Fig 2: MATIIα and VCIP135 are essential for HCC proliferation.a, b Quantification of different metabolites of VCIP135 knockdown in MHCC-97H cells (a) and MHCC-LM3 cells (b). Mean ± s.e.m. of n = 3 biologically independent experiments, one-way ANOVA test. c, d Quantification of different metabolites of MAT2A knockdown in MHCC-97H cells (c) and MHCC-LM3 cells (d). Mean ± s.e.m. of n = 3 biologically independent experiments, one-way ANOVA test. e VCIP135 putback recovers VCIP135 knockdown-induced reduction of cell proliferation in MHCC-LM3 cells. Mean ± s.e.m. of n = 3 biologically independent experiments, one-way ANOVA test. f MAT2A putback recovers VCIP135 knockdown-induced reduction of cell proliferation in MHCC-LM3 cells in normal medium. Mean ± s.e.m. of n = 3 biologically independent experiments, one-way ANOVA test. g The effects of MAT2A and VCIP135 double knockdown on proliferation in vitro. Mean ± s.e.m. of n = 3 biologically independent experiments, one-way ANOVA test. h MTX and FIDAS-5 synergistically repress cell proliferation. Mean ± s.d. of n = 3 biologically independent experiments, one-way ANOVA test. n.s. donates for no significance, *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001

Fig 3: DUB VCIP135 stabilizes MATIIα to promote cell proliferation by sensesing folic acid. a VCIP135 endogenously interacts with MATIIα. b VCIP135 knockdown increases MATIIα ubiquitylation. HEK293T cells were transfected with the indicated plasmids. c VCIP135 WT but not enzymatic-dead mutant C218S decreases MATIIα ubiquitylation. HEK293T cells were transfected with the indicated plasmids. d, e VCIP135 WT but not enzymatic-dead mutant stabilizes MATIIα upon folate deprivation for 72 h. MHCC-97H cells were cultured under folate-deprived condition, followed by CHX treatment. Mean ± s.d. of n = 3 (d) biologically independent experiments, one-way ANOVA test. f MG132 rescues MTX-reduced MATIIα protein level in multiple HCC cell lines. g Folate rescues MTX-reduced MATIIα protein level in MHCC-97H and Huh7 cells. h Working model. VCIP135 deubiquitylates MATIIα in response to folate and induces MATIIα accumulation in protein level. Data (a–c and e–g) are representative of three independent experiments. n.s. donates for no significance, ***P < 0.001 and ****P < 0.0001

Fig 4: VCPIP1 forms a positive feedback loop and regulates the Hippo/YAP axis.VCPIP1 interacts with YAP and reduces the K48-linked ubiquitination of the YAP protein. As a result, it stabilizes the YAP protein and enhances the transcription of downstream target genes. VCPIP1 was identified as one of the target genes of the YAP/TEAD complex. Transcriptionally activated VCPIP1, in turn, stabilizes YAP, forming a positive feedback loop that ultimately contributes to pancreatic cancer progression.

Fig 5: VCPIP1 is required for PAAD progression.A PAAD cells were transfected with either siControl or siVCPIP1#1/#2 at a concentration of 50 nM. The knockdown efficiency of VCPIP1 was subsequently measured via qRT‒PCR. B The mRNA expression levels of CTGF and CYR61 in PAAD cells after transfection with siControl or siVCPIP1 were measured via qRT‒PCR. C, D A luciferase reporter gene assay was used to detect TEAD transcriptional activity in AsPC-1 and PANC-1 cells after transfection with siControl or siVCPIP1. E The protein levels of VCPIP1 and YAP in PAAD cells after transfection with siControl or siVCPIP1 were measured via Western blotting. Proliferation rates of PAAD cells were evaluated by CCK-8 (F) and EdU (G, H) assays after transfection with siControl or siVCPIP1. Nuclei in the EdU assay were stained with Hoechst 33342. The scale bar is 200 μm. Transwell assays were performed to assess the migration (I) and invasion (J) abilities of PAAD cells after transfection with siControl or siVCPIP1. K, L A wound healing assay was performed to assess the migration ability of PAAD cells after transfection with siControl or siVCPIP1. M, N PAAD cell apoptosis was evaluated via flow cytometry after the cells were transfected with siControl or siVCPIP1 and then stained with PI and FITC. O–Q PANC-1 cells stably transfected with shControl or shVCPIP1 were inoculated subcutaneously into nude mice. Tumor weight (O) and volume (P) were evaluated. An IHC assay was used to evaluate Ki-67, YAP and VCPIP1 expression (Q). The percentage of Ki-67-positive cells was calculated. n = 6. The scale bar is 200 μm. The experiments were performed in triplicate. All the data are presented as the means ± SDs. Statistical methods: Student’s t test for (A–D, G–O, Q); two-way ANOVA for (F, P). *P < 0.05; **P < 0.01; ***P < 0.001.