Fig. 2: Generation and multi-omics characterization of TG2 knock-out melanoma B16F10 cells.

a Schematic representation of the employed strategy. B16F10 TG2 KO clones were generated by means of the CRISPR/Cas9 genomic editing tool. After obtaining the clones, they were subjected along with the B16F10 WT cell line to Proteomics profiling and RNA-seq analyses. b Immunoblot analyses showing the obtained TG2 KO clones, namely TG2 KO 1 and TG2 KO 2. Actin was used as loading control. c TGM2 expression evaluated by qRT-PCR analysis in B16F10 WT cells and TG2 KO clones (number of independent biological replicates = 8). Statistical significance was calculated with One-Way ANOVA and specified with asterisks (^****p < 0.0001). Data are represented as mean ± SEM. d Venn diagrams showing the differentially expressed proteins from comparative Proteomic analyses of the TG2 KO clones. Comparisons were divided in Up and Down-regulated Proteomics targets (hits and candidate proteins). Areas of overlap indicate shared protein targets. Statistically significant targets were defined based on adj.p < 0.05 (adjusted p < 0.05). Proteins were annotated as “hits” with FDR < 5% and a fold change of at least 100% and as “candidates” with FDR < 20% and a fold-change of at least 50%. e Bar plot representative of the GO enrichment analyses of the top 11 downregulated Biological Processes (BPs). Bar color represents the adj.p-value (dark blue= most significant). Bar lengths refer to the proportion of enriched proteins for each term. f Heat map of comparative proteomic analysis of the melanogenesis related proteins was generated using pheatmap R package. g Bar plot representative of the GO enrichment analyses of the top 15 upregulated Biological Processes (BPs). Bar color represents the adj.p-value (dark blue= most significant). Bar lengths refer to the proportion of enriched proteins for each term. h Heat map of comparative proteomic analysis of the migration and adhesion proteins was generated using pheatmap R package. Dot plots representative of down (i) and up (l) regulated GO of Biological Processes analyses performed on significantly differentially expressed genes (DEGs) obtained from RNAseq profiling of the TG2 KO 2 clone (FDR < 0.01). Bubble colors represent the adj.p-value (red=most significant). The rich factor refers to the proportion of enriched genes for each term.