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( A ) Quantified levels of IL-1α from naïve fibroblast (Naïve fibro), inflammatory fibroblast (Inflam fibro), and ectopic basal cell (Basal) conditioned media as determined by ELISA. ( B ) Saa3 and Ccl2 gene expression by RT-qPCR for fibroblasts cultured on 0.5kPa plates and treated with a 5-fold titration of IL-1α added to control media, including the level determined in (A). ( C ) Graphic illustrating the strategy used to deplete conditioned media of endogenous IL-1α with antibodies, magnetic beads, and column-based separation. ( D ) Saa3 and Ccl2 gene expression by RT-qPCR for fibroblasts cultured on 0.5kPa plates in control or conditioned media depleted using <t>IgG</t> (CTRL IgG or CM IgG) or IL-1α nAb (CTRL nAB, CM nAb) and supplemented with additional IL-1α nAb after depletion (+Spike). [(B) and (D)] Data shows mean fold change ± SD and are pooled from 3 independent experiments (n=3 mice). P values were calculated using either (B) a ratio paired t -test or (D) a one-way ANOVA with post-hoc Turkey multiple comparison test: * P < 0.05, ** P < 0.01, **** P < 0.0001, ns: not significant.
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( A ) Quantified levels of IL-1α from naïve fibroblast (Naïve fibro), inflammatory fibroblast (Inflam fibro), and ectopic basal cell (Basal) conditioned media as determined by ELISA. ( B ) Saa3 and Ccl2 gene expression by RT-qPCR for fibroblasts cultured on 0.5kPa plates and treated with a 5-fold titration of IL-1α added to control media, including the level determined in (A). ( C ) Graphic illustrating the strategy used to deplete conditioned media of endogenous IL-1α with antibodies, magnetic beads, and column-based separation. ( D ) Saa3 and Ccl2 gene expression by RT-qPCR for fibroblasts cultured on 0.5kPa plates in control or conditioned media depleted using <t>IgG</t> (CTRL IgG or CM IgG) or IL-1α nAb (CTRL nAB, CM nAb) and supplemented with additional IL-1α nAb after depletion (+Spike). [(B) and (D)] Data shows mean fold change ± SD and are pooled from 3 independent experiments (n=3 mice). P values were calculated using either (B) a ratio paired t -test or (D) a one-way ANOVA with post-hoc Turkey multiple comparison test: * P < 0.05, ** P < 0.01, **** P < 0.0001, ns: not significant.
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( A ) Quantified levels of IL-1α from naïve fibroblast (Naïve fibro), inflammatory fibroblast (Inflam fibro), and ectopic basal cell (Basal) conditioned media as determined by ELISA. ( B ) Saa3 and Ccl2 gene expression by RT-qPCR for fibroblasts cultured on 0.5kPa plates and treated with a 5-fold titration of IL-1α added to control media, including the level determined in (A). ( C ) Graphic illustrating the strategy used to deplete conditioned media of endogenous IL-1α with antibodies, magnetic beads, and column-based separation. ( D ) Saa3 and Ccl2 gene expression by RT-qPCR for fibroblasts cultured on 0.5kPa plates in control or conditioned media depleted using IgG (CTRL IgG or CM IgG) or IL-1α nAb (CTRL nAB, CM nAb) and supplemented with additional IL-1α nAb after depletion (+Spike). [(B) and (D)] Data shows mean fold change ± SD and are pooled from 3 independent experiments (n=3 mice). P values were calculated using either (B) a ratio paired t -test or (D) a one-way ANOVA with post-hoc Turkey multiple comparison test: * P < 0.05, ** P < 0.01, **** P < 0.0001, ns: not significant.

Journal: bioRxiv

Article Title: Dysplastic Epithelial Repair Propagates Chronic Pathology Through the Paracrine Transformation of Pulmonary Fibroblasts

doi: 10.64898/2026.04.02.716135

Figure Lengend Snippet: ( A ) Quantified levels of IL-1α from naïve fibroblast (Naïve fibro), inflammatory fibroblast (Inflam fibro), and ectopic basal cell (Basal) conditioned media as determined by ELISA. ( B ) Saa3 and Ccl2 gene expression by RT-qPCR for fibroblasts cultured on 0.5kPa plates and treated with a 5-fold titration of IL-1α added to control media, including the level determined in (A). ( C ) Graphic illustrating the strategy used to deplete conditioned media of endogenous IL-1α with antibodies, magnetic beads, and column-based separation. ( D ) Saa3 and Ccl2 gene expression by RT-qPCR for fibroblasts cultured on 0.5kPa plates in control or conditioned media depleted using IgG (CTRL IgG or CM IgG) or IL-1α nAb (CTRL nAB, CM nAb) and supplemented with additional IL-1α nAb after depletion (+Spike). [(B) and (D)] Data shows mean fold change ± SD and are pooled from 3 independent experiments (n=3 mice). P values were calculated using either (B) a ratio paired t -test or (D) a one-way ANOVA with post-hoc Turkey multiple comparison test: * P < 0.05, ** P < 0.01, **** P < 0.0001, ns: not significant.

Article Snippet: The working concentration for the following reagents can be found in the text and figure legends: Human IL-1RA Recombinant Protein (IL1Ra) (PeproTech®, 200-01RA), Mouse IL-1 alpha Recombinant Protein, (PeproTech®, 211-11A), Mouse IL-1α Neutralizing Antibody (Invivogen, Anti-mIL-1α-mIgG1 clone 6H7, mIL-1α-mab9-02), Recombinant Mouse IgG1 Isotype Control Antibody (Invivogen, Anti-β-Gal-mIgG1 clone T9C6, bgal-mab9-02), Human TGF-β 1 Recombinant Protein (PeproTech®, 100-21), Human GDF15 Recombinant Protein (PeproTech®, 120-28C), Human VEGF Recombinant Protein (PeproTech®, 450-32), Human OPN Recombinant Protein (PeproTech®, 120-35), Murine CXCL1 Recombinant Protein (PeproTech®, 250-11), Human IGFBP3 Recombinant Protein (PeproTech®, 100-08), Human TNFRSF11B Recombinant Protein (PeproTech®, 450-14), Murine CXCL5 Recombinant Protein (PeproTech®, 250-17), and Muring CCL20 Recombinant Protein (PeproTech®, 250-27).

Techniques: Enzyme-linked Immunosorbent Assay, Gene Expression, Quantitative RT-PCR, Cell Culture, Titration, Control, Magnetic Beads, Comparison