CQ increased the expression of LC-3 and p62 in GBC cells after treatment with 5-FU. (A) Time course detection of LC-II and p62 following treatment of GBC cells with 5-FU for 24 hours or 48 h by western blot. The lower panels represent the densitometric values obtained from the bands of the western blot (*, p < 0.05 vs. control, n = 3). (B) Representative electron microscopy images of control SGC-996 cells (upper left), 5-FU-treated (5 μM) for 24 hours (upper middle) or 48 hours (upper left). Obvious autophagic vacuoles were highlighted by white arrows in higher magnification (lower), with typical double-layer membrane containing organelle remnants present in 5-FU treated cells rather than untreated cells. (C) Western blot analysis of LC-II and p62 from lysates of GBC cells treated with 5-FU alone for 48 h or after 12 hours pre-treatment with CQ. GAPDH was used as a loading control and the expressions of autophagy-related proteins (LC3-II, p62) were quantified (*,p < 0.05 vs. control, n = 3). (D) SGC-996 cells were transfected with vectors expressing either GFP or GFP-LC3, followed by pre-treatment of 100 μM CQ and/or 5 μM 5-FU as described. The GFP or GFP-LC3 staining patterns were analyzed by fluorescence microscopy. The GFP control cells display diffuse GFP distributed throughout the cytoplasm, coincident with GFP-LC3 patterns of SGC-996 vehicle control cells (lower left panels). Both 5-FU and CQ (lower middle panels) induced punctuate patterns in GFP-LC3 patterns, while the latter was more bright and clear. 5-FU combined with CQ (lower right panels) showed a similar diffuse GFP-LC3 but notably bright punctuate patterns. Images are representative of at least three independent experiments and quantification of green dots is shown in graph as mean ± SD (*,p < 0.05, n = 5, Scale bar = 10 μm).