Fig 1: RASSF1A expression is associated with disorganized ECM and abrogated stiffness in vitro and in vivo Bar graph representing quantification of stiffness measured by atomic force microscopy (Young modulus) of ECM area generated by H1299control or H1299RASSAF1A cells in vitro.Representative atomic force microscopy (AFM) images showing organization of extracellular matrix generated by H1299control and H1299RASSF1A cells in 3D collagen matrices (2 mg/ml).Bar graph representing quantification of stiffness by atomic force microscopy (Young modulus) of n = 5 H1299control and n = 3 H1299RASSF1A primary lung tumours on day 30.Representative topographic images of stiffness map of H1299control and H1299RASSF1A lung primary tumours provided by AFM.AFM images of ECM fibre organization within primary lung tumours generated by H1299control and H1299RASSF1A cells on day 30.Second harmonic generation (SHG) representative images and quantification (bars) showing deposition and organization of collagen fibres produced by H1299control and H1299RASSF1A spheroids embedded in collagen I matrix (2 mg/ml), treated with siNT, siRNA against P4HA2 or 4 μM DPCA to restrict P4HA2 activity. Red arrowheads show highly organized, long collagen fibres. Scale bars: 20 μm.SHG images of organization and quantification (bars) of collagen fractions in primary lung tumours and metastases at day 30. Scale bars: 10 μm.Representative H&E and quantification (bars) of picrosirius red staining of two independent regions of n = 5 H1299control and n = 3 H1299RASSF1A primary lung tumours. Scale bars: 100 μm; zoom: 20 μm.Data information: All statistical analyses were performed using Student's t‐test (two‐tailed) of n = 3 for in vitro experiments, and error bars represent the mean ± SEM.
Fig 2: Expression of P4HA2 is associated with poor prognosis in solid cancers Kaplan–Meier plots showing overall survival prognosis in breast cancer patients with high and low mRNA expression of TFPI2 and TIMP1 proteins revealed by proteomics study from extracted ECM produced by H1299RASSF1A lung cancer cells. Data show that breast cancer patients with high mRNA expression of TFPI2 and TIMP1 have better prognosis. The P values were derived from a log‐rank test.Clinical outcome and percentage of survival in patients across various cancers show effect of low versus high expression levels of mRNA P4HA2. Data collected from TCGA. The P values were derived from a log‐rank test.Quantification of fluorescence intensity of P4HA2 expression in H1299 cells with or without P4HA2 knockdown, 1.4DPCA treatment or combination of both. Bottom graph: Representative immunofluorescence images showing different expression of P4HA2 and collagen I in H1299control or H1299RASSF1A re‐expressing cells. Treatment of H1299control cells with siRNAP4HA2, P4HA inhibitor 1.4‐DPCA (inh.) or combination of both shows decreased collagen I expression. Scale bars: 10 μm.RT–PCR analysis of relative mRNA expression levels of P4HA2 in H1299 cells validating its after siP4HA2 knockdown.
Fig 3: P4HA2‐mediated collagen synthesis attenuates cancer cell differentiation Western blot analyses of expression levels of TTF‐1 and Mucin 5B in H1299control and H1299RASSF1A cells with band intensity quantification ratio determine by ImageJ analyser.Representative images of H&E and immunohistochemical staining for lung differentiation markers TTF‐1 and Mucin 5B for at least two independent regions of n = 4 H1299control and n = 2 H1299RASSF1A primary tumours at day 17 with quantification (right) of TTF‐1 based on nuclear intensity. Scale bars: 100 μm. Statistical significance was determined by Student's t‐test (two‐tailed). Error bars represent the mean ± SEMRepresentative images of immunofluorescence staining of spheroids grown in collagen matrix (2 mg/ml) for differentiation markers TTF‐1 (Green) and Mucin 5B (Red). Scale bars: 50 μm; Zoom: 10 μm.Bright‐field images of H1299control and H1299RASSF1A spheroid differentiation on Matrigel matrix after 24 h when 3D spheroids were seeded on three‐dimensional substrate. Scale bars: 200 μm. Source data are available online for this figure.
Fig 4: YAP regulates Nanog and OCT4 expression via P4HA2 in H1299control cells Relative mRNA expression levels of NANOG, OCT4 and SOX2 in H1299control and H1299RASSF1A lung adenocarcinoma cells.Representative immunofluorescence images of NANOG and YAP in the presence of siNT or siYAP in H1299control and H1299RASSF1A cells embedded and grown in three‐dimensional collagen matrix (2 mg/ml). Scale bars: 10 μm.Western blots of H1299control and H1299RASSF1A lysates for NANOG, SOX2 and OCT4 in the presence of siNT or siYAP.Relative H1299control mRNA expression levels of NANOG, OCT4 and SOX2 in H1299control cells in the presence of siNT or siYAP.Representative immunofluorescence images of H1299control and H1299RASSF1A spheroids embedded and grown in collagen matrix (2 mg/ml) and stained for NANOG (Green) and YAP (Red) with or without 4 μM DPCA. Scale bars: 10 μm.Western blots of H1299control lysates for NANOG, SOX2 and OCT4 in the presence of siNT, siP4HA2 or 4 μM DPCA.Data information: Statistical significance was determined by Student's t‐test (2‐tailed) of n = 3 experiments unless otherwise stated. Error bars represent the mean ± SEM.Source data are available online for this figure.
Fig 5: RASSF1A methylation is associated with increased P4HA2 and collagen I deposition Mass spectrometry analysis of proteins purified from extracellular matrix isolated from H1299control and H1299RASSF1A lung adenocarcinoma cell lines with summary of results. Only proteins identified with two or more peptides were taken into consideration. The resulting list of proteins was restricted to ECM proteins (n = 3).Kaplan–Meier plots depicting prognosis in TCGA_LUAD (lung adenocarcinoma) with high and low mRNA expression of ECM proteins revealed by proteomics. Significance derived from log‐rank test.Representative images of H&E and IHC staining for P4HA2, and quantification (bars) of two independent regions of n = 5 H1299control and n = 3 H1299RASSF1A primary lung tumours. Scale bars: 100 μm.Hydroxyproline assay (Cell biolabs Inc.) for total hydroxyproline activity of cells.Western blot indicating levels of collagen I in H1299control and H1299RASSF1A cells.Western blot indicating levels of collagen I in H1299control cells in the presence of (4 μM) prolyl 4‐hydroxylase inhibitor (DPCA), siRNA against P4HA2 or combination.Quantification of immunofluorescence staining of collagen I in H1299control cells in the presence of (4 μM) prolyl 4‐hydroxylase inhibitor (DPCA), siRNA against P4HA2 or combination. H1299RASSF1A cell line was used as negative control for comparison. Representative images in Fig EV2C.Immunofluorescence staining for collagen I and P4HA2 and quantification (right graph) in HOP92 cells after siRNA against RASSF1A. Scale bars: 20 μm.Western blot analyses in HOP92 cells showing increased expression of collagen I after siRNA against RASSF1A.Immunofluorescence staining for collagen I and P4HA2 and quantification (right graph) in HeLa cells after siRNA against RASSF1A. Scale bars: 20 μm.Data information: Unless otherwise indicated, all statistical analyses were performed using Student's t‐test (two‐tailed) of n = 3 experiments, and error bars represent the mean ± SEM.Source data are available online for this figure.
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