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. Author manuscript; available in PMC: 2020 Dec 21.
Published in final edited form as: Oncogene. 2020 Jun 21;39(31):5390–5404. doi: 10.1038/s41388-020-1370-9

Figure 6: Androgen-induced autophagy requires integrin α6 and Bnip3.

Figure 6:

A) Laminin-adherent C4–2 cells stably expressing an shRNA targeting integrin α6 (shITGα6) or a control scrambled shRNA (shScrm) treated with (+) or without (-) 10nM R1881 for 24 h in the absence (-) and presence (+) of 100ng/mL BafA1 during the last 2 h. Levels of Bnip3, LC3B-I/II, and tubulin as assessed by immunoblotting. B) Laminin-adherent C4–2 cells transfected with siRNA targeting Bnip3 (siBnip3) or a control scrambled siRNA (siScrm) treated with (+) or without (-) 10nM R1881 for 24 h in the absence (-) or presence (+) of 100ng/mL BafA1 during the last 2 h. Levels of Bnip3, LC3B-I/II, and GAPDH as assessed by immunoblotting. C) Laminin-adherent C4–2 cells stably expressing Tet-inducible shRNA targeting Bnip3 (Tet-shBnip3) were treated with (Dox) or without (Veh) 100ng/ml doxycycline for 48 h and then treated with (+) or without (-) 10nM R1881 for 24 h in the absence (-) or presence (+) of 100ng/mL BafA1 during the last 6 h. Levels of Bnip3, LC3B-I/II, and β-actin as assessed by immunoblotting. D) Laminin-adherent C4–2 cells stably expressing Tet-inducible shRNA targeting Bnip3 (Tet-shBnip3) were treated with (Dox) or without (Veh) 100ng/ml doxycycline for 48 hours, and then treated with (+) or without (-) 10nM R1881 for 24 h and in the absence (-) or presence (+) of 100ng/mL BafA1 during the last 6 h. Endogenous LC3-positive puncta were visualized by immunostaining and quantified. ***p<0.005, n=100 cells, error bars = SD. E) Laminin-adherent C4–2 or C4–2B cells treated with (+) or without (-) 10nM R1881 for 24 h. Levels of activated (P-AMPK T172), total AMPK, activated (P-ULK S555), and total ULK as assessed by immunoblotting. F) Laminin-adherent C4–2 cells stably expressing Tet-inducible shRNA targeting Bnip3 (Tet-shBnip3) treated with (Dox) or without (Veh) 100ng/ml doxycycline for 48 hours and then stimulated with (+) or without (-) 10nM R1881 for 24 h in the presence (+) or absence (-) of 100ng/mL BafA1 during the last 6 h. Levels of activated ULK (P-ULK-S555), total ULK, and β-actin as assessed by immunoblotting. G) C4–2 cells adherent to laminin treated with 10nM R1881 were further treated with increasing concentrations of NAC. Levels of activated AMPK (P-AMPK-T172) and tubulin as assessed by immunoblotting.