CDK6 mRNA (NM_001259) was amplified from 10 933 to 11 119, with primers: forward 5′-ctttcccaagaggcagatga-3′ and reverse 5′-gggtcacaaagcatccctta-3′. BAY 80-6946 mouse CDK2 mRNA (NM_001798) was amplified from 1903 to 2027, with primers: forward 5′-cctgatcccattttcctctg-3′ and reverse 5′-ttttacccatgccctcactc-3′. Cyclin D2 mRNA (NM_001759)

was amplified from 3617 to 3831, with primers: forward 5′-gtttttcccctccgtctttc-3′ and reverse 5′-ttgaaaacccgaccgtttag-3′. Cyclin D3 mRNA (NM_001760) was amplified from 615 to 774, with primers: forward 5′-ggacctggctgctgtgattg-3′ and reverse 5′-gatcatggatggcgggtaca-3′. Cyclin E1 mRNA (NM_001238) was amplified from 1625 to 1777, with primers: forward 5′-tacaccagccacctccagac-3′ and reverse 5′-tacaacggagcccagaacac-3′. Cyclin A2 mRNA (NM_001237) was amplified from 1366 to 1587, with primers: forward 5′-ttattgctggagctgccttt-3′ and reverse 5′-ctggtgggttgaggagagaa-3′. SKP2 mRNA (NM_005983) was amplified from 711 to 924, with primers: forward 5′-catttcagcccttttcgtgt-3′ and reverse 5′-gggcaaattcagagaatcca-3′. CKS1B mRNA (NM_001826) was amplified from 532 to 723, with primers: forward 5′-ccagatgagtgctctgtgga-3′ and reverse 5′-ccgcaagtcaccacacatac-3′. TBP mRNA (NM_003194) was amplified from 29 to 219, with primers: forward 5′-cggctgtttaacttcgcttc-3′ and reverse 5′-ttcttggcaaaccagaaacc-3′. RPL13A mRNA (NM_012423) was amplified from 540 to BAY 73-4506 768, with

primers: forward 5′-agctcatgaggctacggaaa-3′ and reverse 5′-cttgctcccagcttcctatg-3′. Data are the mean ± standard deviation (SD) of three independent experiments. Statistical significance was determined using Student’s t-test. P-values < 0·05 were considered statistically significant. Engagement of the TCR together with the costimulatory

receptor CD28 programmes naïve T cells to proliferate in response to autocrine IL-2.23 When purified CD4+ CD25− human naïve selleck chemicals T cells (Fig. 1a) were stimulated with anti-CD3 plus anti-CD28 antibodies, a time-dependent induction of DNA synthesis was observed, which was inhibited in a concentration-dependent manner by nIL-2. At 4 μg/ml, nIL-2 abrogated T-cell proliferation. nIL-2 effects were reproduced by the two IKK inhibitors, BMS-345541 and PS-1145, at increasing concentrations. At 3 μm, both inhibitors reduced cell proliferation by over 90%, at all times tested (Fig. 1b). Analysis of DNA content showed that BMS-345541 and PS-1145 inhibited cell-cycle progression before DNA synthesis. Inhibition of cell proliferation was not caused by pro-apoptotic effects, as shown by the absence of hypodiploid DNA peaks left of the G0/G1 peak (Fig. 1c). CD3/CD28 costimulation of human naïve CD4+ T cells was associated with a marked decrease in I-κBα levels. I-κBα was significantly stabilized in cells pretreated with BMS-345541 or PS-1145 (Fig. 2a). CD3/CD28 costimulation also resulted in noticeable nuclear translocation of both p50 and p65-RelA, which was markedly reduced by pretreatment with either drug (Fig. 2b-e).