Animals
All experiments were performed with either newborn or adult male C57BL/6 mice, weighing 20-25 g. All animal experiments were performed with the approval of the Institutional Animal Care and approved by the Animals Ethics Committee of Jilin University of China (10 February 2014, NO. 2014-277).
Cell culture and reagents
Astrocytes were obtained from the cerebral cortices of one-day-old C57BL/6 mice. The cells were cultured in DMEM (Gibco, 11995065) containing 10% fetal bovine serum (Gibco, 10099141) and 1% penicillin/streptomycin (Gibco, 15140122) for 10 days. HeLa (American Type Culture Collection) were cultured in DMEM (Gibco, 11995065) containing 10% fetal bovine serum (Gibco, 10099141) and 1% penicillin/streptomycin (Gibco, 15140122). All cells were tested for mycoplasma contamination bimonthly using the PlasmoTest kit (InvivoGen).
The following drugs were used: baf A1 (Selleck, S1413, 400nM, 6h).
OGD injury to astrocytes
Astrocytes were washed with PBS three times and cultured in DMEM (no glucose) (Gibco, 11966025). Cells were designed to grow in an incubator with a mixture of 95% N2, and 5% CO2 (hypoxia) inlet for 3 h or 6 h.
SiRNA, plasmids and antibodies
We purchased short interference RNAs for mouse Atg5, PINK1, OPTN, NDP52 and negative siRNA from Gene Pharma (Suzhou, China). All siRNA sequences have been listed in Supplementary Table 2. pCDNA3 HA-Ub and pCDNA3 PINK1-Myc were obtained from Miaoling (Wuhan, China). RFP-OPTN, RFP-NDP52, GFP-Cx43, GFP-Cx43368A and GFP-Cx43247A+265A plasmids were designed by Gene Pharma (Suzhou, China) and these are all pGCMV plasmids. All plasmids sequences have been listed in Supplementary Table 1.
Primary antibodies: Cx43 (Abcam, ab79010), Cx43 (Millipore, AB1728-25UG), p-Cx43(S368) (Cell Signaling Technology, 3511S), p-Cx43(T265) (Invitrogen, PA5-37584), Atg5 (Abcam, ab108327), Beta-actin (Abcam, ab8226), OPTN (GeneTex, GTX132575), NDP52 (GeneTex, GTX115378), GFP(Abcam, ab6556), LC3B (Abcam, ab192890), PINK1(Novus Biologicals, BC100-494), phospho-Ubiquitin (Ser65) (Cell Signaling Technology, 62802S), HA-Tag (Cell Signaling Technology, 3724T), RFP (Abcam, ab62341) and Myc-Tag (Cell Signaling Technology, 2272S). Secondary antibodies: Goat anti-rabbit IgG (Cell Signaling Technology, 7074S), Goat anti-mouse IgG (Cell Signaling Technology, 7076S), Mouse anti-rabbit IgG (Conformation Specific) (L27A9) (Cell Signaling Technology, 5127S), Goat anti-mouse IgG (H+L) (Alexa Fluor 488) (Abcam, ab150113) and Goat anti-rabbit IgG (H+L) (DyLight 633) (Invitrogen, 35562).
Transfections
Primary astrocytes were transiently transfected with siRNA using LipofectamineTM RNAiMAX Transfection Reagent (Invitrogen, 13778100) following manufacturer’s instructions, with siRNA at 30 nM final concentration. HeLa cells were transiently transfected with plasmdis using LipofectamineTM 2000 Transfection Reagent (Invitrogen, 11668019) following manufacurere’s instructions.
Western blotting
Cell lysates were retrieved using RIPA lysis buffer (Abcam, ab156034) supplemented with Protease Inhibitor Cocktail (Thermo Scientific, A32955) and PhosSTOP (Roche, 4906845001). Then cells were sonicated on ice and centrifuged at 4°C at 14000 g for 20 min, followed by a BCA assay (Thermo Fisher Scientific, 23225) for protein concentrations. Next All cell lysates were boiled with 4× LDS sample buffer (Invitrogen, NP0007), 30–40 μg of total proteins were run out on a 4-12% Sure PAGE Bis-Tris gel (Genscript, China, M00654) and transferred to PVDF membranes (Thermo Scientific, 88585). Membranes were probed with the indicated primary antibodies overnight at 4°C, followed by the appropriate HRP-conjugated secondary antibodies for 2h at room temperature. The blots were imaged on the ChemiDoc developer system (Bio-Rad). All band detection was in the linear range. Related Information was provided in in Supplementary Figure 2.
Co-immunoprecipitation (Co-IP)
The cell lysates were extracted from the treated cells using cell lysis buffer (Cell Signaling Technology, 9803S) supplemented with Protease Inhibitor Cocktail (Thermo Scientific, A32955) and PhosSTOP (Roche, 4906845001), followed by a BCA assay (Thermo Fisher Scientific, 23225) for protein concentrations at 1mg/ml and incubated with indicated primary antibodies overnight at 4°C with a constant shaking speed. Then, the complexes were mixed with Protein G Agarose (Roche, 11243233001) and shaken for 3 h at 4°C to capture the antigen-antibody mixture. The beads were then washed five times with cell lysis buffer and boiled in SDS loading buffer. The eluted proteins were analyzed by western blotting.
Immunofluorescence
Slide-cultured cells were treated as indicated in the figure legends. After treatment, the cells were fixed with 4% paraformaldehyde at room temperature for 20 min and washed with PBS for 5 min. Then, the cells were permeabilized with 0.1% Triton X-100 and blocked using 3% goat serum in PBS for 40 min. Next, the cells were incubated with primary antibodies (as indicated in the figure legends) diluted in 3% goat serum overnight at 4°C, and then washed with PBS and incubated with anti-mouse Alexa-Fluor-488-conjugated secondary antibodies and anti-rabbit Dylight-633-conjugated secondary antibodies for 1 h at room temperature. The cells were then washed thrice for 5 min each with 1% Triton X-100 in PBS. During the final wash step, they were incubated with DAPI (Solarbio, C0056) in PBS for 5 min. We used the Leica TCS SP5 confocal microscope and LSM 510 Zeiss confocal microscope for observing the immunofluorescence results.
Flow cytometric analysis
Cytokines in cultured cell supernatants were measured using a cytometric bead array (CBA) mouse Th1/Th2/Th1 Cytokine Kit (BD Biosciences, Cat#560485), and IL-6, IL-10, TNF and IFN-γ were selected as relative cytokines for astrocytes.
Statistics
Experiments were not randomized. All statistical data were calculated and graphed using GraphPad Prism6. All data are presented as means ± SD. Statistical differences were detected using a two-tailed Student’s t-test. A p-values less than 0.05 was considered statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001.