Fig. 3

Impact of glutamine on A549 cells clonogenic capacity and the expression of stem-cell associated molecules. a-b A549 cells were cultured in RPMI 1640 medium with or without glutamine or incubated without or with 1 U/ml L-Asparaginase. Colony numbers were counted after 2 weeks of culturing. The quantitative results of 3 independent experiments are shown in bar graphs showing mean ± SD. Images of representative colonies formed were shown in the lower panels. c Representative photographs of A549 cells cultured in medium with or without glutamine for 72 h, original magnification is 400 ×. d Effect of glutamine on expression of genes ABCG2 and SOX-2. A549 cells were cultured in RPMI 1640 with or without glutamine (300 mg/L) for 48 h, and RNA was isolated for real-time RT-PCR for detection of SOX-2 and ABCG2 expression. β-actin was used as an internal control for normalization. e Western blot analysis of ABCG2 and SOX-2. A549 cells were cultured in RPMI 1640 medium with or without glutamine (300 mg/l) for 72 h, and cell lysates were subjected to western blotting to measure the expression of ABCG2, SOX-2 and β-actin. f Effect of L-Asparaginase on expression of genes ABCG2 and SOX-2. A549 cells were cultured in the absence or presence of 1 U/ml L-Asparaginase for 72 h, and RNA was isolated for real-time RT-PCR analysis of SOX-2 and ABCG2. β-actin was used as an internal control. g A549 cells were cultured in RPMI 1640 medium without or with L-Asparaginase (1 U/ml) for indicated time, and cell lysates were subjected to western blotting to measure the expression of ABCG2 and SOX-2. (H, I) Effect of glutamine and L-Asparaginase on ABCG2 expression. A549 cells were cultured in RPMI 1640 medium with or without glutamine (h) or incubated without or with 1 U/ml L-Asparaginase (i) for the indicated time, and cells were collected and incubated with anti-ABCG2 antibody, detection of membrane protein ABCG2 was measured by flow cytometry analysis. Each Bar represents the mean ± SD of the relative fluorescence intensity from 3 independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001; Gln, glutamine; L-ASP, L-Asparaginase