Fig 2: Expression of podoplanin, CD44 and CD9 on FRCs is coregulated by CLEC-2. (A) Analysis of RNA-seq expression (Martinez et al., 2019) of Pdpn (top row), Cd44 (middle row) and Cd9 (bottom row) in unstimulated (left), or short-term stimulated (6 h recombinant CLEC-2-Fc), control (Ctrl; middle), or podoplanin shRNA knock-down (PDPN KD) (right) FRC cell lines. mRNA expression is analysed as Z-score compared to Pdpn expression in unstimulated Ctrl FRCs. n=4 biological replicates per cell type or stimulation. (B) Western blot analysis of total podoplanin, CD44 and CD9 expression in Ctrl or CLEC-2-Fc-expressing FRC cell lines. Histone H3 is used as loading control. Representative data from n=3 biological replicates is shown. (C) Podoplanin (left), CD44 (middle) and CD9 (right) surface expression on control (Ctrl; dark), CLEC-2-expressing (CLEC-2; lighter) and PDPN KD (light) FRC cell lines as determined by flow cytometry. Protein expression is normalized to the level in Ctrl FRCs for each individual experiment. Data shown as mean±s.d. with dots representing biological replicates (n=3–8). **P<0.01; ns, not significant (Kruskal–Wallis test with Dunn's multiple comparisons). (D) Podoplanin surface expression on indicated FRC cell lines by flow cytometry. Podoplanin expression is normalized to the level in Ctrl FRCs for each individual experiment. Data shown as mean±s.d. with dots representing biological replicates (n=3). ns, not significant (Kruskal–Wallis test with Dunn's multiple comparisons). (E) Left panel, double immunofluorescence staining of podoplanin (magenta) and CD44 (top; green) or CD9 (bottom; green) in control (Ctrl) or CLEC-2-expressing (CLEC-2) FRC cell lines. Maximum Z-stack projections of representative images are shown. Scale bars: 10 μm. Yellow boxes indicate areas shown in the magnifications. Right panel, colocalisation of podoplanin with CD44 (red) or CD9 (blue) in Ctrl and CLEC-2 FRCs as measured by Pearson's coefficient (r) in single sections using JaCoP plugin in Fiji/ImageJ. Data shown as box plots where box represents the 25–75th percentiles, and the median is indicated. The whiskers show the range (n=6–7 fields of view per cell line collated from two biological replicates). *P=0.0476; ns, not significant (unpaired two-tailed t-test). (F) Left panel, triple immunofluorescence staining of podoplanin (magenta), CD44 (yellow) and CD9 (blue) in control (Ctrl) or CLEC-2-expressing (CLEC-2) FRC cell lines. Maximum Z-stack projections of representative images are shown. Scale bars: 10 μm. Yellow boxes indicate areas shown in the magnifications. Right panel, colocalisation of CD44 with CD9 in Ctrl and CLEC-2 FRCs as measured by Pearson's coefficient (r) in single sections using JaCoP plugin in Fiji/ImageJ. Data shown as box plots where box represents the 25–75th percentiles, and the median is indicated. The whiskers show the range (n=4 fields of view per cell line collated from two biological replicates). ns, not significant (unpaired two-tailed t-test).

Fig 3: CD44 and CD9 balance podoplanin-mediated FRC hypercontractility. (A) Immunofluorescence of F-actin (black) and cell nuclei (blue) in indicated FRC cell line cultures. Maximum Z-stack projections of representative images are shown. Scale bars: 50 μm. (B) Proportion of contracted cells in indicated FRC cell line cultures. Data shown as mean±s.d. with dots representing n=11–15 images per cell line collated from three or four biological replicates. **P=0.0023; ***P=0.0009; ****P<0.0001; ns, not significant (P=0.0528) (Kruskal–Wallis test with Dunn's multiple comparisons). (C) Area (µm2) of indicated FRC cell lines. Data shown as median with interquartile range with dots representing individual cells from three or four biological replicates. ****P<0.0001 (Kruskal–Wallis test with Dunn's multiple comparisons). (D) Immunofluorescence of CFP (magenta), GFP (green) and F-actin (white) in the control (Ctrl) FRC cell line transfected with PDPN–CFP alone, or co-transfected with GFP control, CD44–GFP or CD9–GFP. Maximum Z-stack projections of representative images are shown. Scale bars: 80 μm. (E) Proportion of contracted cells in the Ctrl FRC cell line transfected with PDPN–CFP alone (white), or co-transfected with GFP control (grey), CD44–GFP (red) or CD9–GFP (blue). Data shown as mean±s.d. with dots representing n=7–9 images per cell line collated from three or four biological replicates. *P=0.0186 (Kruskal–Wallis test with Dunn's multiple comparisons). (F) Area (µm2) of Ctrl FRCs transfected with PDPN–CFP alone (white), or co-transfected with GFP control (grey), CD44–GFP (red) or CD9–GFP (blue). Data shown as median with interquartile range with dots representing individual cells from three biological replicates. **P=0.0062, ***P=0.0003 (Kruskal–Wallis test with Dunn's multiple comparisons).

