Imd0354

Prior to treatment, the cells were plated at 2.5 × 10⁵ cells/well in 6-well plates overnight. The next day, medium was removed, and the cells were incubated for another 24 h in serum-free DMEM. Medium was then removed again, and cells were treated with various concentrations of PM_2.5 (0.01 – 30 μg/cm²) diluted in DMEM with 1% FBS, for the indicated times. In experiments where cells were treated more than once, medium was removed before each treatment, and cells were washed with PBS before being treated again with the respective concentration of PM_2.5. Control cells in these repeated exposure experiments were also washed and had medium replaced with DMEM with 1% FBS alone. After the exposure period(s), cells were harvested for RNA or protein analysis. Prior to collection, photographic images of cells were taken with the Echo Revolution microscope for live-cell imaging. In some experiments, the cells were pre-incubated with the AhR antagonist CH223191 (10 μM, Tocris Bioscience, Ellisville, MO) or the NF-κB inhibitor IMD0354 (10 nM, Tocris Bioscience) for 30 min prior to addition of PM_2.5. The concentrations of CH223191 and IMD0354 were chosen based on their effective inhibitory concentrations as reported in the literature (Tanaka et al., 2005 (link); Zhao et al., 2010 (link)).

Human MIO-M1 Müller cells were acquired from the UCL Institute of Ophthalmology (London, UK). REDD1 KO MIO-M1 cells were generated by CRISPR-Cas9 genome editing as previously described.³⁶ MIO-M1 cultures were maintained in DMEM (Thermo Fisher Scientific, Waltham, MA, USA) containing 5.6 mM glucose and supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin-streptomycin. MIO-M1 cells stably expressing an shRNA targeting GSK3β were generated as previously described.²⁹ (link)^,37 (link) Cells expressing pLKO.1-TRC were used as an shRNA control (Addgene Plasmid #10879). To model hyperglycemic conditions, culture medium was supplemented with d-glucose to achieve a final concentration of 30 mmol/L glucose for up to 24 h. Medium containing 5.6 mM glucose plus 24.4 mM mannitol was used as an osmotic control. Lipofectamine 2000 (Life Technologies, Carlsbad, CA, USA) was used for cell transfection. Plasmids included pCMV5 vector, pCMV-HA-caGSK3β, and pCMV-HA-REDD1.²⁹ (link) In some studies, cell culture medium was supplemented with 1 µM IMD0354 (Tocris Biosciences, Bristol, UK) or 1 µM VP3.15 (MedKoo Biosciences, Durham, NC, USA).