Figure 5.

H. parasuis-induced resistin in PAMs modulates AMPK/mTOR pathway activity through LKB1 in PAECs. (a) PAECs were co-cultured with wild-type or resistin knockout PAMs, and the PAMs were infected by H. parasuis (100 MOI) for 2 h. Protein levels of phospho-LKB1 (T189), LKB1, phospho-CaMKK2 (S511), and CaMKK2 in PAECs were detected using western blot. (b) Western blot analysis of full-length LKB1 or CaMKK2 expression in PAECs. PAECs seeded on six-well plates were transfected with 2.5 μg/well plasmids pCAGGS-LKB1 or pCAGGS-CaMKK2 by 5 μg/well of lipofectamine 2000. Cells were collected to detect LKB1 and CaMKK2 expression levels using western blot analysis at 8 h post-transfection. The empty vector was used as a negative control. (c, d) pCAGGS-LKB1 or pCAGGS-CaMKK2 were transfected into PAECs, cells were co-cultured with wild-type or resistin knockout PAMs 4 h after transfection, and the PAMs were then infected by H. parasuis (100 MOI) for 2 h. Protein levels of phospho-AMPK (S458) and AMPK (c) or claudin-5 and occludin (d) in PAECs were determined using western blot analysis. β-actin served as a loading control. The antibodies against phospho-LKB1 (T189), LKB1, phospho-CaMKK2 (S511), and CaMKK2 were diluted at 1/1000, and the antibody against β-actin was diluted at 1/50000. β-actin served as a loading control, and the relative protein level was calculated with ImageJ software and normalized to β-actin. **p<0.01 compared with the uninfected group (a, c, d). ^##p<0.01 compared with the HPS-infected group (c, d). Error bars represent the mean ± SEM (n = 3).