Fig. 6
TRIM33 deficiency resulted in accelerated DC apoptosis. A Schematic diagram of the generation of DCs from WT or Trim33 KO DCs in Flt3L cultures. BM cells from Trim33fl/fl and Trim33fl/fl Cre-ERT2 mice were cultured with 200 ng/mL Flt3L, and 1 µM 4-OHT was added on Days 3–4. FL-DCs were FACS-purified on Day 8. B qPCR analysis of Trim33 expression in FL-DCs of the indicated genotypes, n = 3. C, D Survival analysis of cultured WT (Trim33fl/fl) and KO (Trim33fl/fl Cre-ERT2) FL-DCs. A total of 5\(\times\)104 FACS-purified FL-DCs/well were cultured with or without the indicated supplements (20 ng/ml GM-CSF, 1 µM ODN 2216, 100 µg/mL poly(I:C), or 100 ng/mL LPS) for 12 h or 24 h before detection. The numbers in quadrants indicate percentages. D The percentages of viable (Annexin V–7-AAD–) FL-DCs after culture, n = 3. E, F Survival analysis of DC2.4 cells of the indicated genotypes. A total of 2.5\(\times\)105 DC2.4 or DC2.4 Trim33 KO cells/well were seeded in 24-well plates and cultured for 48 h before detection. F The percentages of viable (Annexin V–7-AAD–) cells after culture are plotted as the means of 3 independent experiments. G Western blot analysis of Caspase-3 and PARP in FL-DCs cultured for 12 h. Approximately 1\(\times\)105 cells/sample were loaded. The relative band intensities are shown. Bars represent the means ± SEMs. The data are representative of 3 independent experiments. Statistical significance was determined using an unpaired two-tailed Student’s t test. Ns: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001
