We measured the level of the P-STAT1 in extracts from M14 cells cultured either in absence of IFNγ or after 15 min and 6 h of treatment with this cytokine. As expected, also in this instance blots densitometry revealed a rapid and strong increase of PSTAT1 accumulation after 15 min of stimulation with IFNγ ( Fig. 3 B). However, our analysis showed that IFNγ-dependent STAT1 activation in M14 was much more sustained than the one directed by IFNα, given that

after 6 h of stimulation there was only the 50–60% reduction in quantity of PSTAT1 compared with the amount measured at 15 min. We wondered whether the kinetics PLX4032 price of P-STAT1 activation by IFNα in MHCII-positive human tumor cells was consistent with our hypothesis that the IFNα-initiated negative feedback loop is responsible for the reduction of expression of MHCII molecules on these cells’

surface. We examined the kinetics of expression of factors that drive IFN-induced P-STAT1 activation as well as its attenuation. We compared the effects of treatment with either IFNγ or IFNα on the expression of interferon regulatory factors 1 (IRF1) and 2 (IRF2) (because www.selleckchem.com/products/ABT-263.html of their role in CIITA-PIV promoter activation [34,51]) and on suppressors of cytokine signaling 1 (SOCS1) and 3 (SOCS3) (because of their role in repressing IFNγ-dependent CIITA-PIV transcription [50,52]). We performed qRT-PCR assays using specific primer pairs (see Table 1) on total RNA isolated from Me10538, M14 and U-87 cells collected after 24 h of culture in absence of IFN and in presence of either IFNγ or IFNα. Dolutegravir This interval of time was chosen to first detect any durable activation of these genes. In agreement with similar measurements performed in other systems [53], our results (showed in Fig. 4) indicated upregulation of IRF1 and IRF2 by both IFNs at the concentrations tested. Notably, IFNα relative to IFNγ induced significantly lower ( p < 0.05) accumulation

of both IRF transcripts at 24 h in all cell lines tested. Measurements of the level of SOCS3 transcripts in IFNα-treated cells relative to IFNγ-treated cells revealed that IFNα induced a significantly lower ( p < 0.05) increase of SOCS3 expression in Me10538, M14 and U87. Finally, the measurement of SOCS1 transcript accumulation, which exhibited a very low basal constitutive expression in untreated cells, demonstrated a strong upregulation after 24 h of treatment with IFNγ in treated vs. untreated cells and a significantly stronger ( p < 0.05) upregulation after 24 h of treatment with IFNα. To obtain further information on the kinetics of IRF1 and SOCS1 activation in our system, we analyzed by qRT-PCR the accumulation of IRF1 and SOCS1 transcripts in M14 cells treated with IFNα for various time periods (15 min, 3 h, 24 h, and 48 h) and from untreated cells. As illustrated in Fig.