Culture of MIO-M1 human Müller cells

MIO-M1 Müller cells (RRID: CVCL_0433) were cultured in DMEM media (Invitrogen#11885) containing 4.5 g/L D-glucose, 110 mg/L sodium pyruvate, 1% penicillin/streptomycin and 10% fetal bovine serum (FBS) unless specified. We used Nunc™ EasYFlask™ Cell Culture Flasks (Cat# 156367) and Nunc™ Non-Treated Multidishes (Cat# 150200) to culture MIO-M1 Müller cells. In order to optimize the time points of Notch and TGFβ ligand treatment for activation of these two signaling pathways, Müller cells were cultured in DMEM media until 80% confluence and then incubated in media containing recombinant human Notch ligands including Delta-like 4 (Dll4, R&D Systems, Cat#1506-D4, 50 ng/ml) and Jagged-1 (Jag1, R&D Systems, Cat# 277-JG, 50 ng/ml) or recombinant human TGFβ1 (R&D Systems, Cat#240-B, 10 ng/ml) for 3, 6, 18 and 24 hours. Changes in Notch and TGFβ signaling proteins were studied by Western Blots using antibodies against γ-secretase proteases, Notch downstream effectors, total and phosphorylated Smad 2 and 3 (Table ​Table11).

Antibodies used for Western blots

In order to study the effects of Notch inhibition on the events associated with retinal fibrosis, we also incubated Müller cells in media containing Notch ligands including Dll4 and Jag1 (both 50 ng/ml) and TGFβ1 (10 ng/ml) with or without 1 or 10 µM of the γ-secretase protease inhibitor RO4929097 (Selleckchem, Cat#S1575, stock solution 50 mM dissolved in DMSO). Changes in Notch and TGFβ signaling and ECM proteins were studied by Western blots (Table ​Table11).