4 mug

Senescence-associated β-galactosidase activity was quantified in cell extracts as previously described [90 (link)]. Briefly, MCF7 cells were seeded into 6 cm plates at 0.5×10⁵ cells/plate and grown in media supplemented with indicated combinations of nutlin-3 (1 μM), GSK2830371 (0.5 μM) and doxorubicin (0.05 μM) for 7 days. Cells were washed in PBS, collected to ice cold lysis buffer (5 mM CHAPS, 40 mM citric acid, 40 mM sodium phosphate, 0.5 μM benzamidine and 0.25 mM PMSF, pH 6.0), vortexed and centrifuged for 5 min at 12,000g. Cell extract was mixed 1:1 with 2x reaction buffer supplemented with 4-MUG (1.7 mM, Sigma) and MgCl2 (4 mM) and incubated at 37°C for 0.5 – 4 hours. Reaction was stopped by addition of sodium carbonate (400 mM) and fluorescence signal was measured at excitation wavelength 360 nm and emission wavelength 465 nm using EnVision plate reader. β-galactosidase activity was determined as the rate of 4-MUG conversion to the fluorescent 4-MU and normalized to the protein concentration measured by BCA assay. Alternatively, cells were grown on coverslips, fixed by 0.2 % glutaraldehyde 7 days after treatment with indicated combinations of nutlin-3 (1 μM), GSK2830371 (0.5 μM) and doxorubicin (0.05 μM) and β-galactosidase activity was determined by colorimetric staining as previously [73 (link)].

Restriction enzymes, DNA polymerases and T4 DNA ligase were purchased from New England Biolabs (Beijing, China). Oligonucleotides was synthesized by Life Technologies (Shanghai, China). 4-MU and 4-MUG were purchased from Sigma-Aldrich (St. Louis, USA). (+)-Catechin, daidzein, genistein, genistin and silybin were purchased from Aladdin (Shanghai, China).
All E. coli strains were routinely grown in Luria-Bertani (LB) medium at 37 ^oC. The antibiotics ampicillin (100 μg/mL) and kanamycin (50 μg/mL) were supplemented when necessary. The genomic DNA of S. mutans UA159 was kindly provided by Prof. Xiuzhu Dong from Institute of Microbiology, Chinese Academy of Sciences.

GUS staining of Pro_MdbHLH130::GUS transgenic apple calli and the WT was performed according to Jefferson et al. (1987) (link). The apple calli were transferred to a filter paper and dried at 23°C for 4 h and then immersed into GUS staining buffer at 37°C in the dark overnight. After staining, the samples were de-stained and photographed.
GUS activity was detected as described previously (Jefferson et al., 1987 (link)). The total proteins from the samples were extracted with extraction buffer and reacted with 4-MUG (Sigma-Aldrich, St. Louis, MO, USA) at 37°C. The GUS activity was determined using a VersaFlour spectrofluorometer (Bio Rad Laboratory Inc., Hercules, CA) at an Ex = 365 nm and Em = 450 nm.

Embryos were collected overnight (16 hours), dechorionated in 3% hypochlorite solution for 2 minutes, washed thoroughly with water, and frozen in liquid nitrogen. 20 wandering third instar larvae and six adult flies (3 days post eclosion, 3 males and 3 females), respectively, were collected per replicate and frozen in liquid nitrogen. Frozen samples were homogenized on ice in assay buffer (1 mM MgSO₄, 2% Triton X-100 (v/v), 100 mM Hepes pH 8) supplemented with cOmplete™, EDTA-free Protease Inhibitor Cocktail (Roche). The pH 8 of assay buffer and Hepes were used to select for LacZ protein activity compared to endogenous β-galactosidase^{52 (link), 53 (link)}. Homogenates were spun twice at 15′000 rpm for 10 minutes at 4 °C, each time discarding debris and retaining supernatants. Homogenate protein concentrations were measured using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). 1 µg of protein from homogenates was incubated at 37 °C for 50 minutes in 0.1 ml of assay buffer supplemented with 4-MUG (Sigma, M1633) at 0.9 mM final concentration. ß-galactosidase converts non-fluorescent substrate 4-MUG into 4-MU, a fluorescent product. 4-MU fluorescence was measured using EnVision Multilabel Reader 2104 (PerkinElmer).