Fig. 6
Factor XIIIA in IMs promotes fibrin cross-linking and LUSC invasion. a Relative mRNA expression of F13a1 from sorted IMs and RMs, n = 3 mice. b Immunofluorescent (IF) imaging of IMs and RMs comparing FXIIIA (red) expression. c Dual staining for FXIIIA (red) and CD11b (green) in IMs. Contents of dotted white box are enlarged under each panel. d Confocal imaging of IMs for FXIIIA (red). White arrows point toward podosome-like structures. Scale bar: 5 μm; nuclei were stained with Hoechst (panels b–d). e Fibrin cross-linking patterns by western blotting using a polyclonal anti-human fibrin(ogen) antibody using freshly sorted IMs and RMs (Low = 25k cells, High = 100k cells), with or without the FXIIIA-inhibitor, T101. f Percent invadopodia of LN4K1 cells growing in either unfractionated (UF Fgn, left) or Peak 1 (FXIIIA-depleted) fibrinogen (right). White arrows show evidence of invadopodia. Scale bar 50 μm. Data are averages ± s.e.m. P-values obtained with Student’s t-test. g Schematic of co-culture model (top). Representative images of LN4K1-GFP cells growing in either UF Fgn or Peak 1 with or without low (100k) or high (300k) IMs. FDR = 0.0001 for all statistical comparisons shown. Groups in e + g treated with the T101 were dosed at 50 μM. Scale bar 12.5 μm. Data are averages ± s.e.m. P-values were obtained with Student’s t-test. h Invaded LN4K1-GFP cells per high power field (HPF) at 24 h following seeding into either UF Fgn or Peak 1 Fgn alone or co-cultured with IMs with or without T101 (50 μM). FDR < 0.01 for all statistical comparisons shown. i Representative images (left) of LN4K1-GFP cells co-cultured with IMs from either wild-type or F13a1−/− mice. Number of invadopodia positive LN4K1-GFP cells (right) per HPF at 24 h when growing in UF Fgn or Peak 1 Fgn alone or co-cultured with IMs from either wild-type or F13a1−/− mice. Scale bar 12.5 μm. Data are averages ± s.e.m. P-values were obtained with Student’s t-test. FDR < 0.01 for all statistical comparisons shown. ** P < 0.01, *** P < 0.0001
