Figure 6: Effects of knockdown RKIP or PIAS1 in BEAS-2B and HFL-1 cells and expression and location of RKIP and PIAS1 in AIPI lungs.

BEAS-2B cells were transfected with 40nM si-RKIP or si-NC, while HFL-1 cells were transfected with 40nM si-PIAS1 or si-NC for 24h. The mRNA expression of RKIP and PIAS1 were detected by RT-qPCR (A,C). The si-RKIP-3 was used to knockdown RKIP expression and IL-1β was added to stimulate the BEAS-2B cells. The mix of si-PIAS1-2 and si-PIAS1-3 was used to knockdown PIAS1 expression and IL-4 was added in HFL-1 cells. The protein expressions of RKIP, PIAS1 and PRMT1 were detected after transfection and stimulation (B,D). RKIP and PIAS1 were detected by immunohistochemical staining. Representative images of RKIP (E) and PIAS1 (F) protein expression in control rat lungs (left panel), acute (middle panel) and chronic AIPI rat lungs (right panel) were stained with anti-RKIP (1:1000) and anti-PIAS1 antibody (1:100). IOD (integrated optical density) of RKIP and PIAS1 was determined by Image-Pro Plus 6.0 software to estimate the expression of protein in epithelium and sub-epithelium (n = 6 for each group) (G). Western blotting was shown as representative image and density under the band was measured (ImageJ software) and normalized to β-actin. The results were expressed as mean ± S.E.M of triplicates from three independent experiments and analyzed by One-way ANOVA test. * and ** represent P < 0.05 and P < 0.01 between indicated groups and control group.