Sumoylation of STAT1 desensitizes cells to IFNγ. (A) End point RT-PCR analyses of IFNγ-induced genes in mouse embryonic fibroblasts derived from SUMO-free STAT1 (ΔSUMO) knockin mice or wild-type littermates. Cells were left untreated or treated with 50 U/mL mouse IFNγ for the indicated times, followed by RNA extraction, reverse transcription, and PCR. Shown are the Gapdh-normalized signal intensities of ethidium bromide–stained PCR products. Data are the mean and SEM of 3 independent experiments. (B) Real-time PCR analyses using BMMs from mice expressing SUMO-free STAT1 (ΔSUMO) or wild-type littermates. Cells were left untreated or were treated with mouse IFNγ (50 U/mL) for 6 or 24 hours. Shown are Gapdh-normalized gene expression data (mean ± SEM) of 3 independent experiments. (C) As in panel B, but BMMs were left untreated or were cotreated with IFNγ (50 U/mL) and LPS (1 ng/mL) for 3, 6, or 24 hours. Shown is the Gapdh-normalized expression of Nos2; values are mean ± SEM of 3 independent experiments. (D) Consecutive immunoblot analysis using NOS2-specific antibody (iNOS; BD Biosciences) and then β-actin antibodies with whole cell extracts from macrophages derived from SUMO-free STAT1 (ΔSUMO) knockin mice or wild-type littermates. The cells were treated for the indicated times with IFNγ alone or in combination with LPS. Data are representative of 2 independent experiments. (E) Nitric oxide production of BMMS derived from wild-type (left) or SUMO-free STAT1 knockin mice (ΔSUMO; right). Macrophages were seeded in 96-well plates at a density of 1 × 10⁴ cells/well and kept for 60 hours in L cell–conditioned medium (10% L cell–supplemented DMEM with 10% calf serum) supplemented for the indicated times with IFNγ or LPS or combinations thereof. Half the culture supernatant (50 μL) and Griess reagent were subsequently used to determine absorption at 550 nm, before nitrite concentrations were calculated using a standard curve. Data are representative for 2 independent experiments carried out in duplicate. (F) Corresponding viability of the cells used in panel E, as determined by their ATP content. The value obtained for cells kept in L cell–conditioned medium for 60 hours was set to 100 and used as the reference point.