Fig 1: MAPK/JNK signaling and lipid droplets accumulation were inhibited by SP600125 in a dose-dependent manner. At 24 h post-induction of differentiation, ICP cells were then incubated for an additional 24 h in differentiation medium containing 0, 2.5, 5, or 10 µM SP600125. (A) Images for JNK1, JNK2, p-JNK1, p-JNK2, and ß-actin expressions in cells treated with different concentrations of SP600126 by Western blotting (representative of three independent experiments). Then, the bands intensities were quantified by Image J software. Graphs are plotted as mean ± SE from three independent experiments. Different uppercase letters above columns denote significant differences; (B) images for oil-red O staining of lipid droplets in preadipocytes treated with different concentrations of SP600125 (representative of three independent experiments). Then, oil-red O dye was extracted from the cells treated with different concentrations of SP600125 in order to quantify staining intensity. Graphs are plotted as mean ± SE from three independent experiments. Different uppercase letters above columns denote significant differences.
Fig 2: MAPK/JNK signaling pathway was activated by TCF21 overexpression. At 24 h post-induction of differentiation, lysates from LV-control and LV-TCF21 cells were collected. (A) A schematic overview of the constructs used for the Cignal Finder 45-Pathway Reporter Array. A. The inducible transcription factor-responsive construct expressing firefly luciferase. B. The constitutively expressing Renilla luciferase construct. C. The non-inducible firefly luciferase reporter construct. D. The constitutively expressing GFP construct. E. The constitutively expressing firefly luciferase construct. The negative control is a mixture of C. and B. (20:1). The positive control is a mixture of D., E. and B. Each reporter is a mixture of A. and B. (20:1). (B) A Luciferase activity-based array was used in order to identify those signaling pathways that were responsive to overexpression of TCF21. Graphs are plotted as mean ± SE relative to luciferase activity in LV-control cells from three independent experiments; (C) images for TCF21, JNK1, JNK2, p-JNK1, p-JNK2, and ß-actin expressions in cells by Western blotting; (D) bands intensities were quantified by Image J software. Graphs are plotted as mean ± SE from three independent experiments. ** p < 0.01.
Fig 3: Inhibition of MAPK/JNK signaling attenuates TCF21-mediated enhancement of preadipocyte differentiation. At 24 h post-induction of differentiation, LV-TCF21 and LV-control preadipocytes were then incubated for an additional 24 h in differentiation medium containing either 0 or 10 µM SP600125. (A) Images for JNK1, JNK2, p-JNK1, p-JNK2, and ß-actin expressions in LV-control or LV-TCF21 cells treated with 0 or 10 µM SP600126 by Western blotting (representative of three independent experiments). Then, the bands intensities were quantified by Image J software. Graphs are plotted as mean ± SE from three independent experiments. NS, no significance, * p < 0.05, ** p < 0.01; (B) images for oil-red O staining of lipid droplets in differentiated LV-control or LV-TCF21 preadipocytes treated with 0 or 10 µM SP600125 (representative of three independent experiments). Then, oil-red O dye was extracted from the cells in order to quantify staining intensity. Graphs are plotted as mean ± SE from three independent experiments relative to staining intensity of LV-control treated with 0 µM SP600125. * p < 0.05, ** p < 0.01; (C) expressions of pro-adipogenic genes in differentiated LV-control or LV-TCF21 preadipocytes treated with 0 or 10 µM SP600125 by real-time PCR. Graphs are plotted as mean ± SE from three independent experiments relative to the gene expression in LV-control treated with 0 µM SP600125. NS, no significance, * p < 0.05, ** p < 0.01.
Supplier Page from Abcam for Anti-JNK2 antibody