Fig 1: Overexpression of OTUD5 promoted HBV replication and transcription. HepG2.2.15 cells were infected with OTUD5 lentivirus plasmid and mock vector as negative control with RFP (A). Western blot results showed that the HBc and HBx proteins were elevated when the OTUD5 lentivirus plasmid was transfected into HepG2.2.15 cells (B). The concentrations of HBsAg, HBeAg and HBV DNA were increased in OTUD5 overexpression compared with the mock vector in culture supernatants (C–E). The levels of pg RNA and HBV total RNA were both significantly elevated in HepG2.2.15 cells with OTUD5 overexpression (F, G) (*P < 0.05, **P < 0.01, ***P < 0.001)
Fig 2: The effect of HBV infection on the expression level of OTUD5. Western blot results showed elevated expression of OTUD5 in HepG2.2.15 cells compared with HepG2 cells (A). Levels of OTUD5 mRNA in HepG2.2.15 cells compared with HepG2 cells (B). Expression of OTUD5 in liver tissues based on IHC. (Left) Overexpression OTUD5 was observed in the cytoplasm in chronic hepatitis B liver tissues compared with HBV-negative liver tissues. (Middle) OTUD5 expression as positive control in the human carcinoma of the rectum tissues compared with normal rectum tissues. (Right) HBc expression in HBV-positive patients compared with HBV-negative patients (scale bar 50 μm) (C). OTUD5 levels in HBV negative cohort, HBV carriers and chronic hepatitis B patients (D). OTUD5 concentration at baseline between HBeAg seroconversion group and HBeAg none seroconversion group (E). ROC curve of OTUD5 as a host marker to predict HBeAg seroconversion in the follow-up period (F, G). Analysis of HBeAg seroconversion rate using the Kaplan–Meier method during the follow-up (H) (*P < 0.05, ***P < 0.001)
Fig 3: OTUD5 interacted with hepatitis B precore/core proteins. OTUD5 plasmid with RFP and hepatitis B precore, core and x plasmids with GFP were cotransfected into HEK293T cells, and the interactions between OTUD5 and hepatitis B precore, core and x proteins were observed using confocal microscopy (A). OTUD5 plasmid with myc tag and hepatitis B precore, core and x plasmids with flag tag were co-transfected into HEK293T cells, and the interactions between OTUD5 and hepatitis B precore, core and x proteins were analyzed using anti-flag magnetic beads (B–D). OTUD5 plasmid with myc tag and hepatitis B precore, core and x plasmids with flag tag were co-transfected into HEK293T cells. The interactions between OTUD5 and hepatitis B precore, core and x proteins were analyzed using anti-myc magnetic beads (E–G)
Fig 4: OTUD5 inhibited the hepatitis B core protein degradation through the proteasome pathway. Hepatitis B core plasmids with flag tag and OTUD5-sh or OTUD5 overexpression plasmid with myc tag were co-transfected into HEK293T cells. After transfection, the cells were treated with MG132 or DMSO for 8 h before harvest, then cell lysates were collected and subjected to Western blot (A, B). Hepatitis B core plasmid with flag tag, ubiquitin plasmids with HA tag and OTUD5 knockdown or overexpression plasmid were co-transfected into HEK293T cells. After transfection, the cells were treated with MG132 or DMSO for 8 h. Lysates immunoprecipitation and whole cell lysates analysis of the deubiquitination of HBc were performed by Western blot (C, D)
Fig 5: ERK1/2 signaling was involved in the OTUD5-mediated promotion of HNF4α and HBV. HepG2.2.15 cells were transfected with OTUD5 overexpression lentivirus plasmid or OTUD5 knockdown lentivirus plasmid, and cell lysates were collected and subjected to Western blot using antibodies against ERK1/2, p-ERK1/2, JNK, p-JNK, p38, p-p38, OTUD5, or GAPDH (A, B). HepG2.2.15 cells were transfected with OTUD5 knockdown lentivirus plasmid, 36 h post-transfection, cells were treated with ERK-specific pharmacological inhibitor PD98059 (10 μM), JNK-specific pharmacological inhibitor SP600125 (10 μM) and p38-specific pharmacological inhibitor SB203580 (10 μM) or DMSO for another 24 h, cells were lysed and subjected to Western blot using antibodies against ERK1/2, p-ERK1/2, JNK, p-JNK, p38, p-p38 (C). HepG2.2.15 cells were transfected with OTUD5 knockdown lentivirus plasmid and mock vector as negative control, 36 h post-transfection, cells were treated with PD98059 (10 μM), SP600125 (10 μM) and SB203580 (10 μM) for another 24 h, cells were lysed and analyzed by Western blot using antibodies against HNF4ɑ, HNF1ɑ or GAPDH antibodies (D). HepG2.2.15 cells were transfected with OTUD5 knockdown lentivirus plasmid and mock vector as the negative control, 36 h post-transfection, cells were treated with PD98059 (10 μM) or DMSO for another 24 h, HBsAg, HBeAg and HBV DNA were tested in culture supernatant fluid (E–G), the expression of pgRNA, total HBV RNA and HBV DNA in cells were analyzed by PCR (H–J)
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