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  • SB 203580 br Evaluation of intracellular reactive oxygen spe

    2020-08-12


    2.9. Evaluation of intracellular reactive oxygen species (ROS) generation
    MDA-MB-231 cells (1 × 105 cells/mL), under standard cultural conditions, were seeded in a 6-well plate containing RPMI media and allowed to attach for 24 h. After this, the cells were treated for 72 h with different concentrations of WSPF in a dose-dependent fashion. After removal of media, 2′,7′-dichlorofluoresceindiacetate (DCFH-DA) (5 μM) was added to each well and incubated at 37 °C under dark conditions for 30 min. DCFH-DA undergoes deacetylation by intracellular esterases to non-fluorescent DCFH, which on
    Fig. 1. Electrophoretic and intact mass spectrum analysis of WSPF. (A) Electrophoretic profile with left lane showing the protein ladder and the right lane showing the observed protein bands in WSPF. (B) Intact mass spectrum of WSPF.
    oxidation by intracellular ROS results in the formation of the fluores-cent compound 2′,7′-dichloroflorescein (DCF). In order to remove unbound stain, the incubated cells were washed and maintained in PBS. Intracellular ROS generation, which is directly proportional to fluorescence intensity of DCF, was quantified by flow cytometry.
    2.10. Analysis of SB 203580 phase distribution by flow cytometer
    Under standard cultural conditions, MDA-MB-231 cells of density
    1 × 105 cells/mL, seeded in a 6-well microplate containing complete media, were incubated for 24 h. After this, the cells were treated with different concentrations of WSPF in a dose-dependent fashion. After
    72 h of treatment, cells were harvested by trypsinization and col-lected by centrifugation at 1200 rpm for 5 min at room temperature. Resuspension of cells was carried out in 300 μL of PBS-EDTA, to which
    0. 7 mL chilled 70% ethanol was added in a drop wise manner. After overnight incubation at 0 °C, 20 mg/mL of RNase was added to the cells at a ratio of 1:100 v/v and incubated at 37 °C for 20 min. Follow- ing this, 50 μg/mL propidium iodide dye was added to the cell sus-pension and incubated for 20 min at room temperature. The unbound dye was removed by centrifugation at 1200 rpm for
    5 min. Cells harvested after centrifugation at 1200 rpm for 5 min, were analyzed for cell cycle phase distribution and DNA histogram using flow cytometry.
    2.11. Assessment of morphological changes
    2.11.1. Phase contrast microscopy
    MDA-MB-231 cells of density 1 × 105 cells/mL, seeded in a 6-well plate were allowed to attach for 24 h. Subsequently, the cells were treated with various concentrations of WSPF for 72 h in a dose-dependent fashion. Morphological changes in untreated and WSPF treated MDA-MB-231 cells were observed by using 20× magnification phase contrast microscopy. 
    MDA-MB-231 cells of density 1 × 105 cells/mL, seeded in a 6-well microplate, were allowed to attach for 24 h. After 72 h of treatment of cells with different concentrations of WSPF in a dose-dependent fash-ion, exhausted media from each well was removed by washing twice with PBS and the cells were fixed by 10% formalin for 30 min at room temperature. After fixation, DAPI stain (2.5 μg/mL) was applied on fixed cells for 10 min at room temperature. Unbound DAPI stain was re-moved by washing the cells with PBS. Following this, the cells were an-alyzed for any change in nuclear morphology including chromatin
    Fig. 2. Dose dependent WSPF-induced inhibition of proliferation of different cancer cell lines. Experiments were carried out in triplicates.
    Fig. 3. Flow cytometric analysis of WSPF-induced cell cycle arrest in MDA-MB-231 cells. Figure shows cell cycle phases distribution in untreated (panel A) and WSPF treated MDA-MB-231 cells with WSPF concentration of 50 μg/mL (panel B), 100 μg/mL (panel C) and 200 μg/mL (panel D).
    condensation, apoptotic body formation etc. by using DAPI staining method.
    ditions, were seeded in a 6-well plate containing RPMI media and allowed to attach for 24 h. After this, the cells were treated for 72 h with different concentrations of WSPF in a dose-dependent fashion.
    After removal of media, 2 mL of AO/EtBr dye mixture (0.15 mg/mL of AO and 0.5 mg/mL of EtBr in 1× PBS) was added. The stain treated cells were then allowed to incubate at room temperature for few mi-nutes. In order to remove unbound stain, the cells were washed and maintained in PBS. The stained cells were immediately visualized using fluorescence microscopy. Representative images were captured from random fields of view using filters appropriate for the specific fluo-rescent dyes.
    Fig. 4. Mitochondrial membrane potential loss in untreated and WSPF treated MDA-MB-231 cells. (A) Flow cytometry analysis of WSPF on ΔΨm in MDA-MB-231 cells. (B) % Loss in ΔΨm verses different concentrations of WSPF in untreated and WSPF treated cells. (**) p ≤ 0.05 and (***) p ≤ 0.01, compared with the untreated cells.