Fig 2: COX5B modulates UHMK1 expression in hepatoma. (A) The heat map representation of the relative gene expression levels in hepatoma cells receiving control treatment (Ctrl) and knockdown of COX5B (KD). The average linkage method was used for hierarchical clustering. The distances between rows and columns were computed by Pearson method. (B) RT-qPCR and western blot were performed for validation of UHMK1 expression changes under depletion of COX5B. All the in vitro cell-based assays shown in (B) were conducted in duplicates. (C) IHC staining of paraffin-embedded tissues derived from Figure 2E. The expression relationships between COX5B and UHMK1 in (D) protein and (E) mRNA levels in samples derived from hepatoma patients. The p value was derived from paired two-tail student t-test. *, p < 0.05; **, p < 0.01; ***, p < 0.001. The western blot images in (B,D) were all acquired by exposing the luminescent signals to X-ray films. (F) The Kaplan-Meyer analysis of recurrence-free and metastasis-free survival in hepatoma patients stratified according to the T/N ratio of COX5B and UHMK1 mRNA levels.

Fig 3: The COX5B-UHMK1-ERK cascade regulates hepatoma cells proliferation and migration. (A) The western blot analysis of the phosphorylation status of ERK and Stathmin with or without the treatment of ERK inhibitor U0126 in hepatoma cells. The western blot images in (E) were all acquired by using chemStudio PULS imaging system. (B) The cell proliferation curves of hepatoma cells receiving the indicated treatments. The transwell-based assays to examine cell migration ability under the indicated treatments in (C) J7, (D) Huh7 and (E) Mahlavu cells (left panel). The quantitative results were shown in right panel. The p value was derived from paired two-tail student t-test. *, p < 0.05; **, p < 0.01; ***, p < 0.001. All the in vitro cell-based assays shown in this figure were conducted in duplicates. (F) The schematic depiction summarizing the key findings of this study. X and Y indicated the unknown factors involved in the regulatory events downstream of COX5B. The dotted lines indicated possible routes affected by COX5B, which remained to be characterized in the future.

Fig 4: COX5B-UHMK1 axis is crucial for regulating cell proliferation and migration in hepatoma cells. (A) The western blot analysis of UHMK1 in 3 independent hepatoma cell lines receiving the indicated treatments. The western blot images in this panel were acquired by exposing the luminescent signals to X-ray films, except for those of Mahlavu cell, which were obtained by using chemStudio PLUS imaging system. (B) The growth curves of hepatoma cells receiving the indicated treatments. (C) The transwell-based assays to evaluate cell migration capability under the indicated treatments. The quantitative results were shown in (D). The p value was derived from paired two-tail student t-test. *, p < 0.05; **, p < 0.01; ***, p < 0.001. (E) The western blot analysis of the potentially phosphorylated candidates downstream of UHMK1 in hepatoma cells. The western blot images in (E) were all acquired by using chemStudio PULS imaging system. All the in vitro cell-based assays shown in this figure were conducted in duplicates.