Fig 1: ZFP91 K48-ubiquitinates and degrades hnRNP A1. (A) Proteins that interacted with ZFP91 were identified by co-IP and mass spectrometry assays using HEK293T cells as a cell model. (B, C) ZFP91-Flag and hnRNP A1-HA plasmids were transfected into HCC cells HCC MHCC-LM3 (B) and SK-hep1 (C), ZFP91-Flag (B) and hnRNP A1-HA (C) complexes were co-immunoprecipitated with anti-Flag and HA antibodies, and hnRNP A1 and ZFP91 were detected, respectively. (D) MHCC-LM3 HCC cells were transfected with ZFP91-Flag plasmid, hnRNP A1 protein level was determined. (E) SK-hep1 HCC cells were transfected with anti-ZFP91 siRNA, hnRNP A1 protein level was determined. (F) ZFP91 and hnRNP A1 protein levels in six pairs of primary HCC tissues (T) and corresponding N tissues used in Figure 1B were detected. The correlations of ZFP91 with hnRNP A1 were analyzed in the right panel. (G) MHCC-LM3 cells transfected with ZFP91-Flag plasmids for 36 h were incubated with cycloheximide (CHX) for the indicated times. The indicated proteins were analyzed (left panel), and the relative hnRNP A1 protein level is illustrated graphically (right panel). (H) MHCC-LM3 cells were cotransfected with the indicated plasmids for 36 h, followed by treatment with 10 µM MG132; the polyubiquitination level of hnRNP A1 was detected. (I) SK-hep1 cells were cotransfected with the indicated plasmids together with anti-ZFP91 siRNA for 36 h, followed by treatment with MG132; the polyubiquitination level of hnRNP A1 was detected. (J) MHCC-LM3 cells were cotransfected with wild type HA-ub or its mutants together with ZFP91-HA and hnRNP A1-Flag plasmids for 36 h, followed by treatment with MG132; the polyubiquitination level of hnRNP A1 was detected.
Fig 2: ZFP91 regulates the alternative splicing of PKM pre-mRNA to promote PKM1 isoform formation and inhibit PKM2 isoform formation by ubiquitinating at Lys 8 and degrading hnRNP A1. (A-D) HCC MHCC-LM3 and SK-hep1 cells were transfected with ZFP91-Flag plasmids (A, B) or anti-ZFP91 siRNAs (C, D), respectively; the protein levels of PKM1 and PKM2 (A, C) and the PKM pre-mRNA splicing (B, D) were detected. (E, F) HCC SK-hep1 cells were cotransfected with anti-ZFP91 siRNA together with anti-hnRNP A1 siRNA; the protein levels of PKM1 and PKM2 (E) and PKM pre-mRNA splicing (F) were detected. (G, H) MHCC-LM3 cells were cotransfected with the ZFP91-HA plasmids together with wild type hnRNP A1-Flag plasmids or hnRNP A1-Flag K8R mutant; the protein levels of PKM1 and PKM2 (G) and PKM pre-mRNA splicing (H) were detected. (I, J) The protein levels of PKM1 and PKM2 (I) and PKM pre-mRNA splicing (J) were detected in six pairs of primary HCC tissues (T) and corresponding adjacent nontumoral liver tissues (N) used in Figure 1B. The correlations of ZFP91 protein levels with PKM1 and PKM2 protein levels were analyzed in clinical HCC samples (right panel in I).
Fig 3: ZFP91 ubiquitinates hnRNP A1 protein at Lys8. (A) The ZFP91-HA plasmids together with wild type hnRNP A1-Flag plasmids or hnRNP A1-Flag K8R mutant were cotransfected into MHCC-LM3 cells for 36 h, and the hnRNP A1-Flag protein level was determined. (B) MHCC-LM3 cells were cotransfected with the ZFP91 plasmids together with wild type hnRNP A1-Flag plasmids or hnRNP A1-Flag K8R mutant for 36 h and then incubated with CHX for the indicated times. The indicated proteins were determined (top panel), and the relative hnRNP A1-Flag protein level is illustrated graphically (bottom panel). (C) MHCC-LM3 cells were cotransfected with the indicated plasmids for 36 h and then treated with 10 µM MG132. The polyubiquitination levels of hnRNP A1-Flag and hnRNP A1-Flag K8R mutant protein were analyzed.
Fig 4: ZFP91 is frequently downregulated in HCC, and its downregulation is associated with a poor prognosis for patients with HCC. (A, B) The mRNA (A) and protein (B) levels of ZFP91 were detected in primary HCC tissues (T) and corresponding adjacent nontumoral liver (N) tissues. (C) Representative IHC images of ZFP91 protein expression in HCC tissues and their corresponding nontumoral liver tissues. (D) Differences in the ZFP91 protein level between HCC tissues (T) and their corresponding nontumoral liver tissues (N) are presented as a violin plot (n = 90). (E) Association between ZFP91 protein levels and the percentage of patient death in HCC samples. (F, G) The disease-free survival rate (F) and overall patient survival rate (G) for patients with HCC according to ZFP91 expression ratios of HCC/corresponding nontumoral liver tissues.
Fig 5: ZFP91 silencing promotes HCC cell growth, colony formation, migration and invasion in vitro and tumorigenesis and metastasis in vivo. (A-D) BEL7402 and SK-hep1 cells were transfected with anti-ZFP91 siRNAs; the indicated proteins (A), cell growth (B), migration and invasion (C), and colony formation (D) were determined. (E) The in vivo growth of the indicated cell lines stably silencing ZFP91 was examined. Mouse xenograft tumors are shown in the left panel. The weights of the xenograft tumors are presented in the right panel (n = 6). (F, G) Luc-labeled SK-hep1 cells (2 × 106 cells/mouse) were injected into NOD-SCID mice; the luciferase activity was visualized 2 months posttransplantation (n = 5) (F), and the metastatic tumor nodules in mouse lung were observed (G). Data are represented as mean ± SD.
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