Fig 2: Inverse correlation between Fbw7 and LDHA/lactate/miR-223 axis in ABC DLBCL. (A, B) the effects of lactate induction after miR-223 inhibitor was treated in SU-DHL-2 and OCI-LY-10 cells. The expressions of Fbw7, LDHA, pY10-LDHA were examined by western blotting. (C, D) His-LDHA and HA-ubiquitin were treated with miR-223 inhibitor in SU-DHL-2 and OCI-LY-10 cells. After treated with 10 mM MG132 for 6 hours, LDHA was co-immunoprecipitated with anti-His antibody, and the polyubiquitination status of LDHA was examined by immunoblotting. (E) schematic graph of potential mechanism of Fbw7 negatively regulates the positive feedback loop of the LDHA/lactate/miR-223 in ABC DLBCL.

Fig 3: Lactate decreases Fbw7 expression in ABC-DLBCL. (A, B) lactate was added into culture medium of SUDHL-2 and OCI-LY-10 cells in a range of concentrations and Fbw7, LDHA, and pY10-LDHA levels were determined by western blotting analysis. (C) lactate promotes intracellular LDH in SUDHL-2 and OCI-LY-10 cells. The mean ± SD is shown for five independent experiments. **, P <0.01; one-way ANOVA. (D) lactate promotes ATP production SUDHL-2 and OCI-LY-10 cells. The mean ± SD is shown for five independent experiments. **, P <0.01; one-way ANOVA. (E, F) western blotting shows the expression of LDHA and pY10-LDHA after transfection of Fbw7 and then addition of lactate.

Fig 4: Fbw7 impaired LDHA-mediated glucose metabolism reprogramming in ABC DLBCL. (A) QRT-PCR analysis of the relative mRNA expression in OCI-LY-3 and SU-DHL-2 cell lines. Cells were transfected with Flag-Fbw7 and vector control to detect Fbw7 expression. The mean ± SD is shown for three independent experiments. ***P < 0.001; one-way ANOVA. (B) Fbw7 inhibited intracellular LDH in ABC-DLBCL cells. The mean ± SD is shown for five independent experiments. ***P < 0.001; one-way ANOVA. (C) Fbw7 reduced intracellular lactate production in ABC-DLBCL cells. Results are presented as mean ± SD. ***P < 0.001; one-way ANOVA. (D) Fbw7 reduced ATP production. Results are presented as mean ± SD. ***P < 0.001; one-way ANOVA. (E) Western blotting results showed that overexpression of Fbw7 mainly inhibited LDHA expression in Glycolysis relative proteins. And the expression of Fbw7, LDHA, HK2, GLUT1, PDK1 were shown. (F) IHC staining results of Fbw7 and LDHA were shown with representative images from the same tumor case. The scale bar of “100x” indicates for 100μmand the scale bar of “200x” indicates for 50μm. (G) spearman correlation analysis between Fbw7 and LDHA in DLBCL FFPE tissues. Spearman rank correlation. P < 0.05 was considered to indicate a significant difference.

Fig 5: Fbw7 interacts with LDHA and facilitates its ubiquitination and degradation. (A) Western blotting analysis SUDHL-2 and OCI-LY-3 cells transfected with Flag-Fbw7 to detect the expression of LDHA and pY10-LDHA. (B) SUDHL-2 cells were transfected with Flag-Fbw7 and treated with cycloheximide (CHX; 10 mg/ml) for the indicated time intervals to examine the stability of LDHA. Western blotting results were shown(top) and the mean ratios of the indicated proteins was quantified through densitometry in line graph(bottom). Results are displayed as mean ± SD in the line graph for three independent experiments using; **P < 0.01; ***P < 0.001; one-way ANOVA. (C) the similar results were shown in OCI-LY-10 cells. Results are displayed as mean ± SD in the line graph for three independent experiments using; **P < 0.01; ***P < 0.001; one-way ANOVA. (D, E). interaction between endogenous Fbw7 and LDHA in indicated cell lines. Cell lysates from SU-DHL-2 and OCI-LY-10 cells were co-immunoprecipitated with anti-Fbw7 or anti-LDHA antibody and the original results were analyzed by western blotting. IgG was used as a control. (F, G) SU-DHL-2 and OCI-LY-10 cells were transfected with the indicated plasmids. Cells were treated with 10 mM MG132 for 6 hours after transfection, the Fbw7-mediated polyubiquitination status of LDHA was examined by immunoblotting.