Sirt4

Protein was obtained from cells in RIPA lysis buffer (Beyotime Biotechnology) with protease inhibitors and phosphatase inhibitors. Extracted protein was subjected to SDS‐polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes (Bio‐Rad). After blocking at room temperature for 2 hours with 5% non‐fat dried milk, membranes were incubated with primary antibody overnight at 4°C. Membranes were then incubated with the appropriate secondary antibodies for 2 hours at room temperature. The protein band was measured in an electro‐chemiluminescence detection system (Thermo Fisher Scientific). Primary antibodies used were as follows: SIRT4 (Abcam), α‐SMA, Collagen I, TIMP‐1, TGF‐β, AMPK, p‐AMPK, Smad3, p‐Smad3, Smad2, p‐Smad2 and β‐actin rabbit mAbs (1:1000; Cell Signaling Technology). The secondary antibodies, including HRP‐conjugated goat anti‐rabbit immunoglobulin G (IgG) or goat anti‐mouse IgG (Cell Signaling Technology, MA, United States), were diluted 1:2000. The proteins were detected using ECL chemiluminescence (GE Amersham, Arlington Heights, IL).

Retinal tissues and r-MCs were lysed in RIPA buffer containing PMSF and phosphatase inhibitor (Solarbio, China). The protein concentration was measured using a Bradford assay (Beyotime Institute of Biotechnology) according to the manufacturer’s instructions. Protein samples (7 or 15 µg) were separated in 7.5–12% SDS–polyacrylamide gels by electrophoresis at 70 V for 2 h. The proteins were transferred to PVDF membranes using the wet transfer method. The blots were blocked in 5% skimmed milk in Tris-buffered saline/Tween-20 for 1 h, then incubated with the appropriate primary antibodies, followed by incubation with peroxidase-conjugated secondary antibodies (CST) at 1:5000. The following antibodies were used: OPA1 (#612606, BD Biosciences) at 1:1000, DRP1 (#8570, CST) at 1:1000, cleaved caspase 3 (#9664, CST) at 1:1000, SIRT4 (ab10140, Abcam) (#69786, CST) at 1:1000, LC3 (#4599, CST) at 1:1000, SQSTM1/p62 (#16177, CST) at 1:1000, p-mTOR (#5536, CST) at 1:1000, p-AMPK (#4184, CST) at 1:1000, GADPH (HC301-01, TransGen Biotech) at 1:2500, and β-tubulin (HC101-01, TransGen Biotech) at 1:2500. Finally, proteins on membranes were detected.

Formalin‐fixed and paraffin‐embedded liver samples were deparaffinized, rehydrated and subjected to antigen retrieval. Samples were incubated with a primary antibody against α‐SMA (1:500; Cell Signaling Technology) or SIRT4 (1:500; Abcam) overnight at 4°C. Then, the samples were washed with pre‐cooled PBS and incubated with HRP‐polymer‐conjugated secondary antibody at 37°C for 1 hour. 3,3′‐Diaminobenzidine tetrahydrochloride staining was performed to evaluate the expression of α‐SMA. The nuclei were counterstained with haematoxylin.

Streptozotocin (STZ) and metformin (Mef) were acquired from Sigma–Aldrich Biotechnology (St. Louis, MO, USA). The primary antibodies including kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), selenium binding protein-1 (SBP1), pyruvate kinase muscle isozyme M2 (PKM2), collagen-1, E-cadherin, TGF-β1, vimentin, fibronectin, α-tubulin, claudin-1, α-SMA, SIRT1, SIRT3, SIRT4, and β-actin were obtained from Abcam (Cambridge, MA, USA). Immobilon Forte Western HRP substrate (cat. no. WBLUF0100) and polyvinylidene difluoride (PVDF) membrane were procured from Millipore (Burlington, MA, USA). Other chemicals were obtained from Sigma–Aldrich (St. Louis, MO, USA).