3 4 5 dimethylthiazol 2 yl 5 3 carboxymethoxyphenyl 2 4 sulfophenyl 2h tetrazolium mts

The chemical reagents involved in our experiments are listed as follows: glycidyl methacrylate (GMA, Mw 142.2) was purchased from J&K company. N-isopropylacrylamide (NIPAm, Mw, 113.2), copper(I)bromide (CuBr, Mw 143.5), 2,2′-bipyridyl (bPy, Mw 156.2), ω-mercaptoundecyl bromoisobutyrate (MUDBr), sodium azide (NaN₃, Mw 65.01), N,N-dimethyl formamide (DMF, Mw 73.1), l-ascorbic acid (VC, Mw 176.1) and gelatin were all purchased from Sigma-Aldrich. Methanol (Mw 32.05) and dichloromethane (Mw 84.93) were purchased from Duksan Ltd. Toluene (Mw 92.1), acetone (Mw 58.1), ethanol (Mw 46.1) and 2-propanol (Mw 60.1) were all purchased from VWR-BDH Co. Ltd. Phalloidin-Rhodamine, DAPI and MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) were purchased from Sigma-Aldrich. Alkaline phosphatase (ALP) Kit and Bicinchoninic Acid (BCA) Kit were from Beyotime Bio Company, while the BMP-2 ELISA kit (QuantiCyto®) was purchased from Neobioscience Co. Ltd. All cell-culture related reagents were purchased from Gibco Co. Ltd.

The human colon cancer cell line Caco2 was obtained from the American Type Culture Collection (Manassas, VA, USA). Dulbecco’s modified Eagle’s medium (DMEM), penicillin–streptomycin mixture, trypsin-EDTA, and fetal bovine serum (FBS) were obtained from Gibco Life Technologies (Milano, Italy). Lipofectamine 2000 was purchased from Invitrogen (Carlsbad, CA, USA). The protein assay kit was bought from Bio-Rad (Berkeley, CA, USA). An enhanced chemiluminescence kit, and rabbit monoclonal antibodies against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and EGFR were purchased from Proteintech (Chicago, IL, USA) and Abcam (Cambridge, UK), respectively. Goat anti-rabbit immunoglobulin G was obtained from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] (Sigma-Aldrich, Gillingham, UK) and EGFR Human Gene Knockout Kit [clustered regularly interspaced short palindromic repeats (CRISPR)] were purchased from Sigma-Aldrich and OriGene (Rockville, MD, USA), respectively. The Caco2 cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin–streptomycin at 37% and 5% CO₂ in humidified atmosphere. The culture medium was changed every other day, and the cells were subcultured using trypsin-EDTA. We obtained 5-FU from Sigma-Aldrich Co.

Mouse calvariae origin osteoblastic cell line subclone 4 (CRL-2594) was purchased from American Type Culture Collection (ATCC) Cell Bank (Manassas, VA, USA). Cell culture reagents- Alpha modified minimal essential medium (α-MEM)), penicillin, streptomycin and fetal bovine serum were sourced from Gibco Laboratories (Grand Island, New York, USA). Ascorbic acid, β-glycerophosphate and MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium) dyes were purchased from Sigma–Aldrich, USA. Eurycoma longifolia Jack (EL) was sourced from Faculty of Pharmacy, University Sains Malaysia (Malaysia). Enzyme-linked immunosorbent assay (ELISA) kits for the expression of ALP, Runx-2, AR, OPN, OCN, BMP-2, collagen type 1, and TGF-β were purchased from Elabscience Biotechnology Co., Ltd. (Wuhan, China). All other chemicals used were of analytical grade and were sourced from pharmacology and cell culture laboratories of Universiti Kebangsaan Malaysia (Malaysia).