Cells were Dounce homogenized and nuclei were collected by centrifugation at 750 × g for 5 min. Cell

extracts were kept at 4°C for 5 min and the remaining intact nuclei were collected by a further centrifugation at 750 × g for 5 min. The supernatant was recovered and a crude membrane fraction was obtained by centrifugation at 43,000 × g for 20 min. The leftover supernatant represented the cytoplasmic fraction. Nuclear and membrane fractions were than separated on SDS-PAGE, transferred to nitrocellulose membrane (GE Healthcare) and analyzed by western blot with the appropriate antibodies. Statistics All experiment unless indicated were performed at least three times. All experimental results were expressed GSK1120212 clinical trial as the arithmetic mean ± standard deviation (s.d.). Student’s t-test was used for statistical significance of the differences between treatment groups. Statistical analysis was performed using analysis of variance at 5% (p < 0.05) or 1% (p < 0.01). Results and discussion KSHV-latent infection of monocytic cell line THP-1 results in an increase of AKT phosphorylation that persisted after bortezomib c-Met inhibitor treatment THP-1 monocytic cells, infected with KHSV for 48 hours, were subjected to immunofluorescence analysis

and, as shown in Figure 1A, the expression of latent associated nuclear antigen (LANA) was detected in about 35% of the cells, compared to mock infected cells. No expression of lytic antigens was found (data not shown), in accordance to previous reported studies [12], indicating that KSHV establishes a latent infection in THP-1 cells. Next, we investigated the impact of KHSV-infection on AKT phosphorylation in THP-1 cells. Western blot analysis showed that THP-1-infected cells displayed increased phosphorylation of AKT, in comparison to THP-1 mock-infected cells (Figure 1B). This is in agreement with other studies showing that KSHV proteins are able to activate PI3K/AKT pathway or down-regulate AKT phosphatases such as PTEN in several cell types [14, 20]. The activation of

AKT pathway has been also reported for other oncoviruses Edoxaban [32]. As bortezomib has been shown to interfere with the activation status of AKT [27, 33], we then investigated if bortezomib-treatment could affect AKT phosphorylation in THP-1 cells. We observed that bortezomib (Bz, 10 nM for 48 hours) strongly down-regulated AKT phosphorylation in mock-infected cells, while KSHV infection impaired such effect (Figure 1B). This might be due to KSHV-induced inhibition of PTEN, demonstrated in other studies [20], that could counteract the bortezomib-mediated up-regulation of this phosphatase [34]. As expected, AKT phosporylation was completely abolished by pre-treatment with AKT inhibitor LY294002, both in mock and viral-infected cells (Figure 1B).