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. 2020 Jan 25;12(2):289. doi: 10.3390/cancers12020289

Figure 6.

Figure 6

GPER activation inhibits Suit2-007 invasion through basement membrane mimics. (a) Immunofluorescence images of a decellularised mesentery extracted from mice, stained for laminin 111 (green) and perlecan (magenta). Image from single plane (top down view) and maximum intensity projection from side view, indicating presence of bilayer. Scale bar = 20 µm. (b) Suit2-007 cells beginning invasion of mesentery (24 h). White arrows indicate filopodia. Scale bar = 20 µm. (c) Top down and side view of control and G1 (1 µM) treated cells on mesenteries after 1 day. Scale bar = 10 µm. (d) Top down and side view of control and G1 (1 µM) treated cells on mesenteries after 10 days. Scale bar = 10 µm. (e) Mesh representations of invading cells after 1, 5, or 10 days from volume analysis. G1 (1 µM) + RhoA rescue mesh representation on Day 1 only. Blue mesh = cells, grey plane = top layer of mesentery. Scale bar = 2 μm. Arrow represents direction of invasion. (f) Quantification of cell area below top layer of mesentery for 1, 5 and 10 days. For control, n = 53, 34 and 55 cells for day 1, 5, and 10 respectively. For G1, n = 56, 39 and 27 cells for day 1, 5, and 10 respectively. For G1 + RhoA, n = 80 cells. * represents Mann–Whitney test between control and G1-treated cells for each individual time point, *** p < 0.001. (g) Cumulative count of cells invaded through mesenteries attached to bottom of well in 24 well plate for control and G1 (1 µM) treated Suit2-007 cells. Mesenteries were transferred to a new well each day, and cells attached to the bottom of the old well, where mesenteries had been for 24 h, were counted. Cell count was normalised depending on the amount of mesenteries in a well, each with the same amount of cells seeded on top of them. Each point is the sum of the mean values for each day, with standard error for each day calculated as the sum of standard errors for all the days used in summation. For Day 1-10, control = 13, 21, 23, 13, 21, 10, 17, 12, 13, 16 regions. For Day 1–10, G1 = 22, 19, 13, 11, 12, 10, 5, 5, 5, 7 regions. p = 0.00032 for straight line slope comparison.