Fig 2: Differentially expressed genes in H9C2 cells after RhoE-knockout. (A) Volcano plot. The x-axis represents log2 conversion of the fold change values, whilst the y-axis represents the corrected significance level after base log10 conversion. Red dots in the figure indicates all probes with significance levels <0.05. (B) Scatter plot indicating the top five upregulated and downregulated genes. (C and D) Data from Chip assay were used to generate the maps using the GraphPad Prism 8.0 software. Heatmap of (C) the top 20 upregulated and (D) top 20 downregulated genes. Compared NC with KO, genes in red color were indicated to be up-regulated and genes in blue color were indicated to be downregulated. NC, negative control; KO, knockout.

Fig 3: Knockout of RhoE expression in H9C2 cells using CRISPR/Cas9 technology. (A) Representative images of the DNA gels from H9C2 cells showing the fragments of PCR products. PC represents the Cdc42 gene positive control, which was proven to be correctly recognized by Detecase, with the product at 456 bp and the fragments at 151 and 303 bp after Cruiser digestion. NC-1 represents cells infected with the scrambled sgRNA-overexpressing viruses, where primer pairs for sgRNA-1 were used; NC-2 represents cells infected with the scrambled sgRNA-overexpressing viruses, where primer pairs for sgRNA-3 were used. (B) Western blot analysis of RhoE protein expression in the mix clone population of H9C2 cells. (C) Heatmap representing the mRNA the expression levels of RhoE and the internal control GAPDH in H9C2 cells. (D) Chip results of RhoE mRNA expression in H9C2 cells from the knockout and NC groups. FC, fold change; KO, knockout; PC, positive control; NC, negative control.

Fig 4: Classification of diseases and functions. (A, C and D) Categories are shown in terms of the -log (P-value), as represented by the left y-axis and the number of differentially expressed genes counted, represented by the right y-axis. (A) A total of 24 representative classification of diseases and functions possibly mediated by RhoE are plotted. (B) Heatmap representing the classification of diseases and functions. This heatmap was drafted according its Z-score values, where higher Z-scores represented by orange indicates activation, whilst lower Z-scores represented by blue indicates inhibition. (C) Classification of pathophysiological processes possibly mediated by RhoE in cardiovascular disease. (D) Classification of possible functions of RhoE in cardiovascular system development and function.

Fig 5: Gene interaction network map. This network consists of the top ranked network found associated with the role of RhoE in neurological disease, hereditary disorder and organismal injury and abnormalities.