Analysis of gene expression changes upon topobexin treatment and TOP2B-knockout in hiPSC derived cardiomyocytes

Figures

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  Fig. 1.. WT and TOP2B null hiPSCs can efficiently differentiate into beating cardiomyocytes. (A) The differentiation protocol resulted in the conversion of monolayers of WT or TOP2B null (line 18D, BKO) hiPSC cells into layers of beating cardiomyocytes in 8-12 days. However, on average this took 1 day longer for BKO than WT hiPSC cells. Each spot represents a separate hiPSC to CM differentiation experiment, four biological replicates for WT and five biological replicates for the BKO. Line=mean. Statistical significance was determined using a two-tailed unpaired t-test, *P<0.05. (B) Principal component analysis of RNA-seq analysis comparing WT and TOP2B null (BKO) hiPSC and derived CM (quadruplicate biological replicates). (C) Scree plot showing the variance of PC1-PC5 (D) Volcano plots to visualize gene expression changes upon differentiation of WT and BKO hiPSC cells to CM. Red lines represent cutoffs of >2-fold change in transcript abundance with a Padj of <0.05. Numbers in the corners of the plots highlight the number of up- or downregulated protein-coding genes in the CM compared to the hiPSC cells, using the above cutoffs.

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  Fig. 2.. Transcriptome comparison of WT and BKO hiPSC-derived CM cell, and effect of TOP2 inhibitors topobexin and ICRF-187. (A) Volcano plot illustrating the number of genes up- or downregulated in BKO versus WT CM. (B) Effect of topobexin gene expression (transcript abundance) in WT CM. (C) Effect of ICRF-187 on gene expression (transcript abundance) in WT CM. For volcano plots, cut-offs (red-lines) were as in Fig. 1. Representative genes are highlighted. (D) Principal component analysis of transcript abundance comparing in four conditions, WT and TOP2B null (BKO) CM, WT CM treated with topobexin and CM treated with ICRF-187 (quadruplicate biological replicates). (E) A scree plot of PC1-PC10.

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  Fig. 3.. Comparison of gene expression changes in WT versus BKO CM and topobexin treated versus untreated CM. Differential gene expression tables were filtered to include only genes that change significantly (Padj <0.05) in expression in both TOP2B null CM versus WT cells and after treatment of WT CM with topobexin. The gene list was further filtered to only include protein-coding genes. Log2 FC values are plotted on the x and y axes. r=Pearson correlation coefficient.

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  Fig. 4.. TPM levels in the RNA seq datasets for each of the six conditions analyzed.

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  Fig. 5.. Gene set enrichment analysis. Sets of transcripts whose abundance changed (≥2 fold) in either TOP2B−/− (BKO) CM or topobexin-treated CM compared to untreated WT CM were examined by g:GOSt functional enrichment analysis (https://biit.cs.ut.ee/gprofiler/gost). The top 41 biological pathway (BP) terms for (A) TOP2B−/− (BKO) CM and (B) topobexin-treated CM were plotted by negative log10(adjusted P-value). Numbers in red correspond to the intersection size. (C) Venn diagram illustrating the degree of overlap between the top 200 (by statistical significance) BP terms highlighted for each condition.