Fig. 5.

Increased expression of SΔE-1/KLHDC8A in ΔEGFR-independent U373MG cells and GBMs and effect of knock-down on tumorigenicity. (A) Western blot analysis of lysates from ΔEGFR-dependent or -independent escaper tumors as in Fig. 4 with a polyclonal rabbit anti-SΔE-1 antibody. Cos7 cells transfected with SΔE-1 were used as a positive control for the antibody. (B Inset) Two tet-regulatable U373MG cell lines established from escaper tumors, Esc-0 and Esc-1, were infected with shRNA-expressing retroviruses targeting SΔE-1/KLHDC8A (No. 1 or No. 2), a nonspecific sequence (N.S.), or Luciferase (Luc), and stable cell lines were established. qPCR analysis of SΔE-1/KLHDC8A gene expression was performed in the cell lines expressing the indicated retroviral shRNA constructs. Each bar represents the mean ± SD of three replicates. y axis represents the relative gene expression level. These cells (3 × 10⁶) were injected s.c. into the flanks of nude mice fed with dox-containing drinking water. Tumor volumes were measured at the indicated times to generate the growth curve of tumors with knockdown of SΔE-1/KLHDC8A expression. n = 8 mice for all groups, mean ± SEM.