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  • br caption on next page br Fig aPKC

    2022-05-23


    (caption on next page)
    Fig. 3. aPKCι competes with Nrf2 for Keap1 binding through the DLL motif. (A) The protein levels of aPKCι and Nrf2 were determined by western blotting in negative control or aPKCι knockdown NOZ ATPγS with or without MG132 treatments (20 μM for 6 h). (B) The ubiquitinated Nrf2 (Ub-Nrf2) was measured by IP in NOZ cells after aPKCι overexpression or knockdown. (C) The protein levels of aPKCι, Nrf2, Keap1 and p62 were examined in aPKCι overexpression GBC cells with or without Keap1 knockdown. (D) Co-IP assays were performed to detect the interaction between Flag-tagged aPKCι and Myc-tagged Keap1 in HEK293T cells. (E) The interaction between aPKCι and Keap1 was determined by Co-IP in GBC cells with the treatment of gemcitabine for 24 h. MG132 (20 μM) was added 6 h before cells were collected. (F) The interaction among aPKCι, Nrf2 and Keap1 was examined by Co-IP in vitro translation systems with aPKCι (0, 2, 4, or 8 μl), Nrf2 (4 μl), and Keap1 (6 μl). (G) Left panel, sequence alignment of Keap1-recognizing motif in aPKCι from chicken to human. Right panel, schematic description showed the aPKCι wild-type or mutants. (H) Co-IP assays were used to evaluate the interaction of aPKCι and Keap1 in HEK293T cells transfected with indicated plasmids. (I) Diagrams of the Keap1 wild-type or deletion mutants. (J) The interaction of aPKCι with Keap1 wild-type or deletion mutants was analyzed by Co-IP assays in HEK293T cells transfected with indicated plasmids.
    Fig. 4. aPKCι promotes GBC cells tumorigenesis both in vivo and in vitro. (A) Cell proliferation ability was analyzed by the CCK-8 assay in GBC cells with indicated treatments. (B) Anchorage-independent growth was evaluated by soft agar growth assay in the indicated GBC cells. Scale bar, 50 μm. (C) Images of subcutaneously transplanted tumors from the nude mice injected with NOZ cells following the indicated treatments (n = 5 per group). (D) Tumor volume and tumor weight of NOZ xenografts with indicated treatments. (E) Expression of aPKCι, Nrf2 and Keap1 proteins in samples derived from xenograft tumors were measured by western blotting assay. (F) The mRNA levels of Nrf2 and its target genes in samples derived from xenograft tumors were analyzed by qPCR. *P < 0.05, **P < 0.01. Data are derived from three independent experiments and presented as means ± SDs.
    Fig. 5. aPKCι-mediated ROS inhibition enhances gemcitabine resistance in GBC. (A) The cell viability was measured by the CCK-8 assay in NOZ cells. (B) The cell viability of NOZ cells with indicated treatment was evaluated at 24, 48 and 72 h. (C) Relative ROS levels were determined by DCFH-DA in the indicated NOZ cells in the presence or absence of gemcitabine. (D) Expression levels of Nrf2, Keap1, Bcl-2 and Bax proteins were detected by western blotting assay in NOZ cells with or without gemcitabine treatment. (E) The mRNA levels of Nrf2 and its target genes were measured by qPCR in NOZ cells with the indicated treatments. (F) Negative control and aPKCι knockdown NOZ cells were subcutaneously injected into the upper back of nude mice with or without gemcitabine treatment (n = 5 per group). NS, sodium chloride. (G) Tumor volume and tumor weight of NOZ xenografts with indicated treatments. *P < 0.05, **P < 0.01. Data are derived from three independent experiments and presented as means ± SDs.
    3.5. aPKCι-mediated ROS inhibition enhances gemcitabine resistance in GBC
    Next, we further investigated whether aPKCι would influence the sensitivity of GBC cells to commonly used chemotherapeutic drugs gemcitabine. Given that ROS has been reported to participate in che-moresistance, we speculated that aPKCι may involve in this process. We treated aPKCι or Nrf2 silencing GBC cells with various concentrations of gemcitabine for 72 h to analyze the cell viability by CCK-8 assays. Indeed, when aPKCι was knocked down, the cells exhibited more sen-sitivity to gemcitabine than the negative control group, with a drama-tically decreased of IC50 (Fig. 5A and S4A). Then, we examined the viability of the two GBC cell lines with the indicated dose of