Cyquant ldh assay

To delete CHOP in TIB-73 cells (RRID:CVCL_4383), gRNAs targeting the N-terminus of CHOP were cloned into the pX458 plasmid backbone that also expresses Cas9 and GFP separated by a P2A cleavage site (Ran et al, 2013 (link)). TIB-73 cells were transfected with either empty vector or gRNA-containing vector using Lipofectamine 3000 (Thermofisher, USA). Forty-eight hours after transfection, GFP positive cells (from both groups) were sorted by flow cytometry and plated on 96-well plates (1 cell per well). After obtaining single cell-derived colonies, cells were trypsinized and expanded, and screened for CHOP expression by treatment with TM (5 μg/ml for 24 h) followed by western blot. For GADD34 knockdown, MEFs were seeded on 60 mm cell culture dish at 0.7 × 10⁶ cells/dish. Cells were allowed to attach overnight before siRNA transfection. siRNA transfection was performed following manufacture’s protocol (MEF Avalanche Transfection Reagent, EZ biosystems, EZT-MEFS-1) using either scrambled negative control siRNA (IDT, 51-01-14-04) or validated GADD34 siRNA (IDT, mm.Ri.Ppp1r15a.13.2). Cells were treated with TG 24 h after transfection. For LDH assay, cells were treated with vehicle or TG for 24 h and cytotoxicity was assessed using the CyQuant LDH Assay (ThermoFisher) according to the manufacturer’s protocol.

Culture supernatants from cervical epithelial monolayer cell infections were used to assess cytotoxicity using the CyQUANT LDH assay (Thermo Fisher Scientific) according to the manufacturer’s protocol. LDH activity was measured by recording absorbance values at 490 nm and 680 nm and the percentage LDH activity was calculated according to the equation: $\frac{\text{sample LDH activity}}{\text{lysed control LDH activity}}\times 100.$ The assay was performed using three independent biological replicates.