Fig 1: CCAAT/enhancer-binding protein delta (CEBPD) overexpression confers mTORC1-driven metabolic reprogramming and leads to glucose addiction. The potential relationship between CEBPD and proteins associated with the mTOR pathway was explored by immunoblotting analysis. (A) pMAPK3/1, pPI3K, pAKT1, pMTOR, pRPS6, pEIF4EBP1 and SKP2 were significantly upregulated, while cleaved CASP3 was notably decreased in CEBPD-overexpressing BFTC909 and TCCSUP cells treated with culture medium containing glucose compared to mock-expressing cells. (B) 2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl) 2H-tetrazolium-5-carboxanilide (XTT) assay indicated that cell viability was notably inhibited in mock-expressing BFTC909 and TCCSUP cells under glucose starvation (culture medium without glucose) for 72 h compared to mock-expressing cells in a complete cell culture medium. Furthermore, CEBPD overexpression further exacerbated glucose withdrawal-induced cell viability suppression in these two cell lines. (C) Immunoblotting data indicated that glucose deficiency suppresses the effect of CEBPD on the upregulation of pAKT1, pMTOR, pRPS6, pEIF4EBP1 and SKP2 protein and the downregulation of CASP3 in these two distinct UC-derived cells. However, the protein levels of pMAPK3/1 and pPI3K were not comparable in CEBPD-overexpressing BFTC909 and TCCSUP cells under glucose starvation conditions. Glucose uptake assay (D), lactate level analysis (E), Seahorse XFp Analyzer assessment (F–G), OCR, and MitoDsRed-tagged fluorescent mitochondrial staining (H) were performed to evaluate the cellular glucose uptake, lactate production, oxygen consumption rate (OCR), extracellular acidification rate (ECAR) and mitochondrial status. The data showed that stable overexpression of CEBPD notably increased glucose uptake (D), lactate production (E), the ECAR (F) and mitochondrial fragmentation/fission (H) but decreased the OCR (G) in BFTC909 and TCCSUP cells compared to mock-expressing cells. To clarify the metabolic switch from mitochondrial oxidative phosphorylation (OXPHOS) to glycolysis under CEBPD regulation, the relationship between CEBPD and hexokinase 2 (HK2) was estimated through analysis of the Gene Expression Omnibus (GEO) database (GSE13507 dataset, n = 188), indicating a significantly positive correlation between the mRNA levels of CEBPD and HK2 in bladder cancer specimens (I). The result was validated by quantitative reverse transcription-polymerase chain reaction (RT-PCR) (J), promoter activity assay (K), and immunoblotting (L) and indicated that the transcript (J) and protein (L) levels of HK2 were markedly upregulated in CEBPD-overexpressing BFTC909 and TCCSUP cells versus mock-transfected cells via the upregulation of HK2 promoter activity (K). Additionally, CEBPD overexpression increased the protein level of SLC2A1 but not LDH A/C (L). All experiments were performed in triplicate and data was represented as the mean ± SE. For immunoblot assay and fluorescent image data, one representative image is displayed. GAPDH was served as a loading control for immunoblot assay. Statistical significance: * p < .05
Supplier Page from Abcam for Anti-SKP2 antibody [EPR3306(2)]