Fig. 5: TG2 sustains cell survival by forming a signalosome platform and hyperactivating the PI3K p-AKT S473 and p-mTORC2 signalling axes.

A NB4 WT and TG2-KO cells (1 × 10⁷) were intravenously injected via the suborbital vein into NOD/SCID female mice, followed by intraperitoneal injection of ATRA 1.0 mg/kg + DEX at 50 µg, ATO at 0.75 mg/kg, or ATRA + ATO every two days. At day 14, blood samples were collected from the heart and analysed for total numbers of NB4 WT and TG2-KO cells by FACS and human-ALU-based RT-qPCR analyses. Graphs show the mean ± SD values after 14 days (n = 4). Statistical analysis was conducted by two-way ANOVA (Bonferroni post hoc test; *p < 0.05, **p < 0.01 and ***p < 0.001, ****p < 0.0001). B Representative western blot showing expression of TG2, p-AKT (S473, T308), AKT, p-mTOR (2481, 2448), mTOR, pro-caspase3/truncated forms, and GAPDH proteins in the total cell lysate or in a cell population sorted by Annexin-V labelling (n = 7). C Representative IP-western blot showing plasma membrane CD18 and TG2 protein expression levels following ATRA treatment of NB4 WT cells (n = 5). D Representative co-immunoprecipitation-Western-blot showing plasma membrane and total cell lysate TG2, CD18, SRC, p-mTOR (2481, 2448), p-AKT (S473, T308), p85, p-p85, p110, PTEN, and p-PTEN protein expression levels in NB4 WT cells following upon ATRA, ATRA + NC9 (30 μM), or ATRA + PP2 (20 μM) treatment (n = 3). E TG2-dependent signalosome formation and its inhibition by NC9 or PP2. F Interaction map of the bait and prey proteins in plasma membrane fractions of differentiated NB4 WT cells detected by modified far western blot (black dots) and by LC-MS/MS method (orange dots). Representative co-immunoprecipitation-western-blot showing plasma membrane containing TG2, mTOR, p-mTOR (2481, 2448), AKT, p-AKT (S473, T308), p85, p110, PTEN, CD18, SRC protein expression levels in NB4 WT cells following upon ATRA treatment for 5 days (n = 3). G Interaction map of the bait and prey proteins in plasma membrane fractions of differentiated NB4 WT CD18^−/− cells detected by modified far western blot. The left panel’s representative western blots show the expression of the detected prey proteins with TG2 antibody in the absence of CD18. In contrast, the right panel’s western blot result shows the same PVDF membrane, developed with CD18 antibody (n = 3).