EMBO/SourceData huggingface dataset:
Figure 2qPCR validation (C) of indicated genes related to fatty acid metabolism of WT+NI (n = 6), Ehmt2Ksp+NI (n = 6), WT+I/R 3D (n = 6) and Ehmt2Ksp+I/R 3D (n = 6) groups.qPCR validation (E) of indicated genes related to fatty acid beta-oxidation of WT+NI (n = 6), Ehmt2Ksp+NI (n = 6), WT+I/R 3D (n = 6) and Ehmt2Ksp+I/R 3D (n = 6) groups.G Representative CES1 immunostaining (left) with semi-quantitative analysis (right) of human kidneys. AKI (acute kidney injury), n = 3; Con (control), n = 6.H Representative immunoblots (left) with quantitative results (right) of Ces1 level in the kidneys of WT (n = 3) and Ehmt2Ksp (n = 3) mice at I/R 3D.
Figure 3B Representative immunoblots (left) with quantitative results (right) of G9a and H3K9me1/2 in cisplatin-injured mouse kidneys.C Representative H&E images (left) with quantitative pathological scores (right) of indicated groups.H mRNA levels of fatty acid oxidation related genes of indicated groups.representative immunoblots with quantitative results of Ces1 (K) of indicated groups.M Representative immunostaining for F4/80 and quantitative results of indicated groups.
Figure 4C mRNA levels of Kim1 and Ngal of PBS+NI (n = 6), A366+NI (n = 5), PBS+I/R 3D (n = 6) and A366+I/R 3D (n = 5) groups.Representative renal TG (triglyceride) and TC (cholesterol) levels (E) of PBS+I/R 3D (n = 12) and A366+I/R 3D (n = 12) groups.F mRNA levels of fatty acid oxidation related genes of PBS+I/R 3D (n = 6) and A366+I/R 3D (n = 5) groups.K Representative immunostaining for CD3, Ly6G, and F4/80 and quantitative results of PBS+I/R 3D (n = 6) and A366+I/R 3D (n = 5) groups.Representative immunoblots (left) with quantitative results (right) of p-Rip3 for PBS+I/R 3D (n = 3) and A366+I/R 3D (n = 3) groups.
Figure 5protein (H) levels of Ces1 in Ad-NC or Ad-G9a transfected mPTECs. n = 4 cultures.Representative images (M) with quantitative results of lipid droplet areas (N) and percentage of different lipid droplet size (O), in labeled-PA treated mPTECs isolated from WT and Ehmt2Ksp mice at one hour after treatment. The results were obtained from three biological replicates; a total of 20 cells were imaged in each group for quantification of lipid droplet area (N, O)Q-S Seahorse studies (Q) with quantitative results of basal (R) and maximal (S) respiration in PA-treated mPTECs isolated from WT and Ehmt2Ksp mice. OCR, oxygen consumption rate; n = 4 cultures.
Figure 6E Representative Oil Red O staining with quantification of WT+Veh (n = 5), WT+WWL (n = 4), Ehmt2Ksp+Veh (n = 4) and Ehmt2Ksp+WWL (n = 4) groups.F mRNA levels of fatty acid oxidation related genes of WT+Veh (n = 5), WT+WWL (n = 4), Ehmt2Ksp+Veh (n = 4) and Ehmt2Ksp+WWL (n = 4) groups.G Representative Cpt1a immunostaining with quantitative analysis in the kidneys of WT+Veh (n = 5), WT+WWL (n = 4), Ehmt2Ksp+Veh (n = 4) and Ehmt2Ksp+WWL (n = 4) groups. Scale bar = 50 μm. IOD, integral optical density.Representative immunostaining for CD3 and F4/80 (K) of WT+Veh (n = 5), WT+WWL (n = 4), Ehmt2Ksp+Veh (n = 4) and Ehmt2Ksp+WWL (n = 4) groups.Representative CD3 and F4/80 quantitative results (L) of WT+Veh (n = 5), WT+WWL (n = 4), Ehmt2Ksp+Veh (n = 4) and Ehmt2Ksp+WWL (n = 4) groups.M Representative immunoblots (left) with quantitative results (right) of p-Mlkl for WT+Veh (n = 3), WT+WWL (n = 3), Ehmt2Ksp+Veh (n = 3) and Ehmt2Ksp+WWL (n = 3) groups.
