Figure 7

Sirt6 is required to maintain Rspo1 expression of intestinal epithelial cells under inflammation status. (A and B) YAMC cells (n=3 in each group) were transfected with Sirt6 siRNA (siSirt6, 50 nM) or negative control siRNA (siControl, 50 nM) using lipofectamine 2000. Forty-eight hours after transfection, cells were cultured with medium containing TNF-α (100 ng/mL) or medium alone for an additional 24 h. At the end of treatment, cells were harvested to measure expression of (A) Rspo1 mRNA with quantitative real-time RT-PCR and (B) Rspo1 protein with Western blot. (C) Sirt6^IEC-KO mice and their control littermates (male, 8–10 wks old, n=4 in each group) were intraperitoneally injected with either LPS (2 mg/kg) or saline (100 µL, sham control). Twenty-four hours after treatment, mice were euthanized by CO₂ inhalation. Then, colons were collected and processed to determine levels of Rspo1 protein by Western blot. (D) Western blot examination of Rspo1 protein expression in Sirt6^IEC-KO mice and their control littermates (male, 8–10 wks old, n=4 in each group) with DSS-induced colitis. Colonic tissues were collected on d 7 after induction of colitis with 3.5% DSS drinking water. Results are expressed as mean ± SEM and represent the average of findings from three independent experiments. *, P < 0.05 versus control; **, P < 0.01 versus control; ***, P < 0.001 versus control.