Non targeting control sirna

PASMCs were seeded in 6-well plates and cultured till reaching 30–50% confluence. Then cells were transfected with 100 nM NFATc4 siRNA, or non-targeting control siRNA (GenePharma, Shanghai, China) using Lipofectamine™ 2000 Reagent (Invitrogen) according to the manufacturer’s protocols. Briefly, siRNA and Lipofectamine were diluted separately in serum-free DMEM, and incubated for 5 min at room temperature. Next, the diluted siRNA was gently mixed with the diluted Lipofectamine and incubated for 20 min at room temperature. Then, the complex of siRNA and Lipofectamine was added into cells. The cells were incubated in serum-free DMEM containing the siRNA and Lipofectamine complex for 6 h, after which the medium was replaced with fresh DMEM containing 10% FBS. Effect of siRNA transfection was analyzed using immunoblotting following a further 48 h culture in a 37 °C, 5% CO₂ humidified incubator.

For transient knockdown of the TP53 gene, 2 TP53 siRNAs and non‐targeting control siRNA (GenePharma, Shanghai, China) were transfected into U2OS cells using Lipofectamine RNAiMAX Transfection Reagent (Thermo, Waltham, Massachusetts, USA) according to the manufacturer's instruction. The siRNA targeting sequences of P53 are: 5′‐GACUCCAGUGGUAAUCUAC‐3′ and 5′‐GUAGAUUACCACUGGAGUC‐3′.

siRNA screen was performed using 14 siRNA pools (GenePharma) targeting 14 rate-limiting enzymes/proteins that are involved in SUMOylation process. MEFs cultured in 384-well white plates were transfected with 50 nM siRNA by reverse transfection method using RNAiMax (Invitrogen) according to a protocol from the manufacturer. At 48 h after the transfection, the cells were treated with 100 nM 5Z-7-Oxozeaenol (5z7) and TNFα (1 ng/ml) and cultured for an additional 12 h to induce RDA. Viability was measured using luminescence-based ATP levels as a surrogate marker in surviving cells using CellTiterGlo ATP assay (Promega). Positive control (RIPK1) and negative control (nontargeting control siRNA, GenePharma) were present in the same plate. The screen was performed in duplicate. Z-scores were calculated based on plate median (negative controls excluded) and median absolute deviation, with Z-score = (cell ATP value - median plate ATP value)/(plate median absolute deviation × 1.4826)^{66 (link)}. The screen hits were selected based on the median Z-score of the duplicate plates with cut-offs set at Z-score >2 or < −2 (p < 0.05).