Figure 7B mRNA levels of Kim1 and Ngal of indicated groups.Representative Oil Red O staining with quantification (C of indicated groups.E mRNA levels of fatty acid oxidation related genes of indicated groups.representative immunoblots (H, left) with quantitative results (H, right) of Ces1 and G9a, of indicated groups.J, K Representative immunostaining for CD3, Ly6G, and F4/80 (J) and quantitative results (K) of indicated groups.L Representative immunoblots (left) with quantitative results (right) of p-Rip3 and p-Mlkl for indicated groups.
Figure 8A Representative H&E staining (left) and pathological scores (right) of injured kidneys from mice received intervention treatment of saline (n = 4) or atorvastatin (20 mg/kg, i.g., first dose at 6-hours after ischemia, n = 5, or 14-hours after ischemia, n = 5).C, D Representative Oil Red O staining with quantification (C, scale bar = 100 μm), renal triglyceride and cholesterol levels (D) of saline (n = 4), 6 h-ATO (n = 5) and 14 h-ATO (n = 5) groups.E mRNA levels of fatty acid oxidation related genes of saline (n = 4) and 6 h-ATO (n = 5).representative immunoblots (H, left) with quantitative results (H, right) of Ces1 and G9a of saline (n = 4) and 6 h-ATO (n = 5).Representative CD3, Ly6G, and F4/80 quantitative results (K) of indicated groups.L Representative immunoblots (left) with quantitative results (right) of p-Rip3 and p-Mlkl for saline (n = 4) and 6 h-ATO (n = 5).
Figure 1(a) Haematoxylin and eosin (H & E) staining of kidneys from control (Flcnflox/flox), Flcn KO, Flcn/Tfeb DKO, Flcn/Tfe3 DKO, Flcn/Tfe3 DKO; Tfeb-HET and Flcn KO; Tfeb-HET mice at p18 (replicated three times). Scale bars, 3 mm (upper panels). Boxed areas are magnified in the bottom panels. Arrowhead indicates tubular papillary atypical hyperplasia. Scale bars, 100 μm (lower panels).(b) Blood urea nitrogen (BUN) levels in mice of the indicated genotypes at p18 (mean± SD, n=3). One Way ANOVA and the Tukey's HSD posthoc test (corrected for multiple comparisons) was applied. Significance for each comparison is provided in Methods.(c) Kaplan-Meyer survival analysis of the indicated genotypes (n=20 for each genotype); log-rank test, p-value < 0.0001.(d) Representative immunohistochemical (IHC) analysis of Tfeb or Tfe3 and Cadherin-16 (Cdh16) on adjacent kidney sections from mice of the indicated genotypes at p2. Tfeb and Tfe3 were stained in DAB, Cdh16 was stained in teal. Nuclei were stained with haematoxylin II (blue). Magnification: 10x, scale bar:100 µm. Magnification: 20x (in inset image), scale bar:25 µm. (e) Quantification of TFEB or TFE3 levels relative to cadherin 16 (indicated as ratio); values represent mean ± S.E.M (n=4 biological replicates for each genotype). A Welch's One Way ANOVA test with the Dunnett's T3 multiple comparisons test was applied. Significance for each comparison is provided in Methods. *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 2(a) Abdominal imaging, H&E staining and immunohistochemical staining for TFE3, TFEB, GPNMB and NPC1 in Hybrid RCC tumors from BHD patients 1 and 2, BHD Chromophobe RCC tumor (patient 3), BHD papillary/clear RCC (patients 4, 5 and 6), a BHD patient with clear/eosinophilic/papillary RCC (patient 7, origin of the BHD RCC cell line UOK257) and an area of normal kidney from the BHD patient 3. In the row 1-7, the arrows point to the tumor in the right or left kidney of BHD patients 1-7; in the bottom row, the arrow points to uninvolved, normal kidney in BHD patient 3.(d) Correlation between transcriptomic and proteomic analysis of BHD-associated kidney tumor samples (n=7 biological replicates) relative to control kidney tissues (n=5 biological replicates).
