Fig. 3: SmD2 depletion enhances the accumulation of DNA damage and sensitivity to PARP inhibitors in HCC. | Nature Communications

Fig. 3: SmD2 depletion enhances the accumulation of DNA damage and sensitivity to PARP inhibitors in HCC.

From: Acetylation-dependent regulation of core spliceosome modulates hepatocellular carcinoma cassette exons and sensitivity to PARP inhibitors

Fig. 3

a Gene Ontology analysis of genes (n = 2636, p < 0.05) downregulated by inducible shSNRPD2 Knockdown in HCCLM3 cells, compared to control cells. b Gene Set Enrichment Analysis (GSEA) indicating that DNA repair pathways are significantly downregulated in HCC cells with SNRPD2 knockdown (DOX+) and in HCC samples from The Cancer Genome Atlas (TCGA) with low expression of SNRPD2 (coding SmD2). c Volcano plot of differentially expressed genes after DOX-induced SNRPD2 knockdown, with |Log2 (fold change)| = 0.5 and –log10(P-value) = 6 as thresholds; genes with y > 250 are shown as y = 250. Genes involved in DNA repair pathways such as FANCA, FANCD2, BRCA1 are notably downregulated. d Quantitative RT-PCR validation of RNA-seq data showing reduced mRNA levels of several DNA repair genes with SNRPD2 knockdown. (n = 4 independent experiments). e Immunoblot showing BRCA1, FANCA, FANCD2 and γH2A.X expression in inducible shSNRPD2 cells. The experiment was repeated three times with similar results. f DNA damage accumulation with or without silencing of SNRPD2 detected using γH2A.X immunostaining. g Percentage of cells with ≥10 γH2A.X foci/nucleus, analyzed in random cells (n = 5 independent experiments). h Levels of PARylation and PARP1 expression measured by immunoblot in inducible shSNRPD2 cells. The experiment was repeated three times with similar results. i Olaparib dose-response curves on inducible shSNRPD2 HCC cell lines over 72 hours (n = 4 biologically independent samples). j Representative 3D culture plot of transfected inducible shSNRPD2 HCCLM3 cells after DOX and Olaparib (IC20) treatment. k Diameter of the cell spheres (n = 22 biologically independent samples) counted from j. l Inducible shSNRPD2 HCCLM3 cells were seeded into male nude mice. Doxycycline (20 mg/kg, orally, daily) and Olaparib (50 mg/kg, intraperitoneally, daily) were administered from day 10. Each line represents an individual tumor. m Tumor volume reduction in HCCLM3 xenografts after inducible SNRPD2 knockdown and further inhibition by Olaparib treatment, with representative images. n Statistical analysis of tumor volume data from (m) (n = 5). Mean values ± SEM. Two-sided t-tests were used to determine statistical significance, **P = 0.0022, and ***P < 0.001. Source data are provided as a Source Data file.

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