Figure 5. Pharmacological STAT3 inhibition does not inhibit IL-6-induced LC3B accumulation in C4-2 or C4-2B cells. (A) C4-2B cells were treated with 50 ng/ml IL-6, or vehicle control, in the presence of 0–30 μM Cpd188 for 3 d. Total protein was isolated and analyzed by western blot and normalized to β-actin loading control. Total LC3B and phospho-STAT3 accumulation were normalized to β-actin (LC3B/β-actin, pSTAT3/β-actin). LC3B and phospho-STAT3 levels were calculated relative to the vehicle control, 0 μM Cpd188. (B) C4-2 (top) and C4-2B (bottom) cells were treated with 50 ng/ml IL-6 for 3 d (first panel) or with 50 ng/ml IL-6 for 1 d, followed by treatment with 30 μM Cpd188 for an additional 2 d (second panel). (Third panel) One day after the IL-6 + 30 μM Cpd188 treatment, the media was removed and replaced with fresh, untreated media. Cells were imaged using brightfield (20× magnification, scale bar = 100 μm). Arrows points to examples of cell process extension/branching. (C and D) C4-2 and C4-2B cells were treated with 50 ng/ml IL-6 ± 30 μM Cpd188 for 1 d. (C) Western blot, three biological replicates were analyzed by western blot. Total LC3B and phospho-STAT3 were normalized to β-actin and levels calculated relative to the first biological replicate of the 0 μM Cpd188 control. LC3B, p value = 0.04 (C4-2) or 0.90 (C4-2B), phospho-STAT3, p value = 0.05 (C4-2) or 0.006 (C4-2B). (D) Image, C4-2 (top) and C4-2B (bottom) cells were imaged using brightfield (20x magnification, scale bar = 100 μm). Arrows point to examples of cell process extension/branching. For each treatment, the percentage of cells displaying cell process extension and/or branching was determined for 3–5 microscopy fields, n = 121–250 total cell count, and percentage is denoted on the image.