Figure 6.
Allelic loss of beclin1 promotes drug resistance mediated by gene amplification in vitro. (A,B) Quantitation with standard deviation (SD) of PALA-resistance for beclin1+/+ and beclin1+/− iMMECs with Bcl-2 treated with PALA at 5× LD50. The P-value was calculated by unpaired t-test. (C) PCR of genomic DNA from several stable cell lines derived from independent PALAR colonies using primers for an 800-bp fragment of the CAD gene. (D) beclin1+/+ and beclin1+/− iMMECs with Bcl-2 (WT3.B3, BLN2.B4) and stably expressing EGFP-LC3 after 0, 24, and 48 h of PALA treatment at LD50. (Left panel) Fluorescence (F). (Right panel) γ-H2AX IF. (E) WT3.B3 and BLN2.B4 stably expressing EGFP-LC3 following 0, 24, and 48 h of PALA treatment at LD50. (Left panel) Quantitation of autophagy (percentage EGFP-LC3 translocation). (Right panel) Quantitation of γ-H2AX activation (percentage γ-H2AX-positive cells as number of cells with bright red nuclei/total number of cells). Three independent experiments were performed, and the mean values with standard deviation are presented. P-values were calculated by unpaired t-test. (F) Western blot analysis of γ-H2AX, GRP-78, and actin in WT3.B3 and BLN2.B4 stably expressing EGFP-LC3 following 0, 24, and 48 h of PALA treatment at LD50.