TG2 influences the expression of ATRA-regulated genes in differentiation of NB4 cell. (A) Genes (830) regulated by ATRA on day 3 belonging in the 4 most affected biological process categories (Table 1) were further investigated in the respect of TG2 effect. The Venn diagram illustrates the number of genes regulated by ATRA (830), the genes influenced by TG2 (313), and the number of the common part of these 2 gene sets (197). (B). The ratios of normalized transcript levels of 3-day ATRA-treated control NB4 cells versus untreated control NB4 cells were calculated in case of selected genes (effect of differentiation). Similarly, the ratios of normalized transcript levels of 3-day ATRA-treated TG2-KD NB4 cells versus 3-day ATRA-treated control NB4 cells were determined (effect of TG2 knockdown). The 2 ratios of transcript levels were plotted against each other resulting in a scatter plot that shows the relationship of transcriptional changes caused by the differentiation process and the TG2 knockdown effect. Down-regulation of 59 genes is behind (cluster 1) and the expression of 8 genes (cluster 4) is suppressed at higher degree than it occurs in ATRA-treated NB4 cells. Whereas 1 gene is more up-regulated by ATRA in TG2-KD cells than normally expected (cluster 3), 132 genes are less induced in TG2-KD NB4 cells (cluster 6) as compared with the control. TG2-influenced genes regulated by ATRA are highlighted, and genes that belong to a certain cluster are indicated. (C) Gene sets regulated by TG2 during ATRA-induced differentiation of NB4 cells. The heat map represents the differentially expressed genes in control and TG2-KD NB4 cells upon ATRA-treatment at undifferentiated, 2-day, and 3-day differentiated stages. Cluster 1 contains the genes (59 genes) that are down-regulated by ATRA in normal case, but remained significantly at higher level in TG2-KD NB4 cells. Cluster 2 (48 genes) and cluster 5 (69 genes) contain those genes whose expression change did not reach the 2-fold threshold upon ATRA-treatment, but were up-regulated or down-regulated in TG2-KD cells, respectively. In cluster 3 (1 gene) and cluster 4 (8 genes) the suppressed TG2 expression further increased the up- or down-regulating effect of ATRA, respectively. Cluster 6 represents the genes (132 genes) significantly induced by ATRA, but showing lower expression in TG2-KD NB4 cells. Heat maps were generated by GeneSpring 10.1 (Agilent) using hierarchical clustering algorithm in all cases of gene lists. Fold change differences are indicated by colors.