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. 2021 Dec 2;22(23):13052. doi: 10.3390/ijms222313052

Figure 5.

Figure 5

OLFML3 promotes glioma cell migration and invasion. (A) Representative images of glioma cell (GL261) migration and invasion following treatment with human recombinant OLFML3 (rhOLFML3; 1 ng/mL, 10 ng/mL); scale bar 20 µm. (B) Exposure to rhOLFML3 induced a concentration-dependent increase in GL261 migration relative to vehicle-treated (0) cells (1: 1 ng/mL, 10: 10 ng/mL; 48 h); * p < 0.05, ** p < 0.01. (C) GL261 invasion was increased following exposure to 1 ng/mL, but not 10 ng/mL, rhOLFML3 relative to vehicle-treated cells (48 h); * p < 0.05, ** p < 0.01. (D) Cell viability was not affected by rhOLFML3 (1 ng/mL, 10 ng/mL; 48 h). (E) Migration was similar between GL261 cells exposed to conditioned media (CM) from vehicle-treated isogenic control and Olfml3-/- microglia (48 h). However, exposure to CM from isogenic control, but not Olfml3-/-, microglia pre-treated with β1 increased GL261 migration (48 h; 5 ng/mL); * p < 0.05, **** p < 0.0001. (F) GL261 invasion was increased following exposure to CM from isogenic control microglia pre-treated with β1 (48 h; 5 ng/mL) relative to CM from vehicle-treated isogenic control and Olfml3-/- microglia (48 h). Exposure to CM from Olfml3-/- microglia pre-treated with β1 increased GL261 invasion relative to CM from vehicle-treated Olfml3-/- microglia; *** p < 0.001, **** p < 0.0001. (G) GL261 viability was not affected by exposure to microglia CM under any condition (p = 0.4925). (H) Interleukin-6 (IL-6) was reduced in the media of Olfml3-/- microglia relative to isogenic control microglia following exposure to vehicle and β1; * p < 0.05, *** p < 0.001. (I) Secretion of platelet factor 4 (PF4) was attenuated in Olfml3-/- microglia following exposure to vehicle and TGFβ isoforms; * p < 0.05. (J) Loss of microglial Olfml3 abolished the TGFβ-induced increase in Pdgfa mRNA (48 h; 5 ng/mL); * p < 0.05, ** p < 0.01, *** p < 0.001. Comparisons based on one-way ANOVA with Tukey’s Multiple Comparison Test. Bars represent group mean with standard error of the mean (SEM); data represent one of three independent experiments.