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. 2020 May 25;123(3):449–458. doi: 10.1038/s41416-020-0885-8

Fig. 4. Effects of proteasome and autophagy on GMI-inhibited CD133 expression.

Fig. 4

a Total cell lysates of A549/A400 cells (2 × 105 cells of 60 mm dish) after co-treating with GMI and chloroquine for 24 h were analysed by Western blot assay. b A549/A400 cells (2 × 105 cells of 60 mm dish) were treated with MG132 for 6 h after treating with GMI for 18 h. Western blot assay was performed to detect the expression of CD133. c After pre-treating with 3-MA, A549/A400 cells (2 × 105 cells of 60 mm dish) were treated with GMI for 24 h. The total protein was analysed by Western blot assay. d Total protein from A549/A400 shLuc and A549/A400 shATG5 cells was analysed by Western blot and detected by CD133, ATG5, LC3B, and β-actin antibodies. β-actin served as a loading control. Software ImageJ was used to quantify the band intensities of CD133, LC3B-II, and ATG5. Data shown are the relative expression standardised by the β-actin protein level. The ratio without treatment in A549/A400 or A549/A400 shLuc cells was set at 1. e After co-treating with GMI and chloroquine for 24 h, total cell lysates of A549/A400 cells (6 × 105 cells of 100 mm dish) were used to investigate the interaction between CD133 and autophagosome protein (p62 and LC3B) by co-IP.