Figure 7. Mutual regulation of SIRT5 and TRIM21 influences IL‐1β production in LPS‐activated macrophages and DSS‐induced colitis in mice.

• A, B
  SIRT5 ^−/− macrophages (BMDMs) were transfected with HA‐SIRT5 WT or mutants in the absence or presence of Flag‐TRIM21 as indicated. Macrophages were stimulation with 100 ng/ml LPS for 4 h. mRNA expression of IFN‐β (A) and IL‐1β (B) was determined by qRT‐PCR analysis.
• C, D
  TRIM21 ^−/− macrophages (BMDMs) were transfected with Flag‐TRIM21 WT or mutants in the absence or presence of HA‐SIRT5 as indicated. Macrophages were stimulation with 100 ng/ml LPS for 4 h. mRNA expression of IFN‐β (C) and IL‐1β (D) was determined by qRT‐PCR analysis.
• E
  Genotyping of TRIM21 ^−/−, SIRT5 ^−/− and TRIM21 ^−/− SIRT5 ^−/− mice. DNA derived from the tail of wild‐type and knockout animals was analyzed by PCR. Protein expression of TRIM21 and SIRT5 in BMDMs derived from mice were also analyzed by Western blotting.
• F–I
  Wild‐type (WT, n = 5), TRIM21 ^−/− (n = 5), SIRT5 ^−/−(n = 5), and TRIM21 ^−/− SIRT5 ^−/− (n = 5) mice were orally administrated with 2.5% (w/v) DSS in drinking water for 7 days and regular drinking water for another 2 days. Mice body weight (F) and Colon‐length (G) changes were measured. The colon tissues were examined microscopically for pathological changes (H, left panel, scale bar: 100 μm), which were further semiquantitatively scored (H, right panel). (I) IL‐1β mRNA expression in colon tissues determined by qRT‐PCR.

Data information: Data are means ± SD. In (A–D), n = 3 biological replicates, in (F–I), n = 5 biological replicates. Western blots in (E) represent three independent experiments. P‐values were determined by unpaired two‐tailed Student's t‐tests. ns, not significant; ***P < 0.001, ****P < 0.0001.