Figure 1.
Modulation of noncanonical Wnt signaling in mMSCs with the supplementation of Wnt5a, SP600125 or GF109203X in general culture conditions.
The p-PKC (pan) (β II Ser660), p-PKCα/β II (Thr638/641), PKC pan, p-SAPK/JNK (Thr183/Tyr185), SAPK/JNK, p-CamK II, CamK II β/γ/δ, and nuclear β-catenin levels in mMSCs cultured in 10% FBS-DMEM/F12 media added with different concentrations of Wnt5a (A) or 500 ng/ml Wnt5a plus 5 µmol/L SP600125 or 2.5 µmol/L GF109203X for 2 hours were evaluated through western blotting (B). (n = 3; *P<0.05 vs Control; #P<0.05, ##P<0.01vs Wnt5a 0 ng/ml or DMSO Control; &P<0.05 vs DMSO + Wnt5a + GF109203X- SP600125-).
Figure 2.
Regulation of noncanonical Wnt signaling in mMSCs with the supplementation of Wnt5a, SP600125 or GF109203X in differentiation conditions into AT II cells.
The p-PKC (pan) (β II Ser660), p-PKCα/β II (Thr638/641), PKC pan, p-SAPK/JNK (Thr183/Tyr185), SAPK/JNK, p-CamK II, CamK II β/γ/δ, and nuclear β-catenin levels in mMSCs co-cultured with MLE-12 cells and SAGM with the supplementation of 500 ng/ml Wnt5a or 500 ng/ml Wnt5a plus 5 µmol/L SP600125 or 2.5 µmol/L GF109203X for 10 days were evaluated through western blotting. (n = 3; #P<0.05, ##P<0.01 vs DMSO control; &P<0.05 vs differentiation + DMSO + Wnt5a + GF109203X- SP600125-).
Figure 3.
The activity of noncanonical Wnt signaling during the differentiation of mMSCs into AT II cells.
The levels of the noncanonical Wnt pathway-related proteins, p-PKC (pan) (β II Ser660), p-PKCα/β II (Thr638/641), PKC pan, p-SAPK/JNK (Thr183/Tyr185), SAPK/JNK, p-CamK II, and CamK II β/γ/δ, in mMSCs on the first, third, seventh and tenth days of differentiation into AT II cells were detected through western blot. (n = 3, #P<0.05, ##P<0.01 vs 0 d).
Figure 4.
The effect of noncanonical Wnt pathway on the expression of specific markers of alveolar epithelial cells in mMSCs differentiating into AT II cells.
The expression of pro-SPC protein (A) and SPB, SPC, SPD and AQP5 mRNA (B) in mMSCs after 10 days of differentiation driven by co-culture with MLE-12 cells and SAGM with supplementation of 500 ng/ml Wnt5a plus 5 µmol/L SP600125 or 2.5 µmol/L GF109203X were evaluated with western blotting and qRT-PCR. (n = 3; *P<0.05 vs Control; #P<0.05, ##P<0.01 vs DMSO control; &P<0.05, &&P<0.01 vs differentiation + DMSO + Wnt5a + GF109203X- SP600125-).
Figure 5.
Role of noncanonical Wnt signaling in the proliferation of mMSCs.
The proliferation of mMSCs was evaluated using MTT assay after incubation in 2% FBS-DMEM/F12 media supplemented with increasing concentrations of Wnt5a (A) or 500 ng/ml Wnt5a plus 5 µmol/L SP600125 or 2.5 µmol/L GF109203X (B) for 72 h. (n = 4; #P<0.05 vs Control).
Figure 6.
Role of noncanonical Wnt pathway in the H2O2-induced cellular toxicity of mMSCs.
The viability of mMSCs after treatment with increasing concentrations of H2O2 supplemented in 2% FBS-DMEM/F12 growth media for 12 hours was determined using an MTT assay (A, n = 4). The expressions of p-PKCα/β II (Thr638/641), p-PKC (pan) (β II Ser660), p-SAPK/JNK (Thr183/Tyr185), p- CamK II, PKC pan, SAPK/JNK, CamK II β/γ/δ in mMSCs with 0.2 mmol/L H2O2 treatment for 12 hours were analyzed using western blotting (B, n = 3). The effect of pretreatment mMSCs with 500 ng/ml Wnt5a plus 5 µmol/L SP600125 or 2.5 µmol/L GF109203X for 1 hour on mMSC survival (C, n = 4) and the expression of Bcl-2 and Bax (D, n = 3) influenced by 0.2 mmol/L H2O2 for 12 hours were analyzed using MTT assay and western blotting. (#P<0.05, ##P<0.01 vs control, &P<0.05 vs H2O2 Vh, *P<0.05 vs DMSO+ H2O2+ Wnt5a- SP600125- GF109203X-).
Figure 7.
Role of noncanonical Wnt pathway in the migration of mMSCs.
The horizontal migration of mMSCs incubated in 2% DMEM/F12 media supplemented with 500 ng/ml Wnt5a or 500 ng/ml Wnt5a plus 2.5 µmol/L GF109203X or 5 µmol/L SP600125 was examined by wound healing assay (A ×200; n = 3; #P<0.05 vs DMSO control; &P<0.05 vs DMSO+ Wnt5a+ GF109203X- SP600125-). The vertical migration of mMSCs incubated in 2% DMEM/F12 media supplemented with 500 ng/ml Wnt5a or 500 ng/ml Wnt5a plus 2.5 µmol/L GF109203X or 5 µmol/L SP600125 towards 10% FBS-DMEM/F12 media (10% FBS-GM) (B ×200; n = 3; #P<0.05 vs DMSO control; *P<0.05 vs DMSO+ Wnt5a+ GF109203X- SP600125-) or conditioned media from normal (NL-CM) or ARDS mouse-derived lung tissue (ARDSL-CM) (C ×200; n = 3; *P<0.05 vs NL-CM; #P<0.05 vs ARDSL-CM; &P<0.05 vs ARDSL-CM+ Wnt5a+) was examined through Transwell inserts migration assay.
Figure 8.
Wnt5a ligand in lung tissue of normal or ARDS mice.
The expression of Wnt5a in normal or ARDS mouse-derived lung tissue was evaluated through western blotting analysis. (n = 3; #P<0.05 vs NL).