d.). f GFP-tagged SUM149-PT cells were cultured under non-adherent conditions for 24 days (three serial passages) to form tertiary mammospheres (MPS). In total, 10,000 SUM149-PT cells derived from tertiary MPS were then treated with DMSO (control) or 1 μM A37 (ALDH inhibitor) for 8 days. MPS area was measured using the NIH Image-J software. Graph showing the average of MPS area from three independent experiments (±s.d.).