Figure 2. Tsc1^IEC-KO mice exhibit disrupted intestinal homeostasis.

(A and B) Immunoblots of purified colonic epithelial lysates from Tsc1^fl/fl and Tsc1^IEC-KO mice for p-RIPK1 (S166), p-RIPK3 (S232), p-MLKL (S345), p-S6 (S235/236), or p–4E-BP1 (Thr27/46). Numbers under Western blot bands represent relative quantifications over actin. (C) IF staining of colon sections (n = 6) from various genotypes. Epcam/TUNEL–double positive cells were counted over 5 high-power fields (×400) per sample (n = 30). Scale bars: 20 μm. (D) Mice (n = 6–8) fasted for 4 hours were gavaged with FITC-dextran and bled in 4 hours. (E) Quantitative PCR (qPCR) analysis of inflammatory cytokine and chemokine mRNAs (normalized to β-actin) in steady-state colons (n = 7). Primers are listed in Supplemental Table 2. (F) IF staining and quantification of various immune cells in steady-state colons (n = 6). Scale bars: 20 μm (F4/80 and Ly6G), 50 μm, (CD3). Data were pooled from 2 independent experiments (D and E) or are representative of 3 independent experiments (A–C and F) and are shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, by unpaired Student’s t test (E and F) or 1-way ANOVA (C and D).