br using the QuickMutation Kit Beyotime Hangzhou China with
using the QuickMutation™ Kit (Beyotime, Hangzhou, China) with full-length PRKCI expressing plasmid as a template. The plasmids used in this study are listed in Supplementary Table S1. All mutants were va-lidated by sequencing.
2.3. Western blotting
Briefly, cells or tissues were lysed by ice-cold RIPA Lysis Buﬀer plus protease and a phosphatase inhibitor cocktail (Beyotime, Hangzhou, China). The cytoplasmic and nuclear fractions were isolated from cells by using the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, Hangzhou, China) according to the manufacturer's protocol. Equal amount of proteins were analyzed on 10% SDS-PAGE gels. β-actin was used as a loading control for cytoplasmic fraction, whereas LaminB was used as a loading control for the nuclear fraction. All blots were vi-sualized by ECL (Boster, Wuhan, China). The intensity of bands was evaluated by Image Lab (Bio-Rad, California, USA). The experiments were repeated independently for three times. The HG-9-91-01 used in this study are listed in Supplementary Table S2. Redox Biology 22 (2019) 101149
Co-immunoprecipitation (Co-IP) assay was performed to analyze protein-protein interactions. In brief, the cells were lysed by RIPA buﬀer and divided into parallel groups named input or IPs. Then, the primary antibody or IgG was added to the lysates for incubation over-night at 4 °C. Subsequently, the Protein A + G agarose beads were added to the mixture at 4 °C for 3 h. After centrifugation at 15,000×g for 15 min, the beads were collected and washed 5 times with RIPA buﬀer. The immunoblotting was performed with the indicated anti-bodies as previously reported.
2.5. Patients and specimens
In total, 72 human GBC tissues and paired normal gallbladder tis-sues (5 cm distant from tumor) were collected from patients undergoing resection at the Department of Biliary and Pancreatic Surgery, Tongji Hospital (Wuhan, China) between January 2009 and December 2016. Ethical approval for the use of human samples was obtained from the Tongji Hospital Research Ethical Committee. None of the patients had received any adjuvant therapy before surgery. All cases were diagnosed by two independent pathologists. The GBC samples were staged ac-cording to the 7th edition of the American Joint Committee on Cancer (AJCC) Cancer Staging Manual. The detailed clinicopathological char-acteristics of the 72 patients with GBC are listed in Supplementary Table S3.
2.6. Immunohistochemistry (IHC)
GBC samples or xenograft tumor tissues were fixed in 4% paraf-ormaldehyde, paraﬃn embedded, and sectioned. The expression of aPKCι, Nrf2 and Keap1 was detected by immunohistochemistry as previously reported . The positively stained cells were scored, with scores ranging from to 12. The total score ≤4 was considered as low expression and > 4 as high expression .
Six-week-old female BALB/c-nude mice were used in all animal experiments and housed under specific pathogen-free (SPF) conditions in Central Animal Laboratory, Tongji Medical College. For the first animal experiment, 20 mice were randomly divided into four groups (n = 5 per group). A total of 2 × 106 NOZ cells transfected with len-tivirus empty vector, aPKCι overexpression, si-neg, or si-aPKCι vectors were injected subcutaneously in the upper back of mice, respectively. For the second animal experiment, the same number of mice were randomly divided into 2 groups (n = 10 per group). A total of 2 × 106 NOZ cells transfected with lentiviral si-neg or si-aPKCι vectors were injected subcutaneously in the upper back of mice, respectively. One week later, each group was randomly regrouped two subgroups (n = 5 per group) to receive intraperitoneal injection of gemcitabine (15 mg/ kg) or 0.9% sodium chloride (NS) every 3 days. The diameter of tumors and the weight of the mice were measured every 3 days. The volume of tumors was calculated using the formula: 1/2 (length × width2). All mice were sacrificed 3 weeks later, and the tumors were dissected out for immunohistochemistry, western blot assay or quantitative real-time PCR (qPCR). All animal experiments were conducted according to the ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines and were approved by the Committee on the Ethics of Animal Experiments of the Tongji Medical College, HUST.
Additional experimental procedures are provided in detail in the Supplementary data.
2.8. Statistical analyses
Statistical analyses were performed using SPSS 22.0 software (IBM
Fig. 1. aPKCι inhibits ROS in a kinase-independent manner. (A–B) qPCR and western blotting were performed to detect the expression levels of aPKCι in NOZ and GBC-SD cells with lentivirus-mediated aPKCι overexpression (aPKCι)/knockdown (si-aPKCι) or re-expression. β-actin was used as a loading control. neg, negative control. N.S. no significance. (C–D) ROS levels were measured by DCFH-DA in GBC cells with the treatment of aPKCι overexpression, knockdown, re-expression or gemcitabine. (E) Protein levels of aPKCι, phosphorylated-Nrf2 (p-Nrf2) and Total Nrf2 (T-Nrf2) were tested in GBC cells after aPKCι overexpression with or without PSI treatments (10 μM). (F) Relative ROS levels were detected in GBC cells after aPKCι overexpression with or without PSI treatments (10 μM). *P < 0.05, **P < 0.01. Data are derived from three independent experiments and presented as means ± SDs.