Figure 4

High-content cellular images of kinases. (a,b,c). Multicolor cellular imaging showing simultaneous monitoring of the upregulation/downregulation of five kinases in HepG2 cells. Five kinases: p-GSK3β, p-p38α, p-IRS1ser307, p-FOXO1, and p-IRS1tyr. (a) The positive control group was stimulated with TNF-α; whereas, in the negative control, TNF-α treatment was absent. For inhibitor studies, HepG2 cells were plated in serum-free DMEM media for 24 h prior to testing. P38 inhibitor was added 30 min before stimulation with 10 ng/ml TNF-α (for 5 h), and the cells were treated with 100 nM insulin. The cells were treated with formaldehyde for fixing the cells following permeability enhancement using saponin. The QDot-antibody conjugate (p-p38α, p-JNK1, p-IKKβ, p-IRS1ser, p-IRS1tyr, p-GSK3β, and p-FOXO1) treatment was done. Quantitative estimation and monitoring of the phosphorylation of the five kinases were simultaneously carried out according to emission wavelengths mentioned in the experimental section. 1: Positive Control, 2: Negative Control, 3: p38 inhibitor (SB203580), 4: Aspirin treatment, 5: Indomethacin treatment, 6: Amygdalin treatment, 7: Cinnamic acid treatment. (b) 1: Positive Control, 2: Negative Control, 3: JNK-1 inhibitor (SP600125), 4: Aspirin treatment, 5: Indomethacin treatment, 6: Amygdalin treatment, 7: Cinnamic acid treatment. (c) 1: Positive Control, 2: Negative Control, 3: IKKβ inhibitor, 4: Aspirin treatment, 5: Indomethacin treatment, 6: Amygdalin treatment, 7: Cinnamic acid treatment. (d) Cellular images of HepG2 cells treated with non-conjugated QDots (QDot525, QDot565, QDot605, QDot655, QDot705). (e) Western blot analysis of upregulation/downregulation of seven different kinases in HepG2 cells. TNF-α induction was absent in the negative control (NC). The positive control (PC) was stimulated with TNF-α. AS: Aspirin, IM: Indomethacin, AD: Amygdalin, CA: Cinnamic acid.

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