3 4 5 dimethylthiahiazo z y1 3 5 diphenytetrazoliumromide mtt

SWCNTs (XFS05) were purchased from Nanjing XFNANO Materials Tech Co., Ltd., 3-(4,5)-Dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT) and rapamycin (V900930) were purchased from Sigma-Aldrich (St. Louis, USA). Dulbecco's modified Eagle’s medium (DMEM) and foetal bovine serum (FBS) were purchased from HyClone (Logan, USA), and goat serum was purchased from Boster Biological Technology Co. Ltd. (Wuhan, China). 3-MA was from (19312) MedChem Express (New Jersey, USA). Rabbit monoclonal anti-α-SMA (ab32575) and mouse monoclonal anti-β-actin (ab20272) were purchased from Abcam (Cambridge, MA). Rabbit monoclonal anti-p21 (64016S), rabbit monoclonal anti-autophagy related 5 (Atg5) (12994S), rabbit monoclonal anti-p62 (39749) and mouse monoclonal anti-LC3B (83506) were purchased from Cell Signaling Technology (Beverley, MA). Mouse monoclonal anti-COL I (sc-59772) and mouse monoclonal anti-p16 (sc-1661) were purchased from Santa Cruz (California, USA). Rabbit polyclonal anti-SP-C (DF6647) was purchased from Affinity Biosciences (Cincinnati, USA). ELISA kits were obtained from Calvin Biotechnology Co., Ltd. (Suzhou, China). Haematoxylin, eosin, Masson’s trichrome and senescence-associated-β-galactosidase (SA-β-Gal) staining kits were purchased from Solarbio Science & Technology (Beijing, China).

MCF-7 cells were seeded in 96-well plates (3 × 10³ cells/well), followed by 12 h or 24 h of treatments. The media containing Asc was removed before being subjected to MTT assay, because the oxidative products of Asc interfere with the MTT assay. 100 μL of serum-free DMEM medium was applied into each well, and then 20 μL of 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide (MTT) (Sigma, Germany) was added to each well. Followed by 4 h of incubation at 37°C, all media were removed and then 150 μL of dimethyl sulfoxide (DMSO) (Sigma, Germany) was added to each well; after 10 min of shaking, the value of OD₄₉₀ was recorded by a Varioskan Flash (Thermo Fisher, Finland).

Cell culture medium and fetal bovine serum were purchased from Invitrogen (Carlsbad, CA, United States). Antibodies to the following proteins were used: PARP (BD Biosciences, San Jose, CA, United States, Cat#556494); Jab1 (Santa Cruz, CA, United States, Cat#sc-13157); p-Akt (p-Thr308) (Cell Signaling, Danvers, MA, United States, Cat#13038), T-Akt (Cell Signaling, Danvers, MA, United States, Cat#2920) and β-actin (Cell Signaling, Danvers, MA, United States, Cat#3700). Western Lightning Chemiluminescence Plus reagent was from Thermo Scientific Pierce (Rockford, IL, United States, Cat# 34087). Annexin V/PI kit was from BD (Cat# 556547). AZA (Cat# 95054), DMSO (Cat# D2650), and 3(4,5)dimethylthiahiazo(zy1)3,5diphenytetrazoliumromide (MTT) (Cat# M2128) were from Sigma.

Insulin assay kit was obtained from Millipore (USA). Dulbecco's modified Eagle's medium (DMEM), phenol red-free DMEM, and fetal bovine serum (FBS) were from Gibco (USA). Penicillin, streptomycin, and 0.25% trypsin were purchased from Invitrogen (USA). The Annexin V-FITC&PI kit was obtained from Merck Millipore (USA). The 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide (MTT) was obtained from Sigma (USA). Metformin was purchased from Squibb Pharmaceutical (Shanghai, China). Blood sugar meter and blood sugar test paper were purchased from Sannuo Biosensors (Changsha, China). Assay kits of triglyceride, total cholesterol, and free fatty acids were from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Palmitic acid was from Sigma (USA).The primary antibodies against ERK1/2, p- ERK1/2, MEK1/2, p-MEK1/2, cleaved-caspase-3, and GAPDH were from Abcam (USA). The secondary antibodies were purchased from Beyotime Biotechnology (China). The herbs in WSZYF were purchased from Tongrentang Pharmacy (Tangshan, China). Wushenziye formula (freeze-dried powder) dissolved in DMEM was deposited in -20°C.

Niclosamide was purchased from (Xiyashiji Chemical Co., LTD, ChengDu, SiChuan, China). Purity (98.5%) was measured by high-performance liquid chromatography (HPLC) analysis. For all in vitro studies, Niclosamide was prepared initially as a 20 mM stock solution in dimethyl sulfoxide (DMSO) and stored at −20°C. Then the stock solution diluted in the relevant assay media, and 0.1% DMSO served as a vehicle control. For in vivo assays, Niclosamide was prepared in 12.5% (v/v) aqueous Cremophor EL (CrEL) containing 2.5% (v/v) DMSO and dosed at 0.1 ml/10 g of body weight.
3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT), Triton X-100, DMSO, Hoechst 33342 were from Sigma Chemical Co.(St. Louis, MO). The primary antibodies against STAT3/P-STAT3^Tyr705, focal adhesion kinase (FAK)/p-FAK ^Tyr925, Src/p-Src^Tyr416, VEGF, cleaved caspase-3 (CC-3), Bcl-2, Mcl-1, Survivin, β-actin were purchased from Cell Signaling Technology (Beverly, MA). Mouse monoclonal anti Ki-67 and Rabbit polyclonal anti-CD31 were purchased from Merck-Millipore. FITC-CD11b, PE-Gr1 conjugated antibodies were obtained from BD Biosciences. The Annexin V-FITC Apoptosis Detection Kit was purchased from KeyGen Biotech (Nan-jing, China).

GL-V9 (C₂₄H₂₇NO₅, MW: 409.47, purity > 99%) was kindly provided by Dr. Bin Di (China Pharmaceutical University, China) and dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, USA) to 0.1 M as a stock sulution and stored at -20°C. For cellular experiment, GL-V9 was dissolved in sterilized dimethylsulfoxide (DMSO) and diluted with cell culture medium to different final concentrations. The final DMSO concentration did not exceed 0.1% v/v (volume-to-volume ratio) throughout the study. The PI3K/Akt signaling activator Insulin-like growth factor-1 (IGF-1) and the specific PI3K inhibitor LY294002 was obtained from Beyotime Biotechnology Corporation, Shanghai (Shanghai, China).
Primary antibodies against PI3 Kinase p85, Akt, p-Akt (Ser473), p38 MAPK, p-p38 MAPK (Thr180/Tyr182), ERK1/2, p-ERK1/2 (Thr202/Tyr204), MMP-2, MMP-9, and β-actin were obtained from Cell Signaling (Danvers, MA, USA) and were used at a 1:1000 dilution. HRP-conjugated secondary antibodies were obtained from Cell Signaling (Danvers, MA, USA) and were used at a 1:2000 dilution. MTT (3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide) was obtained from Sigma-Aldrich (St. Louis, MO, USA).