Finally, the fluorescence intensity was quantified by ImageJ software. 2.9. cultured in Medium 199 (M199) (Gibco) supplemented with 10% fetal bovine serum (HyClone) at 37 C in a humidified incubator with 5% CO2. In the process of the whole experiment, the cell’s density was around 90% before doing all experiment, dissolved plump cells with 0.25% or 0.05% trypsin (Sangon Biotech) in phosphate buffered saline (PBS), the decentralized cells were seeded onto 6-well plates, 12-well plates, 24-well plates, 96-well plates or other appropriate dishes at a density of 20?000 cells per ml pre-incubated 12 h before adding the compound PBQC. HeLa cells and A549 cells were purchased from your ATCC (American Type Culture Collection), U87 cells were provided by Bing Yan (Institute of Analytic Chemistry, School of Chemistry and Chemical Engineering, Shandong University or college, Jinan, China), and luciferase HeLa cells were provided by Qing Ye and Fan Jiang (The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Health, Qilu Hospital, Shandong University or college, Jinan, China). Human umbilical vein Diazepinomicin endothelial cells (HUVEC) were provided by People’s Liberation Army 456 Hospital, Jinan. 2.3. Luciferase assay Luciferase HeLa cells that can stably express an Nrf2-responsive reporter plasmid (pGL4-3 ARE) were seeded into 96-well plates at the density of 40?000 cells per ml and consistently cultured for 12 h. About twelve hours, we respectively incubated luciferase HeLa cells with PBQC (concentration, 0, 1, 5 and 10 M) for 6, 12, 24 and 48 h. And then we added and collected the reporter gene cell lysis answer (RG-005-1), examined the luciferase activity by Lucifer Reporter Gene Assay Kit (RG005, Beyotime, Shanghai). Finally, the luciferase activity was normalized to cell viability which assessed by SRB assay.33 2.4. Cell morphological observation HeLa cells, A549 cells, U87 cells and HUVECs were seeded into 24-well plates and treated with 0.1% DMSO as a vehicle control or 1, 5 and 10 M PBQC for 6, 12, 24 and 48 h, taken the microscopic photographs (200) under the inverted phase contrast microscope (Nikon, Japan). 2.5. Cell viability assay (SRB) According to the relevant reports, HeLa cells, A549 cells, U87 cells and HUVECs were seeded into 96-well plates. After 12 h, cells were respectively treated with 0.1% DMSO as a relevant control or compound PBQC (concentration, 1, 5 and 10 M) for specified time durations. And then cell viability was evaluated by sulforhodamine B (SRB) assay, according to the method of Skehan. In short, discarded Diazepinomicin the original medium and added 100 L of chilly 10% trichloroacetic acid (TCA) into the 96-well plates to fix cells and incubated for 1 h at 4 C. Poured off the supernatant and washed Diazepinomicin the cell five occasions with ultrapure water. After drying the 96-well plates at room heat, added 50 L of 0.4% (w/v) SRB answer in 1% acetic acid into each hole and shook 10 min on microtiter Diazepinomicin plate shaker. And then washed the 96-well plates five occasions with 1% acetic acid and subsequently added 100 L of 10 mM unbuffered Tris base (pH = 10.5) to dissolve the original dye after again dried the 96-well plates at room heat. Shook 10 min on microtiter plate shaker, and finally measured the light absorption at the wavelength of 540 nm used a SpectraMAX190 microplate spectrophotometer (GMI Co, USA). 2.6. Co-immunoprecipitation HeLa cells were lysed in IP buffer (Beyotime, P0013). And the main components of IP buffer (without inhibitor) are 20 mM Rabbit Polyclonal to OR10A4 Tris (pH 7.5), 150 mM NaCl, 1% Triton X-100, without Diazepinomicin protease, phosphatase and other inhibitors. The lysates were pre-cleared with protein A/G agarose beads (Beyotime, P2012) for 1 h at 4 C. After centrifugation (3000 rpm), the supernatant was collected and incubated with 10 L specific antibodies or normal corresponding IgG, then with protein A/G beads overnight at 4 C. The beads were rinsed with IP buffer 3 times and eluted with 2 SDS loading buffer. The immunoprecipitated proteins were detected by western blot assay.34 2.7. Immunofluorescence microscopy For the next immunofluorescence assay, we seeded HeLa cells (20?000 cells per ml) into confocal dishes (20 mm) (SPL, Korea) pre-incubated 12 h and used PBQC to treat cell. After treatment, discarded the culture medium and rinsed the cells softly with 1 PBS, then the cells were fixed in 4% paraformaldehyde for 15 min, after washing three times with 1 PBS, permeated cells with 0.1C0.2% Triton X-100 for 5 min, then washed and blocked with donkey serum (1?:?30 dilution in 0.1 M PBS) for 20 min at room temperature. Discarded the enclosed liquid, incubated cells.