B5002 100mg

U87 and LN229 cells were grown in 24-well plate and cultured overnight. After treatment with either DMSO or the compound-1H for 48 h, cells were incubated with 10 μg /mL BrdU (Sigma, B5002-100MG, USA) for another 30 min, then fixed in 4% paraformaldehyde (PFA) for 15 min. Followed by the treatment with 1 mol/L HCl and the blockage with 10% goat serum, cells were respectively incubated with primary antibody against BrdU (1:2000, ab6326, Abcam, MA, USA) and Alexa FluorR^® 594 secondary antibody for 2 h. DAPI was used for nuclear staining. Finally, the BrdU signal was captured with the Olympus IX73 fluorescence microscope. And then at least 10 microscopic fields were used for the calculation of BrdU percentage.

In order to fluorescently label DNA, WI-38 and MDA-MB-231 cells were grown in DMEM medium containing BrdU (Sigma Chemicals USA, Catalogue No. B5002-100MG) at a final concentration of 10 μM for 24 h. Cells were washed ×3 with PBS, and the labeled HMW and sDNAs were isolated as described above. Bacteria, grown in Luria Broth were labeled with BrdU for 30 min at the above concentration of BrdU and labeled HMW and sDNA were extracted. Thirty minutes of labeling produced satisfactorily observable fluorescent signals under confocal microscope. Unlabeled DNA was not quantified. Since we did not grow plant cells in culture, DNA from plant was non-enzymatically labeled in vitro after HMW DNA extraction. We used Platinum Bright™ 550 Red/Orange Nucleic Acid Labelling Kit (Kreatech Diagnostics, The Netherlands. Catalogue No. GLK-004) in 50 μl reactions. Labelling was done as per manufacturer’s protocol followed by sonication.

For in vivo analysis of proliferation and migration, mice were pulse-labeled with BrdU (5-bromo-2-deoxyuridine) (Sigma, B5002-100MG) at a concentration of 50 mg/kg body weight that was applied i.p. to pregnant female mice on E14.5. Embryos were sacrificed two days later on E16.5.