Additional file 1 of An oncogenic KRAS-driven secretome involving TNFα promotes niche preparation prior to pancreatic cancer onset

Supplementary Material 1: Supplementary Figure 1. (A) Brightfield images of PDLOs ± Dox treatment. Scale bar = 250 µm. (B) Flow cytometry quantifying mCherry reporter expression after Dox treatment. (C) Dot plot showing expression of cell type markers in PDLO scRNA-seq data set. (D) KRAS copy numbers for hESC lines, PDAC lines and patient-derived organoids (PDOs) using probes targeting either the 3’ untranslated region (3’UTR) of the endogenous locus or exon 3, which detects the respective sequence in the endogenous locus and in the inserted piggyBac KRASG12D construct. Copy numbers are shown relative to the respective KRAS wild-type cell lines (parental hESCs, BxPC3 and PDO#38188). BU3 = monoallelic BU3 NGST-TetOn:KRASG12D line[92]. (E) Scatter plot showing correlation between KRASG12D expression and module scores of different pathways over the time of Dox treatment within the scRNA-seq data set. R = Pearson’s correlation coefficient. (F) Protein expression of phospho-ERK1/2 and ERK1/2 in PDLOs after indicated time-points of Dox treatment, in PDAC lines and PDOs. For quantification, phospho-ERK1/2 levels were normalized to ERK1/2 levels for each sample. n = 3 for KRAS-PDLOs, n = 2 for parental PDLOs and n = 1 for PDAC lines and PDOs. (G) Cell cycle analysis of PDLOs after indicated time-points of Dox treatment assessed by EdU quantification via flow cytometry. n = 4. (H) PCA plot demonstrating variance of chromatin accessibility between ATAC-seq samples. (I) Heatmap showing mean gene expression of TFs, overlapping in ATAC- and scRNA-seq. Supplemental Figure 2. (A) Proportion of the KRAS clusters within the individual time points of the scRNA-seq analysis. (B) Expression of the top 10 DEGs from each KRAS cluster compared to all other clusters. (C) Overlapping and unique DEGs of KRAShighand KRASintermediate compared to the KRASlow cluster with corresponding KEGG pathway analysis of unique DEGs. (D) Overall survival of TCGA-PAAD patients grouped based on KRAS cluster DEGs by gene set variance analysis (GSVA). (E) Pie charts showing the distribution of grouped GO terms from DEGs of KRAShigh and KRASintermediate compared to KRASlow. (F) Bar plot showing the relative abundance of proteins after 7 days of Dox treatment. Mean ± SD, n=1 in triplicates. Unpaired t-test **, p