Ag490

A549, H1299, and H157 cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and cultured in RPMI 1640 with 10% FBS. CD133+ CSCs were cultured in DMEM/F12 medium supplemented with ITS (insulin, transferin, selenium, Invitrogen), 20 ng/ml EGF (Invitrogen), and 20 ng/ml FGF (Invitrogen). All cells were maintained in a humidified 5% CO₂ environment at 37°C.
For inhibition studies of sphere formation of CD133+ cells, we applied the inhibitors LY294002 (10 μM, Sigma, St Louis, MO), SB203850 (10 μM, Sigma, St Louis, MO), AG490 (5 μM, Cell Signaling, Danvers, MA), U0126 (10 μM, Cell Signaling, Danvers, MA), ABT737 (0.1 μM, Selleckchem Houston, TX), and cyclopamine (5 μM, Selleckchem Houston, TX) that inhibit Akt, MAPK, and JAK/Stat3, Erk/MEK, Bcl-2, and Hhg signaling pathways, respectively.

A549 and H157 cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and cultured in RPMI 1640 with 10% FBS. All cells were maintained in a humidified 5% CO₂ environment at 37°C. For inhibition studies, LY294002 (10 μM) (Sigma, St Louis, MO), SB203850 (10 μM) (Sigma, St Louis, MO), AG490 (5 μM) (Cell Signaling, Danvers, MA), and U0126 (10 μM)(Cell Signaling, Danvers, MA) that inhibit the Akt, MAPK, JAK/Stat3, and MEK/Erk pathways, respectively, were added into the culture before cisplatin treatment.

At 24 hours after seeding, RGCs were stressed by the addition of 25 µM hydrogen peroxide (H₂O₂; Sigma-Aldrich, St. Louis, MO, USA) for 24 hours. The concentration of H₂O₂ was based on the concentration required to reduce the number of the surviving RGCs by 50% when oxidative stress was applied (Supplementary Fig. S3). Recombinant rat CNTF (PeproTech, London, UK) was added at a concentration of 0 to 100 ng/mL for 24 hours. After that, CNTF was treated with RGCs for 24 hours at a concentration of 50 ng/mL, which was thought to be the concentration for maximum effect. To assess the role of each signal transduction pathway, pathway inhibitors were administered in the same environment, including the PI3K/AKT pathway inhibitor LY294002 (50 µM; Cell Signaling Technology), MAPK/ERK pathway inhibitor PD98059 (50 µM; Cell Signaling Technology), or JAK/STAT3 pathway inhibitor AG490 (10 µM; Cell Signaling Technology), 4 hours before recombinant CNTF treatment, at concentrations reported in previous studies.²⁴ (link) RNA sequencing was performed under the same conditions without oxidative stress.