Fig 2: Sgo2 specifically localizes B56α to centromeres.(A-G) The effect of Sgo1 and/or Sgo2 knockdown on YFP-B56α localisation in Flp-in HeLa cells. Representative images (A, C, F) and quantifications (B, D, G) of relative kinetochore intensity of B56α in cells arrested in prometaphase with nocodazole after knockdown of Sgo2 (A, B), Sgo1 (C, D), or Sgo1 +Sgo2 (F, G). (E) shows line plots of Sgo2 and B56α localisation following Sgo1 knockdown; 5 kinetochore pairs were analysed per cell, for a total of 10 cells per experiment. Graphs represent the mean intensities (±SD) from 3 independent experiments. Intensity is normalized to the maximum signal present in each channel within the endogenous B56α experiment. (H–M) Flp-in HeLa cells expressing YFP-B56α or YFP-B56γ were transfected with the CB-Sgo2 (H–K) or gChr7 +Cas9 DARPIN (L, M) and analysed for B56 recruitment in cells arrested in prometaphase with nocodazole. (H), (L), and J). are representative images; I) and K) are quantifications of relative centromere/kinetochore intensity of the indicated antigen; and M) is quantification of intensity of Sgo2 over B56 at the Chr7 locus. For all centromere/kinetochore intensity graphs, each dot represents a cell and 10 cells were quantified per experiment for at least 3 independent experiments. The spread of dots indicates the biological variation between individual cells and the errors bars display the variation between the experimental repeats (displayed as -/+SD of the experimental means). Asterisks indicate significance (Mann-Whitney test); ns p>0.05, *p≤0.05, ****p≤0.0001. Scale bars, 5µm.

Fig 3: SGOL2 knockdown inhibited HCC growth and metastasis in vivo. A-B Xenograft model was set up to study the effects of SGOL2 on HCC tumor growth in vivo. Mice were divided into two groups and inoculated with SK-HEP-1 shNC or SK-HEP-1 shSGOL2 cells (s.c. n = 3, i.v. n = 7). Images of the isolated livers and tumors from sacrificed mice are presented, and the hepatic replacement area (HRA%) and the tumor volumes and tumor weights of the indicated groups were analyzed and compared. Loss of SGOL2 in SK-HEP-1 cells contributed to the reduction in tumorigenesis. C-D, HE staining of metastatic tumors in liver and lung tissues. Representative images and quantitative analysis results are shown

Fig 4: A C-terminal loop in B56 regulates binding to Sgo2 and LxxIxE motifs to specify centromere/kinetochore localisation.(A-D) Flp-in HeLa cells expressing either YFP-B56α WT or TKHG were transfected with the CB-Sgo2 and analysed for B56 recruitment (A, B) or gChr7 +dCas9 DARPIN to assess YFP-B56α:Sgo2 co-localisation (C, D). Representative images (A, C) and quantification of relative kinetochore intensity (B) or intensity of Sgo2 over B56α at the Chr7 locus (D). For the intensity graphs in B) and D), each dot represents a cell and 10 cells were quantified per experiment from at least 3 independent experiments. The spread of dots indicates the biological variation between individual cells and the errors bars display the variation between experimental repeats (displayed as -/+SD of the experimental means). (E) Immunoblot of the indicated antigens following immunoprecipitation of YFP from nocodazole-arrested Flp-in HeLa cells expressing YFP- B56γ, YFP-B56α WT or YFP-B56α-TKHG. (F) Quantification of the mean normalised intensity (+SD) of indicated antigens in B56α WT or B56α TKHG immunoprecipitates, relative to B56γ, from at least 4 experiments. (G) Crystal structure of PP2A-B56γ [accession code 2NPP (Xu et al., 2006)] with annotation to indicate the regions that specify localisation to centromeres or kinetochores (see Figure 6—figure supplement 1 for details of the additional centromere/kinetochore specification regions). Note, the structure is meant only as a guide because the critical regions may be present within B56α, which has not been structurally solved. The B56α WT values are also used in some of the points plotted in Figure 2m. Asterisks indicate significance (Mann-Whitney test, except Figure 6f: Welch’s t -test, unpaired); ns p>0.05, *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001. Scale bars, 5µm.

Fig 5: Downregulation of SGOL2 expression inhibited the malignant behaviors of HCC cells in vitro. A The mRNA level of SGOL2 in liver and HCC cell lines. B-C, SK-HEP-1, and HEP3B cells were transfected with shNC or shSGOL2 lentivirus, and the knockdown of SGOL2 at the mRNA and protein levels was validated by RT–PCR and Western blots, respectively. GAPDH was used as a loading control. D-G Invasion, migration, sphere formation, and colony formation assays of the SGOL2-downregulated HCC cells were detected and analyzed. H-I Downregulation of SGOL2 expression induced cell cycle arrest in the G1/S phase and activated the apoptosis of HCC cells. J SK-HEP-1, and HEP3B cells were transfected with shNC or shSGOL2, and the proliferation of HCC cells was detected at Days 0, 1, 2, and 3 by CCK-8 assays. K Effect of SGOL2 on the EMT in HCC cell lines. SK-HEP-1 and HEP3B cells were transfected with shNC or shSGOL2, and Western blots were used to detect the levels of E-cadherin, N-cadherin, β-catenin, Vimentin, fibronectin, and MMP9. The results are presented as the mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001