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: Housing conditions affect the mode of enterocyte shedding/death. ( A , B ) Evaluation of enterocyte shedding using regenerated villi (RV) cultures. To evaluate enterocyte shedding, shed cells (indicated by * in A ) from each villus (indicated by a white line) were counted. Cells fully detached from the villus (black arrows in B ) and those in the inter-villous region (white arrow in B ) were NOT counted. In ( B ), the area surrounded by a white rectangle is shown enlarged in the inset to the right. Shed cells directly associated with a villus (indicated by a smaller * in the inset in B ) were counted, but those indirectly associated (indicated by a larger * in the inset in B) were NOT counted, because of aiming to count only newly shed cells. More than 30 villi were counted. ( C , D ) Nec-1 inhibited enterocyte shedding in RV culture derived from mice housed with αDri. ( C ) Counts of shed cells; data are the mean ± SEM from three independent experiments. In ( D ), all plots of the three experiments are shown (vehicle: n = 128, Nec-1: n = 118); the horizontal line represents the mean. ( E , F ) Nec-1 did not inhibit enterocyte shedding in RV culture derived from mice housed with αDri + Shepherd Shack. ( E ) Counts of shed cells; data are the mean ± SEM from three independent experiments. In ( F ), all plots of the three experiments are shown (vehicle: n = 152, Nec-1: n = 185); the horizontal line represents the mean. ( G , H ) Nec-1 did not inhibit enterocyte shedding in RV culture derived from mice housed with Soft Chip. ( G ), Counts of shed cells; data are the mean ± SEM from three independent experiments. In ( H ), all plots of the three experiments are shown (vehicle: n = 118, Nec-1: n = 134); the horizontal line represents the mean. For ( C , E , G ), P values were calculated using two-tailed unpaired t -tests. For ( D , F , H ), P values were calculated using Mann–Whitney U -tests. * P < 0.05, ** P < 0.01, n.s., not significant.

Article Snippet: To examine the enterocyte turnover rate in the small intestine of mice housed with αDri (Shepherd Specialty Papers, TN, USA) and to compare the enterocyte turnover rate of mice housed with αDri with that of mice housed with αDri plus a Shepherd Shack (Shepherd Specialty Papers), we used cages (CLEA Japan, width, 143 mm; depth, 293 mm; height, 148 mm; not ventilated) with 40 g of αDri with or without a Shepherd Shack (width, 83 mm; depth, 146 mm; height, 63 mm).

Techniques: Derivative Assay, Two Tailed Test, MANN-WHITNEY

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: Corticosterone changes the mode of cell death in RV culture. ( A ) Plasma corticosterone concentration at Zeitgeber time 0 in WT mice housed with αDri (n = 3) compared with that of sex-matched littermate WT mice housed with αDri + Shepherd Shack (n = 3). ( B , C ) Nec-1 inhibits enterocyte shedding in corticosterone-treated RV culture derived from mice housed with αDri + Shepherd Shack. ( B ) Villi regenerated for 21 h in the absence or presence of 50 ng/mL of corticosterone were treated with vehicle alone or 20 μM Nec-1 for 3 h. The figures show a representative plot of the number of shed cells in RV culture treated with vehicle (n = 61), and Nec-1 (n = 59), corticosterone (50 ng/mL)/vehicle (n = 61), and corticosterone (50 ng/mL)/Nec-1 (n = 56) (left two panels). For comparison, a data set of vehicle without or with corticosterone and that of Nec-1 without or with corticosterone are shown (right two panels). In ( C ), another data set of RV culture treated with corticosterone (50 ng/mL)/vehicle (n = 44) and corticosterone (50 ng/mL)/Nec-1 (n = 45) is shown. ( D , E ) Nec-1 inhibits enterocyte shedding in corticosterone-treated RV culture derived from mice housed with Soft Chip. ( D ), Villi regenerated for 21 h in the absence or presence of 100 ng/mL of corticosterone were treated with vehicle alone or 20 μM Nec-1 for 3 h. Cells shed in these experiments were counted; data are the mean ± SEM from three independent experiments. In ( E ), all plots of the three experiments are shown (vehicle: n = 149, Nec-1: n = 150); the horizontal line represents the mean. For ( A , D ), P values were calculated using two-tailed unpaired t -tests. For ( B , C , E ), P values were calculated using Mann–Whitney U -tests. * P < 0.05, ** P < 0.01, n.s., not significant.

