Fig. 5.

The PRRX1 pmx‐1b isoform reduces the CD133‐positive population of GICs. Cells were treated with or without BMP‐4 (30 ng·mL⁻¹) for 72 h, except for panel (B). (A) Immunoblot analysis of enforced expression of FLAG‐tagged pmx‐1a or pmx‐1b in TGS‐01 and TGS‐04 cells. (B) Phase‐contrast images showing the morphology of EGFP‐ and FLAG‐pmx‐1a‐ or FLAG‐pmx‐1b‐expressing TGS‐01 and TGS‐04 cells treated with or without BMP‐4 (30 ng·mL⁻¹) for 5 days under serum‐free condition. Scale bars: 200 µm. (C) Downregulation of PROM1 mRNA by ectopic expression of the PRRX1 pmx‐1b isoform in TGS‐01 and TGS‐04 cells (n = 3 biological replicates). (D) Surface expression of CD133 in TGS‐01 and TGS‐04 cells evaluated by flow cytometric analysis. Ectopic expression of the PRRX1 pmx‐1b decreased the CD133‐positive population (n = 3 biological replicates). (E, F) In vivo tumorigenic activity of GICs expressing firefly luciferase and EGFP, pmx‐1a, or pmx‐1b (n = 7 mice for each group). The in  vivo bioluminescent imaging analysis was performed 2 weeks after intracranial injection (E). Kaplan–Meier survival curves of mice injected with TGS‐01 cells expressing EGFP, pmx‐1a, or pmx‐1b (F). The graphs in panels (C–E) represent mean ± SD of biological replicates. The P‐values were determined by Tukey’s test (C–E). In panel (F), the P‐value was determined by two‐tailed log‐rank test and then adjusted by Bonferroni correction.