Fig. 3

MAGI3 regulates c-Myc activation through degradation of c-Myc by the ubiquitin pathway. a, Enrichment plots of GSEA showed that the gene signatures of c-Myc targets were significantly enriched in low MAGI3 expression CRC specimens. b, MAGI3 inhibited c-Myc transcriptional target genes expression. Heatmap of c-Myc target genes expression modulated by MAGI3 in RKO or HT29 cells (three independent experiments). c, Overexpression of MAGI3 decreased, whereas knockdown of MAGI3 increased the protein levels of c-Myc. c-Myc protein levels were detected by western blot in HT115 or RKO cells over-expressed MAGI3 (left), and in HT29 or SW480 cells silenced MAGI3 (right). d, MAGI3 had no detectable effect on the c-Myc expression at mRNA level. qPCR analysis of the c-Myc mRNA expression in RKO or HT29 cells with the indicated MAGI3 modulation, three independent experiments, data were presented as the mean ± SEM, a 2-tailed, unpaired t test was used to determine statistical significance. ***P < 0.001, NS, no significance. e, MAGI3 and c-Myc had no correlation (by Pearson’s) at transcriptional level in CRC specimens from TCGA (left, n = 375) and GSE40967 (right, n = 566). f and g, Overexpression of MAGI3 decreased, whereas knockdown of MAGI3 prolonged the half-life of c-Myc. The effect of MAGI3 on the half-life of c-Myc were detected by western blot in CRC cells treated with CHX. The protein half-life curves were obtained by quantifying three independent experiments (right). h, MAGI3 regulated c-Myc turn over through proteasome pathway. The levels of c-Myc protein were detected by western blot in CRC cells transfected with MAGI3 and treated with MG132 (10 μM) for 6 h before harvesting. i and j, Overexpression of MAGI3 promoted, whereas knockdown of MAGI3 inhibited c-Myc ubiquitin. Ubiquitination assays of c-Myc in lysates from RKO or HT29 cells with the indicated MAGI3 manipulation. k, MAGI3 regulated c-Myc ubiquitin in Lys48-linked poly-ubiquitination. Ubiquitination assays of c-Myc in RKO cells transfected with HA-Ub or HA-Ub/K48R