Fig 2: CUL4B inhibits miR-143-3p expression mediated by FUS.A FUS protein level determined using western blotting after treating Huh7 and LM3 cells with MLN4924 at indicated concentrations for 24 h. B FUS protein level determined using western blotting after CUL4B knockdown in Huh7 and LM3 cells, with quantification normalized to β-actin loading control. C Half-life of FUS in MLN4924-treated Huh7 cells. Huh7 cells were treated with 50 μg/mL CHX along with DMSO or MLN4924 (1 μM) and then harvested for western blotting analysis. FUS protein expression was quantified using ImageQuant TL software, with levels normalized to that of β-actin for comparison (mean ± SD, n = 3). D Half-life of FUS in CUL4B-knockdown cells. Huh7 cells were transfected with control or CUL4B siRNA for 72 h and then treated with 50 μg/mL CHX at the indicated time points before being subjected to western blotting analysis. FUS protein expression was quantified using ImageQuant TL software, with levels normalized to that of β-actin for comparison (mean ± SD, n = 3). E Effect of CUL4B on FUS ubiquitination. Vectors encoding Flag-tagged FUS and HA–Ub were transfected into CUL4B-knockdown Huh7 cells for 48 h, as indicated. All cells were treated with 10 μM MG132 for 6 h before being subjected to immunoprecipitation with anti-Flag M2 affinity resin and western blotting analysis. F Level of miR-143-3p expression determined using qRT-PCR following FUS downregulation in Huh7 and LM3 cells by siRNA interference for 72 h (mean ± SD, n = 3). G Effect of FUS on miR-143-3p expression level in CUL4B-knockdown Huh7 and LM3 cells. CUL4B-knockdown Huh7 and LM3 cells were transfected with control or FUS siRNA for 72 h and then harvested for qRT-PCR (mean ± SD, n = 3). H Effect of FUS on the proliferation of Huh7 cells. Huh7 cells were transfected with control or FUS siRNA. Cell proliferation was measured using a CCK8 growth assay for 4 days (mean ± SD, n = 3). I Effect of FUS on the proliferation of CUL4B-knockdown Huh7 cells. CUL4B-knockdown Huh7 cells were transfected with control or FUS siRNA. Cell proliferation was measured using a CCK8 growth assay for 4 days (mean ± SD, n = 3). Two-tailed, unpaired t-test was used for (C, D) One-way ANOVA/LSD test was used for (F, H). Two-way ANOVA/LSD test was used for (G, I). Data are presented as the mean ± standard deviation derived from a minimum of three independent experiments. Statistical significance was assessed using the appropriate testing methods, with **p < 0.01, and ***p < 0.001 denoting levels of significance.

Fig 3: FUS is a ubiquitin substrate of CRL4BDTL E3 ligase.A Structural model of CRL4B E3 ligase. B FUS protein level in DDB1-knockdown cells. Huh7 and LM3 cells were transfected with control or DDB1 siRNA for 72 h and then harvested for western blotting analysis. C FUS protein level in RBX1-knockdown cells. Huh7 and LM3 cells were transfected with control or RBX1 siRNA for 72 h and then harvested for western blotting analysis. D FUS protein level in DCAFs-knockdown Huh7 cells. Huh7 cells were transfected with control or DCAF siRNAs for 72 h and then harvested for western blotting analysis. E Half-life of FUS in DTL-knockdown cells. Huh7 cells were transfected with control or DTL siRNA for 72 h and then treated with 50 μg/mL CHX at the indicated time points before being subjected to western blotting analysis. FUS protein expression was quantified using ImageQuant TL software, with levels normalized to that of β-actin for comparison (mean ± SD, n = 3). F The interaction among CUL4B, DTL, DDB1, RBX1, and FUS. Vectors encoding Flag-tagged CUL4B were transfected into Huh7 cells. Cells were then subjected to immunoprecipitation with anti-Flag M2 affinity resin and western blotting analysis. G, H Molecular docking model of CRL4BDTL and FUS based on AlphaFold3 protein prediction. I Root Mean Square Deviation (RMSD) of CRL4BDTL-FUS complex structure during 100 ns MD simulation. J Root Mean Square Fluctuation (RMSF) of CRL4BDTL-FUS complex residues during 100 ns MD simulation. K Radius of Gyration (RG) of CRL4BDTL-FUS complex structures during 100 ns MD simulation. Two-tailed, unpaired t-test was used for (E). Data are presented as the mean ± standard deviation derived from a minimum of three independent experiments. Statistical significance was assessed using the appropriate testing methods, with **p < 0.01 denoting levels of significance.

Fig 4: CUL4B/FUS/miR-143-3p axis regulates KRAS signaling.A TargetScan and miRDB databases predict KRAS as a potential target of miR-143-3p. B, C KRAS mRNA level determined using qRT-PCR after transfecting Huh7 and LM3 cells with mimic-miR-143-3p (B) or inhibitor-miR-143-3p (C) for 72 h (mean ± SD, n = 3). D KRAS protein level determined using western blotting after transfecting Huh7 and LM3 cells with mimic-miR-143-3p or inhibitor-miR-143-3p for 72 h. (E, F) KRAS mRNA and protein levels determined using qRT-PCR and western blotting, respectively, after CUL4B knockdown (mean ± SD, n = 3). G, H KRAS mRNA and protein levels determined using qRT-PCR and western blotting, respectively, after FUS knockdown (mean ± SD, n = 3). I Levels of phosphorylated AKT and phosphorylated ERK determined using western blotting after transfecting FUS-knockdown cells with miR-143-3p mimic for 72 h. J, K Levels of phosphorylated AKT and phosphorylated ERK determined using western blotting after transfecting CUL4B-knockdown cells with FUS siRNA or miR-143-3p inhibitor for 72 h. Two-tailed, unpaired t-test was used for (B−E). One-way ANOVA/LSD test was used for (G). Data are presented as the mean ± standard deviation derived from a minimum of three independent experiments. Statistical significance was assessed using the appropriate testing methods, with *p < 0.05, **p < 0.01, and ***p < 0.001 denoting levels of significance.