Fig 1: Laminin α5 was required for IL-6-induced osimertinib resistance.a Experimental workflow of the proteomic experiments (biological triplicates per group). b Volcano plot of the differentially expressed proteins in PC-9GROR cells compared to PC-9GR cells, including significantly (FDR < 5%) upregulated (red dots), downregulated (blue dots), and unchanged (Gray dots) proteins. X axis = log2 fold change; Y axis = −log10 p-value. Cutoff of p = 0.05 and 1.5-fold change were marked by horizontal dashed line and vertical dashed line, respectively. Proteins in four IL-6 downstream pathways, MAPK signaling pathway, JAK-STAT pathway, NF-kB pathway, and PI3K-AKT pathway were marked, and Laminin α5 (LAMA5) was highlighted with an arrow. c Heat map of four indicated signaling pathways. Each row corresponds to a certain protein involved in the indicated pathway with its name in the left panel. The colors indicate regulation intensity (upregulated or downregulated using Z-score normalization of quant). d Network of interactions between proteins that are significantly (1.5 fold-change) regulated. Red nodes are upregulated proteins (>1.5-fold), and the blue nodes are downregulated proteins (>1.5-fold). Node size is proportional to the degree of protein-protein interaction. Greater the node is, more proteins interact with it. Each dashed circle represents a functional category. LAMA5 in the focal adhesion category was highlighted with an arrow. e Western blot showing the expression levels of certain proteins in cell lines as indicated. IL-6 (20 ng/ml) was added to the culture medium for 48 h. β-tubulin served as loading control. f The levels of Laminin α5 and STAT3 in PC-9GROR cells, IL-6-GFP PC-9 cells, and IL-6-GFP PC-9GR cells after transfection with LAMA5 siRNAs, respectively. β-tubulin served as loading control. g Cell viability CCK-8 assay for indicated cells transfected with control or LAMA5 siRNAs, respectively, and treated with increasing concentrations of osimertinib for 48 h. Data are shown as mean ± SEM (n = 3 biologically independent experiments).
Fig 2: Laminin α5/FAK pathway specifically mediated IL-6-induced osimertinib resistance.a Schematic overview of generation of osimertinib-resistant single clones. Increasing concentrations of osimertinib was applied to osimertinib-sensitive cells to generate resistant cells. Then, limiting dilution method was applied and osimertinib-resistant single-cell clones were further selected; b histogram showing IC50 values of the selected osimertinib-resistant single-cell clones (*p < 0.01 compared with parental cells, respectively). n = 3 biologically independent experiments; c IL-6 levels in osimertinib-resistant single-cell clones detected by ELISA. d Western blot showing the expression levels of Laminin α5, total and phosphorylated FAK in indicated cell lines. β-tubulin served as loading control; e representative images of Laminin α5 and phosphorylated FAK in indicated cell lines by fluorescent detection. Cells were counterstained with DAPI. Scale bars: 50 μm. f Western blot showing the levels of Laminin α5, total and phosphorylated STAT3 in H1975OR2 cells and H1975OR7 cells transfected with control or LAMA5 siRNAs. β-tubulin served as loading control. g Cell viability CCK-8 assay for H1975OR2 cells and H1975OR7 cells transfected with control or LAMA5 siRNAs, respectively, and treated with osimertinib for 48 h (n = 3 biologically independent experiments). Histogram shows IC50 values in the indicated groups (*p < 0.01 by Student’s t-test). h The levels of FAK, total and phosphorylated STAT3 in H1975OR2 cells and H1975OR7 cells after transfection with PTK2 siRNAs, respectively. β-tubulin served as loading control. i Cell viability CCK-8 assay for indicated cells transfected with control or PTK2 siRNAs, respectively, and treated with osimertinib for 48 h (n = 3 biologically independent experiments). Histogram shows IC50 values in the indicated groups (*p < 0.01 by Student’s t-test).
Fig 3: Laminin α5 mediated IL-6-induced osimertinib resistance through phosphorylation of FAK.a Protein–protein network of the four indicated signal pathways. Red nodes are upregulated proteins (>1.5-fold), and the blue nodes are downregulated proteins (>1.5-fold). Node size is proportional to the degree of protein–protein interaction. The dashed circles frame the corresponding pathways. LAMA5 and PTK2 were highlighted with circles. b Representative images of Laminin α5 and phosphorylated FAK in indicated cell lines by fluorescent detection. Cells were counterstained with DAPI. Scale bars: 30 μm. c Western blot showing the expression levels of FAK and phosphorylated FAK in cell lines as indicated. IL-6 (20 ng/ml) was added to the culture medium for 48 h. Experiments were performed in triplicates, and β-tubulin served as loading control. d Western blot showing the levels of total and phosphorylated PTK2 in PC-9GROR cells after transfection with LAMA5 siRNAs, respectively. β-tubulin served as loading control. e The levels of total and phosphorylated FAK in PC-9GROR cells after transfection with PTK2 siRNAs, respectively. β-tubulin served as loading control; f cell viability CCK-8 assay for PC-9GROR cells transfected with control or PTK2 siRNAs, respectively, and treated with increasing concentrations of osimertinib for 48 h. Data are shown as mean ± SEM (n = 3 biologically independent experiments). Histogram shows IC50 values in the indicated groups (*p < 0.01 by Student’s t-test). g Immunohistochemistry analysis of IL-6, Laminin α5, and phosphorylated FAK on tumor sections from two representative patients upon osimertinib resistance. HE, Hematoxylin and Eosin staining. Scale bars: 100 μm.
Supplier Page from Abcam for Anti-Laminin alpha 5/LAMA5 antibody [EPR18919]