Fig. 3
ISP I targets to SELH. A Scheme of a drug affinity responsive target stability (DARTS) assay. B The western blots corresponding to thermo-stability assay of ISP I binding to SELH extracted from LN229 cells. DMSO serves as a control. C Surface plasmon resonance (SPR) analysis of the interaction of ISP I and SELH synthesized in bacteria. D Western blots of SELH in LN229 cells treated with ISP I for 24 h. E Western blots of SELH in glioblastoma cell lines (T98G, U118, LN229, and U251) treated with ISP I (10 μM) for 24 h. F and G Cycloheximide (CHX) pulse chase assay shows SELH protein half-life in ISP I-treated LN229 cells by western blots. Quantification of the western blots was listed in G. H Western blots (left) show SELH expression in SELH knockout (KO) LN229 cells, KO #1 and KO #2. Dose–response curve of cell viability was measured by CCK-8 assay (right) in SELH-deficient LN229 cells (KO #2) or wild-type LN229 cells (WT). IC50 was calculated and listed. I Western blots show expression of SELH in the LN229 cells two days after SELH siRNA transfection. CCK-8 assay was performed to measure cell proliferation. LN229 cells transfected with scramble siRNA serve as a control. ***p < 0.001 by two-way ANOVA. J and K Cell-cycle (J) and Annexin-V apoptosis (K) analysis in SELH-deficient LN229 and U251 cells. Summarized results from 4 independent wells are shown. L Survival analysis of xenograft NSG mice intracranially inoculated with 3 × 105 SELH-deficient LN229 cells (KO #2) or wild-type LN229 cells (WT). Median survival of KO #2 vs WT inoculated mice: 51 vs 28 days, ***p < 0.001 by log-rank test. N = 10 for per group. M Western blots show undetectable expression of SELH in B16 cells. N C57BL/6 mice received tail vein injection of 2 × 105 wild-type or SELH-deficient B16 cells (N = 5 per group). Representative lungs of mice corresponding to wild-type or SELH-deficient B16 cells. O Quantification of lung tumor nodules. Expression of GAPDH serves as an internal control in (D, E, F, H, I and M). All data are shown as mean ± SEM. P value: ***p < 0.001