Figure 3Plots show tumor volumes in mice injected with UOK257/shLuc (n=8) or UOK257/shTFEB (n=10) (b) p value = 0.036 for plot in (b)Kaplan-Meier analysis of mice survival relative to the two experimental groups. Long-rank test, p < 0.0001. The median survival time of mice injected with UOK257/shLuc is 219.5 days in (c)Plots show tumor volumes in mice injected with UOK257/shLuc (n=8) or UOK257/shTFE3 (n=9) (e) *p= 0.023 for plot in (e)Kaplan-Meier analysis of mice survival relative to the two experimental groups. The median survival time of mice injected with UOK257/shLuc is 244.5 days in (f).
Figure 1B) SUMO2 immunoprecipitation using crosslinked SUMO2 antibody beads and liver samples of wild-type C57BL6/J mice in the fasted (16 h) or re-fed (16 h fasted and re-fed 2 h) states. Eluates analyzed by immunoblotting using anti-Prox1 antibodies and ponceau staining was used as loading control. Data information Data: mean ±SEM. Significance was determined by one-way ANOVA with Dunnett´s multiple comparison test relative to samples collected at ZT 12. *** P≤0.001.C) 8 weeks old C57BL/6N male mice were fasted (food removed at ZT 12) or re-fed (fasted from ZT4 to ZT12 for synchronization; food re-introduced at ZT 12). Tissue samples were collected at ZT 12, 15, 18 and 21 (n=4). Liver lysates analyzed by immunoblotting using anti-Prox1 and anti-P_S6K(Thr389) antibodies; β actin was detected as an input control. The quantification of SUMOylated Prox1 (%) and total Prox1 protein expression as well as Prox1 mRNA levels analyzed by qPCR are shown. qPCR data is presented as relative fold change normalized to the housekeeping gene TBP. Data information: Every dot represents one individual mouse. Data: mean ±SEM. Significance was determined by one-way ANOVA with Dunnett´s multiple comparison test relative to samples collected at ZT 12. *** P≤0.001.
Figure 2B) HepG2 cells overexpressing HA-tagged mouse wild-type Prox1 (wt), Prox1 K556R mutant or Prox1 E558A mutant. Cell lysates analyzed by immunoblotting using anti-HA antibodies; tubulin was detected as an input control.C) Mouse un-tagged Prox1 (mProx1) was purified from HEK293T cells. Purified mProx1 was incubated with recombinant E1 and E2 enzyme together with either SUMO1 or SUMO2. The enzymatic reactions were started with ATP and incubated for 15, 30 and 60 min. A reaction with the Prox1 K556R mutant (KR) was used as a control. The 0 min sample was incubated without ATP. Samples were analyzed by immunoblotting using anti-Prox1 antibodies.
Figure 3A) 6 weeks old C57BL/6N male mice were fed either a 0% cholesterol diet (ctrl) or a 2 % high cholesterol diet (chol 2%) for 6 weeks. B) 6 weeks old C57BL/6N male mice were fed either a standard chow diet or a 60 % high fat diet (HFD) for 8 weeks. C) 6 weeks old C57BL/6N male mice were fed either a standard chow diet or a 45% high fat + 20% w/v fructose diet (HF/hfD) for 12 weeks. (A-C) Liver samples were collected at ZT 15 in the fasted (11 h) and re-fed (fasted 8h and re-fed 3 h) states (n=4-5). Liver lysates were analyzed by immunoblotting using anti-Prox1 antibodies; β actin was detected as an input control. The quantification of SUMOylated Prox1 and total Prox1 protein expression are shown. Data information: Every dot represents one individual mouse. Data: mean ±SEM. Significance was determined by two-way ANOVA with Sidak´s multiple comparison test between different groups and conditions. *P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001.
