Figure 4

Effect of MIF on LCC α1C subunit protein and Src activity in HIT-T15 cells. (A) Expression of LCC α1C subunit in HIT-T15 cells was decreased following rMIF-treatment in a concentration dependent manner. 40 nmol/L rMIF treatment showed the most obvious suppression effect on LCC α1C subunit expression. Top: representative Western blot analysis of LCC α1C protein in treated cells and densitometric analysis of Western blot analysis result. Bottom: LCC α1C subunit mRNA level was also decreased along with rMIF treatment. (B) Protein tyrosine kinase inhibitors antagonized the effect of MIF on Scr activity. Top: representative Western blot analysis of Scr activity in treated cells and controls; GAPDH was the internal control. Bottom: densitometric analysis of Src protein in treated cells and controls. (C) Protein tyrosine kinase inhibitors antagonized the effect of MIF on down-regulating LCC α1C protein level. Top: representative Western blot analysis of LCC α1C protein in treated cells; GAPDH was the internal control. Bottom: densitometric analysis of LCC α1C protein in treated cells and controls. (D) Insulin secretion assay on islets and HIT-T15 cells treated with rMIF with or without co-treatment of Src inhibitors. The inhibitory effect of rMIF on insulin secretion was eliminated by Src inhibitors. (E) In the indirect co-culture method of islets with PC cell lines, LCC α1C subunit expression and Src activity was evaluated through Western blot analysis. Results showed that MIF mediated the regulatory effect of PC cell lines on islets, the LCC α1C subunit expression level was in consistent with Src activity. Sh-PANC-1: shRNA MIF-knockdown PANC-1 cell line; Sh-Capan-2: shRNA MIF-knockdown Capan-2 cell line; MIF-HPDE6: MIF-transgene overexpression HPDE6 cell line; pGIZ-PANC-1, pGIZ-Capan-2, and pLOC-HPDE6 are null vector transfected cell lines as controls to exclude the effect of vector on expression of potential genes coding proteins which might regulate insulin releasing.