Fig. 6. The FECD133 potently inhibits glioma progression.

a GLT8D1 physically interacts with the FECD133 as assessed by GST pull-down assay. GST and GST-FECD133 were expressed and purified in E.coli BL21 (DE3), and then visualized by Coomassie blue staining. b Co-IP assay examining the interaction between GLT8D1 and CD133 in the presence of different doses of the FECD133 (40 nM, 200 nM). GST (200 nM) proteins were used as control. GSC11 cells were lysed, and then different doses of the FECD133 were added into the indicated cell lysates. After incubation for 30 min, cell lysates were immunoprecipitated with GLT8D1 antibodies and then subjected to IB with the indicated antibodies. c Evaluation of the efficacies of the FECD133 and TMZ in regulating cell viability using SRB assay in indicated cells. The IC₅₀ in each cell line was indicated. d GSC11 cells were treated with different doses of the FECD133 (30 nM, 60 nM), and the indicated cell lysates were analyzed by IB with the indicated antibodies. e The indicated doses of the FECD133 that inhibit the expression of c-Myc and Axin2 examined by qRT-PCR. f β-catenin proteins were reduced upon different doses of the FECD133 (0, 30, 60 nM) treatment. g The FECD133 alone or with TMZ induces GSC11 and GBM1 cellular apoptosis. GST (40 nM), TMZ (200 μM), the FECD133 (40 nM), or TMZ plus FECD133 for 48 h. h GSC11 cells were treated under the indicated conditions, and the cell lysates subjected to IB with the indicated antibodies. GST (40 nM), TMZ (200 μM), FECD133 (40 nM), or TMZ plus FECD133 for 48 h. i–k Xenograft tumors after subcutaneously injecting the indicated cell lines and treated by the indicated conditions, mice received tail vein injections of GST (5 mg kg⁻¹), TMZ (60 mg kg⁻¹), the FECD133 (5 or 50 mg kg⁻¹) alone, or the FECD133 (5 mg kg⁻¹) with TMZ (60 mg kg⁻¹), once every week for five 5 weeks. Xenograft tumors were harvested and photographed (i), tumor weights (j) and volumes (k) were plotted. l Schematic representation of the intracranial mouse model. GSC11 cells (5 × 10⁵ cells per mouse) were intracranially injected into NOD-SCID mice. Mice were in situ injected with GST (100 μg kg⁻¹), the FECD133 (100 μg kg⁻¹), TMZ (1 mg kg⁻¹) or the FECD133 with TMZ. m The survival of mice was evaluated (n = 5, Kaplan–Meier model with a log-rank test). P values were analyzed by comparing TMZ or the FECD133 alone versus the combination of TMZ with the FECD133. The X-axis represents days after cell injection. n Representative images of H&E staining for tumor formation following indicated treatments. Scale bar: 2 mm. o PDXs tumors treated by GST (5 mg kg⁻¹) or FECD133 (5 mg kg⁻¹) after tail vein injection were harvested and photographed, tumor weights and volumes were plotted. Mean  ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, t-test.