Additional file 3 of c-Rel drives pancreatic cancer metastasis through fibronectin-integrin signaling-induced isolation stress resistance and EMT

Supplementary Material 3. Supplementary Figure 3. A) Representative fluorescence images of c-Rel nuclear localization in CKP cells after TNF-α and leptomycin-B treatment for 20 minutes. B) Co-immunoprecipitation analysis of RelA, RelB, c-Rel and rabbit IgG as negative controls. Ft: flow through, Ip: immunoprecipitate. The antibodies used for immunoprecipitation (IP) are listed above, whereas the antibodies used for immunoblotting are listed on the left. C) Total cell lysates were immunoblotted for the expression of multiple NF-κB proteins. Each cell line was isolated from a separate mouse autochthonous tumor (mPDAC) with the CK or CKA (p65/RelA knockout) genotype. The CKA model expresses an NLS-truncated RelA protein. Signal is normalized to β-actin. On the graph t-test p-values are given. D) Immunoblot analyses of the given NF-κB proteins in cell lysates obtained from CCKP, CKP and GCKP cells cultured under basal conditions. Signal is normalized to β-actin and plotted (p-values: RelA, RelB, p50, p100 n.s., p105 p=0.0025, p52 p=0.0214, Ikbα p=0.0138, Ikbβ p=0.0052) E) Nuclear fractionation was performed with cell lines isolated from CK, CKA, CKP and CKPA tumors (n= 2 per genotype) and immunoblotted for the given proteins. Nuclear signal is normalized to Lamin A/C for quantification. F) Nuclear fractions were isolated from CCKP, CKP and GCKP cells and immunoblotted for the given NF-κB proteins. Signal is normalized to Lamin A/C (p-values: RelA p=0.0129, RelB p=0.0019, p50 p=0.0235, p52 n.s.) G) With pdacR, multiple human PDAC RNA-seq datasets are analyzed for REL mRNA correlation to the Moffitt normal vs activated stroma top 25–100 and 250 gene signatures. The top correlation graph represents data from the TCGA cohort. The Pearson-R values and their p values are given in the table below. H) A human PDAC proteomics dataset was analyzed for c-Rel and FN1 expression in tumor cells or stroma in samples stratified on the basis of their sub-TME types. A one-way ANOVA test for a linear trend is displayed in the figure. I) Tumor cell c-Rel protein expression vs FN1 protein expression in the tumor stroma correlation plot. J) Selected GSEA results for the GCKP vs CKP and GCKP vs CCKP pairs on the basis of RNA-seq analyses. K) Human PDAC RNA-seq datasets were analyzed for KEGG-ECM receptor interaction pathways. The samples were divided into medians on the basis of REL expression. L) Two human PDAC scRNA-seq datasets were analyzed for REL expression in multiple given cell types. UMAPs showing REL levels in various cell types. The cell types are determined on the basis of the expression matrix on the left. Violin plots showing the relative REL values in the given cell types. Dot plots at the bottom display integrin expression levels and the fraction of cells expressing them in the epithelial cell compartment. Epithelial cells are divided into HIGH and LOW groups on the basis of REL expression.