Coverslips had been mounted on glass slides with Prolong Gold w DAPI Antifade reagent and analyzed by epifluorescence microscopy. Four dual channel photos were captured from every single sample utilizing a 60x goal lens. Image evaluation was carried out applying NIS Factors software program v3. 1. Mean fluorescence intensity per cell was calculated by the fol lowing, To assess p21 nuclear accumulation, p21 fluorescence was also measured within discrete nuclear areas as defined applying a DAPI intensity threshold. Down regulation of p21 by compact interfering RNA CWR22Rv1 had been transfected with val idated p21 small interfering RNA or Stealth siRNA detrimental control utilizing Lipofectamine 2000 transfection re agent following the manufac turers instruction. Six hr post transfection, cells had been cultured with RPMI 1640 media containing 10% FBS over evening.
Right after recovery, media was replaced with 0. 05% FBS media containing car or Zyflamend for 24 hr at 37 C. The total RNA was harvested for quantita tive actual time polymerase chain reaction and cell number was determined. Overexpression of p21 pRc CMV p21, containing complete length wild kind p21 cDNA, was utilized to overexpress p21. CWR22Rv1 cells were plated overnight. pRc CMV p21 or pRc CMV purchase CP-690550 was transfected working with Lipofectamine 2000 reagent in serum totally free RPMI 1640 media. Transfected cells have been picked by treatment for two weeks with neomycin and subjected to your MTT cell proliferation assay. p21 protein expression within the transfected cells was examined by Western blot. RNA isolation and quantitative RT PCR Complete RNA was isolated from CWR22Rv1 cells using Trizol reagent followed by chloroform extraction.
The aqueous phase was precipi tated in 100% isopropanol along with the pellet was washed in 75% ethanol prior to re suspension in RNase totally free water. Contaminating DNA was removed from RNA samples making use of Turbo DNA free of charge kit then the concentration of total RNA was measured working with TGX221 NanoDrop one thousand. Complete RNA from just about every sample was mixed with MultiScribe Reverse Transcriptase, RNase Inhibitor, dNTP Mixture, random hexamers, RT buffer, MgCl2 solution and incubated at 25 C for 10 min, 48 C for thirty min and 95 C for five min to reverse transcribe to cDNA applying TaqMan reagent kit. cDNA samples were used for quantita tive RT PCR. cDNA was utilised being a template for qPCR amplification with primer sets of p21 sense, were examined.
Amplification was carried out using a conventional thermo cycle system beginning with an first temperature at 94 C for one min followed by 30 cycles of 94 C for 15 sec, 50 C for thirty sec and 72 C for 2 min. Each sam ple was examined in triplicate and also the quantities of PCR product were normalized with 36B4, because the internal handle. The relative amounts of all mRNAs have been calculated using the comparative CT method as previously described with 36B4 since the invariant manage.