Article Snippet: To examine the enterocyte turnover rate in the small intestine of mice housed with αDri (Shepherd Specialty Papers, TN, USA) and to compare the enterocyte turnover rate of mice housed with αDri with that of mice housed with αDri plus a Shepherd Shack (Shepherd Specialty Papers), we used cages (CLEA Japan, width, 143 mm; depth, 293 mm; height, 148 mm; not ventilated) with 40 g of αDri with or without a Shepherd Shack (width, 83 mm; depth, 146 mm; height, 63 mm).

Techniques: Concentration Assay, Derivative Assay, Comparison, Two Tailed Test, MANN-WHITNEY

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: 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. .

Article Snippet: To examine the enterocyte turnover rate in the small intestine of mice housed with αDri (Shepherd Specialty Papers, TN, USA) and to compare the enterocyte turnover rate of mice housed with αDri with that of mice housed with αDri plus a Shepherd Shack (Shepherd Specialty Papers), we used cages (CLEA Japan, width, 143 mm; depth, 293 mm; height, 148 mm; not ventilated) with 40 g of αDri with or without a Shepherd Shack (width, 83 mm; depth, 146 mm; height, 63 mm).

Techniques: Inhibition, Western Blot, Control, Comparison

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: Housing conditions affect enterocyte turnover and shedding. ( A ) Housing materials used in this study. The image of a Shepherd Shack is from the website of EP Trading Co. Ltd. (Tokyo, Japan). ( B ) Evaluation of turnover rate. The distance from the villus-crypt junction to the leading BrdU-labeled cell in each villus of the duodenum (distance between arrows, indicated by a white dotted line; hereafter designated as Distance ) was measured and plotted. More than 25 plots (usually around 30 plots) are shown; a bracket indicates a crypt region, and the horizontal line represents the mean. In each turnover rate comparing experiment, we did not notice the difference of villous length between the groups. ( C ) Enterocyte turnover rate in WT mice housed with αDri (n = 3). Distance was plotted; the horizontal line represents the mean. ( D ) Enterocyte turnover rate in WT mice housed with Soft Chip (n = 3). Distance was plotted; the horizontal line represents the mean. ( E ) The mean value in αDri (data from C ) was compared with that in Soft Chip (data from D ). Data are the mean ± SEM from three independent experiments. ( F ) Enterocyte turnover rate in WT mice housed with αDri was higher than that in sex-matched littermate WT mice housed with αDri + Shepherd Shack. Three pairs of mice were used. Distance was plotted; the horizontal line represents the mean. ( G ) When mice were housed with αDri, enterocyte turnover rate was higher in WT than in Ripk1 + /− mice. A pair comprising a WT and a sex-matched littermate Ripk1 + /− mouse was used. Distance was plotted; the horizontal line represents the mean. ( H ) When mice were housed with Soft Chip, enterocyte turnover rate was not different between WT and Ripk1 + /− mice. Three pairs comprising a WT and a sex-matched littermate Ripk1 + /− mouse were used. The data of the WT mice are the same as those shown in ( D ). Distance was plotted; the horizontal line represents the mean. For ( C – H ), P values were calculated using two-tailed unpaired t -tests. *** P < 0.001, n.s., not significant.

Article Snippet: To examine the enterocyte turnover rate in the small intestine of mice housed with αDri (Shepherd Specialty Papers, TN, USA) and to compare the enterocyte turnover rate of mice housed with αDri with that of mice housed with αDri plus a Shepherd Shack (Shepherd Specialty Papers), we used cages (CLEA Japan, width, 143 mm; depth, 293 mm; height, 148 mm; not ventilated) with 40 g of αDri with or without a Shepherd Shack (width, 83 mm; depth, 146 mm; height, 63 mm).

Techniques: Labeling, Two Tailed Test