Ldh release assay

Cell viability was measured by lactate dehydrogenase (LDH) release assay (Cat# C0016, Beyotime), according to the manufacturer's instructions. The culture supernatants were collected and centrifuged at 12000 g for 5 min to remove cell debris. LDH release in the supernatants was measured at OD 490.

Cell Titer-Glo ® Luminescent Cell Viability Assay (Promega, Madison, WI, USA) was used to monitor the ATP levels in SiO₂ NP-treated ARPE-19 or R28 cells according to the manufacturer’s instructions. Briefly, cells were cultured in 96-well plates and hatched overnight. After the cells were adhered, 15 and 50-nm SiO₂ NPs were added in a dose-dependent manner (0, 5, 10, 20, 40, and 80 μg/mL) into the culture medium and cultured for 12 or 24 h. After washed off the culture medium with PBS, 100 μL of Cell Titer-Glo reagent was added per well. Cells were incubated at 37 °C in 5% CO₂ for 10 min, and luminescence was recorded using a Synergy H4 Hybrid microplate reader (BioTek, Winooski, USA). Cellular ATP levels were calculated by comparing the luminescence intensity of the treated cells to that of the vehicle control. The cytotoxicity of SiO₂ NPs was assessed using the LDH Release Assay (Beyotime, Beijing, China) as our previous method [34 ]. The absorbance was detected at a wavelength of 490 nm by using a Synergy H4 Hybrid microplate reader and data are presented as relative values compared to control.

Zearalenone (ZEA), and 4-phenylbutyrate (4-PBA), were purchased from Sigma–Aldrich (St. Louis, MO, USA); Fetal bovine serum (FBS) and DMEM/F-12 medium were obtained from Gibco (Grand Island, NY, USA); the cell counting kit-8 (CCK8) was purchased from Dojindo Laboratories (Kumamoto, Japan); the LDH release assay, ROS assay kit (S0033) and ATP assay kit were obtained from Beyotime Institute of Biotechnology (Shanghai, China); and the cell cycle assay caspase 3 activity assay kit and Annexin V/propidium iodide (PI) were purchased from Becton Dickinson Company (BD; Franklin Lakes, NJ, USA). Dorsomorphin dihydrochloride (HY-13418) was purchased from Medchem express (Shanghai, China).

Zearalenone (ZEA) was purchased from Sigma–Aldrich (St. Louis, MO, USA); resveratrol (RSV) was purchased from Solarbio (Beijing, China, SR8070); DMEM/F-12 medium was obtained from Gibco (Grand Island, NY, USA, 12500-062); fetal bovine serum (FBS) was obtained from Gemini (California, USA, 900-108). RIPA lysate and protease inhibitor complex were purchased from Pulilai (Beijing, China, C1053); GAPDH and SIRT1 antibody were purchased from Cell Signaling Technology (Boston, Massachusetts, USA); GLUT1, LDH, and MCT4 antibodies were purchased from Abcam (Cambridge, MA, USA); LDH release assay was obtained from Beyotime Institute of Biotechnology (Shanghai, China, C0017); the lactic acid assay kit and the pyruvate assay kit were purchased from Nanjing Institute of Biological Research (Nanjing, China, A019-2, A081); and all other reagents and chemicals were analytical grade and were obtained commercially.

CHON-001 cells were treated with a series of concentrations of nintedanib (0, 0.75, 1.5, 7.5, 15, 75, and 150 μM) for 24 h. Cytotoxicity of the drug on CHON-001 cells was assessed using the LDH release assay, which was performed using a commercially available kit (Beyotime, Shanghai, China) to detect the LDH release level in the medium according to the manufacturer’s protocol [16 (link),17 ].

HCEC-B4G12 cells were collected and plated in 96-well plates at a density of 1 × 10⁴ cells/well and cultured overnight in a CO₂ incubator. Next, the cells were exposed to Na-Mt, H-Na-Mt, C-H-Na-Mt, Ca-Mt, and MMt having different concentrations (1.56–100 μg/mL) for 24 and 48 h. As recently described [30 (link)], the ATP levels were measured by using the CellTiter-Lumi™ Plus Luminescent Cell Viability Assay (Beyotime, Beijing, China), while the LDH Release Assay (Beyotime, Beijing, China) was used to assess the membrane integrity of HCEC B4G12 cells, with cell viability examined by applying the Cell Titer 96 Aqueous One Solution Cell Proliferation Assay (Promega Corporation, Madison, WI, USA). Luminescence and absorbance were respectively recorded in a microplate reader (SpectraMax ID3, Molecular Devices, USA). Finally, morphological changes of HCEC-B4G12 cells were also examined under a phase-contrast microscope (Leica DM16000B, Germany).