Figure 6: DNA repair-dependent autophagy induction upon 5-FU treatment in human cells.

(a) Western blots showing autophagy induction by monitoring LC3 levels in whole-cell extracts from U2OS cells treated or not with 5-FU alone or in combination with Mx for the indicated time. Staurosporin was used as a positive control for apoptosis induction shown by the appearance of cleaved fragments of caspase-3 (17 and 19 kDa) and poly (ADP-ribose) polymerase 1 (PARP1; ΔPARP, 85 kDa). α−Actin was used as loading control. (b) Quantification of LC3-II levels in cells treated as in a. Intensities of the lower LC3 band (LC3-II) was normalized to those of actin. (c) Autophagy induction demonstrated by accumulation of LC3-positive puncta in U2OS cells treated or not with 5-FU alone or in combination with Mx for 48 h. The cells were fixed and prepared for immunofluorescence of LC3 (green). DAPI staining is shown in blue. Scale bar, 20 μm. (d) Quantification of LC3-positive puncta in cells treated as in c. In each separate experiment, 200–1,600 cells were scored per condition. (e) Western blots showing absence of LC3-II accumulation in U2OS cells transfected with siRNA against MSH-2. The same blot probed with anti-MSH-2 antibodies confirm efficient knockdown. (f) Quantification of LC3-II levels in cells transfected with control or MSH-2 siRNA with or without FU treatment for 24 h. (g) Immunofluorescence showing reduced accumulation of LC3 puncta in U2OS cells transfected with siRNA against MSH-2. (h) Quantification of LC3-positive puncta in cells treated as in g. (i) Quantification of normalized RPS3 protein level (black bars) or RPS3 mRNA level (white bars) in U2OS cells after 48 h 5-FU treatment. RPS3 mRNA levels were determined by quantitative real-time reverse transcriptase–PCR. Graphs (b,d,f,h,i) show mean values±s.e.m. quantified from at least three independent experiments. Student’s t-tests were performed to assess the significance between treatment groups in all panels as indicated; *P<0.05; **P<0.01.