Fig. 4: Deconvolution of molecular pathways underpinning different phenotypes. | Nature Communications

Fig. 4: Deconvolution of molecular pathways underpinning different phenotypes.

From: Traject3d allows label-free identification of distinct co-occurring phenotypes within 3D culture by live imaging

Fig. 4

a Schema of PC3 subline pairs. Pair 1 is PC3 selected for epithelial shape (PC3-Epi) then made to undergo EMT by co-culture with macrophages (PC3-EMT). Pair 2 is PC3 FACS sorted for high surface E-cadherin (E-cad + ) vs PC3 harvested from a liver metastasis following in vivo selection (GS689.Li). Two PC3-EMT lines stably expressing ZEB1 shRNAs were also examined to identify ZEB1-influenced transcripts. Venn diagram summarizes RNAseq profiling which identifies 36 ZEB1-responsive genes upregulated in both Pair 1 and 2 and depleted upon ZEB1 shRNA-induced knockdown. b Comparison of transcript levels (shown as Log2 Fold Change) in Cell Pair 1 between epithelioid (PC3-Epi) vs invasive (PC3-EMT) samples. p-values shown as -Log10 (Negative Binomial GLM fitting and Wald statistics using DESeq2). c MetaCore analysis of pathways maps from RNAseq profiling of PC3-Epi and PC3-EMT (Pair 1) and E-cad+ and GS689.Li (Pair 2) identified pathways commonly upregulated across both cell pairs in PC3-EMT (vs PC3-Epi) and in GS869.Li (vs E-Cad + ). Molecular processes enriched in these data sets are ranked by p value (MetaCore version on 2017-10-26, -Log10). d Comparison of expression of EMT transcription factors (TFs; GRHL1-3, ZEB1, SNAI1-3, TWIST1) between PC3-Epi and PC3-EMT (Pair 1) and E-cad+ and GS689.Li (Pair 2) cells indicates that ZEB1 is the most upregulated EMT TF in both cell pairs (in PC3-EMT and GS689.Li). Data are presented in the heatmap as Log2 Fold Change from green to magenta. e Comparison of the top and bottom 50 most differentially expressed genes that were concordant between both cell pairs revealed that within the Top 50 epithelioid-associated genes, 20% of these transcripts were altered in expression upon shRNA targeting the transcriptional repressor ZEB1. ZEB1-responsive genes are indicated by red asterisk or red text. f Western blot analysis of PC3 sublines was performed using anti-E-cadherin, N-cadherin, vimentin, ZEB1, ESRP1, ESRP1/2 and GAPDH antibodies. GAPDH blot is loading control for vimentin and sample integrity control for other blots. Representative of n = 2 independent experiments. Note that ZEB1 had inverse expression compared to E-cadherin and ESRP1/2 in all PC3-derivative cell lines, except TEM2-5. Note that N-cadherin expression was not concordant with ZEB1.

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