Fig 1.
The Impact of FAT10 on IL-12 expression in DC2.4 cells.
(A, B) DC2.4 cells were treated with TNF (400 U/ml)/IFN-γ (200 U/ml), IFN-γ (200 U/ml) in combination with (indicated +LPS) or without (indicated -) LPS (2 μg/ml) for 1 day. The mRNA level of FAT10 (A) and IL-12 (B) were detected by real-time RT-PCR. (C, D) DC2.4 cells and DC2.4-mFAT10 cells (stably expressing mouse FAT10) were treated with (indicated +LPS) or without (indicated -) LPS (2 μg/ml) for 1 day. The mRNA level of FAT10 (C) and IL-12 (D) were detected by real-time RT-PCR. (A-D) The y-axis depicts relative mRNA levels normalized to GAPDH. Data was analyzed by one-way ANOVA with Tukey’s multiple comparisons test. P values are indicated. Data are depicted as mean ± SDs. Each symbol in the graph represents an individual experiment. A, B: n = 5; C: n = 17, D: n = 15.
Fig 2.
No impact of FAT10 on IL-12 expression in BMDCs.
(A-C) BMDCs were generated from C57BL/6 wild type mice or FAT10-/- mice. BMDCs were treated with IFN-γ (200 U/ml) for 1 day, followed by LPS (1 μg/ml) treatment for 1 further day (indicated IFN-γ + LPS) or were left untreated (indicated untreated). (A, B) The mRNA level of FAT10 (A) and IL-12 (B) were detected by real-time RT-PCR. The y-axis depicts relative mRNA levels normalized to GAPDH. (C) The secretion of IL-12 into the supernatant was measured by ELISA. (A-C) Data was analyzed by one-way ANOVA with Tukey’s multiple comparisons test. P values are indicated. Data are depicted as mean ± SDs. Each symbol in the graph represents BMDCs derived from an individual mouse. n = 6 per group.
Fig 3.
FAT10 does not affect Th1 differentiation.
CD4+ T cells were magnetically purified from the spleen of C57BL/6 wild type mice or FAT10-/- mice. Purified cells were cultured under Th0 or Th1 skewing condition for 72 h. Cells were restimulated with PMA/ionomycin in the presence of brefeldin A for 4 h, stained for CD4 and intracellular IFN-γ, and analyzed by flow cytometry. (A) Representative dot plots. (B) Bar graph depicting percentage of IFN-γ + CD4 cells. Data are depicted as mean ± SDs of CD4+ cells isolated from 6 different mice per group (n = 6) statistically analyzed by unpaired Student’s t-test.
Fig 4.
FAT10 does not modulate the IL-12 signaling pathway.
(A-D) CD4+ T cells were magnetically purified from spleens of C57BL/6 wild type mice or FAT10-/- mice. (A, B) CD4+ T cells were treated with TNF (400 U/ml)/IFN-γ (200 U/ml) (indicated +) for 1 day or were left untreated (indicated -). pSTAT1, STAT1, and GAPDH were analyzed by western blot. (B) Quantification of pSTAT1 normalized to GAPDH. Data are depicted as mean ± SDs derived from 7 different experiments (n = 7). (C, D) CD4+ T cells were treated with TNF (400U/ml)/IFN-γ (200 U/ml) for 1 day or left untreated, followed by a treatment with IL-12 (10 ng/ml) for 2 h. Treatment regime is indicated. pSTAT4 and GAPDH were analyzed by western blot. (D) Quantification of pSTAT4 normalized to GAPDH. Data are depicted as mean ± SDs derived from 5 different experiments (n = 5).