![]() ![]() ![]() Extracts were centrifuged at 4,000 × g for 15 min at 4☌. Chloroform (2.89 ml at −20☌) was added to each tube and vortexed for 10 min at 4☌. After adding 1.74 ml of ice water, the cells were scraped with a cell lifter and collected in 15-ml conical tubes. The saline was aspirated, and cells were quenched with 2.89 ml of −20☌ HPLC-grade methanol. After incubation, the medium was aspirated and each plate was rinsed with 10 ml ice-cold saline. The membrane signal was subsequently analyzed by the LiCOR Odyssey system (LiCOR Biosciences, Frankfurt, Germany).įor carbon tracing through central carbon metabolism, cells were cultured for approximately 24 h in 10-cm plates in glucose- and glutamine-free DMEM (Sigma D5030, Sigma-Aldrich) containing 10% dialyzed FBS, 1% streptomycin/penicillin, naturally labeled 4 mM glutamine or 25 mM glucose, and the appropriate tracer, glucose or glutamine (Cambridge Isotopes Laboratories, Inc., Cambridge, MA, USA). Membranes were then washed with TBS-T, incubated with the appropriate secondary antibody for 1 h at room temperature and then washed with TBS-T. Antibodies included E-cadherin (BD Biosciences, San Jose, CA, USA), β-actin (Abcam, Cambridge, MA, USA), Complex II Western Blot Antibody Cocktail that detects SDHB (30 kDa) and Complex Va (60 kDa) (Abcam, Burlungame, CA, USA), Methyl-Histone H3 Antibody Sampler Kit (Cell Signaling, Danvers, MA, USA), and SDHB (Santa Cruz Biotechnology, Dallas, TX, USA). The membrane was then blocked in Odyssey blocking buffer (LiCOR, Lincoln, NE, USA) for 30 min and incubated with the appropriate antibody overnight at 4☌. ![]() Semi-dry protein transfer to a PVDF nitrocellulose membrane (BioRad) was performed using the Transblot Turbo transfer system (BioRad). Fifty micrograms of lysate was loaded onto a gradient gel (BioRad, Hercules, CA, USA) and subjected to gel electrophoresis. Protein concentrations were quantified by the BCA protein assay (Thermo Scientific, Rockford, IL, USA). This has led to the definition of SDH as a mitochondrial tumor suppressor and a key player in cancer cell differentiation.Ĭell lysates were prepared using RIPA lysis buffer (Sigma-Aldrich) containing a protease inhibitor cocktail (Roche, Basel, Switzerland). Furthermore, the SDH regulators SDHAF1 and SIRT3 have been identified as tumor suppressor genes in lung and breast cancer, respectively. Interestingly, it has been shown that SDHx mutations are associated with hallmarks of EMT in PCC and PGL as a result of alterations to the epigenome. SDHx loss-of-function mutations have been found to predispose individuals to hereditary pheochromocytoma (PCC), paraganglioma (PGL), gastrointestinal stromal tumor (GIST), and renal cell carcinoma. SDH assembly requires two factors, SDHAF1 and SDHAF2, while its function is influenced by the deacetylase activity of SIRT3. SDH is a holoenzyme consisting of four essential subunits: SDHA (a flavoprotein), SDHB (iron-sulfur protein), and two membrane anchor units SDHC and SDHD. In the TCA cycle, SDH catalyzes the oxidation reaction where succinate is converted to fumarate, which is coupled to the reduction of ubiquinone to ubiquinol in the ETC. Also known as mitochondrial respiratory Complex II, SDH is the only enzyme to participate in both the TCA cycle and electron transport chain (ETC.). For example, mutations in the mitochondrial enzyme succinate dehydrogenase (SDH) have been reported in a wide variety of cancers. However, mitochondrial metabolism is also important for cancer proliferation. Alterations in metabolism have been implicated in cancer, with the main focus on the Warburg effect, a phenomenon in which cancer cells upregulate glycolysis and lactate production while decreasing glucose contribution to the citric acid (TCA) cycle in the mitochondria, even in the presence of sufficient oxygen. ![]()
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |