Figure 8.

Human cultured Müller glia (MIO-M1) cells respond to S1P by increased actin assembly and increased N-cadherin production. A) MIO-M1 cells treated with 0.5 μM S1P and BSA that serves as a control. S1P, after 30 min of treatment, increases cell size and actin assembly. B) 4 hours after treatment, BSA treated cells remained spindly with small contacts between neighboring cells, while S1P-treated cells had broad contact with other cells. Representative images from n=3 replicates. C) N-cadherin was increased in MIO-M1 cells 12 hours after S1P treatment. Representative images from n=3 replicates. D) Structured illumination microscopy of MIO-M1 cells showed an increase of N-cadherin puncta in S1P treated cells. Villous actin positive protrusions from the cells were increased in S1P treated cells. Actin positive protrusions in S1P treated cells were decorated with N-cadherin puncta (arrows) seen in the enlargements below. N-cadherin decoration was not seen in the BSA treated cells. Representative images from n=3 replicates. E) Western blot of MIO-M1 cells probed for N-cadherin. There is a significant increase of N-cadherin after treatment with 1 uM S1P for 12 hours (3 replicates, normalized to vimentin loading control, two-tailed Student’s t-test, α=0.05, p=0.02, error bars SD).