Z vad fmk

To detect whether viral replication was required for caspase activity, two caspase inhibitors, Z-VAD-FMK (caspase inhibitor; Beyotime, Shanghai, China) and Ac-DEVD-CHO (caspase-3 inhibitor; Sigma, St. Louis, MO, USA), were used to inhibit cellular caspase activity. SeFB or Ha-E cells were seeded in 6-well plates and infected with HvAV-3h. At 24 and 48 hpi, Z-VAD-FMK or Ac-DEVD-CHO was added to the medium to obtain final concentrations of 10 μM and 20 μM, respectively. At 24 and 48 h postexposure to caspase inhibitors, the cells were collected. Immunoblotting was performed, and the grayscale of each immune band was analyzed as described above. Quantitative PCR was performed to detect viral DNA copies under different treatments, as described above. The differences in protein expression levels or viral DNA copies between different samples were analyzed by one-way ANOVA and compared using the LSD method in SPSS 22.0.

The reagents and solvents were purchased commercially and used without further purification unless otherwise noted. 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), Hoechst 33258, 2-(6-Amino-3-imino-3H-xanthan-9-yl) benzoic acid methyl ester, RIPA Lysis Buffer and Annexin V-FITC/PI Apoptosis Detection Kit were purchased from Sigma. The Caspase Activity Assay Kit, caspase-3 inhibitor Z-DEVD-FMK, caspase-9 inhibitor Z-LEHD-FMK and caspase inhibitor Z-VAD-FMK, LDH Release Assay Kit were purchased from Beyotime.

All breast cancer samples were obtained from newly diagnosed patients with prior patient consent and the approval of the Institutional Clinical Ethics Review Board in the 1^st Affiliated Hospital of Dalian Medical University. Samples were kept in liquid nitrogen for protein extraction.
HEK293T cell and human breast cancer cells (SKBR-3, BT-549) were cultured in DMEM medium (Invitrogen) or RMPI 1640 medium (Invitrogen) supplemented with 10% (v/v) fetal bovine serum (FBS, HyClone). The immortalized breast epithelial cell MCF-10A was cultured in DMEM/F12 medium (Invitrogen) supplemented with 5% (v/v) horse serum (HS, HyClone), 20ng/ml EGF, 100ng/ml cholera toxin, 0.01mg/ml insulin and 500ng/ml hydrocortisone. All cell lines were purchased from American Type Culture Collection (ATCC) and incubated at 37°C in humidified 5% CO₂ incubator.
Reagents used were purchased as followed: VX680 (Selleck Chemicals, 639089-54-6), 3-Methyladenine (3-MA, Sigma, M9281), rapamycin (Sigma, 37094), SB216763 (Sigma, S3442), z-VAD-FMK (Beyotime, C1202), lithium chloride (LiCl, Sangon Biotech, LDB0307), doxorubicin (KeyGEN BioTECH, KGA8182). Nutrient deprivation was performed using Hank's balanced salt solution (HBSS, HyClone). To avoid any supplementary stress, HBSS was preheated at 37°C before added into cells.

Berberine was purchased from Chengdu Must Bio-Technology Co. (Chengdu, China) and was stored in ddH₂O as a 4 mM stock solution at 4° C in dark. The control group received an equivalent volume of medium.
To perform different inhibitory analyses, 10 mM autophagy inhibitor 3-methyladenine (3MA; Sigma-Aldrich Co.), 50 μM of the autophagy inhibitor chloroquine (Sigma-Aldrich Co.), 20 μM apoptosis inhibitor z-VAD-FMK (Beyotime Biotechnology; Beijing, China), 2 μM caspase 3 activator Apoptosis Activator 2 (Selleck Chemicals; Houston, TX, USA), 10 mM SIRT1 inhibitor nicotinamide (NAM, Beyotime Biotechnology), 10 mM HDAC inhibitor trichostatin A (TSA, Beyotime Biotechnology), 200 μM IDO1 inhibitor 1-methyl-L-tryptophan (1MT, Sigma-Aldrich Co.), 200 μM QPRT inhibitor phthalic acid (PA, Sigma-Aldrich Co.), 10 μM NAMPT inhibitor FK866 (Beyotime Biotechnology), 5 μM PI3K inhibitor LY294002 (Selleck Chemicals), 5 μM PI3K agonist insulin-like growth factor-1 (IGF-1, R&D Systems, Inc., Minneapolis, USA), 5 μM AKT inhibitor triciribine (Selleck Chemicals), and 1 μM mTOR inhibitor rapamycin (Rapa, Selleck Chemicals) were used in combination with Berberine.

H9c2 cells were incubated with a normal medium in a cell incubator for 24 h. Cells were then exposed to hypoxic conditions (oxygen deprivation, 1% O₂) for 24 h in a culture medium with lower glucose and 1% FBS. After hypoxia, the cells were oxygenated under a normal oxygen concentration (reoxygenation) for 24 h in a normal medium. According to the previous study, propofol (Sigma-Aldrich, United States) was added respectively to the cells 1 h before and during the hypoxia-reoxygenation with different concentrations (5, 10, and 20 μM) (Zhao et al., 2015 (link)). U0126 (Beyotime, Haimen, China), MEK1/2 inhibitor, was added to the cells during OGD/R at a concentration of 20 μM. After H9c2 cells were incubated with a normal medium in cell incubator for 24 h, propofol and caspase inhibitor Z-VAD-FMK (Beyotime, Haimen, China) were respectively added to the cells for 24 h.

Oleandrin was obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). The molecular structure of Oleandrin is showed in the Supplementary file (Figure S1). The purity was approximately 99%, as analyzed by HPLC. A 1 mmol/L stock solution (Mr = 576.73) was prepared by dissolving Oleandrin in 100% DMSO (Sigma-Aldrich) and was stored at −80 °C. All subsequent dilutions were made in the medium. The final concentration of DMSO was confirmed to be less than 0.1%. The pan-caspase inhibitor with cell membrane permeability, z-VAD-fmk, was purchased from Beyotime (Beyotime Biotech, Nanjing, China). The caspase-9 inhibitor, z-LEHD-fmk, was obtained from Sigma (Sigma-Aldrich Chemical Co.). The Fas blocking antibody (Abcam, Cambridge, MA, USA) was kindly donated by Wei Zhang from the Institute of Zoology, Chinese Academy of Science.

The activity of caspase-3 was determined using the caspase-3 activity kit, based on the ability of caspase-3 to change acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA) into a yellow formazan product p-nitroaniline (pNA). Caspase inhibitor Z-VAD-FMK (100 μM) and caspase-3-like inhibitor Ac-DEVD-CHO (100 μM) were used according to the manufacturer's instructions (Beyotime, Haimen, China). Lysates were centrifuged at 12,000 g for 10 min, and protein concentrations were determined by Bradford protein assay. Cellular extracts (30 μg) were incubated in a 96-well microtitre plate with 20 ng Ac-DEVD-pNA for 4 hours at 37°C. OD405 values were read with a 96-well plate reader. An increase in OD405 indicated the activation of caspase-3.