Figure 1. HS5 BMSC CM induces accumulation of LC3B and GFP-LC3B in C4-2 or C4-2B bone metastatic PCa cells. (A) C4-2 or C4-2B cells were treated with conditioned media (CM) for 1–3 d. Total protein was isolated from cells and analyzed by western blot for LC3B accumulation. β-actin is a loading control. Densitometry was used to measure protein band intensity. Total LC3B (LC3B-I and LC3B-II) accumulation was normalized to β-actin (LC3B/β-actin) and LC3B levels were calculated relative to the PCa CM control. (B and C) C4-2 and C4-2B cells transiently expressing GFP-LC3B or GFP-LC3B (G120A) transgenes were treated with CM for 2 d. (B) Images, C4-2 (left) and C4-2B (right) were imaged using fluorescence microscopy (FITC, 40x magnification, scale bar = 50 μm). (C) Graph, the percentage of cells with GFP-LC3 puncta was calculated for 3–10 microscopy fields, n = 183–820 total cell count. (D) C4-2 or C4-2B cells were treated for 2 d with CM ± 20 μM chloroquine (CQ) and total protein was analyzed by western blot for LC3B accumulation.