Figure 3.

ATRT-specific SIRT1 upregulation suppresses NF-κB and p53 activity, which can be reversed through gemcitabine treatment

(A) mRNA expression levels of SIRT1 in normal brain, cerebellum, and various tissues (in green, GSE: 11882, GSE: 3526, GSE: 7307) compared to adult and pediatric CNS tumor tissues (in blue, GSE: 7696, GSE: 16011, GSE: 26576, GSE: 19578, GSE: 74195, GSE: 64415) and ATRT tissues (in red, GSE: 70678).

(B) Western blot analysis depicting SIRT1 expression in VUMC-ATRT-01 and VUMC-ATRT-03 cells after gemcitabine treatment (0, 5, and 10 nM) for 24 h.

(C) Volcano plot depicting −log10 false discovery rate (FDR)-corrected p value of differential expressed genes between DMSO (n = 6) and gemcitabine-treated (n = 6) ATRT cell cultures VUMC-ATRT-01, VUMC-ATRT-03, CHLA-02, CHLA-05, CHLA-06, and CHLA-266. Downstream targets of SIRT1 (SHH, PTCH1, TP73, CDKN1A, NFKB2, RELB, RELA, NFKB1, FOXO3, TP53, E2F1, XRCC6, FOXO1, PPARG, LXR, and PPARGC1A) are marked in red.

(D) Untreated (blue) vs. gemcitabine (red)-treated mRNA expression of NFKB1 (black) and NFKB2 (magenta) in VUMC-ATRT-03 and a panel of pediatric HGG models.

(E) Heatmap representation illustrating mRNA expression of the KEGG NF-κB gene set in VUMC-ATRT-01 and VUMC-ATRT-03 cells before and after gemcitabine treatment (PAGE FDR p < 0.05).

(F) Western blot analysis depicting p65-NF-κB and phospho-p65-NF-κB (pNF-κB) expression in VUMC-ATRT-01 and VUMC-ATRT-03 cells after gemcitabine treatment (0, 5, and 10 nM) for 24 h.

(G) Western blot analysis depicting p53 expression in VUMC-ATRT-01, VUMC-ATRT-03, CHLA-ATRT-02, CHLA-ATRT-04, CHLA-ATRT-05, and CHLA-ATRT-06 cells after gemcitabine treatment (0, 10, and 20 nM) for 24 h.

(H) Volcano plot depicting −log10 FDR-corrected p value of differential expressed genes between DMSO (n = 6) and gemcitabine-treated (n = 6) ATRT cell cultures VUMC-ATRT-01, VUMC-ATRT-03, CHLA-02, CHLA-05, CHLA-06, and CHLA-266. All 69 genes of the “KEGG p53_signaling_pathway” are highlighted in red.