Supplementary Materials Image_1. RGC loss, attenuated Bax expression, and improved mitochondrial health and mitochondrial surface area. Parkin expression and the number of mitophagosomes were upregulated in OPA1 overexpressed RGCs under glutamate excitotoxicity. While knockdown of OPA1 by siRNA decreased protein expression of parkin in RGCs under glutamate excitotoxicity. Two weeks after intraocular pressure (IOP) elevation, the LC3-II/I ratio and the LAMP1 expression were increased in OPA1 overexpressed optic nerve. These findings suggest that OPA1 overexpression may protect RGCs by ways of enhancing mitochondria GSK1120212 inhibition fusion and parkin mediated mitophagy. Interventions to promote mitochondrial fusion and mitophagy may provide a useful strategy to battle against glaucomatous RGC loss. and standard rodent diet. Cultured Retinal Ganglion Cell Culture and Treatment Retinal ganglion cells were isolated and cultured as previously described (Hu et al., 2017). Briefly, retinas from 2- to 3-day-old Sprague-Dawley rats were dissociated in 5 mg/ml of papain solution (Worthington Biochemical, Lakewood, NJ, United States). The retinal suspensions were then sequentially incubated with a petri dish coated with rabbit anti-macrophage antibody (Millipore Corp., Billerica, MA, United States) and mouse anti-Thy1.1 antibody (Abcam, Cambridge, MA, United States). RGCs were seeded into appropriate plates coated with 0.01% poly-D-lysine (Sigma-Aldrich, St. Louis, MO, United States). Adenovirus, designed and packaged by (Sunbio, Shanghai, China) as previously described (Hu et al., 2017), were diluted in cell culture medium for 48 h to infect RGCs. The RGCs were then incubated with 100 M glutamate (Sigma-Aldrich) to induce excitotoxicity model. Small interfering RNA (siRNA) targeted against OPA1 was designed and packaged by Genomeditech Co., Ltd. (Shanghai, China). The sequences used were as follows: 5- GACAUCUUUUCAGCAAUUC-3. Transfection was performed with RNAi max (Thermo Fisher Scientific, Shanghai, China) according to the manufacturers instruction. Seventy-two LRP2 hours after transfection, the RGCs were then incubated with 0 M GSK1120212 inhibition or 100 M glutamate as described above. Quantitative PCR Total RNA from RGCs (= 3 groups) was extracted with Trizol (Invitrogen, Carlsbad, CA, United States) according to the manufacturers instructions. The target gene was amplified GSK1120212 inhibition by qPCR (SYBR; Takara, Tokyo, Japan) with a program (95C for 15 s, and 60C for 30 s for 45 cycles). GAPDH was used as endogenous reference. The data were analyzed using the 2-= 3 per group) were lysed in RIPA buffer (Beyotime, China) and ultrasonically smashed on ice to get homogenized solutions. RGCs (= 3 per group) were mixed with RIPA buffer. Equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the electrotransferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, United States). After blocking with 5% non-fat dry milk at room temperature for 1 h, the membranes were incubated with polyclonal rabbit anti-OPA1 (1:1000; Abcam), polyclonal rabbit anti-GFAP antibody (1:10000; Abcam), polyclonal rabbit anti-Parkin (1:1000; Abcam), polyclonal rabbit anti-Bcl-2 (1:500; Abcam), monoclonal rabbit anti-Bax (1:1,000; Abcam), monoclonal rabbit anti-LC3 (1:2000; Abcam), polyclonal rabbit anti-LAMP1 (1:1000; Abcam) and polyclonal rabbit anti-GAPDH (1:2000; Yesen, China) in primary antibody dilution (Beyotime, China) overnight at 4C. After the membranes were washed several times, secondary antibody peroxidase-conjugated goat anti-rabbit IgG (1:5000; Jackson) was applied. Proteins were visualized by a Kodak Image Station 4000MM PRO (Carestream, Rochester, NY, United States) using chemiluminescence detection (SuperSignal West Femto Substrate Trial Kit, Thermo Fisher). Band intensity was analyzed with Image J (National Institutes of Health). Immunohistochemistry Analysis Immunohistochemical staining of 7-um frozen sections of full-thickness retina was prepared. Three sections per frozen block from each group (= 3 retinas/group) were used for immunohistochemical analysis. The RGCs (= 3 per group) on the coverslips were fixed with 4% PFA in PBS for 20 min, rinsed with PBS, and then were used for immunohistochemical analysis. The samples were permeabilized with 0.1% Triton X-100 in PBS for 20 min at room temperature, sequentially, blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature, incubated by 16 h at 4C with the following primary antibodies: monoclonal mouse anti-GFAP antibody (1:200; Abcam), polyclonal rabbit anti-OPA1 (1:200; Abcam), and monoclonal rabbit.