Fig 2: TGFβ induces the formation of podosome rosettes in normal human lung fibroblasts (NHLFs).Serum starved, sub-confluent (70–80%), primary NHLFs were stimulated with recombinant human TGFβ (10 ng/ml) for 24 h; a representative experiment out of 4 successful independent ones is shown. a, b TKS5 and COL1A1 mRNA expression was interrogated with Q-RT-PCR (r2 = 0,94/0,92; E = 98,3%/93% respectively) in two NHLF clones (cl.l, cl.2). Values were normalized to the expression values of the housekeeping gene B2M and presented as fold change over control; n = 4/5/4/4; statistical significance was assessed with two-tailed Welch’s test (a/cl.1) and two-tailed Mann Whitney test (a/l.2); **p = 0.0012, *p = 0.0159 respectively. b Two tailed pearson correlation plot of COL1A1 expression in the same samples (*p = 0.0116; r = 0.79). c–j Representative composite images from double immunostaining, and respective quantifications, for: c F-actin/TKS5 (red/green), e F-actin/Cortactin (CTTN; red/green), g F-actin/MMP9 (red/green), h TKS5/MMP9 (green/red). Cells are counterstained with DAPI; scale bars 50 μm; arrows indicate representative podosomes; separate images and proof of colocalization of signals can be found at Supplementary Fig. 5. d, f. Quantification of the number of podosome-containing cells per optical field (n = 6); statistical significance was assessed with two-tailed t-test; **p = 0.0011, ***p = 0.0009. i Representative images of the TGFβ-induced degradation (black holes) of a fluorescein-conjugated gelatin substrate. j Quantification of gelatin degradation area, normalized to control (n = 5); statistical significance was assessed with two-tailed t-test; **p = 0.0016. Source data for all relative panels (a, b, d, f, j) are provided as a Source Data file.

Fig 3: Tks5 haploinsufficiency in mice attenuates bleomycin (BLM)-induced pulmonary fibrosis (PF).a Schematic presentation of the BLM-induced PF model. b. Kaplan Meyer survival curve post BLM administration. c. Weight change post BLM administration; n = 4/7/4/6. Statistical significance was assessed with two-tailed one-way ANOVA; **p = 0.031. d Total protein concentration in bronchoalveolar lavage fluids (BALFs), as determined with the Bradford assay; n = 4/7/4/7. Statistical significance was assessed with two-tailed one-way ANOVA; ****p < 0.0001, ***p = 0.0009. e Inflammatory cell numbers in BALFs, as counted with a hematocytometer; n = 4/6/4/6. Statistical significance was assessed with two-tailed one-way ANOVA; ****p < 0.0001, ***p = 0.0002/0.0008. f Soluble collagen levels in the BALFs were detected with the direct red assay; n = 4/6/4/7. Statistical significance was assessed with two-tailed Kruskal Wallis; *p = 0.0124. g, h Tks5 and Col1a1 mRNA expression was interrogated with Q-RT-PCR; n = 4/5/3/7; values were normalized over the expression of the housekeeping gene B2m and presented as fold change over control. Statistical significance was assessed with two-tailed one-way ANOVA; **p = 0.0012, *p = 0.0207. h Two tailed pearson correlation plot of Col1a1 expression in the same samples; *p = 0.0342; r = 0.91/0.54. i Representative images from lung sections of murine lungs of the indicated genotypes, stained with Fast Green/Sirius Red (green/red; first row), from Hematoxylin & Eosin (H&E)-stained Precision cut lung slices (PCLS) (second row) and H&E-stained lung sections (third row); scale bars 50 μm. j. Quantification of fibrosis severity in H/E stained lung sections via Ashcroft scoring; n = 4/5/4/5. Statistical significance was assessed with two-tailed one-way ANOVA; ****p < 0.0001. k Rrs, mean respiratory system resistance as measured with Flexivent; n = 4/6/4/6. Statistical significance was assessed with two-tailed one-way ANOVA; ***p = 0.0008/0.0004. l Ers, mean respiratory system elastance as measured with Flexivent; n = 4/6/4/5. Statistical significance was assessed with two-tailed one-way ANOVA; ***p = 0.0002, **p = 0.0081. m Cst, mean static lung compliance as measured with Flexivent; n = 4/7/4/6. Statistical significance was assessed with two-tailed one-way ANOVA followed by Welch’s correction; ****p < 0.0001. In all panels cumulative results from 2 different experiments are shown; all samples are biologically independent; boxplots visualize the median of each distribution; upper/lower hinges represent 1st/3rd quartiles; whiskers extend no further than 1.5 *IQR from the respective hinge. Source data for all relative panels (a–h, j–m) are provided as a Source Data file.

Fig 4: Tks5 haploinsufficiency in mouse lung fibroblasts (LFs) disrupts extracellular matric (ECM) homeostasis, that critically controls podosome formation and ECM invasion.a ECM-related gene ontology components are enriched for genes down-regulated in TGFβ stimulated Tks5+/- LFs compared to their WT TGFβ treated counterparts. Gene-set enrichment analysis (GSEA) on expression data pre-ranked according to their fold change values. b Collagen containing extracellular matrix is the term most enriched in down-regulated genes according to gene-set enrichment analysis (GSEA). c Serum starved WT and Tks5+/- LFs were immunostained for Col1a1 (green) and counter stained with DAPI (blue). Representative images are shown; scale bars = 50 μm. d, e Serum starved WT primary LFs were cultured in post saline (pSAL) and post bleomycin (pBLM) aECM. Tks5 and Col1a1 mRNA expression was interrogated with Q-RT-PCR; n = 4; values were normalized over the expression of the housekeeping gene B2m and presented as fold change over control. Statistical significance was assessed with two-tailed Mann-Whitney test; *p = 0.0286. e Two tailed pearson correlation plot of Col1a1 expression in the same samples; ***p = 0.0004, r = 0.99. f, g Representative composite images from double immunostaining for F-actin (red) and Cortacin (Cttn; green; f) or Col1a1 (g; green) counter stained with DAPI (blue); arrows indicate representative podosomes; scale bars = 50 μm. In all panels, representative experiment out of 2 successful independent ones are shown; all samples are biologically independent; boxplots visualize the median of each distribution; upper/lower hinges represent 1st/3rd quartiles; whiskers extend no further than 1.5 *IQR from the respective hinge. Source data for all panels are provided as a Source Data file.