Fig. 4: Glypican-4 is critical for proper Areg signaling in primary Areg-responsive cells and is upregulated on Col14-LMC in the context of viral lung infection.

A Flow cytometry using antibodies targeting Sdc1, Sdc2, Sdc3, Sdc4, Gpc1, Gpc3, Gpc4, or Gpc6 on cultured bulk LMC (negative bead enrichment for CD45⁻CD31⁻Epcam⁻ cells, plated without sorting [see Supplementary Fig. 2]; non-adherent cells washed away/media changed at 16–18 h, then cultured for 48 h). Gated on CD45⁻CD31⁻Epcam⁻Pdgfra⁺ cells post-culture. Representative flow cytometry plots shown. Gating based on IgG controls. Percent staining positive displayed in plots. n = 4 per target, graph contains all values from two experiments. B Flow cytometry using antibodies targeting Sdc2 or Gpc4 on cultured bulk LMC, treated with non-targeting (NT) siRNA (n = 4 for Sdc2 siRNA control, n = 3 for Gpc4 siRNA control) or siRNA targeting either Sdc2 (n = 4) or Gpc4 (n = 4) (negative bead enrichment as in A; non-adherent cells washed away/media changed after 16–18 h, then treated with 25–50 nM siRNA for 48 h). Gated on CD45⁻CD31⁻Epcam⁻Pdgfra⁺ cells post-culture. Representative flow cytometry plots shown. Graphs contain all values from two to three experiments. C Western blotting for phospho-EGFR (Y1068) and β-actin of bulk LMC treated with NT siRNA, Sdc2 siRNA, or Gpc4 siRNA as in B (50 nM, 48 h), then stimulated (15 min) with mouse AREG (500 ng/ml) or EGF (100 ng/ml). AREG phosphorylation level quantification was done by adjusting to EGF phosphorylation controls. Representative western blots shown. n = 3 per condition, graph contains all values from three experiments. D Gpc4 protein expression determined by flow cytometry (MFI median fluorescence intensity), using a Gpc4-directed antibody, on freshly harvested LMC subsets, from either saline-treated (n = 6) or IAV-infected (n = 7) (300 TCID50) lungs (8 d.p.i). Gating strategy in Supplementary Fig. 2 (total lung). Graph contains all values from two experiments. E Sdc2 protein expression determined by flow cytometry (MFI median fluorescence intensity), using a Sdc2-directed antibody, on freshly harvested LMC subsets, from either saline-treated (n = 6) or IAV-infected (n = 7) (300 TCID50) lungs (8 d.p.i). Gating strategy in Supplementary Fig. 2 (total lung). Graph contains all values from two experiments. Statistical analysis done for western blot/flow cytometry data using two-tailed unpaired t-tests. Mean and standard error displayed on graphs; ns not significant, *0.01 < p < 0.05, ***0.0001 < p < 0.001, ****p < 0.0001. Source data are provided as a Source Data file.