Fas ligand

J774 cells were induced to undergo apoptosis by incubation for 6 h at 37 °C, with 2 µg/ml actinomycin D, RAW cells were induced using 1 µg/ml actinomycin D for 4 h. THP-1 cells were induced to undergo apoptosis by incubation with 1 µg/ml of staurosporine (calbiochem) for 12 h in culture. Hela cells were induced to undergo apoptosis by 300 ng/ml Fas ligand (sigma) for 24 h and 300 ng/ml TRAIL (sigma) for 12 h in complete RPMI. Neutrophils were induced to undergo apoptosis under physiological conditions by culturing them in complete RPMI for 18–20 h [65 (link)] and also by infection with Mycobacterium tuberculosis (M.tb H37Rv) as described previously [66 (link), 67 (link)]. Briefly cells were incubated with M.tb bacilli for 30 mins and then cultured for 18 h in fresh medium. In all cases apoptosis was confirmed by detecting PS exposure on cell membrane using Annexin V staining while necrotic cells were identified as those staining positive with 7-aminoactinomycin D (7AAD, Invitrogen #A1310) using flow cytometry as described below.

RIPA lysis buffer was used to extract proteins from tissues or cell lines and protein samples (50 μg) were resolved by SDS-PAGE and then probed with indicated antibodies. CYB561D2, FAS ligand (FASLG), TGF-β2 (TGFB2), CD70, PD-L1, PD-L2, CCL2, TDO2, total STAT3, p-STAT3 (Tyr705) antibodies were from Sigma-Aldrich (SAB2500281), abcam (ab15285), abcam (ab36495), abcam (ab77868), abcam (ab205921), abcam (ab187662), abcam (ab186421), abcam (ab123403), CST (4904) and CST (9131), respectively. STAT3 inhibitor C188-9 was from selleckchem (S8605) [16 (link)]. The band density was analyzed with Image J and the density of target protein is normalized by Tubulin band. There are four biological replicates for each WB experiment.

Stress inducers and inhibitors: PD173074, staurosporine, anisomycin, etoposide, brefeldin A, thapsigargin, doxorubicin and Fas ligand were from Sigma-Aldrich (MO, USA). Actinomycin D and camptothecin were from Santa Cruz Biotechnology (TX, USA). ABT 737, BTSA-1, BGJ398, ARQ087 were from Selleckchem (TX, USA), Apo2/TRAIL was from Merck (Darmstadt, Germany). The following primary antibodies were used: rabbit anti-PARP1/2 (9542), rabbit anti-BAX (D2E11) (5023), rabbit anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (9101) from Cell Signaling Technology (MA, USA), mouse anti-gamma tubulin (T6557) from Sigma-Aldrich, goat anti-FGF1 (sc-1884), mouse His-tag (H3) (sc-8036) and mouse anti-p53 DO-1 (sc-126) from Santa Cruz Biotechnology. Rabbit polyclonal antibodies against FGF1 (OPA1) were generated by Davids Biotechnologie GmbH (Regensburg, Germany) by immunizing rabbits with purified FGF1. The following HRP-conjugated secondary antibodies from Jackson Immuno Research Laboratories (PA, USA) were used: peroxidase affinipure goat anti-rabbit IgG (111-035-144) and anti-mouse IgG (115-035-003).