Fig 1.
IGFBP2 expression levels in MSCs.
(A) Real-time RT-PCR revealed that lower IGFBP2 expression in WJCMSCs than that in PDLSCs, BMSCs, and ASCs. (B-E) Increased IGFBP2 expression after adipogenic induction in PDLSCs (B), BMSCs (C), ASCs (D), and WJCMSCs (E). GAPDH was used as an internal control. **p < 0.01. w: week; PM: proliferation medium; AM: adipose-inducing medium.
Fig 2.
IGFBP2 overexpression enhances adipogenic differentiation in WJCMSCs.
(A) Flag-IGFBP2-infected WJCMSCs showed IGFBP2 overexpression by real-time RT-PCR. GAPDH was used as an internal control. (B) Overexpression of IGFBP2 was verified by Western Blot analysis. (C-D) Oil Red O staining and quantitative analysis showed that IGFBP2 overexpression prompted formation of lipid deposits. Scale bar: 100 μm. (E-H) Real-time RT-PCR showed that overexpression of IGFBP2 upregulated expressions of PPARγ (E), LPL (F), CD36 (G), and CEBPA (H) in WJCMSCs at 0, 1, and 2 weeks after induction. GAPDH was used as an internal control. *p < 0.05. **p < 0.01. α: anti; w: week.
Fig 3.
IGFBP2 activates JNK and Akt signaling pathways.
(A) Western Blot analysis demonstrated that IGFBP2 overexpression caused an increase in p-JNK and p-Akt in WJCMSCs, however, the total amounts of JNK, ERK, p38, and Akt proteins were not affected; The phosphorylated p38 protein was not found. (B) Quantitative analysis of p-ERK, p-JNK, and p-Akt based on Western Blot results for the WJCMSC-Flag-IGFBP2 cells and WJCMSC-Vector cells. Total ERK, JNK, and Akt were used as internal control respectively. **p < 0.01. α: anti.
Fig 4.
Effect of JNK inhibitor on IGFBP2-induced adipogenic differentiation of WJCMSCs.
(A) Western Blot analysis showed a reduction of p-JNK in WJCMSC-Flag-IGFBP2 after treatment with a JNK inhibitor, SP600125 (10 μM, 20 μM, 50 μM or 100 μM in DMSO) for 48 h during adipogenic induction. (B) Quantitative analysis of p-JNK based on Western Blot results. Total JNK was used as internal control. The expression levels that are indicated with the same letter do not differ significantly. (C-D) Oil Red O staining and quantitative analysis revealed that 20 μM SP600125 effectively suppressed IGFBP2-mediated enhancement of lipid formation. Scale bar: 100 μm. (E-F) Real-time RT-PCR results showed downregulated expressions of PPARγ (E) and LPL (F) in WJCMSC-Flag-IGFBP2 cells following 20 μM SP600125 treatment during adipogenic induction at 1, 2, and 3 weeks. GAPDH was used as an internal control. **p < 0.01. α: anti; w: week.
Fig 5.
Effect of Akt inhibitor on IGFBP2-induced adipogenic differentiation of WJCMSCs.
(A) Western Blotting results showed a reduction of p-Akt in WJCMSC-Flag-IGFBP2 following treatment with an Akt inhibitor, LY294002 (10 μM, 20 μM, 50 μM or 80 μM in DMSO) for 1 h during adipogenic induction. (B) Quantitative analysis of p-Akt based on Western Blot results. Total Akt was used as internal control. The expression levels that are indicated with the same letter do not differ significantly. (C-D) Oil Red O staining and quantitative analysis showed that 10 μM LY294002 effectively inhibited IGFBP2-mediated adipogenic differentiation. Scale bar: 100 μm. (E-F) Real-time RT-PCR results showed downregulated expressions of PPARγ (E) and LPL (F) in WJCMSC-Flag-IGFBP2 cells following 10 μM LY294002 treatment during adipogenic induction at 1, 2, and 3 weeks. GAPDH was used as an internal control. **p < 0.01. α: anti; w: week.
Fig 6.
IGFBP2-mediated Akt activation is abrogated by JNK inhibitor.
(A) Western Blot results indicated that administration of 20 μM SP600125 decreased JNK activation and abrogated Akt phosphorylation in WJCMSC-Flag-IGFBP2 cells. (B) Quantitative analysis of p-JNK and p-Akt based on Western Blot results for the WJCMSC-Vector cells, WJCMSC-Flag-IGFBP2 cells, and WJCMSC-Flag-IGFBP2 + 20 μM SP600125 cells. Total Akt and JNK were used as internal control respectively. (C) Western Blotting results showed administration of 10 μM LY294002 decreased the level of p-Akt activation, while had no effect on JNK phosphorylation in WJCMSC-Flag-IGFBP2 cells. (D) Quantitative analysis of p-Akt and p-JNK based on Western Blot results. Total Akt and JNK were used as internal control respectively. **p < 0.01. α: anti.
Fig 7.
BCOR decreases IGFBP2 expression and weakens adipogenic differentiation in WJCMSCs.
(A) Flag-BCOR-infected WJCMSCs showed BCOR overexpression, as determined by Western Blot analysis. GAPDH was used as an internal control. (B) Real-time RT-PCR analysis showed that BCOR overexpression suppressed the expression of IGFBP2 in WJCMSCs. GAPDH was used as an internal control. (C-D) Oil Red O staining and quantitative analysis showed that BCOR overexpression inhibited the formation of lipid deposits. Scale bar: 500 μm (a, b), 100μm (c, d). **p < 0.01. α: anti; w: week.