Figure 3: HuR inhibition sensitizes IDH1 mutant cells to a mutant IDH1 inhibitor, AGI-5198.

A, Cell viability (PicoGreen DNA quantitation) of Mut.IDH1 cell lines treated at the indicated doses of AGI-5198. IC₅₀ values are provided. B, Representative qPCR analysis of HuR and IDH1 mRNA expression in HT1080.HuR(+/+) and HT1080.HuR(−/−) cells (HuR-knockout by CRISPR gene editing); representative immunoblots for HuR and IDH1 of HT1080.HuR(+/+) and HT1080.HuR(−/−) whole cell lysates, **P ≤ 0.01 and ***P ≤ 0.001. C, Immunoblot analysis of fractionated lysates from HT1080.HuR(+/+) cells upon glucose withdrawal, AGI-5198 treatment (0.3 μM), and a combination of both conditions for 24 hours. Lamin A/C and α-Tubulin were used as controls to determine the integrity of nuclear and cytosolic lysates respectively. D, Immunofluorescence demonstrates HuR subcellular localization to the cytoplasm (green cytoplasmic signal) in HT1080 cells upon glucose withdrawal, AGI-5198 treatment, and a combination of both conditions for 24 hours. Magnification 40×. E, Drug sensitivity measured by PicoGreen DNA quantitation, in HT1080 cells under the indicated culture conditions, and with varying doses of AGI-5198. IC₅₀ values are provided. F, Trypan blue staining in HT1080 and BT054 cells after HuR silencing or CRISPR gene editing, cultured under high or low glucose conditions, with or without AGI-5198 treatment (0.3 μM). G, Long-term cell survival assessed by colony formation in soft agar. HT1080 cells were cultured under the indicated conditions. AGI-5198 was dosed at 0.3 μM for 4 weeks, as indicated. Each data point represents the mean ± SEM of three independent experiments. N.S. non-significant; * p < 0.05; ** p < 0.01; *** p < 0.001.