Figure 2. Effect of JIB 04 and CPI 455 on TMZ resistant GB cell lines and primary cultures and effect of JIB 04 on gene expression and H3 methylation.

(Panel A) Effect of JIB 04 on two TMZ resistant derivatives of the GBM3 stem-enriched culture obtained independently at a distance of one year. Treatment time 96 hours. Each datapoint represents the mean of triplicate assays. Significance of the difference of TMZ resistance between native and TMZ R cells was determined by ANOVA with the Bonferroni's correction for multiple comparisons and is indicated for each point (*P = 0.05; **P = 0.01; ***P = 0.001). (Panel B left): sensitivity of GB cells to the selective KDM5 inhibitor CPI 455 evaluated by MTS. Treatment time 120 hours. The significance of the difference between treated and control cells was determined by ANOVA as described for Panel A. (Panel B right): sensitivity of two independent TMZ resistant derivatives of the GBM3 stem-enriched culture to CPI 455 evaluated by MTS. Treatment time 120 hours. The significance of the difference between TMZ-resistant and native cells treated with the same concentration of CPI 455 was determined by ANOVA as described for Panel A. (Panel C) Expression of CCNB1, PCNA, DEPP and DDIT4 genes in A172 and U251 cells exposed to JIB 04 for 4 hours normalized against untreated cells (fold-change = 1.0). (Panel D) Chromatin immunoprecipitation of A172 cells exposed to JIB 04 for 4 hours with an antibody against H3K4 me3 and with an irrelevant antibody. The immunoprecipitated was then amplified by qPCR with a set of primers for the promoter region of DEPP and the results show a > 30 fold enrichment for H3K4me3 over the irrelevant antibody only in the JIB 04 treated cells.