Z atad fmk

NPC076 and NPC039 are the isolated nasopharyngeal squamous carcinoma cell lines used in this study [10 (link)]. The two cell lines were cultured in DMEM/F-12 (Invitrogen, Carlsbad, CA, USA) supplemented with 5% FBS at 37 °C under 5% CO₂/95% air. The cell lines were incubated and conditioned with the following reagents for experiment control: Z-ATAD-fmk (1–3 μM, BioVision, San Francisco, CA, USA), a cell permeable caspase 12 inhibitor. BMS345541 (1–5 μM, #16667, Cayman, Ann Arbor, MI, USA), a cell permeable IKKα/β inhibitor. Multiple antibodies were purchased and applied for either immunofluorescence staining, western blotting, or coimmunoprecipitation. Antibodies for a variety of proteins are listed as follows: antibody for Casp12 (#55238-1-AP, Proteintech, Rosemont, IL, USA), IκBα, IKKα’IKKβ, p-IκBα, and p-p65 (#4812, #2682, #8943s, #9246, #3033, Cell Signaling, Danvers, MA, USA), IKKα, NEMO (#sc14A231, #sc8032, Santa Cruz, Dallas, Texas, USA), or GFP tags (#GTX628528, GeneTex, Hsinchu City, Taiwan). P-IkB Human Casp12 (hCasp12), Casp12 (GFP-tagged), is from the human Casp12 cDNA (#RG231175, OriGene, Rockville, MD, USA).

AF cells were seeded into 96-well culture plates at a density of 5 × 10³ cells and maintained in complete medium (DMEM/F12 with 10% fetal bovine serum; Gibco/Invitrogen, Carlsbad, CA, USA). When the AF cells reached to 90% confluence, the medium was replaced with complete medium consisting of different concentrations of H₂O₂ (50, 100, 200 μmol/L; 30% solution, Sangon Biotech, Shanghai, China) for various time points (0.5, 1, 2, and 4 h). Each different concentration was repeated in 5 wells. To observe the influence of TGF-β1 addition on AF cells, different concentrations of TGF-β1 (5, 10, 20 ng/mL; Peprotech, Rocky Hill, NJ, USA) were added when the above pre-designed concentrations of H₂O₂ were reached. The cells were pretreated by various chemical blockers, including EK1/2 inhibitor U0126 (Calbiochem, San Diego, CA, USA), p38 inhibitor SB203580 (Calbiochem, San Diego, CA, USA), JNK inhibitor SP600125 (Calbiochem, San Diego, CA, USA), autophagy inhibitor Bafilomycin A (Baf A; Biotime, Shanghai, China), apoptosis inhibitors Z-VAD-FMK (BioVision, California, USA), Z-ATAD-FMK (BioVision, California, USA), and NS3694 (Calbiochem, Billerica, MA, USA), at 2 h before the addition of H₂O₂ and/or TGF-β1.

Saikosaponin (SSa; Nacalai Tesque Inc., Kyoto, Japan) was dissolved in DMSO (Sigma Chemical Co., St. Louis, MO) to form a 10-mM stock solution and stored at –20°C until use. Propidium iodide (PI), RNase A, Hoechst 33342, cytochalasin-B, Giemsa stain, and 4’,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma. Caspase colorimetric assay kits were purchased from BioVision (Palo Alto, CA). Caspase-4 inhibitors, z-LEVD-fmk and Ac-LEVD-CHO, were obtained from Enzo Life Sciences (Plymouth Meeting, PA). The caspase-12 inhibitor, z-ATAD-fmk, was obtained from BioVision. z-DEVD-fmk (caspase-3 inhibitor), z-VDVAD-fmk (caspase-2 inhibitor), and z-IETD-fmk (caspase-8 inhibitor) were obtained from Calbiochem (La Jolla, CA). The annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit was purchased from BD Biosciences (Franklin Lakes, NJ, USA).

Human nasopharyngeal carcinoma cell lines NPC076, and NPC039 are isolated from nasopharyngeal squamous cell carcinoma [35 (link)]. The cells are maintained in basal medium (DMEM/F-12 at 1:1, v/v; Invitrogen, Carlsbad, CA) supplemented with 5% fetal bovine serum in a humidified incubator at 37°C under 5% CO₂/95% air. The Z-ATAD-fmk is a specific inhibitor for Casp12 purchased from BioVision or ProteinTec. The antibody for IκBα was purchased from Santa Cruz (SC-371). Other antibodies or reagents were purchased from Cell Signaling, ABcam, or Sigma Aldrich. A plasmid for cDNA-Caspase 12 (pC12) was a gift from Junying Yuan (Addgene plasmid # 35574) [11 (link)].