Figure 3

Rottlerin induced autophagy in breast CSCs. (a) Breast CSCs were grown in complete stem cell culture medium and treated with Rott (2 μM) for 0, 24, 48 and 72 h. Representative blots showing the concentration dependent effect of Rott on breast CSCs. Rott regulates autophagy-related genes in breast CSCs. Conversion from LC3-I to LC3-II by Rott. The Western blot analysis was performed to measure the expression of LC3. β-actin was used as a loading control. (b) Breast CSCs were grown in complete stem cell culture medium and treated with Rott (0, 0.5, 1 and 2 μM) for 48 h. The Western blot analysis was performed to measure the expression of Atg12, Beclin-1, LC3 and β-actin. (c) Breast CSCs were pre-incubated with Baf (10 nM) for 2 h, followed by treatment with Rott (2 μM) in complete stem cell culture medium for 48 h. The Western blot analysis was performed to measure the expression of p-AMPK, AMPK, Atg12, Beclin-1, LC3 and β-actin. (d) Breast CSCs were pre-incubated with CHX (10 μg/ml) for 2 h, followed by treatment with Rott (2 μM) for 48 h. The Western blot analysis was performed to measure the expression of p-AMPK, AMPK, Atg12, Beclin-1, LC3 and β-actin. (e) Breast CSCs were pre-incubated with 3-MA (10 mM) for 2 h, followed by treatment with Rott (2 μM) for 48 h. The Western blot analysis was performed to measure the expression of p-AMPK, AMPK, Atg12, Beclin-1, LC3 and β-actin. (f) Atg7 or Beclin-1 shRNA inhibits Rott-induced autophagy. pEGFP-LC3-positive breast CSCs were transduced with scrambled, Atg7 shRNA or Beclin-1 shRNA and treated with Rott (2 μM) for 24–48 h. Autophagy was measured as described in Figure 2. Data are reported as the mean ± standard error (SE). n = 5, *P < 0•05.