Transcription factor 21 (TCF21) could promote chicken preadipocytes differentiation at least in part via activating MAPK/JNK pathway.
Supplementary Figure S1. Detection of TCF21 over-expression efficiency and its effect on lipid
droplets accumulation. Oleic acid was used to induce the differentiation of LV-control and LV-TCF21
preadipocytes for 0 - 120 h. (A) The mRNA expression of TCF21 in LV-control and LV-TCF21
detected by real-time PCR. (B) Images for the protein expression of TCF21 in LV-control and
LV-TCF21 detected by western blot (representative of three independent experiments). (C) The
quantification of protein bands by Image J. (D) Images of the accumulation of lipid droplets in
LV-control and LV-TCF21 cells by Oil red-O staining (representative of three independent
experiments). (E) The Oil red-O dye was extracted from stained LV-control and LV-TCF21
preadipocytes at the indicated time points in order to quantify staining intensity. Graphs are plotted as
mean ±SE from three independent experiments. NS, no significance, *p<0.05, ** p< 0.01
Pathway |
LV-control(mean±SE) |
LV-TCF21(mean±SE) |
P-value |
Amino acid deprivation |
0.41 ± 0.37 |
0.062 ± 0.043 |
0.44 |
Androgen |
/ |
/ |
/ |
Antioxidant response |
/ |
/ |
/ |
ATF6 |
0.015 ± 0.0048 |
0.013 ± 0.0027 |
0.72 |
C/EBP |
/ |
/ |
/ |
cAMP/PKA |
/ |
/ |
/ |
Cell cycle |
/ |
/ |
/ |
DNA damage |
/ |
/ |
/ |
EGR1 |
/ |
/ |
/ |
ER stress |
0.95 ± 0.72 |
0.90 ± 0.60 |
0.96 |
Estrogen |
/ |
/ |
/ |
GATA |
/ |
/ |
/ |
Glucocorticoid |
/ |
/ |
/ |
Heat shock |
/ |
/ |
/ |
Heavy metal |
0.090 ± 0.065 |
0.11 ± 0.078 |
0.87 |
Hedgehog |
/ |
/ |
/ |
HNF4 |
/ |
/ |
/ |
Hypoxia |
/ |
/ |
/ |
Interferon regulation |
/ |
/ |
/ |
Type 1 interferon |
/ |
/ |
/ |
Interferon-r |
/ |
/ |
/ |
KLF4 |
/ |
/ |
/ |
Liver X |
/ |
/ |
/ |
MAPK/Erk |
0.12 ± 0.043 |
0.11 ± 0.044 |
0.88 |
MAPK/Jnk |
0.028 ± 0.0060 |
0.19 ± 0.014 |
0.000423 |
MEF2 |
/ |
/ |
/ |
Myc |
/ |
/ |
/ |
Nanog |
/ |
/ |
/ |
Notch |
/ |
/ |
/ |
NFκB |
0.081 ± 0.037 |
0.20 ± 0.11 |
0.36 |
Oct4 |
/ |
/ |
/ |
Pax6 |
/ |
/ |
/ |
PI3K/Akt |
/ |
/ |
/ |
PKC/Ca+2 |
/ |
/ |
/ |
PPAR |
/ |
/ |
/ |
Progesterone |
/ |
/ |
/ |
Retinoic acid |
/ |
/ |
/ |
Retinoid X |
/ |
/ |
/ |
Sox2 |
/ |
/ |
/ |
SP1 |
0.3 ± 0.28 |
0.097 ± 0.067 |
0.52 |
STAT3 |
/ |
/ |
/ |
TGF-β |
/ |
/ |
/ |
Vitamin D |
/ |
/ |
/ |
Wnt |
/ |
/ |
/ |
Xenobiotic |
/ |
/ |
/ |
Negative control |
0.00057 ± 0.000067 |
0.000625 ± 0.000062 |
0.59 |
Figure 1. 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 β-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.
Figure 2. 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 β-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.
Figure 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 β-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.