M the skin tumor tissue of female patient. SCC-13 cells have been isolated in the skin squamous cell carcinoma from a female patient. Cryptolepine was dissolved in DMSO with final concentration of DMSO in media was not greater than 0.1 (v/v). Equal volume of DMSO was added in handle group of cells. 4.three. Preparation of Topoisomerase (I II) Extract from Cells Cells from treated and untreated groups have been harvested and collected into 5 mL medium in 15 mL tubes. Suspensions had been centrifuged at 800g for 3 min at 4 C. Cell pellets had been resuspended in three mL of ice cold TEMP buffer (10 mM Tris-HCl, pH 7.five, 1 mM EDTA, 4 mM MgCl2 and 0.five mM PMSF). Suspensions have been centrifuged at 800g for three min at four C and cell pellets had been resuspended in three mL of ice cold TEMP buffer and left on ice for ten min. Following incubation, suspensions were homogenized by 80 strokes in Dounce tight fitting homogenizer. Homogenates were centrifuged at 1500g for 10 min at 4 C to pellet the nuclei. Nuclear pellets had been resuspended in 1 mL of cold TEMP buffer within a microcentrifuge tube. Tubes have been centrifuged at 1500g for 2 min at 4 C, pellets collected and resuspended inside a compact volume ( three pellet volume) of TEP buffer (same as TEMP buffer but lacking MgCl2 . An equal volume of 1M NaCl was added, vortexed and left on ice for 60 min. Right after incubation, tubes were centrifuged at 15,000g for 15 min at 4 C. The supernatants had been collected as topoisomerase (I II) extracts. Topoisomerase activity assays have been performed on identical day or extracts were aliquoted and stored at -80 C. RS-1 medchemexpress protein concentration was determined applying a Bio-Rad protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). 4.four. Topoisomerase I Enzyme Activity Assay To determine the topoisomerase I enzyme activity, cell extracts containing ten protein from treated or untreated groups had been mixed with two of 10x topoisomerase I assay buffer (100 mM Tris-HCl pH 7.9, ten mM EDTA, 1.five M NaCl, 1 bovine serum albumin, 1 mM spermidine, 50 glycerol) and 1 (0.25 / ), and supercoiled DNA (25 in one hundred TE buffer; ten mM Tris-HCl pH 7.5, 1 mM EDTA) as a substrate. Reaction volumes have been produced as much as 20 employing Bentiromide web distilled water. Reaction mixtures were incubated for 30 min at 37 C. Reaction was stopped by adding 5 of two SDS. Proteinase K (0.5 mg/mL) 5 was added and incubated for 15 min at 37 C. Reaction mixture with 10x loading dye (0.25 bromophenol blue, 50 glycerol) was loaded to 1 agarose gel in TAE buffer. Gel was run at 1.five V/cm for 2 h. Gel was stained with 0.5 /mL ethidium bromide and destained in distilled water for 15 min at room temperature and photographed using UV transilluminator from Bio-Rad. Comparative reactivity with the enzyme amongst different groups is represented by the band intensity. four.five. Topoisomerase II Enzyme Activity Assay To determine the topoisomerase II enzyme activity, cell extracts equivalent to ten protein every single from treated or untreated groups have been mixed with four of 5x total topoisomerase II assay buffer prepared freshly by mixing equal volume of Buffer A (0.5 M Tris-HCl pH eight, 100 mM MgCl2 , 5 mM dithiothreitol, 300 bovine serum albumin/mL) and Buffer B (200 mM ATP in sterile distilled water). A single (0.25 / ) supercoiled pHOT1 DNA (150 ng to final volume) was added as a substrate. Reaction volumes were produced as much as 20 employing distilled water. Reaction mixtures have been incubated for 30 min at 37 C. Reaction was stopped by adding five of 2 SDS. Proteinase K (0.five mg/mL) five was added and.