Fig. 5.

RNA sequencing analysis of isogenic CRPC lines. (A) Schematic representation of gene comparisons for RNA sequencing result analysis. Dot plot analysis of significant C4–2R downregulated (B), upregulated (C), MR49F downregulated (D), and upregulated (E) pathways. Methods: The RNA-seq differential expression (DE) analysis involved the following comparisons and cell lines: (1) metformin versus controls in the C4–2 cell line; (2) metformin versus controls in the C4–2R cell line; and (3) Combo (combination) versus controls in the C4–2R cell line. Additionally, 3 more comparisons were performed in parallel with the above 3: (4) metformin versus controls in the LNCaP cell line; (5) metformin versus controls in the MR49F cell line; and (6) combo versus controls in the MR49F cell line. In this setup, the LNCaP cell line serves as a parallel to the C4–2 cell line, and the MR49F cell line serves as a parallel to the C4–2R cell line. For each comparison, RNA-seq DE analysis was conducted using the R package “edgeR,” with the control group chosen as the reference. In comparisons 1–3 and 4–6, respectively, significantly upregulated DE genes were identified by a log₂(fold change) > 0.5 and a q-value < 0.05, whereas significantly downregulated DE genes were identified by a log₂(fold change) < −0.5 and a q-value < 0.05. We then isolated the significant DE genes that were found in the combo versus control comparison but not in the metformin versus control comparison, identifying genes that responded exclusively to the combo treatment. Using this subset of upregulated or downregulated DE genes, we performed KEGG pathway enrichment analysis with the R package “clusterProfiler,” which uses Fisher’s exact test. The output of the enrichment analysis reveals the KEGG pathways that are enriched specifically in response to the combo treatment. Figure 5 illustrates the top 20 upregulated and downregulated KEGG pathways with the smallest P values for the cell lines C4–2/C4–2R and LNCaP/MR49F, respectively.