Fig 2: Role of mitotic arrest deficient 1 (MAD1) in PRAP1-mediated MCC assembly inhibition and cisplatin-resistant CRC cells. (A) HCT-116 and HCT-116/DDP cells were transfected with EGFP-PRAP1 and siPRAP1 plasmid, respectively, MAD1 expression was measured by western blotting assay. (B) Quantitative analysis of panel A is shown on the right. (C) Colcemid-challenged HCT-116/DDP cells were transfected with siPRAP1 or MAD1 overexpression plasmid, the interaction of MAD2 with MAD1, and with BUBR1 as explored by Co-IP. (D) Representative photographs of colcemid-challenged HCT-116/DDP cells with siPRAP1 or MAD1 overexpressing plasmid transfection which were examined using Livecyte Cell Analysis System. The rounded-up cell morphology was accepted to be under mitotic arrest (the top picture). (E) Expression and distribution of pH3-positive and apoptotic cells as monitored by IF staining. Red: pH3, Blue: Hoechst, Scale bar: 20 µm. (F) Multidrug resistant protein as estimated via western blotting and quantitative analysis as displayed below. *P<0.05; **P<0.01.

Fig 3: Effects of PRAP1 on cisplatin-resistance of colorectal carcinoma in vivo. (A) All mice were sacrificed by cervical dislocation and the tumors of mice from the four groups were harvested and images captured (n=6 per group). (B) The tumor growth curve of xenograft mice after initial cell injection and drug-treatment. Cisplatin was taken orally on day 15 after initial cell injection. (C) The differences in tumor weight among the four groups. (D) IHC staining of tumors with Ki67 antibody in the four groups. Relative quantitative analysis of Ki67-positive cell number is shown in the left panel. Scale bar, 50 µm. (E) Drug resistance related protein levels including MDR1 and MAD1, and protein level of PRAP1 in tumors as determined by western blotting. Relative quantitative analysis of protein levels is shown on the left panel. (F) The interaction of MAD2 with MAD1, and with BUBR1 in tumors as explored by Co-IP. ns, no significant. * P<0.05, ** P<0.01.