Fig. 1

Analysis of TRIB3's role in RMS. Expression data obtained from patients' datasets on the R2 platform were used. The data are presented as the mean ± 95% confidence interval. a TRIB3 was found to be overexpressed in RMS datasets compared to muscle healthy counterpart. One-way ANOVA was performed, and Tukey's multiple comparisons test was used as the post hoc test. Significance versus Hofman is indicated by †, versus Asmann by #, and versus Gordon by*. b Tumors harboring PAX3-FOXO1 or PAX7-FOXO1 fusion gene displayed higher TRIB3 expression levels. One-way ANOVA with Dunnett's post hoc test was used to determine significance. c Proliferation was assessed using the inducible model and the crystal violet assay at different IPTG concentrations. A two-way ANOVA followed by Dunnett's multiple comparisons test was performed to compare each IPTG concentration with the non-induced condition at each time point. d Apoptosis was analyzed by determining apoptotic markers through WB. e Analysis of fusion protein and its phosphorylation status by WB, along with MYCN and myogenin proteins as fusion protein target genes, confirmed that PAX3-FOXO1 function is affected after TRIB3 knockdown. Akt signaling pathway inhibition after TRIB3 knockdown was also analyzed by WB. Phosphorylation status of Akt and its main downstream effectors (PRAS40 and rpS6) was assessed. f Immunoprecipitations immobilizing TRIB3 (left panel) or Akt (right panel were carried out using cell lysates from RH4 and RH30 cells. Negative control was included (beads only and no antibody), and isotype-matched IgG was used as a control. FP: fusion protein. P: phosphorylated. IP: immunoprecipitation