Fig. 3

MCT4 promotes cell proliferation and glycolysis via activation of NF-κB and c-Myc pathway. (A) Western blotting analysis of NF-κB pathway activation in MCT4-knockdown and control astrocytes, cultured alone, or with 50 ng/mL TNF-α and IL-1β for 24 h. The protein level of phospho-p65 is normalized to β-actin. (B) Effect of MCT4 inhibitor CHCA on the expression of phosphorylated p65 in NF-κB pathway was measured by western blotting. Primary astrocytes were pretreated with 1 mM CHCA for 24 h, followed by stimulation with TNF-α and IL-1β for 24 h. The protein level of phospho-p65 is normalized to β-actin. (C) Western blotting analysis of phosphorylated c-Myc and total c-Myc in MCT4-knockdown and control astrocytes. The protein level of phospho-c-Myc is normalized to β-actin. (D) Effect of MCT4 inhibitor CHCA on activation of c-Myc signaling was measured by western blotting. The protein level of phospho-c-Myc is normalized to β-actin. (E) Effect of c-Myc inhibitor and NF-κB inhibitor on the proliferation of astrocytes was measured by EdU assays. Cells were treated with NF-κB inhibitor JSH-23 for 2 h, or with c-Myc inhibitor 10,058-F4 for 24 h at indicated concentration. (F) EdU⁺ cells were quantified in each group indicated. (G) Effect of c-Myc inhibitor and NF-κB inhibitor on lactate production of astrocytes were measured. Scale bar: 100 μm. Data are from at least 3 independent experiments (A-D). Data are represented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 using one-way ANOVA with Dunnett’s multiple comparison test