Fig 2: RSF1 depletion impairs kinetochore localization of PLK1 in early mitosis.(a) HeLa cells were transfected with siRNAs, and floating mitotic cells were obtained after nocodazole treatment for 4 h and subjected to chromosome spread immunostaining. Images were obtained from representative mitotic cells: PLK1 (green), ACA (red) and 4,6-diamidino-2-phenylindole (DAPI; blue). The graph represents relative intensity of PLK1 against ACA at kinetochores. At least 100 kinetochores of prometaphase cells were analysed in three independent experiments. Data are represented as means±s.e.m. (n=3). **P<0.01 versus control siRNA by Student's t-test. Scale bar, 5 µm. (b) HeLa cells transfected with RSF1 or SNF2H siRNA were treated with paclitaxel for 16 h, and floating mitotic cells were obtained. Whole-cell lysates (WCLs) and chromatin-bound fractions were subjected to immunoblotting. (c) Cells stably expressing the RSF1 shRNA plasmid were treated with paclitaxel for 16 h. Chromatin-bound fractions were subjected to immunoblotting. CDK1-specific phosphorylation of BubR1 on Ser670- and PLK1-specific phosphorylation on Ser676 were determined. (d) Restoration of PLK1 localization by RSF1: RSF1 KO HeLa cells were transfected with WT-V5 or RSF1-C2-V5 for 48 h. Mitotic cells were analysed by immunofluorescence staining: PLK1 (green), RSF1 (red) or DAPI (blue). Scale bar, 5 µm. (e) Immunoblotting of chromatin fractions in RSF1 shRNA cells after reintroduction of RSF1-WT or truncated forms of the RSF1 gene. See full blots Supplementary Fig. 8.

Fig 3: miR-1224-5p inhibited the proliferation, migration and invasion of gastric cancer cells via downregulating RSF1. (A) Bioinformatics and luciferase assay clarified that miR-1224-5p can directly binding to the RSF1 3'-UTR region. (B) The miR-1224-5p mimics and luciferase reporter plasmids with wild-type or mutant RSF1 3'-UTR were co-transfected into SNU-16 cells. Dual luciferase reporter gene assay was performed to verify the direct binding relationship between miR-1224-5p and RSF1. (C) SNU-16 was transfected with mimics NC, miR-1224-5p mimics, inhibitor NC, miR-1224-5p inhibitor, and then qRT-PCR was taken to evaluate relative expression levels of miR-1224-5p and RSF1. (D) Cell proliferation was tested by CCK-8 assay. (E) Cell invasion was detected by transwell assay. (F) The number of invaded cells. (G) Cell migration was detected by wound healing assay. (H) The distance of the cell migration detected by wound healing assay. Data are shown as mean ± SD. **P < 0.01.

Fig 4: RSF1-deficient cells suffering from chromosome-alignment defects are rescued by RSF1 that retains the ability to bind PLK1.(a) After RSF1 shRNA cells stably expressing the RSF1 shRNA plasmid were co-transfected with H2B-GFP and indicated RSF1-V5 constructs, cells were synchronized at the G1/S boundary by adding 2 mM of thymidine for 24 h and released from the thymidine block for 8 h. Time-lapse images were acquired every 5 min. Scale bar, 5 µm. (b) Quantification of aligned or unaligned chromosome (%) in the acquired images in a. At least 30 cells on average were traced in three independent experiments (n=30). Data are represented as means±s.e.m. (n=3). **P<0.01, ***P<0.001 by Student's t-test. (c) A proposed model for recruitment of PLK1 to the kinetochore by RSF1.

Fig 5: RSF1 localizes at mitotic kinetochores and directly interacts with PLK1.(a) RSF1 knockout (KO) HeLa cells were transfected with pcDNA or RSF1-V5. Floating mitotic cells were obtained after nocodazole treatment for 4 h and subjected to chromosome spread immunostaining. Immunofluorescence images of human epithelial RPE1 and HeLa cells are shown: RSF1 (green), ACA (red) and 4,6-diamidino-2-phenylindole (blue). Scale bar, 5 µm. (b) Chromatin fractions of asynchronously growing HeLa (I, interphase) and paclitaxel-arrested HeLa cells (M, mitotic phase) were obtained after centrifugation and washing with 0.5 M NaCl. Proteins were eluted as soluble fractions and chromatin-bound fractions were analysed by immunoblotting with the indicated antibodies. Topo IIa and GAPDH were used as controls for chromatin and soluble fractions, respectively. (c) In vitro binding assays: recombinant RSF1-V5 was incubated with immunopurified Myc-PLK1 or HA-Aurora B for 2 h at 4 °C. RSF1-V5 bound to immobilized Myc-PLK1 or HA-Aurora B was detected by immunoblotting. Recombinant RSF1-V5 was stained with Coomassie blue. (d) Recombinant RSF1-V5 was incubated with recombinant His-PLK1 purified from insect cells, and His-PLK1 bound to immobilized RSF1-V5 was detected by immunoblotting. Recombinant RSF1-V5 and His-PLK1 were visualized by Coomassie blue staining. (e) Recombinant GST-RSF1 proteins were incubated with Myc-PLK1 expressing mitotic lysates and subjected to immunoblotting. N1: amino acids 1–627, N2: 1–871, C2: 982–1441 and PHD (plant homeodomain): 628–973. (f) Immunopurified Flag-PLK1 was incubated with recombinant RSF1-V5. WT, wild type; KD, kinase dead; N, amino acids 1–401; C, amino acids 350–603. (g) Recombinant GST-RSF1 proteins were incubated with Flag-tagged PLK1 WT, PLK1-PBDW414F or PLK1-PBDH538A/K540M expressing mitotic lysates and subjected to immunoblotting. Recombinant GST-RSF1 was stained with Coomassie blue. (h) In vitro binding assays: recombinant RSF1-V5 was incubated with immunopurified Flag-PLK1 WT or Flag-PLK1 PBD mutants for 2 h at 4 °C. RSF1-V5 bound to immobilized Flag-PLK1 WT or Flag-PLK1 PBD mutants was detected by immunoblotting. Recombinant RSF1-V5 was stained with Coomassie blue. See full blots Supplementary Fig. 8.