sb525334  (MedChemExpress)

Journal: bioRxiv

Article Title: Bidirectional fibrogenic cross-talk revealed in a human iPSC-derived epithelial-mesenchymal co-culture model of pulmonary fibrosis

doi: 10.64898/2026.01.30.702837

Figure Lengend Snippet: a: Schematic representation of computational crosstalk analysis using NicheNet. First, ligands and receptors upregulated upon co-culture (vs single lineage cultures) are identified. Second, ligand-receptor pairs enriched in co-cultures with mutant SFTPC I73T iAT2s (vs corrected iAT2s) are identified. Analysis is performed defining either iLM as sender and iAT2s as receiver, or iAT2s as sender and iLM as receiver. b: Expression of top 20 prioritized ligands in iLM co-cultured with SFTPC I73T mutant iAT2s compared to iLM cultured alone. (iLM = sender; SFTPC I73T mutant iAT2s = receiver). c: Differential crosstalk analysis. Left panel: Top 20 prioritized ligands in co-cultured SFTPC I73T mutant iAT2s (sender) vs co-cultured corrected iAT2s (sender), with fold change expression of their top prioritized receptors in co-cultured iLM (receiver). Right panel: Top 20 prioritized ligands in iLM co-cultured with SFTPC I73T mutant iAT2s (sender) vs iLM co-cultured with corrected iAT2s (sender), with fold change expression of their top prioritized receptors in SFTPC I73T mutant or corrected co-cultured iAT2s (receiver). LFC = log fold change in gene expression. d: SFTPC tdTomato and ACTA2 GFP fluorescence in live co-cultures of SFTPC I73T mutant iAT2s and iLM treated with 1 μM SB525334 or 2 μM Bexotegrast for 1 week, as compared to vehicle control (treatment starting on the first day of co-culture). Scale bars = 400 μm. e: Immunofluorescence staining for KRT17 protein in co-cultures of SFTPC I73T mutant iAT2s and iLM treated as in (d). Scale bars = 100 μm. Hoechst = nuclei. f: RT-qPCR analysis showing fold change expression (normalized to iLM co-cultured control) of fibrotic markers in EPCAM- sorted cells from co-cultures of iLM with SFTPC I73T mutant iAT2s or iLM cultured alone, and treated as in (d). N = 5 for SB525334, N = 6 for Bexotegrast, symbol shapes indicate experimental replicates, i.e. multiple wells of the same experiment. Data points are representative of results from 3 repeated independent experiments for SB525334 and 2 repeated independent experiments for Bexotegrast, performed with iAT2s of varying ages and passages. Bars represent mean ± SD, * p<0.05, ** p<0.01, *** p<0.001, ns = not significant, as determined by paired two-tailed Student’s t test. SB5 = SB525334, Bex = Bexotegrast, ctrl = vehicle control, co = co-cultured, alone = cultured alone. g: RT-qPCR analysis showing fold change expression (normalized to iAT2 co-cultured control) of KRT17 in EPCAM+ sorted cells from SFTPC I73T mutant iAT2s co-cultured with iLM or cultured alone, and treated as in (d). N = 5 for SB525334, N = 6 for Bexotegrast, symbol shapes indicate experimental replicates, i.e. multiple wells of the same experiment. Data points are representative of results from 3 repeated independent experiments for SB525334 and 2 repeated independent experiments for Bexotegrast, performed with iAT2s of varying ages and passages. Bars represent mean ± SD, * p<0.05, ** p<0.01, *** p<0.001, ns = not significant, as determined by paired two-tailed Student’s t test. h: Schematic representation of fibrogenic crosstalk between SFTPC I73T mutant iAT2s and iLM.

Article Snippet: Medium was replaced with fresh CK+DCI medium every 48 h. For SB525334 or Bexotegrast treatment of co-cultures, 1 μM SB525334 (Sigma, #S8822-5MG) or 2 μM Bexotegrast (MedChemExpress, #HY-137561) was added to the co-cultures at the time-point of iAT2 plating and kept in the culture medium until the time-point of cell collection.

