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. 2018 Feb 12;8(7):1808–1823. doi: 10.7150/thno.23177

Figure 6.

Figure 6

PHGDH contributes to acquired resistance of erlotinib by regulating the transcripts associated with DNA damage repair and nucleotides metabolism in NSCLC cells. (A) RNA-Seq analysis. The transcripts were differentially regulated in PC9ER4 cells and PC9 cells after 20 nM siPHGDH#4 transfection for 72 h. (B) The KEGG pathway cluster analysis based on the 1011 genes significantly changed in PC9ER4 cells as described in (A). (C) Immunoblotting of γH2AX in the indicated cells after 20 nM siPHGDH#4 transfection for 72 h. M, negative siRNA as mock control; #4, siPHGDH#4. (D) Representative immunofluorescence staining. PC9ER4 cells and PC9 parental cells were stained for γH2AX after 72h transfection of 25 nM siPHGDH#4 and siPHGDH#5 or mock control. ×200 magnification. (E) PC9ER4 cells were transfected with 25 nM siPHGDH#4 and siPHGDH#5 for 36 h followed by 10 mM NAC for 6 h. The level of ROS was analyzed by DCFH-DA staining. (F) Immunoblotting of γH2AX and PHGDH after cells were treated by siPHGDH#4 and siPHGDH#5 for 72 h followed by NAC (10 mM) for 1 h. β-actin as loading control. (G) PC9ER4 cells were treated with NCT-503 (50 μM) for 1 h or pre-treated with NAC (10 mM) for 1 h, then the level of ROS was analyzed by DCFH-DA staining. (H) Immunoblotting of γH2AX after cells were treated as described in (G), Ku86 as loading control. (I) GSH/GSSG ratio of PC9 and PC9ER4 cells after treatment of NCT-503 (50 μM) for 1 h. Results were shown in mean ± SEM of triplicates. *, p < 0.05; **, p < 0.01; ****, p < 0.0001.