These results suggest that the effect of TChal on breast cancer growth is mediated at least in part by HO-1. Open in a separate window Fig. Tchal structure. b BT-20 and MDA-MB-231 cell lines were treated with TChal at the indicated concentrations for 24?h. Cell morphology was observed by microscope. c Cytotoxicity assay was?performed on CX7 LZR using DAPI and PI staining. The top panel represents DMSO treated samples from BT-20 and MDA-MB-231 cells and the bottom graph represents quantification of PI staining after TChal treatment in the cells (* em p /em ? ?0.05, ** em p /em ? ?0.01, *** em p /em ? ?0.001) Cell toxicity assay by high content testing The toxicity assay was performed using the CellInsight CX7 LZR High Content Screening (HCS) platform (Thermo Fisher Scientific, Waltham, MA, USA). Briefly, BT-20 KPNA3 and MDA-MB-231 cell lines were harvested and split at a concentration of 10,000 cells/well in 96-well culture plates in triplicate. After 24?h of incubation, TChal was added at 1, 10, and 30?M for 24?h, with DMSO (0.1%) as a vehicle. The media were removed and the cells were washed twice with 1??PBS, followed by the addition of 200?L of propidium iodide working answer (333 ng/ml; Invitrogen, Carlsbad, CA, USA) into each well and an incubation period of 5?min at room heat. The cells were washed with 1??PBS twice and treated with 100% methanol for 15?min to permeabilize the cells, before washing again with PBS. Finally, 4,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, St. Louis, MO, USA) answer at 1?g/mL was added into each well for 10?min and the cells washed with 1??PBS for 3 times. The samples were analyzed using the CX7 LZR High Content Screening (HCS) platform with the cell toxicity assay method. Antibody array Proteome Profiler Human XL Oncology (R&D Systems, Minneapolis, MN, USA) can identify 84 different human cancer-related proteins around the membrane. Experiments were performed according to the manufacturers protocol. Briefly, protein extraction was performed using RIPA buffer following the manufacturers instructions. Then, two membranes (one membrane for control lysates and another for TChal WAY-100635 Maleate treated lysates) were blocked with array buffer for 1?h on a rocking shaker, and each cell lysate (200?g) was added and incubated with membrane overnight at 4?C. WAY-100635 Maleate Then, both membranes were washed with 1??wash buffer at least 3 times for 10?min, followed by the addition of the detection antibody cocktail answer for 1?h, before washing 3 times for 10?min. Streptavidin-HRP answer was then incubated for 30?min at room heat and on a rocking platform shaker, followed by washing actions. The membranes were visualized by chemiluminescence detection using ECL Western Blot Detection Reagent (Amersham Biosciences, Piscataway, NJ) in Alliance Q9 mini (UVITEC, Cambridge, England, UK). Western blotting Cells were washed with 1??PBS and collected in RIPA buffer supplemented with proteinase inhibitors. The cells were kept on ice for 30?min followed by vortex for 5?min to promote the cell lysate. The cell lysate was then centrifuged at 13,000?g for 20?min at 4?C. The supernatant was collected, and the total protein concentration was measured using a Pierce BCA Protein Assay Kit (Thermo WAY-100635 Maleate Scientific, Rockford, IL, USA). Then, 30?g of each protein was used in electrophoresis with 12% SDS-PAGE gel before being transferred to a nitrocellulose membrane. The membranes were blocked with TBST buffer (25?mM Tris, 3?mM KCI, 0.14?M NaCl, 0.05% Tween-20) containing 5% skim milk at room temperature for 1?h. Subsequently, the membranes were incubated with main antibodies diluted in TBST-5% non-fat milk (1:1000) overnight at 4?C. Afterwards, the membrane was washed with TBST and incubated with the secondary antibody for 2?h at room temperature, then washed with TBST. The proteins were visualized by chemiluminescence detection using ECL Western Blot Detection Reagent (Amersham Biosciences, Piscataway, NJ, USA) in Alliance Q9 mini (UVITEC). Quantitative real-time polymerase chain reaction Total RNAs were extracted using TRIzol? Reagent (Ambion?, Carlsbad, CA, USA). One g of RNA was used as template to reversely transcribe into.