Figure 4. The inhibition of autophagy initiation by 3-MA rescues the metastatic potential of the Gαh-silenced MDA-MB231 cells in vitro and in vivo. (A) The results from the Western blot analysis for the LC3-I/II, p62 and GAPDH proteins derived from MDA-MD231 cells without (NS) or with Gαh knocked down (KD) in the absence or presence of the autophagy inhibitor 3-MA (3 or 10 mM). GAPDH was used as an internal control of protein loading. The protein intensities of representative blots from three independent experiments were normalized by GAPDH levels and presented as a ratio to the control group. (B–C) Giemsa staining (B) and cell number (C) of the invaded MDA-MD231 cell variants shown in A. Data obtained from three independent experiments are presented as the mean ± SEM. Letters indicate the significant differences at p<0.01 analyzed by nonparametric Friedman test. (D and E) H&E stained lung tissues (D) and the number of lung tumor colonies tumors (E) derived from the mice (n=5) transplanted with MDA-MD231 cell variants, shown in A, through tail vein injection for 4 weeks. Tumors are shown in red circles. Statistical significance was determined by nonparametric Mann-Whitney U test.