Figure 8. Effect of LPS on occludin expression, localization and study of signal molecules related to occludin expression by LPS.

Primary Sertoli cells A, B. and TM4 Sertoli cells C, D. were either not stimulated or stimulated with LPS (100 ng/ml). Cells were harvested for RNA or protein after 0, 12, 24 and 48 hours. (A, C) Cell lysates were subjected to western blot analysis for occludin and β-actin. Immunoblots were scanned, and the intensity of bands was quantified by the image program. Data were normalized to the control and expressed as the percentage of maximum activation of stimulation (a1, c1). (B, D) Cells were harvested for RNA, and realtime PCR was carried out using primers specific to occludin and β-actin. E. LPS (100 ng/ml) was added to the medium for 30 and 60 minutes. Cells were immunostained with an anti-occludin antibody (red fluorescence), and internalized protein vesicles were found to colocalize with clathrin (green fluorescence) at 60 minutes after LPS treatment. TM4 Sertoli cells were incubated with U0126 (10 μM), SB203580 (10 μM), JNK II(10μM) and Bay 11-7082 (10 μM) as indicated for 30 minutes prior to stimulation with LPS (100 ng/ml). Cells were harvested for RNA and protein, realtime PCR F. and western blot G. analysis were carried out using primers and antibodies specific to occludin and β-actin. Data were normalized to the control and the ratio was expressed as fold change (F) or the percentage of maximum activation of stimulation (g1) with respect to the control sample. * p<0.05.