Fig 1: COX5B acts as a potent growth-promoting gene in hepatoma. (A) The GSE63898 and GSE76427 datasets were employed to analyze the expression levels of various cytochrome c oxidase genes in hepatoma, tumorous versus nontumorous parts. T, tumorous part. N, nontumorous part. ***, p < 0.001; **, p < 0.01. (B) The prognosis of hepatoma patients included in GSE76427 dataset was analyzed by Kaplan-Meyer method with the mean of mRNA levels in the tumorous parts as cutoff. (C) The representative IHC sections in patient with higher COX5B intensity in the tumorous tissues (tumor > nontumor, left upper panel) or higher in nontumorous tissues (tumor ? nontumor, left lower panel) in hepatoma. The black scale bar represented 100 µm. The Kaplan-Meyer analysis of the prognosis of hepatoma patients stratified according to the intensity of the COX5B IHC staining (right three panels, overall survival, disease-free survival and metastasis-free survival). The (D) mRNA and (E) protein levels of COX5B in tumorous and nontumorous parts of hepatoma retrieved from our tissue bank. The images in this panel were all acquired by exposing the luminescent signals to X-ray films. The p value was derived by paired two-tail student t-test. T, tumorous part. N, nontumorous part. (F) The Kaplan-Meyer analysis for prognosis of hepatoma patients divided by the mean of the tumorous/nontumorous ratios of COX5B mRNA (left two) and protein (right two) levels.
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: COX5B promotes hepatoma cell growth and migration. Assessment of growth curves in 4 independent hepatoma cell lines was conducted under (A) depletion or (B) over-expression of COX5B. The western blot images in (A,B) were all acquired by exposing the luminescent signals to X-ray films. The transwell assays were performed when (C) depletion or (D) over-expression of COX5B. (E) Representative images showing the xenografted tumors 3–6 weeks after implantation of the cells receiving control treatment (shLacZ) or knockdown of COX5B (shCOX5B) in SK-Hep1 or J7 cells. The tumors were weighted and statistically compared (right panel). All the in vitro cell-based assays shown in (A–D) were conducted in duplicates. (F) The growth curve of the tumor volume over time was statistically compared. The p value was derived from paired two-tail student t-test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Fig 4: Knockdown of COX5B suppresses cell proliferation through induction of cell senescence but not programmed cell death. This figure was used to supplement Figure 2 in the main text. (A) The beta-galactosidase staining assay to examine the status of cell senescence. The black Scale bar represented 50 µm. The quantitative results were shown in (B). ***, p < 0.001. (C) The western blot analysis of cell senescence marker p21 levels in HCC cells with or without depletion of COX5B. The western blot images in this panel were all acquired by using chemStudio PULS imaging system. (D) The TUNEL assay to determine the status of cell apoptosis. The white scale bar represented 50 µm. The quantitative results were shown in (E). All the in vitro cell-based assays shown in this figure were conducted in duplicates.
Fig 5: Loss-of-function in COX5B leads to dysfunction of mitochondria and COX. The function of mitochondria was assessed by measuring (A) ROS, (B) ATP and (C) AMP levels with or without COX5B depletion in hepatoma cells. (D) The COX activity to oxidize cytochrome c was measured using mitochondria solution isolated from indicated hepatoma cells with or without COX5B silencing. The p value was derived from paired student t-test (two tails). *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Supplier Page from Abcam for Anti-COX5B antibody [EPR14439(B)]