2 7 dichlorofluorescin diacetate

A549 cells were obtained from the Institute of Radiation Medicine, Peking Union Medical College (China), and cultured in RPMI‐1640 containing 10% fetal bovine serum (FBS) and antibiotics. The cells were incubated at 37°C in a humid environment with 5% CO₂. The above cell culture reagents were purchased from Gibco (Grand Island, USA). MPPa, Cell Counting Kit‐8, 2′,7′‐dichlorofluorescin diacetate and Hoechst 33342 were obtained from Sigma‐Aldrich. Annexin V/PI double staining and JC‐1 mitochondrial membrane potential detection kits were manufactured by Keygen Biotech (Nanjing, China).
Rabbit monoclonal antibodies against human caspase‐3 and ‐9, Bcl‐2, and Bax, respectively, were manufactured by Cell Signaling Technology (Danvers, MA). Anti‐β‐actin and anti‐cytochrome‐c primary antibodies as well as secondary antibodies were purchased from Abcam (Cambridge, UK). The PDT equipment was manufactured by Chongqing Jingyu Laser Technology Co. Ltd. (Chongqing, China).

DNA polymerase, dNTP, restriction enzymes, DNA ligase, and T-vector pMD20 were obtained from Takara Clontech (Japan). Isopropyl-β-thiogalactopyranoside (IPTG) and kanamycin were purchased from Duchefa Farma B.V. (The Neterlands); TALON metal affinity resin was obtained from Qiagen (Germany). Steroid substrates containing testosterone, nandrolone, cortisone, and prednisone were purchased from Tokyo Chemical Industry Co., Ltd. (Japan). Rotenone, H₂O₂, and 2’,7’-dichlorofluorescin diacetate were purchased from Sigma Chemical (USA). Fetal bovine serum (FBS) and RPMI 1640 medium were purchased from HyClone (USA); 96-well collagen IV-coated and white plates were purchased from BD Biosciences (USA). CellTiter Aqueous One Solution Cell proliferation assay kits (MTS) were purchased from Promega Co. (USA). Luminescence ATP and JC-1 mitochondrial membrane potential detection kits were obtained from PerkinElmer (USA) and Biotium (USA). All other high-grade reagents were obtained from commercial sources.

Tamoxifen, 4-hydroxy-Tamoxifen, Dimethylsulfoxide, 2’,7’-dichlorofluorescin diacetate, 5,5’-dithio-bis (2-nitrobenzoic acid), colchicine, valspodar (PSC-833) and Tween-20 (P9416) were all purchased from Sigma Aldrich. Lipofectamine 2000, the microBCA kit and the Pierce™ ECL Western Blotting Substrate Kit were all products of Thermo Fisher. Propidium iodide and the HRP-conjugated goat anti-mouse were purchased from Invitrogen, and the IncuCyte® is a product of Essen BioScience.

Calcium oxalate (Sigma, 455997), 3-methyladenine (Sigma, M9281), rapamycin (Sigma, R0395), 4′,6-diamidino-2-phenylindole (Sigma, D8417), N-acetyl-L-cysteine (Sigma, A7250), catalase (Millipore, 219261-100KU), 2′,7′-dichlorofluorescin diacetate (Sigma, D6883), Lipofectamine 3000 (Invitrogen, L3000008), and rabbit anti-LC3B (Sigma, L7543) for western blot (WB) (1:2000), mouse anti-BECN1 (Cell Signaling Technology, 3495) for WB (1:1000). rabbit anti-LC3B (Abcam, ab51520) (1:1000) and mouse anti-BECN1 (Abcam, ab114071) were used for immunohistochemistry (IHC) (1:500). Mouse monoclonal anti-GAPDH (Proteintech, 60004-1-Ig) was used for WB (1:5000). Mouse and rabbit HRP-conjugated antibodies were obtained from Zhongshan Golden Bridge Biotechnology (ZB-2305, ZB- 2301).

Transwells™ were obtained from Corning (Schiphol, The Netherlands). Lucifer yellow was purchased from Thermo Fisher. Protease inhibitor cocktail Set III from Calbiochem (Merck, Darmstadt, Germany). Oligofectamine™ Reagent and Pierce BCA Protein Assay were from Thermo Fisher Scientific Inc. (Rockford, IL). For Western blotting and immunofluorescence, antibodies directed against occludin, claudin-5, and ZO-1 were purchased from Invitrogen (Basel, Switzerland), β-actin antibody from Sigma–Aldrich (Buchs, Switzerland), and β-catenin antibody from Chemicon (Millipore, Billerica, MA). Anti-phosphotyrosine antibody agarose conjugate, clone 4G10®, was purchased from Milipore. Secondary antibodies for Western blotting and immunofluorescence were obtained from Jackson ImmunoResearch (Suffolk, UK) or Invitrogen. For the glutathione (GSH) and ROS assay, 5,5-Dithio-Bis-(2-Nitrobenzoic acid) (DTNB) and 2′,7′-Dichlorofluorescin diacetate were obtained from Sigma–Aldrich (Buchs, Switzerland). Isotopic labeled ³⁴S¹⁵N-cysteine was designed and synthesized by Dr. T. Sawa (Graduate School of Medical Sciences, Kumamoto University) [54 (link)]. γ-glutamyl cysteine ligase catalytic subunit (GCLc) siRNA (ON-TARGET plus SMART pool) and negative control siRNA (ON-TARGET plus non-targeting pool) were purchased from Dharmacon.

Intracellular ROS generation of R. quercus-mongolicae and R. solani was observed using the ROS-sensitive dye 2′,7′-dichlorofluorescin diacetate (≥97, Sigma-Aldrich, MO, USA). Nutrient agar (NA) (Difco, IL, USA) plates were prepared in Petri dishes (diameter 90 mm). Agar-mycelial plugs (diameter 5 mm) of R. querqus-mongalicae or R. solani were placed in the center of the dishes for incubation at 25 °C (one day for R. solani and four days for R. querqus-mongalicae). On agar-free lids, paper discs (diameter 8 mm, Advantec, Tokyo, Japan) with 10 mg carvacrol and thymol were placed. Controls received only 25% DMSO and 1% Tween 80. After treatment, treated and control NA plates were incubated for one day at 25 °C. After incubation, plugs from treated and control NA plate were stained with 3 μM 2′,7′-dichlorofluorescein diacetate for 1 h at 37 °C in the dark. Stained plugs were washed with phosphate-buffered saline (PBS, Sigma-Aldrich, MO, USA) diluted 10 times with distilled water, and observed with a confocal laser scanning microscope (CLSM) (excitation: 488 nm, emission: 490-560 nm, SP8X, Leica Microsystems, Germany)..