D3072

AsiSI-MEFs were described before^{28 (link)}. WT MEFs were obtained from R. Baer (Columbia University). MEFs (WT MEFs and AsiSI-MEFs) were cultured in high-glucose Dulbecco’s modified Eagle’s medium supplemented with l-glutamine, 10% fetal bovine serum and 1% penicillin-streptomycin. ER-AsiSI-MEF cell lines were developed as previously described^{28 (link)}. Cells were treated with doxycycline (Sigma-Aldrich D3072, 3 μg ml⁻¹) for 24 h to induce AsiSI expression. 4OHT (Sigma-Aldrich H7904, 1 μg ml⁻¹) was added for the last 6 h of doxycycline treatment to induce AsiSI translocation. Cells were cotreated with DMSO, CK-666 (Sigma-Aldrich SML-006, 100 μM), NU7441 (Selleckchem S2638, 10 μM), or wiskostatin (Sigma-Aldrich W2270, 3 μM) and incubated at 37 °C for 6 h. For the DNA-PKc inhibitor experiments, cells were pretreated with 10 μM NU7441 for 1 h before induction of damage with 4OHT. MEF cells were transfected with plasmids using Neon Transfection System (1,350 V, 30 ms, one pulse). For flag-actin experiments, AsiSI-MEF cells were transfected with Flag-actin^R62D–NLS 24 h before induction of AsiSI expression. For cas9 experiments, WT MEF cells were transfected with single-guide RNA (5′-CCCTGTCCCAGCGATCGCGC-3′) targeting Chr. 2 48 h before cell lysis.

Retinal organoids were infected by different lentiviruses in conjunction with LV-rtTA three times between days 23 and 40. TetO promoter induction was carried out by adding doxycycline (Dox) to a final concentration of 2 μg/ml (Sigma-Aldrich, D3072) according to experimental designs as indicated in the results.