Pkcζ specific pseudosubstrate inhibitor

Cell proliferation was evaluated by CCK-8 assay (7Sea Biotech) according to the instructions provided by the manufacturer. Briefly, the cells were seeded in a 96-well plate at a density of 4.0×10³ cells/well and incubated at 37°C for 24 h. The cells were then treated with or without PKCζ-specific pseudosubstrate inhibitor (10 µM; Invitrogen), After 3 h, the inhibitor mixture was removed and replaced with fresh medium alone or with TNFα (rhTNFα, 10 or 100 ng/ml; Shanghai Bioleaf Biotech Co., Ltd.) for 24, 48 and 72 h. At the indicated time point, the medium was removed and supplemented with the medium containing CCK-8 reagent (10 µl/well) and the cells were cultured for a further 1–1.5 h at 37°C, 5% CO₂. Subsequently, the absorbance was measured at 450 nm using a Tecan Infinite M200 Pro Nano Quant micro-plate reader (Tecan).

The cell counting kit-8 (CCK-8) (7Sea Biotech, Shanghai, China) was used to examine the cytotoxicity according to the instructions provide by the manufacturer. The RAW264.7 cells were seeded in a 96-well plate at a density of 5.0×10³ cells/well and incubated at 37°C for 12 h. The cells were then treated with fresh medium alone or with LPS (100 ng/ml; Sigma, Santa Clara, CA, USA) with or without PKCζ-specific pseudosubstrate inhibitor (1, 5 and 10 µM; Invitrogen, Carlsbad, CA, USA) for 48 h. Subseqently, 10 µl CCK-8 reagent were added to each well and the cells were cultured for a further 2 h at 37°C, 5% CO₂. The absorbance was measured at 450 nm using a Tecan Infinite M200 Pro Nano Quant microplate reader (Tecan, Männedorf, Switzerland).