Techniques: Co-Culture Assay, Mutagenesis, Expressing, Cell Culture, Gene Expression, Fluorescence, Control, Immunofluorescence, Staining, Quantitative RT-PCR, Two Tailed Test

Journal: bioRxiv

Article Title: Bidirectional fibrogenic cross-talk revealed in a human iPSC-derived epithelial-mesenchymal co-culture model of pulmonary fibrosis

doi: 10.64898/2026.01.30.702837

Figure Lengend Snippet: a: Percentage of ACTA2 GFP high cells among EPCAM- cells, and mean ACTA2 GFP fluorescence intensity of EPCAM- cells in iLM co-cultured with SFTPC I73T mutant iAT2s or cultured alone, treated with 1 μM SB525334 or 2 μM Bexotegrast for 1 week, as compared to vehicle control (treatment starting on the first day of co-culture). N = 5 for SB525334, N = 6 for Bexotegrast, symbol shapes indicate experimental replicates, i.e. multiple wells of the same experiment. Data points are representative of results from 3 repeated independent experiments for SB525334 and 2 repeated independent experiments for Bexotegrast, performed with iAT2s of varying ages and passages. Bars represent mean ± SD, * p<0.05, ** p<0.01, *** p<0.001, ns = not significant, as determined by paired two-tailed Student’s t test. SB5 = SB525334, Bex = Bexotegrast, ctrl = vehicle control, co = co-cultured, alone = cultured alone. b: Percentage of tdTomato+ cells among EPCAM+ cells and normalized mean tdTomato fluorescence intensity of EPCAM+/tdTomato+ cells in SFTPC I73T mutant iAT2s co-cultured with iLM or cultured alone, treated as in (a). N = 5 for SB525334, N = 6 for Bexotegrast, symbol shapes indicate experimental replicates, i.e. multiple wells of the same experiment. Data points are representative of results from 3 repeated independent experiments for SB525334 and 2 repeated independent experiments for Bexotegrast, performed with iAT2s of varying ages and passages Bars represent mean ± SD, * p<0.05, ** p<0.01, *** p<0.001, ns = not significant, as determined by paired two-tailed Student’s t test. c: RT-qPCR analysis showing fold change expression (normalized to iLM co-cultured control) of fibrotic markers in EPCAM- sorted cells from co-cultures of iLM with SFTPC I73T mutant iAT2s or iLM cultured alone, and treated as in (a). N = 5 for SB525334, N = 6 for Bexotegrast, symbol shapes indicate experimental replicates, i.e. multiple wells of the same experiment. Data points are representative of results from 3 repeated independent experiments for SB525334 and 2 repeated independent experiments for Bexotegrast, performed with iAT2s of varying ages and passages. Bars represent mean ± SD, * p<0.05, ** p<0.01, *** p<0.001, ns = not significant, as determined by paired two-tailed Student’s t test. d: RT-qPCR analysis showing fold change expression (normalized to iAT2 co-cultured control) of KRT7, NKX2-1 and SFTPC in EPCAM+ sorted cells from SFTPC I73T mutant iAT2s co-cultured with iLM or cultured alone, and treated as in (a). N = 5 for SB525334, N = 6 for Bexotegrast, symbol shapes indicate experimental replicates, i.e. multiple wells of the same experiment. Data points are representative of results from 3 repeated independent experiments for SB525334 and 2 repeated independent experiments for Bexotegrast, performed with iAT2s of varying ages and passages. Bars represent mean ± SD, * p<0.05, ** p<0.01, *** p<0.001, ns = not significant, as determined by paired two-tailed Student’s t test.

Article Snippet: Medium was replaced with fresh CK+DCI medium every 48 h. For SB525334 or Bexotegrast treatment of co-cultures, 1 μM SB525334 (Sigma, #S8822-5MG) or 2 μM Bexotegrast (MedChemExpress, #HY-137561) was added to the co-cultures at the time-point of iAT2 plating and kept in the culture medium until the time-point of cell collection.

Techniques: Fluorescence, Cell Culture, Mutagenesis, Control, Co-Culture Assay, Two Tailed Test, Quantitative RT-PCR, Expressing