Figure 6. Altered signaling, cell migration after injury and mitochondrial functional are dependent on specific caveolin isoform expression following LPS stress.

A. WB baseline data of BV2 and PMG treated with 10ng/ml LPS for 4 and 8 hours. Cav-1 expression increases while Cav-3 does not. IL-1β (33kDa: IL-1β) response to LPS is attenuated when cells are grown without serum at 8 hours. Blots are normalized to GAPDH. B. Left panels are knockdown control WB for corresponding right panels of knockdown functional consequences following LPS treatment with at least a 50% reduction in expression of caveolin. siRNA treated cells were further analyzed by 10ng/ml LPS treatment and WB for IL-1β response. Both siCav-1 and siCav-3, in either BV2 or PMG, show attenuation of maximal IL-1β expression evident as early as 4 hours. For WT PMG cells, antisense morpholinos (mrpl), specific stable oligos, that bind to complementary sequences, for either mRNA Cav-1 or Cav-3 were designed. VC = DMSO vehicle; SCR = mrpl control; mCav-1 5uM and mCav-3 5uM. Each experiment was repeated n=3 and blots are representative of results. C. A wound healing/migration assay was used to assess BV2 migration following siRNA at 0 min, 8 hours and 24 hours. Plates were fixed, stained with methylene blue and imaged. D. Comparison of oxygen consumption rate (OCR; left panels) and extracellular cellular acidification rate (ECAR; right panels) measurements for BV2 cells treated with siRNAfor gene knockdown of Cav-1 and Cav-3. Scrambled siRNA (SCR)) was used as a control. Comparisons of oxygen consumption rate (OCR; left panel bottom) and extracellular cellular acidification rate (ECAR; right panel bottom) measurements for BV2 cells treated with adenovirus for gene overexpression of Cav-1 and Cav-3. GFP adenoviral vectors were used for controls. (*; p≤ 0.05)