The two-chamber Transwell system with an 8-mm size pore (Costar, USA) was applied to evaluate cell migration of MSCs with PDK2 overexpression or knockdown.
Total RNA of MSCs with PDK2 overexpression or knockdown was prepared respectively using Trizol reagent (Invitrogen) and treated with DNase I (Promega, USA).
The Bio-Rad (China) Bis-Tris Gel system was established, in which primary antibodies PDK2 (ab68164), JNK (ab124956), p38 (ab31828), ERK (ab54 230) and the internal control GAPDH were obtained from Abcam.
Transfection efficiency of PDK2 overexpression and knockdown in MSCs
The pBABE-puro and pBABE-PDK2 were transfected to construct PDK2 overexpression and shRNA-PDK2, shNC were transfected to construct PDK2 knockdown with lentivirus in MSCs, respectively.
PDK2 promoted cell viability, adhesion and inhibited cell migration in MSCs
Functionally, to investigate the role of PDK2 in MSCs, cell viability, adhesion and migration were assessed.
Strong accumulation of cell adhesion was detected in response to overexpressing PDK2 compared to the control, whereas silencing PDK2 remarkably lessened the percentage of MSC adhesion (both P<0.05, Figure 2B).
The number of migratory MSCs was significantly downregulated by overexpressing PDK2 and was upregulated by shPDK2 compared with the control (both P<0.05).
Next, the mRNA expressions of chondrogenic differentiation markers were determined via qRT-PCR after MSCs were transfected with pBABE-PDK2, shRNA-PDK2 and their controls, to explore whether PDK2 exerted an effect on chondrogenic differentiation in MSCs.
PDK2 upregulated JNK/MAPK/ERK pathway to promote chondrogenic differentiation of MSCs