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precipitation of the buffer. The slides were then removed from and Jiangsu Province Funds for Distinguished Young Scientists
the lysing solution, drained and placed in a horizontal gel (BK20160033). Part of the work was supported by the Priority
electrophoresis tank side by side, avoiding spaces and with the Academic Program Development of Jiangsu Higher Education
agarose end facing the anode (8–21 slides can be accommo- Institutions (PAPD), Program for New Century Excellent Talents
dated depending upon the size of the tank). The tank was lled in University (NCET-13-1033), College Students Innovation
with fresh electrophoresis buffer (1 mM Na2EDTA and 300 mM Project for the R & D of Novel Drugs (J1030830), and Jiangsu
NaOH) to a level approximately 0.25 cm above the slides. The Shuang Chuang team.
slides were le in the high pH buffer for 20 min to allow
unwinding of the DNA before electrophoresis. Electrophores
was conducted at room temperature for 20 min at 25 V, adjusted
to 300 mA by raising or lowering the buffer level in the tank.
Notes and references
Aer electrophoresis the slides were washed gently to remove
alkali and detergents which would interfere with ethidium
bromide staining, by placing them on a tray and ooding them
slowly with 3* changes of 0.4 M Tris, pH 7.5, each for 5 rain.
Aer neutralization the slides were each stained with 2 mg
mLꢀ1 ethidium bromide in distilled water and covered with
a coverslip. All the steps described were conducted under yellow
light, red light or in the dark to prevent additional DNA damage.
Analysis of slides. For ethidium bromide staining observa-
tions were made using an epi-uorescence microscope equip-
ped with an excitation lter of 515–560 nm from a 100 W
mercury lamp and a barrier lter of 590 nm. Photomicrographs
of single ceils can be taken at 400ꢃ magnication using for
example Kodak T-Max 400ASA black and white 35 mm lm or
alternatively measurements can be made using a calibrated
scale in the ocular of the microscope. Samples were viewed as
soon as possible following staining though preparations can be
1 E. Culotta and D. E. Koshland Jr, Science, 1992, 258, 1862–
1865.
2 W. Xu, L. Z. Liu, M. Loizidou, M. Ahmed and I. G. Charles,
Cell Res., 2002, 12, 311–320.
3 B. Oronsky, G. R. Fanger, N. Oronsky, S. Knox and
J. Scicinski, Clin. Transl. Oncol., 2014, 7, 167–173.
4 R. A. Seram, M. C. Primi, G. H. Trossini and E. I. Ferreira,
Curr. Med. Chem., 2012, 19, 386–405.
5 A. B. Seabra, R. de Lima and M. Calderon, Curr. Top. Med.
Chem., 2015, 15, 298–308.
6 D. Hirst and T. Robson, J. Pharm. Pharmacol., 2007, 59, 3–13.
7 S. Huerta, S. Chilka and B. Bonavida, Internet J. Oncol., 2008,
33, 909–927.
8 R. Scatena, P. Bottoni, G. E. Martorana and B. Giardina,
Expert Opin. Invest. Drugs, 2005, 14, 835–846.
9 J. E. Saavedra, T. M. Dunams, J. L. Flippenanderson and
L. K. Keefer, J. Org. Chem., 1992, 57, 6134–6138.
successfully stored up to a few days in a light proof box con- 10 J. E. Saavedra, P. J. Shami, L. Y. Wang, K. M. Davies,
taining moist (PBS) sponges at 4 ꢁC, before analysis. DNA
migration can be determined on negative photomicrographs
M. N. Booth, M. L. Citro and L. K. Keefer, J. Med. Chem.,
2000, 43, 261–269.
using 0–150 mm digital calipers by measuring the nuclear DNA 11 R. S. Nandurdikar, A. E. Maciag, S. Y. Hong, H. Chakrapani,
and migrating DNA (i.e. comet length (ram)) and head diameter
M. L. Citro, L. K. Keefer and J. E. Saavedra, Org. Lett., 2010,
12, 56–59.
(mm) in a minimum of 25 randomly selected cells per pop-
ulation being studied. Comet area (mm2) can be measured by 12 P. J. Shami, J. E. Saavedra, L. Y. Wang, C. L. Bonifant,
placing a mm2 graph transparency between a light box and the
negative and counting the number of squares over which the
comet extends. Image analysis equipment was, however,
required for comprehensive and large scale comet analysis.
Data analysis. ANOVA one-way and Tukey's test were used in
the test data group, and the P value less than 0.05 was consid-
ered to be signicant difference.
B. A. Diwan, S. V. Singh, Y. Gu, S. D. Fox, G. S. Buzard,
M. L. Citro, D. J. Waterhouse, K. M. Davies, X. Ji and
L. K. Keefer, Mol. Cancer Ther., 2003, 2, 409–417.
13 A. E. Maciag, H. Chakrapani, J. E. Saavedra, N. L. Morris,
R. J. Holland, K. M. Kosak, P. J. Shami, L. M. Anderson
and L. K. Keefer, J. Pharmacol. Exp. Ther., 2011, 336, 313–320.
14 T. Kiziltepe, T. Hideshima, K. Ishitsuka, E. M. Ocio, N. Raje,
L. Catley, C. Q. Li, L. J. Trudel, H. Yasui, S. Vallet, J. L. Kutok,
D. Chauhan, C. S. Mitsiades, J. E. Saavedra, G. N. Wogan,
L. K. Keefer, P. J. Shami and K. C. Anderson, Blood, 2007,
110, 709–718.
Apoptosis analysis. Cells were incubated in six-well plates (1
ꢃ 105 per well) and treated with DMSO (1%), 7, or JS-K for 24 h.
To quantify apoptosis, prepared cells were washed twice with
cold PBS and then resuspended in 500 mL binding buffer at
a concentration of 1 ꢃ 106 cells per mL. 5 mL FITC–Annexin-V 15 P. J. Shami, J. E. Saavedra, C. L. Bonifant, J. Chu, V. Udupi,
and 5 mL PI wereꢁthen added to these cells, which were kept
S. Malaviya, B. I. Carr, S. Kar, M. Wang, L. Jia, X. Ji and
L. K. Keefer, J. Med. Chem., 2006, 49, 4356–4366.
in the dark at 25 C for 10 min. Data acquisition and analysis
were performed in a FACS calibur ow cytometer (Becton 16 R. A. D'Sa, Y. Wang, P. H. Ruane, B. M. Showalter,
Dickinson) and calculated by Cell Quest soware (BD Biosci-
ences, Franklin Lakes, NJ).
J. E. Saavedra, K. M. Davies, M. L. Citro, M. N. Booth,
L. K. Keefer and J. P. Toscano, J. Org. Chem., 2003, 68, 656–
657.
17 D. F. Dourado, P. A. Fernandes, M. J. Ramos and
B. Mannervik, Biochemistry, 2013, 52, 8069–8078.
Acknowledgements
This work was supported by grants from the National Natural 18 H. Kojima, Y. Urano, K. Kikuchi, T. Higuchi, Y. Hirata and
Science Foundation of China (No. 21372261 and No. 81673305)
T. Nagano, Angew. Chem., Int. Ed. Engl., 1999, 38, 3209–3212.
18898 | RSC Adv., 2017, 7, 18893–18899
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