CRISPR-Cas9-Mediated REDD1 Knockout in Müller Cells

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).