Fig 1: CCDC80 knockdown in human preadipocytes differentiated to adipocytes activates known drivers of adipogenesis, and SOD3 knockdown deactivates known drivers of healthy energy homeostasis. We knocked down CCDC80 and SOD3 using siRNA transfection in independent cultures of human SGBS preadipocyte cells (see Methods), and measured expression via RNA-seq at 4 time points during adipogenesis. We then performed a differential expression (DE) analysis on the RNA-seq data between the CCDC80 or SOD3 gene knockdown and scramble conditions at each time point. (a) Results of the CCDC80 knockdown DE analysis. The X-axis represents the log fold-change (logFC) of all 43 genes which were DE in at least one time point during the differentiation; the Y-axis the gene names; and facets the time points of adipogenesis. Blue genes were expressed significantly more in the CCDC80 knockdown than in the scramble conditions, and red genes were expressed less. Yellow represents CCDC80, the knocked down gene. (b) Mean expression of CCDC80 and selected well known examples of adipogenesis genes in the scramble and knockdown samples during differentiation. The X-axis represents the time point of the adipocyte differentiation; the Y-axis counts per million (CPM); facets the gene name; error bars the mean ± one standard deviation; and colours the experimental condition (knockdown or scrambled control). Each condition-timepoint combination within each facet represents n = 3 samples, and the total experiment included n = 28 samples. Annotations indicate the significance of DE between the knockdown and scramble samples for a given timepoint and gene: “***” = adjP < 0.001; “**” = adjP < 0.01; “*” = adjP < 0.05. In the CCDC80 panel only: “+++” = p < 0.001, “++” = p < 0.01, “+” = p < 0.05. The CCDC80 p-values are not adjusted for multiple testing because we directly manipulated CCDC80 expression in the knockdown experiment. (c) Results of the SOD3 DE analysis, with 54 DE genes. Plot elements are analogous to those in (a). (d) Mean expression of SOD3 and selected well known examples of adipogenesis and satiety signalling genes in the knockdown and scramble samples during differentiation. Plot elements are analogous to those in (b).
Fig 2: Treatment of human liver HepG2 cells with the CCDC80 and SOD3 recombinant proteins changes the expression of several known NAFLD-related genes. We treated separate cultures of human liver HepG2 cells with the CCDC80 and SOD3 recombinant proteins for 24 h (see Methods), and measured expression via RNA-seq. We then performed a differential expression (DE) analysis between the recombinant protein treated cells and non-treated control cells. (a) Results of the CCDC80 treatment DE analysis. The X-axis represents the log fold-change (logFC) of the 9 significant DE genes, and the Y-axis represents the gene names. Blue genes were expressed significantly more in the CCDC80 treated cells than in the control cells, and red genes were expressed less. (b) Mean expression of all 9 significant DE genes in the CCDC80 treated cells compared to the control cells. The X-axis represents counts per million (CPM), standardized by the mean and standard deviation of each gene; the Y-axis gene name; error bars the mean ± one standard deviation; and colours the experimental condition (CCDC80 treated HepG2 cells or non-treated control cells). Each row represents 4 samples treated with CCDC80 and 4 control samples, for a total of 8 samples per row. The “*” annotation indicates that the gene was significantly DE between the CCDC80 treatment and control samples (adjP < 0.05 after Benjamini–Hochberg correction (FDR < 0.05)). (c) Results of the SOD3 treatment DE analysis, with 2 significant DE genes. Plot elements are analogous to those in (a). (d) Mean expression of the 2 DE genes in the SOD3 treated cells compared to the control cells. Plot elements are analogous to those in (b).
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