Fig. 2: 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).