Buffer kit 5

For the generation of YbeY deficient HEK293T cell lines, the cells were transiently transfected with CompoZr ZFNs (Sigma-Aldrich) using Cell Line Nucleofector (Lonza), buffer kit V (Lonza) and program A-023. Seventy-two hours after transfection, the cells were single-cloned into 96 well plates and screened using Sanger sequencing to identify indels at the ZFN target site and western blotting. Wild-type Hap1 and YbeY knockout Hap1 cell lines were purchased from Horizon Discovery (Product ID: HZGHC002765c022).

For siRNA tests, THP-1 monocytes were pretreated with NF-kB siRNA or control siRNA using the Cell Line Nucleofector (Lonza, USA) and buffer kit V (Lonza, USA) according to the manufacturer's protocol. After 48 h, treated cells were exposed to IH for 24 h followed by protein samples collection for western blot assays.
To silence the RAGE gene, THP-1 monocytes at a density of 2 × 10⁵ cells/well in a 6-well plate were first suspended in growth media supplemented with 5 μg/mL polybrene and incubated overnight. Then RAGE shRNA lentiviral particles or control shRNA lentiviral particles were added. After 48 h of incubation at 37°C, the viral load was removed by centrifugation. THP-1 monocytes were washed with PBS and cultured in growth media. Successfully transfected THP-1 monocytes were selected by treatment with 10 μg/mL puromycin (Santa Cruz Biotechnology, USA) until all cells in the control flask were confirmed dead.