HASPIN depletion reduces AML proliferation, induces cell cycle arrest, and dysregulates oncogenic transcription. (A) Western blot analysis of HASPIN and β-actin (loading control) in Kasumi-1 cells expressing either nontargeting control (C), or 1 of 2 unique HASPIN-targeting shRNAs (1 and 2). Data are mean ± SD of 3 independent experiments. Representative blot revealed. Significance determined by 1-way analysis of variance (ANOVA) with Holm-Sidak method for multiple comparison correction. ∗∗∗∗P < .0001; ∗∗∗P < .001. (B) Growth curve of Kasumi-1 cells expressing either nontargeting control or 1 of 2 unique HASPIN-targeting shRNAs. Data on curve are mean ± SD of technical triplicates. Representative curve of 3 independent experiments. Significance of day 8 determined by 2-way ANOVA with Holm-Sidak method for multiple comparison correction. ∗∗∗P < .001. (C) Cell cycle analysis of Kasumi-1 cells expressing either nontargeting control or 1 of 2 unique HASPIN-targeting shRNAs. Data are mean ± SD of 4 independent experiments. Significance determined by 2-way ANOVA with Holm-Sidak method for multiple comparison correction. ∗∗∗∗P < .0001. (D) Volcano plot of genes differentially expressed on HASPIN depletion in Kasumi-1 cells. Differentially expressed genes were determined using DESeq2. Points represent genes. Genes meeting a |log2(fold change)| ≥1.0 and Benjamini-Hochberg adjusted P value ≤.05 cutoff (dotted lines) are considered significant and colored red. Nonsignificant genes are colored gray. (E) Selected gene set enrichment analysis plots of genes differentially expressed on HASPIN depletion. NES, FDR q value, and P value are indicated within each plot. shControl, control shRNA; shHASPIN, HASPIN-targeting shRNA.
