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. 2019 Jan 28;30(8):1043–1061. doi: 10.1089/ars.2017.7023

FIG. 2.

FIG. 2.

FPN overexpression induces autophagy. (A) Western blot of LC3B-I and LC3B-II in three independent infections (a–c) of LNCaP cells containing a control vector (Vec) or FPN OE. (B) Western blot of LC3B-I and LC3B-II in LNCaP cells expressing doxycycline-inducible FPN (Tet-FPN). Cells were untreated or treated with 1 μg/mL doxycycline for 12, 24, 48, or 72 h. (C) Western blot of LC3B-I and LC3B-II in LNCaP and PC3 cells untreated or treated with 100 μM DFO for 12, 24, or 48 h. (D, E) Ratio of mCherry/EGFP fluorescence intensity in cells expressing an mCherry-EGFP-LC3B reporter as determined by flow cytometry in (D) LNCaP (Tet-FPN) and (E) PC3 (Tet-FPN) cells treated ± 1 μg/mL doxycycline for 3 days (D top and E) or 4 days (Panel D bottom). Data were analyzed with the FlowJo software (TreeStar, Inc.). (F) Western blot of LC3B-I and LC3B-II in C4-2 (Tet-FPN) cells untreated or treated with 1 μg/mL doxycycline for 6, 12, 24, or 48 h. (A–C, F) GAPDH was used as a loading control. Experiments were repeated at least three times. Uncropped blots are shown in Supplementary Figure S3. DFO, desferoxamine, an iron chelator; EGFP, enhanced green fluorescent protein; LC3B-I, microtubule-associated protein light chain 3 beta; LC3B-II, phosphatidylethanolamine-conjugated microtubule-associated protein light chain 3 beta.