Figure 2.

PIGN loss or suppression results in the differentially disrupted expression of SAC components. (A) CRISPR/Cas9 ablation of PIGN led to MAD1 and MAD2 downregulation in CD34+ mononuclear cells from a healthy donor. (B) Gene and protein expressions of MAD1 and MAD2 were significantly (***p < 0.0001) impacted by PIGN loss in CD34+ mononuclear cells from a healthy donor. (C,D). Complete loss of PIGN via CRISPR/Cas9 ablation (KO) was associated with downregulation of MAD1, MAD2, and MPS1 while causing an upregulated gene (***p < 0.0001) and protein expression of BUBR1 in HEK293 cells. PIGN loss resulted in significant repression (*p < 0.05) of MAD1 and MAD2 gene expression but led to MPS1 gene upregulation (*p < 0.05). (E) PIGN loss (KO) increased the frequency of segregation errors in HEK293 cells. (F) Ectopic overexpression of PIGN restored MAD2 expression in HEK293 PIGN KO cells. (G) RNAi-mediated PIGN suppression resulted in MAD1 and MAD2 downregulation but increased expression of BUBR1 and MPS1 expression in K562 cells. (H) MAD1 suppression was accompanied by a corresponding decrease in PIGN protein expression in K562 cells. (I) CRISPR/Cas9 ablation of PIGN (K562 KO) resulted in MAD1, BUBR1, and MPS1 downregulation in K562 cells. (J) RNAi-mediated MAD1 suppression in K562 cells resulted in MAD1 downregulation while causing an upregulation in BUbR1 and MPS1 expression. (K) AML-MRC patient CD34+ PBMCs (P1 and P2) with PIGN partial intron retentions showed a significant (***p < 0.0001) increase in PIGN and MAD1 gene expressions compared to non-leukemic control cells from a healthy donor. All western blot images were cut within the molecular weight ranges of the protein targets prior to hybridization. Images generated from these cut blots by scanning the developed films. Images were cropped and labeled using Adobe Photoshop CC 2017 (version 18). Where applicable, representative full images of the blots are presented in Figure 2 of the supplementary immunoblotting data.