Figure 3

Autophagy was induced by rhArg in H1975 cells. (a) H1975 cells were incubated with or without 2 U/ml of rhArg for 24 h and TEM was employed to detect the autophagosomes, and the magnified view of the electron photomicrograph exhibited autophagosomes. The left and middle micrographs were taken at × 5000, whereas the right was taken at × 15 000. (b) After stained with Cyto-ID Green dye, the formation of autophagic vesicles in H1975 cells treated with 2 U/ml of rhArg for 24 h was found through laser confocal microscopy. The autophagy inducer rapamycin (50 nM) was chosen as a positive control. Green (Cyto-ID) dots in cells were counted by the ImageJ software (n=3, means±S.D.). Confocal micrographs were taken at × 40. (c) H1975 cells were incubated with different concentrations of rhArg for 24 h, then autophagy associate protein LC3-I/II and p62 was detected by western blot analysis. Densitometric values were quantified using the ImageJ software and normalized to control. The values of control were set to 1. The data are presented as means±S.D. of three independent experiments. (d) H1975 cells were incubated with 2 U/ml of rhArg for different time, then autophagy associate protein LC3-I/II and p62 were detected by western blot analysis. Densitometric values were quantified as described in c