Fig 4: Podoplanin expression is increased in a subset of human melanomas(A) Brightfield (upper panel) and immunofluorescence (lower panel) imaging of WM983A (left) and WM983B treated with vehicle (DMSO; middle) or ROCK inhibitor (1μM GSK269962A) for 4h (top) or 24h (bottom). Cells are stained for F-actin (white). The scale bars represent 100 (upper panel) or 40 (lower panel) microns.(B) Cell morphology (roundness index) of WM983A (light grey) and WM983B (dark grey) cells as presented in panel (A) Kruskal-Wallis test with Dunn's multiple comparisons, ∗∗p = 0.0036, ∗∗∗∗p < 0.0001.(C) Western blot analysis of ppMLC and MLC in WM983A (left) and WM983B treated with vehicle (DMSO; middle) or 1μM ROCK inhibitor (GSK269962A) for 4h (top) or 24h (bottom). Tubulin and GAPDH are used as loading controls.(D) Podoplanin (PDPN) mRNA expression in WM983A and WM983B cell lines measured by qPCR. PDPN mRNA expression is calculated as relative expression of the housekeeping gene GAPDH (2ˆ-Ct(PDPN)/2ˆ-Ct(GAPDH)). Data shown as mean with dots representing n = 3 biological replicates. Unpaired t test, two-tailed, p = 0.1002.(E) Podoplanin (PDPN) mRNA expression in two datasets (Kabbarah et al.GSE46517 and Riker et al.GSE7553) of human melanoma and appropriate control tissues. Data shown as mean with dots representing melanoma tumors from individual patients. Kabbarah et al.: unpaired t test, two-tailed, ∗p = 0.0250; Riker et al.: Mann Whitney test, two-tailed, p = 0.5052.(F) Podoplanin (PDPN) mRNA expression in human melanoma with BRAF and/or NRAS or no (wt) mutation in TCGA dataset (https://www.cancer.gov/tcga). Data are represented as median ± interquartile range.

Fig 5: Loss of podoplanin restores pigmentation and melanocyte differentiation(A and B) Tumors (A) and cells pellets (B) of PDPN+ and PDPN KO B16F10 cell lines.(C) Imaging of pigmentation (brightfield; left) of PDPN+ and PDPN KO B16F10 cell lines, labeled with mOrange (red) or CFP (blue) respectively (middle). The scale bar represents 50 microns.(D) Heatmaps showing expression (Z score) of podoplanin (PDPN) and eight dedifferentiation-associated genes in datasets of primary tumor samples of melanoma patients (top; Riker et al.GSE7553) and metastatic melanoma cultures (bottom; Mannheim cohort, GSE4843; from Hoek et al., 2006). For the Mannheim cohort, each number indicates a separate metastatic melanoma culture, and the BRAF and/or NRAS mutation status is indicated for each.(E) mRNA expression of five melanocyte-associated (Mlana/Mart-1 (p = 0.2045), Tyr (p = 0.9276), Dct (p = 0.9859), Gpnmb (p = 0.1308), Pmel (p = 0.9994); left) and three invasion-associated (Kit (p > 0.9999), Mitf (∗∗∗∗p < 0.0001), Pou3f2 (encodes Brn2; ∗∗∗∗p < 0.0001; right) genes in PDPN+ B16F10 cells. mRNA expression is calculated as fold change of Gapdh expression and normalized to expression in PDPN KO B16F10 cells (set at 1 as indicated by dashed line). Data shown as mean with dots representing n = 3 biological replicates. Two-way ANOVA with Sidak's multiple comparisons; p values indicated above.(F) Western blot analysis of Podoplanin, Melan-A, Tyrosinase and Brn2 (encoded by Pou3f2 gene) in PDPN KO and PDPN+ B16F10 cells. Tubulin and histone H3 are used as loading controls.