Figure 2
A shRNA barcode screen identifies PRC1 as critical mediator of the anti-proliferative effects of RO-3306.
(A) Schematic outline of the RO-3306 barcode screen in U2OS cells. (B) Reconstitution of the RNAi-resistant human PRC1-1 cDNA in PRC1 knockdown cells. The pRS vector (shCTRL) was used as a control. (C) Western blot analysis of protein lysates corresponding to the cell lines shown in (B). shPRC1 Lib#1 targets only endogenous PRC1, while shPRC1 Lib#2 is able to target both endogenous and Venus-NT-PRC1 #1. (D) Western blot analysis of U2OS cells stably expressing individual Venus-PRC1 isoforms after transfection of siRNA duplexes specifically targeting the indicated splice variants. CTRL refers to cells lacking ectopic expression of Venus-PRC1. (E) Depletion of PRC1-1 rescues cell proliferation when Cdk1 activity is compromised. The functional phenotypes of the individual shRNAs targeting the PRC1 variants are indicated by the colony formation assay. The pRS vector (shCTRL) was used as a control. The knockdown efficiency of each individual shRNA was measured by examining the mRNA level of the PRC1-1 and PRC1-2 target genes by qRT-PCR. (Mean ± s.d. n = 3). (F) KIF4 is a PRC1 binding partner. Immunoprecipitations (IPs) with GFP-Trap_A beads were performed on extracts of U2OS WT (CTRL) or U2OS cells stably expressing Venus-PRC1-1 (PRC1-1). Aliquots of the IPs were analysed by SDS-PAGE. (G) Compromised Cdk1 activity in mitosis results in premature binding of PRC1 to KIF4. U2OS cells were synchronised in mitosis using thymidine and nocodazole. Cells arrested in mitosis were obtained by mitotic shake-off, released from the nocodazole and harvested after 60 minutes (G1) or, alternatively, treated with 5 μM MG132 with or without 3 μM RO-3306 and harvested after 30 minutes (M). Cell extracts were subjected to immunoprecipitation using a rabbit anti-PRC1. Aliquots of the immunoprecipitates were analysed by Western blotting. The signal intensity of the KIF4 band in the PRC1 IP was quantified and normalized to 1 for the G1 sample. *p < 0.05, Student’s t test. Western blots in panels (C,D,F,G) have been cropped and full-length gels can be viewed in Supplementary Fig. 6.
