Ml008 02

To monitor p65 translocation, J774A.1 cells were treated with compounds for 1 h, followed by LPS (100 ng/ml) treatment for 30 min. The cells were fixed with 4% paraformaldehyde (Sigma; #158127) in PBS (w/v) for 30 min at room temperature. After washing with 1× PBS (Welgene; ML008-02) three times, the fixed cells were permeabilized with PBS containing 0.1% Triton X-100 for 10 min. The cells were washed with 1× PBS three times and then blocked with 4% bovine serum albumin (MP Biomedicals; #0216006980) in PBS (w/v) for 1 h. The cells were incubated with mouse anti-p65 antibody (Santa Cruz; sc-8008, 1:200 diluted in 1% BSA–PBS) at 4 °C overnight and washed with PBS three times. FITC-labeled goat anti-mouse IgG antibody (Abcam; ab6785, 1:200 diluted in 1% BSA–PBS) was added to the sample and incubated for 1 h at room temperature. The remaining antibodies were finally washed three times with PBS. Nuclei were stained with Hoechst 33342 (Thermo; #62249). The cellular p65 proteins were imaged by fluorescence microscopy (GE Healthcare; Delta Vision) and quantified by the line profiling method.

HeLa cells were seeded 10,000 cells per well at a white 96-well plate (Falcon; #353296). HeLa cells were transfected with a 1 to 1 ratio of 3× AP1pGL3 (Addgene; #40342) and pRL-TK (Promega; E2241) mixture using LTX plus (Invitrogen; #15338100) and Opti-MEM (Gibco, #31985070) according to the manufacturer’s protocol and incubated for 24 h. The transfected cells were treated with 10 μM of cisplatin (Sigma; P4393), 10, 20, 40 μM of TRIP, or 200 nM of thapsigargin (Sigma; T9033) for 24 h. To measure the AP-1 activation, cells were washed with 1× PBS (Welgene; ML008-02) and lysed with 20 μL of 1× passive lysis buffer for 15 min. Luciferase signals were measured by dual-luciferase reporter assay system (Promega; E1980) using a microplate reader (Biotek; Synergy HTX). Expression levels of AP-1 were normalized by the Renilla luciferase signal.