Journal: Scientific reports
Article Title: Housing conditions affect enterocyte death mode and turnover rate in mouse small intestine.
doi: 10.1038/s41598-023-47660-1
Figure Lengend Snippet: Figure 4. Inhibition of TBK1 results in stimulation of RIPK1-dependent enterocyte shedding. (A–F) TBK1i stimulated enterocyte shedding in RV culture, but TAK1i and MK2i did not. RV were treated with 2 µM TBK1i (MRT67307) (A, B), 100 or 300 nM TAK1i (5Z-7-Oxozeaenol) (C, D), or 1 or 10 µM MK2i (PF-3644022) (E, F) for 6 h. In the case of TBK1i, RV were also pre-treated for 30 min with Nec-1s, and then 2 µM TBK1i was added for 6 h. Shed cells were counted; data are the mean ± SEM from three independent experiments (A, C, E). In (B, D, F), all plots of the three experiments are shown (B: vehicle, n = 159, TBK1i, n = 163, TBK1i + Nec-1s, n = 151, D: vehicle, n = 142 , TAK1i [100 nM], n = 127, TAK1i [300 nM], n = 130, F: vehicle, n = 181, MK2i [1 µM], n = 151, MK2i [10 µM], n = 167); the horizontal line represents the mean. (G–I) The amount of TBK1 and the S321-phosphorylation level of RIPK1 were lower in WT mice housed with αDri than in sex-matched littermate WT mice housed with Eco Chips. (G), Western blot analysis of the quantity of TBK1, S321-phosphorylated- RIPK1 (p-RIPK1 [S321]), and total RIPK1 (RIPK1) in the enterocytes of WT mice housed with αDri and sex-matched littermate WT mice housed with Eco Chips. E-cadherin was a loading control. (H), Densitometry of TBK1 was normalized to E-cadherin; then, the ratio (relative to a value of Eco Chips as 1) was calculated in each Eco Chips/ αDri pair. The mean ± SEM from three pairs is shown. (I), Densitometry was performed to obtain the ratio of p-RIPK1 (S321) to total RIPK1 (normalized to Eco Chips as 1) in each Eco Chips/αDri pair. The mean ± SEM from three pairs is shown. For (A–F), P values were calculated using one-way Anova with Dunnett’s multiple comparison tests. For (H, I), P values were calculated using one-sample t-tests. *P < 0.05, **P < 0.01, n.s., not significant. Uncropped Western blot data used in G, H, and I are shown in supplementary Fig. S7.
Article Snippet: Nec-1 and Nec-1s were obtained from Merck (Hessen, Germany); zVAD-fmk, from Peptide Institute (Osaka, Japan); the TBK1 inhibitor (TBK1i) MRT67307, from Selleck (TX, USA); and the TAK1 inhibitor (TAK1i) 5Z-7-Oxozeaenol and MK2 inhibitor (MK2i) PF-3644022, from Merck.
Techniques: Inhibition, Phospho-proteomics, Western Blot, Control, Comparison
Selleck Chemicals is a verified supplier 
Selleck Chemicals manufactures this product 
![Figure 4. Inhibition of <t>TBK1</t> results in stimulation of RIPK1-dependent enterocyte shedding. (A–F) <t>TBK1i</t> stimulated enterocyte shedding in RV culture, but TAK1i and MK2i did not. RV were treated with 2 µM TBK1i <t>(MRT67307)</t> (A, B), 100 or 300 nM TAK1i (5Z-7-Oxozeaenol) (C, D), or 1 or 10 µM MK2i (PF-3644022) (E, F) for 6 h. In the case of TBK1i, RV were also pre-treated for 30 min with Nec-1s, and then 2 µM TBK1i was added for 6 h. Shed cells were counted; data are the mean ± SEM from three independent experiments (A, C, E). In (B, D, F), all plots of the three experiments are shown (B: vehicle, n = 159, TBK1i, n = 163, TBK1i + Nec-1s, n = 151, D: vehicle, n = 142 , TAK1i [100 nM], n = 127, TAK1i [300 nM], n = 130, F: vehicle, n = 181, MK2i [1 µM], n = 151, MK2i [10 µM], n = 167); the horizontal line represents the mean. (G–I) The amount of TBK1 and the S321-phosphorylation level of RIPK1 were lower in WT mice housed with αDri than in sex-matched littermate WT mice housed with Eco Chips. (G), Western blot analysis of the quantity of TBK1, S321-phosphorylated- RIPK1 (p-RIPK1 [S321]), and total RIPK1 (RIPK1) in the enterocytes of WT mice housed with αDri and sex-matched littermate WT mice housed with Eco Chips. E-cadherin was a loading control. (H), Densitometry of TBK1 was normalized to E-cadherin; then, the ratio (relative to a value of Eco Chips as 1) was calculated in each Eco Chips/ αDri pair. The mean ± SEM from three pairs is shown. (I), Densitometry was performed to obtain the ratio of p-RIPK1 (S321) to total RIPK1 (normalized to Eco Chips as 1) in each Eco Chips/αDri pair. The mean ± SEM from three pairs is shown. For (A–F), P values were calculated using one-way Anova with Dunnett’s multiple comparison tests. For (H, I), P values were calculated using one-sample t-tests. *P < 0.05, **P < 0.01, n.s., not significant. Uncropped Western blot data used in G, H, and I are shown in supplementary Fig. S7.](https://pub-med-unpaywalled-images-cdn.bioz.com/pub_med_ids_ending_with_3588/pm37993588/pm37993588__page6_image1.jpg)
![Figure 5. Enterocyte turnover and RIPK1-dependent enterocyte shedding were stimulated in TBK1-deficient mice. (A) The enterocyte turnover rates were higher in Ripk3−/− Tbk1−/− mice than in sex-matched littermate Ripk3−/− mice in three independent experiments. Distance was plotted; the horizontal line represents the mean. (B, C) Enterocyte shedding was stimulated in RV culture from Ripk3−/− Tbk1−/− mice, and this stimulation was Nec-1s-sensitive. (B), Shed cells were counted; data are the mean ± SEM from three independent experiments. In (C), all plots of the three experiments are shown (Ripk3−/−: vehicle, n = 203, Nec-1s, n = 172, Ripk3−/− Tbk1−/−: vehicle, n = 165, Nec-1s, n = 141); the horizontal line represents the mean. For comparison, vehicle and Nec-1s were paired in each genotype and vehicles of each genotype were also paired. (D, E) Western blot showing the levels of S321-phosphorylated-RIPK1 (p-RIPK1 [S321]), total RIPK1 [RIPK1], and TBK1 in enterocytes of Ripk3−/− (n = 3) and Ripk3−/− Tbk1−/− mice (n = 3) (D). E-cadherin is a loading control. (E), Densitometry was performed to obtain the ratio of p-RIPK1 (S321) to total RIPK1. The mean ± SEM from three mice is shown. For (A, E), P values were calculated using two-tailed unpaired t-tests. For (B), P values were calculated using two-way ANOVA with Bonferroni post-tests. For (C), P values were calculated using Mann–Whitney U-tests. *P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant. Uncropped Western blot data used in D are shown in supplementary Fig. S8.](https://pub-med-unpaywalled-images-cdn.bioz.com/pub_med_ids_ending_with_3588/pm37993588/pm37993588__page7_image1.jpg)