Figure 4

Rapamycin interferes with actin cytoskeleton and lamellipodia formation and reduces levels of phospho-FAK in FLS from arthritic rats and RA patients. Rapamycin treatment reduces the linear distribution of the actin cytoskeleton (fluorescein isothiocyanate (FITC), panels E, M versus DMSO in panels A and I) and the formation of lamellipodia (E–H and M–P; arrows on C and K point to lamellipodia), compared with control (DMSO-treated, A–D and M–P), creating more compact and less spread-looking cells. Rapamycin reduced levels (F and N) and to a certain extent the focal localization of phospho-FAK (Tex-red, panels F–H and N–P) in FLS from DA rats, and in FLS from RA patients, compared with DMSO (B-D and J–L). Regions marked by arrows on panels C, G, K and O are magnified for detail on the right-hand panels, respectively (D, H, L and P), showing colocalization (yellow) of phospho-FAK (red) with actin (green) in DMSO-treated cells, and lack of colocalization or formation of lamellipodia in rapamycin-treated cells. Pictures taken from cells stimulated for 24 h (four different DA and three different RA FLS cell lines). Images A, B, C, E, F, G, I, J, K, M, N and O were acquired with a 62× magnification lens. Images D, H, L and P are 3× digital amplifications of details marked with arrows on panels C, G, K and O, respectively.