Fig. 4.
Reducing HSPB8 in MCF7-G11TamR leads to autophagy. (A) Protein expression of HSPB8 was reduced with three of four different targeted hairpins in MCF7-G11TamR compared with vector-expressing scrambled sequences. (B) Silencing HSPB8 reduced cell proliferation of MCF7-G11TamR. The effect was more pronounced in the presence of 4-OHT (1 μM). Resazurin data were normalized against scramble in the presence of puromycin and in absence of 4-OHT after 48 h postdrug treatment (**P < 0.001). (C) Hairpins HSPB888 and HSPB895 reduced cell proliferation compared with scramble in two additional tamoxifen-resistant subclones, MCF7-H9TamR and T47D-G9TamR, in the presence of 4-OHT (**P < 0.001). (D) Immunofluorescence images after silencing HSPB888 in all resistant subclones showed the characteristic redistribution of anti-MAP LC3 into punctuate green structures (B, F, and J, respectively), indicating active autophagy, but scrambled hairpin showed only diffuse staining (A, E, and I). Blue: nucleus by Hoechst; red: actin by Phalloidin; green: autophagy marker anti-MAP LC3. (Scale bars 50 μM.) (E) EM consistently identified a large number of autophagosomes containing organelles undergoing degenerative changes (arrows) in all of the cells where HSPB8 was silenced (D, H, and L). (Scale bars, 500 nm.)