Fig. 3

Changes in cell migration ability following knockdown of S100A4 and inhibition of NMIIA activity in GBM cells. A Western blot analysis for the indicated proteins of total lysates from S100A4-knockdown KS-1 (KS-shS100A4#3 and #5) and control cells (Con). B Left: wound-healing assay with S100A4-knockdown KS-1 and control cells (Con). A scratch was made in the middle of confluently grown cells, and phase -contrast images were taken after 0 h (upper), 24 h (middle) and 48 h (lower). Scale bar = 100 μm. Right: the wound areas were calculated using Image J software version 1.41, with the area at 0 h post-wounding set as 1. The fold wound areas are presented as means ± SDs. C Left: migration rate measured using a transwell assay. The S100A4-knockdown KS-1 and control cells (Con) were seeded in a 24-well transwell plate and incubated for 24 h in medium without serum. Cells were stained by HE and counted using a light microscope. Scale bar = 100 μm. Right: cell numbers are presented as means ± SDs (lower). D Left: KS-1 cells were seeded in a 24-well transwell plate and incubated for 24 h in serum-free medium with 0, 2, 4, and 6 μM blebbistatin. Cells were stained by HE and counted using a light microscope. Scale bar = 100 μm. Right: cell numbers are presented as means ± SDs. E Left and middle: in situ PLA assay for the S100A4-NMIIA interaction in non-Ps and Ps perinecrotic lesions of GBMs. Note the small aggregated dots in cytoplasmic and nuclear compartments of the GBM cells in non-Ps, but not Ps, perinecrotic lesions. Necrotic areas are indicated by asterisks and partitioned by dotted lines. Insets show the magnified views of the boxed area. Original magnification, × 100 and × 400 (inset). Scale bar = 200 μm. Right: PLA score for the combinations of S100A4 and NMIIA in non-Ps and Ps perinecrotic (non-Ps and Ps) and non-necrotic lesions (non). The data are presented as means ± SDs