Fig. 5

FBW7 negatively regulates the stability of IGF2BP2 protein by ubiquitinating IGF2BP2 in lung cancer cells. A A schematic diagram depicting FBW7 consensus motif in IGF2BP2 conserved region. B H1299 and A549 cells were harvested and immunoprecipitated with IgG, IGF2BP2, or FBW7 antibodies. C H1299 and A549 cells were transfected with EV or Myc-IGF2BP2 plasmids for 72 h. Cells were collected and immunoprecipitated with Myc antibody. D and E H1299 and A549 cells were infected with indicated lentivirus. After puromycin selection, cells were harvested for Western blotting and RT-qPCR analyses. n = 3, one-way ANOVA. Data are presented as Mean ± SD. F A549 cells were infected with indicated lentivirus. After puromycin selection, the corresponding groups were treated with MG132 for another 12 h. Cells were harvested for Western blotting analysis. n = 3, one-way ANOVA. Data are presented as Mean ± SD. G, A549 cells were infected with indicated lentivirus. After puromycin selection, cells were treated with Cycloheximide (CHX) and then collected at different time points for Western blotting analysis to detect IGF2BP2 levels. H A549 cells were transfected with indicated plasmids (Myc-IGF2BP2, Flag-FBW7, shFBW7, HA-Ub) for 48 h. Then cells were treated with MG132 for another 12 h. Cells were harvested for Western blotting analysis. I A549 cells were transfected with Flag-FBW7, plasmid expressing wild-type IGF2BP2 (Myc-IGF2BP2-WT), and plasmid expressing IGF2BP2 with Thr306 and Thr310 residues mutated to alanine (Myc-IGF2BP2-AA) for 72h. Cells were collected and immunoprecipitated with Flag antibody for Western blotting analysis. J A549 cells were transfected with indicated plasmids (Myc-IGF2BP2-WT, Myc-IGF2BP2-AA, Flag-FBW7, HA-Ub) for 48 h. Then cells were treated with MG132 for another 12 h. Cells were harvested for Western blotting analysis. K A549 cells were transfected with Myc-IGF2BP2-WT or Myc-IGF2BP2-AA for 72 h. Then cells were treated with Cycloheximide (CHX) and then collected at different time points for Western blotting analysis to detect IGF2BP2 levels. L-N The LUAD tissue microarray (n = 61) was stained with IGF2BP2 and FBW7, respectively. The representative images of IHC for IGF2BP2 and SLC7A5 protein were shown in panel (L). Scale bars, 500 μm for low power field, 50 μm for high power field. The heatmap showing IHC score of IGF2BP2 and SLC7A5 protein was shown in panel (M). The correlation analysis of IHC score among IGF2BP2 and SLC7A5 was shown in panel (N). ns, not significant, P > 0.05; **, P < 0.01; ***, P < 0.001. Abbreviations: BP2, IGF2BP2