Fig 2: 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.

Fig 3: Quantitation of proteins involved in the Wnt pathway upon exposure to Wnt6. Stacked trends of individual donors measuring (A) β-catenin, (B) phosphorylated β-catenin (Y142), (C) RhoA, (D) phosphorylated RhoA (S188), (E) CamKII, and (F) phosphorylated CamKII (T286) at 0, 15, 30, and 60 min after treatment with control, low-, medium-, and high-Wnt6 conditional medium (CM). Treatment with low-Wnt6 CM resulted in immediate increases in β-catenin concentration. Treatment with medium-Wnt6 CM resulted in increases 15 min after exposure. Treatment with high-Wnt6 CM increased β-catenin 60 min after exposure. Activation of β-catenin as determined by phosphorylated β-catenin (Y142) concentrations was observed immediately after treatment with low-, medium-, and high-Wnt6 CM. Control medium also activated β-catenin at 60 min from residual 3T3-expressed Wnt ligands. RhoA expression showed a general reduction, whereas phosphorylation of RhoA was lost immediately upon treatment with medium- or high-Wnt6 CM. An increase in CamKII expression was detected almost immediately after Wnt6 treatment, and maximum levels were detected 30–60 min after Wnt6 treatment. Phosphorylation of CamKII was lost immediately during medium- or high-Wnt6 CM treatment. Each line represents the quantities from individual donors. Donor 1, solid black, 9000 cells/cm2; Donor 2, dotted gray, 3250 cells/cm2; Donor 3, solid gray, 8000 cells/cm2; Donor 4, gray dash, 13,500 cells/cm2. Normalized to GAPDH. Phosphorylated proteins were normalized to the levels of their unphosphorylated counterparts.

Fig 4: RhoA Ser188 mediated the protection of H2S on H/R injury in HNCs.(A–C) Effects of NaHS on cell viability of HNCs transfected respectively with empty, GFP-RhoAwild, and GFP-RhoAS188A plasmids following H/R. (D–F) Effects of NaHS on lactate dehydrogenase (LDH) release from H/R injury HNCs transfected with empty, GFP-RhoAwild, and GFP-RhoAS188A plasmids, respectively. (G–I) Changes of release of nerve-specific enolase (NSE) from H/R injury HNCs transfected with empty, GFP-RhoAwild, and GFP-RhoAS188A plasmids, respectively. Data are shown as the mean ± SEM; n = 6. *P < 0.05, **P < 0.01.

Fig 5: H2S phosphorylates RhoA on Ser188 in vitro.Representative image (A) and summary data (B) of NaHS phosphorylates GST-RhoAwild tested by CBB staining and autoradiography. (C) Representative image of NaHS phosphorylates GST-RhoAS188A. Data are shown as the mean ± SEM, n = 3, **P < 0.01.