Figure 1. An siRNA screen identifies base excision repair pathway members as sensitizers to TMZ in pediatric GBM.

a-b. SJG2 and KNS42 cells were transfected with a 240 DNA siRNA pool library and 24 h post transfections, cells were cultured with or without TMZ (100uM). Cell viability was assessed by the almarBlue cell viability assay 72 h post siRNA transfection. Data were normalized using the standard z-score method by correcting the raw data for plate to plate variation. Significance of potential TMZ sensitizers were determined using z-score cut off values of less than −1.65 (dotted line), which corresponded to a p value of 0.05 in all three biological replicates (rep1-3).

c. Venn diagram of genes common and unique to both cell lines from the siRNA screen.

d. Heat map of cell viability to validate target genes in TMZ and non-TMZ conditions using SJG2, KNS42, NHAs and normal neural stem cells (NSC). TMZ dose used was 100 uM and viability was assessed using almarBlue assay on day 3. *p<0.05 using ANOVA followed by a post-hoc Dunnett’s test. Each heatmap box represents the average of three independent experiments.

e. Immunoblotting to evaluate the protein expression levels of base excision repair pathway members in pediatric glioblastoma cell lines.

f. Immunofluorescence of MPG in SJG2 cells showing nuclear expression of the protein. Scale bar = 16um.

g. Pearson correlation plots of MPG (black) and MGMT (red) protein expression quantified by chemi luminescence densitometry versus IC50 values following 7 days of TMZ in pediatric GBM cell lines from supplemental figure 1a. Strong correlation is seen with MPG protein levels (r= 0.78 at 7 days) but not with MGMT levels (r= 0.03). All experiments were performed in triplicate, error bars represent standard error of the mean.