Fig 1: The key genes and signaling pathways modulated by coptisine were explored by bulk RNA sequencing. (A) Volcano plot displaying differentially expressed genes between coptisine and control groups (n = 3 biological replicates). Significant genes were called via Cuffdiff. The red dots represent the up−regulated expressed transcripts between coptisine and control; the blue dots represent the transcripts whose expression was down−regulated. (B) Significantly enriched modulated KEGG pathways. The depth of the color represents the adjusted p-value, while the area of the circle represents gene counts. The pathway marked in red font is related to CDKN1A. (C) Heat map of differential expression of genes associated with CDKN1A between the control and coptisine groups (n = 3 biologically independent cell samples; FDR < 0.001). Statistical tests were embedded in Cuffdiff. Each row represents a gene; red means an increased gene expression, blue means decreased gene expression, and the darker the color, the more obvious the trend is. Heatmap analysis shows that coptisine could significantly upregulate or downregulate the expression of certain genes compared to the controls. The RNA-Seq data were deposited in GEO (GSE232189) (D,E) MDCK was cultured for 24 h in the presence of different concentrations of coptisine. The total RNA was isolated to detect the expression level of CDKN1A and FOXO1 by quantitative PCR (D). The total protein was collected for Western blot to quantify p21 and FOXO1 (E). Each error bar represents the mean ± SD of triplicate wells assayed for each sample. Statistical significance was analyzed with Dunnett’s test (** p < 0.01; **** p < 0.001, compared with 0 μM).