Fig 2: LANA is required for the recruitment of Polycomb proteins to lytic promoters during de novo infection. (A) Schematic of the KSHV latency locus encoding latent genes. (B) ChIP assays testing the recruitment of PRC2 factor EZH2 and PRC1 factor RING1B onto viral lytic promoters in SLK cells 72 hours after infection with WT or the indicated KSHV mutants. (C and D) ChIP assays for the repressive (H3K27me3) and activating (H3K4me3) histone marks on viral and cellular promoters at 72 hpi. The LANA and actin (ACT) promoters devoid of H3K27me3 and the PRC2-regulated MYT1 promoter served as controls. (E) Immunoblots showing LANA protein levels in mock and KSHV-infected SLK cells at 72 hpi. *Non-specific. (F) ChIP assays for EZH2 and RING1B associations with viral promoters at 72 hpi.

Fig 3: Expression patterns of RNF2 and SIK1 in human HCCA. Relationship between RNF2 amplification and HCC prognosis. Kaplan-Meier curves based on data from TCGA show that RNF2 amplification in HCC was associated with shorter overall survival (P < 0.05). B. RNF2 and SIK1 expression in HCC tissues and paired normal liver tissues. Western blots of RNF2 and SIK1 in six representative paired samples of non-tumor tissue (N) and HCC tissue (T) are shown. β-Actin was used as a control for protein load. C. Expression patterns of RNF2 and SIK1 immunoreactivity in HCC samples and in non-tumor tissues from a representative case. D. RNF2 expression levels in noncancerous liver tissues were lower than those in HCC tissues (left) (P < 0.05). Conversely, SIK1 expression levels in noncancerous liver tissues were higher than those in HCC tissues (right) (P < 0.05). E. Low SIK1 levels were correlated with high RNF2 expression in HCC (P < 0.05). The median expression levels of SIK1 and RNF2 were used as the cutoff values. F. Kaplan-Meier curves show that HCC patients with high RNF2 and low SIK1 immunoreactivity had a poorer prognosis than patients with low RNF2 and high SIK1 immunoreactivity did. The data are expressed as the mean ± SD. The results are representative of three independent experiments. *P < 0.05, **P < 0.01.

Fig 4: Interaction of LANA with PRC2. (A) Gel filtration chromatography of nuclear extracts derived from iSLK and iSLKBAC16 cells. Fractions from 28 to 72 were tested by immunoblot analysis for LANA and polycomb proteins. Molecular weight markers are indicated. (B) Co-immunoprecipitation of LANA-V5 (using V5 antibody) from 293T cells expressing LANA-V5 and the indicated 3xFLAG-tagged PRC2 (EZH2, SUZ12 and EED) or PRC1 (RING1B and BMI-1) factors. Immunoprecipitation and input blots were probed with V5 and FLAG antibodies. (C) Cell lysates derived from de novo KSHV infected iSLK cells (72 hpi) were used for immunoprecipitation with anti-EZH2 rabbit antibody (αEZH2) or normal rabbit IgG (αIgG), followed by immunoblotting with αLANA and αEZH2.

Fig 5: RNF2 knockdown suppresses invasion and metastasis of HCC cellsHCC cell lines infected with RNF2 expression vector or its shRNA or control were used in the following studies. A. The wound-healing assay was used to explore the effect of RNF2 on the motility of HCC cells. The percentage of wound healing is shown in the diagrams (left panel). B. Effects of RNF2 knockdown or over-expression on HCC cell invasion in indicated cells in vitro using transwell assay. The diagrams were shown (left panel). C. Representative immunofluorescence (IF) images indicated that RNF2 has an effect on the expression of EMT genes in HCC cells. D. Effects of RNF2 over-expression on tumor metastasis of indicated cells in nude mice (n = 10 per group): the number of metastatic nodules in the lung (left-panel); representative morphological observation of lung metastases (right-upper panel); and histopathological observation of lung sections (right-lower panel). The results are presented as are means ± SD (n = 3 for each panel). Statistical significance was concluded at *P < 0.05, **P < 0.01, ***P < 0.001; # represents no statistical significance.