Fig. 2: Knocking out HNRNPH1 reduces the expression of cell cycle-associated genes.
From: HNRNPH1 drives glioblastoma progression by regulating the splicing of cell cycle genes
A Schematic representation of the CRISPR/Cas9 strategy to knock out HNRNPH1 in GBM cells. Three spatially coordinated synthetic single guide RNAs (sgRNAs) were designed to induce a fragment deletion between exon 2 and intron 2 of the coding sequence of HNRNPH1 variant 1 (UTR = untranslated region; CDS = coding sequence). B Volcano plot comparing HNRNPH1-knockout U87 cells to control U87 cells. Genes colored in blue are significantly downregulated in the HNRNPH1-knockout cells, and genes colored in red are significantly upregulated in the HNRNPH1-knockout cells. C Dot plots of GSEA Hallmark analysis of enriched gene sets in HNRNPH1-knockout U87 cells compared to control U87 cells. The figure shows the significantly enriched gene sets at FDR q-value < 0.05. A positive Normalized Enrichment Score (NES) value indicates enrichment in the HNRNPH1-knockout cells, and a negative NES indicates enrichment in the control cells. Gene count refers to the number of genes associated with each gene set. D The Venn diagram shows the number of DEGs in HNRNPH1-knockout U87 cells (RNA-seq) relative to the number of genes identified to be directly bound by HNRNPH1 (eCLIP-seq). E The Venn diagram shows the number of DEGs in HNRNPH1-knockout U87 cells (RNA-seq, n = 3 biological replicates) compared to the number of DEGs in HNRNPH1-knockdown patient-derived G62 cells (RNA-seq, n = 2 biological replicates). F, G Enrichment plots for the two most enriched gene sets in control G62 cells from the GSEA Hallmark analysis. Shown are the enrichment plot for E2F targets (F, left panel) and the twenty most downregulated genes in HNRNPH1-knockdown (F, right panel); enrichment plot for G2/M checkpoint (G, left panel) and the twenty most downregulated genes in HNRNPH1-knockdown (G, right panel). H qRT-PCR analysis of HNRNPH1, AURKB, ESPL1, PRC1, MYBL2, CCNF, STMN1, and UBE2S expression in G62 cells transfected with a pool of control siRNAs or a pool of siRNAs targeting HNRNPH1. Data shown as mean ± SD of three replicates. Data were analyzed by unpaired t-test: **p < 0.01, ***p < 0.001. I Western Blot analysis of HNRNPH1, AURKB, MYBL2, CCNF, ESPL1, and PRC1 expression in G62 cells nucleofected with sgRNAs control or sgRNAs targeting HNRNPH1. J The correlation between HNRNPH1 expression and the expression of AURKB, ESPL1, MYBL2, and CCNF in the TCGA GBM dataset.
