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. 2019 May 24;294(27):10530–10543. doi: 10.1074/jbc.RA119.007784

Figure 4.

Figure 4.

PFKFB3 expression modulates response to erlotinib in NSCLC cells. A, H522 and PC9 cells were treated with the indicated concentrations of erlotinib for 24 to 48 h. Whole cell lysates were evaluated by Western blotting with the indicated antibodies. PFKFB3(S461)/PFKFB3 ratios were quantified and normalized to vehicle-treated samples at 24 h. B, PFKFB3 mRNA levels were evaluated by real-time PCR. Error bars, mean ± S.E. of three independent experiments, normalized to vehicle-treated samples (n = 9). C, PC9 cells were treated with either a MEK inhibitor (U0126; 20 μm) or an RSK inhibitor (BI-D1870; 20 μm) in the presence of 0.5 μm erlotinib for 48 h. PFKFB3 mRNA levels were evaluated by real time PCR and normalized to DMSO untreated control. Error bars, mean ± S.E. of three independent experiments (n = 9). D, PC9 cells were treated as described in C and protein levels were evaluated by Western blotting. E, PC9 cells were transfected with either a control siRNA or a pool of CREB1-specific siRNAs and treated with DMSO or erlotinib for 48 h. PFKFB3 mRNA levels were evaluated by real-time PCR and normalized to vehicle-treated control siRNA. Error bars, mean ± S.E. of three independent experiments (n = 9). F, PC9 cells were treated as described in E and protein levels were evaluated by Western blotting. G, ChIP assay experiments were performed with PC9 cells treated with 0.5 μm erlotinib for 24 h using antibodies to CREB1 or control mouse IgG and primers flanking −389 and distal regions (lacking CRE site) of the PFKFB3 promoter. Real-time PCR data were normalized to control IgG signal and shown as -fold enrichment. Error bars, mean ± S.E. of three independent experiments (n = 9). H, PC9 cells were treated with indicated concentrations of erlotinib for 24 h and exposed to a translation inhibitor (4EGI-1; 50 μm) or a proteasome inhibitor (MG132; 50 μm) for 0 to 6 h. Whole cell lysates were collected and immunoblotted with the indicated antibodies. PFKFB3/actin ratios were quantified and normalized to the corresponding time zero. I, H522 and PC9 cells were transfected with PFKFB3 siRNAs (siFB3#1, siFB3#2) followed by treatment with either vehicle or erlotinib (H522, 2 μm; PC9, 0.5 μm) for 48 h. Cell survival was evaluated by trypan blue exclusion. Shown are changes in absolute amounts of viable cells between 24 and 48 h post treatment. Error bars, mean ± S.E. of three independent experiments (n = 12). p values are shown as follows: *, <0.05; **, <0.01, and ***, p < 0.001 compared with vehicle treatment. ##, p <0.01, ###, p< 0.001, compared with indicated erlotinib treatment.