br SPSS Armonk NY USA or GraphPad
SPSS, Armonk, NY, USA) or GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA, USA). The results were presented as mean ± standard deviation (SD). Quantitative data were analyzed by two-tailed in-dependent Student's t tests and analysis of variance. Categorical vari-ables were compared using chi-square tests or Fisher exact tests. Clinical correlations were analyzed using χ2 tests, and survival analysis was conducted by the Kaplan-Meier method with log-rank tests. Diﬀerences with P values of less than 0.05 were considered statistically
3.1. aPKCι inhibits ROS in a kinase-independent manner
To investigate a possible functional link between aPKCι and ROS, we first stably established ectopic aPKCι expression or knockdown GBC
cell lines NOZ and GBC-SD (Fig. 1A and B), which were used in the subsequent experiments. The ectopic expression of aPKCι significantly reduced the cellular ROS levels in GBC cells. Conversely, aPKCι silen-cing triggered the accumulation of ROS. Interestingly, restoring exo-genous aPKCι expression reversed the eﬀects of aPKCι knockdown, suggesting that aPKCι may act as an antioxidative factor (Fig. 1C). Importantly, we further found that aPKCι overexpression also atte-nuated gemcitabine-induced ROS production. aPKCι deletion elevated the cellular ROS levels under this condition, which was reversed by the expression of exogenous aPKCι (Fig. 1D). Therefore, these data showed that aPKCι suppressed the intracellular ROS to control cytoprotective response under both normal and oxidative stress conditions.
Previous studies indicated that aPKC(s) involved in oxidant stress response by phosphorylation of Nrf2 ; however, we found that aPKCι overexpression significantly changed ROS levels in GBC Amiloride HCL treated with PSI, an aPKC peptide inhibitor, while there was weak and no obvious alteration of phosphorylated Nrf2 (Fig. 1E and F). Inter-estingly, aPKCι overexpression led to significant accumulation of the total Nrf2 protein (Fig. 1E). Of note, we also found that PSI reduced the expression levels of phosphorylated aPKCι, but the total aPKCι, phos-phorylated Nrf2 and total Nrf2 proteins had no significant change. The phosphorylation of epithelial cell transforming sequence 2 (Ect2), an aPKCι substrate [20,21], was indeed inhibited by PSI, while there was no obvious alteration of the total Ect2 protein (Fig. S1A). More im-portantly, we constructed Flag-tagged wild-type (WT), constitutively active catalytic domain (CAT) and kinase-inactive (KI) mutants of aPKCι as previously reported . The data showed that these mutants had minimal impact on the expression levels of phosphorylated Nrf2 (Figs. S1B and C). Together, these results suggest that aPKCι-mediated antioxidant eﬀect was independent of its well-studied kinase function.
3.2. aPKCι promotes Nrf2 accumulation and activates antioxidative signaling
Given that the Keap1-Nrf2 complex is the key regulator of anti-oxidative signaling, we determined whether aPKCι induced ROS in-hibition through the Keap1-Nrf2 pathway. Our data showed that aPKCι did not aﬀect the protein expression of Keap1 in GBC cells (Fig. 2A). However, ectopic aPKCι expression significantly increased the protein levels of Nrf2. aPKCι knockdown led to reduction in the level of the Nrf2 protein, which was reversed by restoring aPKCι expression (Fig. 2A). Interestingly, the mRNA levels of both Keap1 and Nrf2 were not altered by aPKCι expression (Fig. 2B). These results indicated that aPKCι positively modulated Nrf2 accumulation, and the process did not occur at the transcriptional level.
Next, we further investigated whether aPKCι regulated the in-tracellular distribution of the Nrf2 protein. Western blotting results showed that aPKCι promoted Nrf2 nuclear location, while aPKCι knockdown prevented the transposition (Fig. 2C). In contrast, there was no obvious change in the level of the Keap1 protein in both the cyto-plasm and nucleus. Previous studies demonstrated that nuclear Nrf2 can bind to the antioxidant response element (ARE) and then activate the downstream genes . To confirm the transcriptional activity of Nrf2, qPCR was performed to detect the mRNA levels of well-established downstream genes such as HMOX1, NQO1, GCLC, GCLM and FTH1. The ectopic aPKCι expression showed an estimated 2.3–3.8 fold increase in GBC cells, whereas aPKCι depletion reduced the expression of these genes by 50% (Fig. 2D). Notably, re-expression of aPKCι reversed the eﬀect of aPKCι knockdown. In addition, we further found that aPKCι-mediated ROS inhibition can be abolished by Nrf2 knockdown (Fig. 2E and F). Therefore, Nrf2 is required for aPKCι-induced antioxidative signaling. Collectively, the above findings suggest that aPKCι promotes Nrf2 accumulation, nuclear location, and lowers the ROS levels.