Figure 4B) 8 weeks old Prox1K556R K.I. (f/f) male mice were injected with a control AAV (Ctrl_AAV), with an AAV to overexpress Cre recombinase (Cre_AAV) or with phosphate-buffered saline (PBS) (n=4). Liver tissue samples were collected 3 weeks later from mice fed ad libitum. Liver lysates were analyzed by immunoblotting using anti-Prox1 and anti-Cre antibodies; Vcp was detected as an input control. The quantification of SUMOylated Prox1 and total Prox1 protein expression as well as Prox1 mRNA levels analyzed by qPCR are shown. qPCR data presented as relative fold change normalized to the housekeeping gene TBP. Data information: (B) Every dot represents one individual mouse. Data: mean ±SEM. Significance was determined by one-way ANOVA with Tukey´s multiple comparison test between groups. *** P≤0.001, **** P≤0.0001.
Figure 5A) 8 weeks old Prox1K556R K.I. (f/f) male mice were injected with a control AAV (Ctrl_AAV) or with an AAV to overexpress Cre recombinase (Cre_AAV) and placed on a high-fat (45%) high-fructose (20% w/v) diet for 18 weeks. Liver samples were collected between ZT 16 and 17 in the fasted (8 h) and re-fed (fasted 8 h and re-fed 4-5 h) states (n=7-8). Liver lysates were analyzed by immunoblotting using anti-Prox1 and anti-Cre antibodies; β actin was detected as an input control. The quantification of SUMOylated Prox1 and total Prox1 protein expression are shown. Data information Every dot represents one individual mouse. Data: mean ±SEM. Significance was determined by two-way ANOVA with Sidak´s multiple comparison test between different groups and conditions. * P≤0.05, ** P≤0.01, **** P≤0.0001.B) Serum analysis using a colorimetric-based serum analyzer. Levels of total cholesterol, high density lipoprotein (HDL) and low density lipoprotein (LDL) are shown. Data information: Every dot represents one individual mouse. Data: mean ±SEM. Significance was determined by two-way ANOVA with Sidak´s multiple comparison test between different groups and conditions. * P≤0.05, ** P≤0.01, **** P≤0.0001.C) Equal amounts of serum were pooled and separated by FPLC, serum lipoprotein cholesterol profiles are shown.
Figure 6C) Heatmap displaying the expression of enriched genes involved in phase I, II and III of bile acid detoxification and bile acid synthesis in the liver of obese mice expressing the Prox1 K556R mutant (Cre_AAV) in comparison to obese mice expressing wild-type Prox1 (Ctrl_AAV) in the fasted state (n=4). The occupation scores according to a published Chip- sequencing analysis on Prox1 Cyp7a1 was used as a reference gene.D) Prox1/LRH-1 co-immunoprecipitation in HEK293A cells overexpressing HA-tagged Prox1 wild-type (wt) or K556R mutant (KR) and Myc-tagged LRH-1.
Figure 1(A) Proliferation analysis of hematopoietic CD34+ precursors for up to 6 days (section i).(A) Neutrophil morphologic differentiation were assessed at day 6 (section ii). Controls were without treatment. White arrows indicate neutrophilnuclear segmentation. A+G, Am80-GCSF combination; CL, chemiluminescence; RLU, relative light units; AUC, area under the curve.(A) Neutrophil bacterial killing were assessed at day 6 (section iii). Controls were without treatment. . A+G, Am80-GCSF combination; CL, chemiluminescence; RLU, relative light units; AUC, area under the curve.(A) Neutrophil ROS production were assessed at day 6 (iv). Controls were without treatment. A+G, Am80-GCSF combination; CL, chemiluminescence; RLU, relative light units; AUC, area under the curve.(B) qRT-PCR assessed RA-target gene expression after culturing CD34+ cells for 1, 2, and 6 days.(C) Flow cytometry analysis of CD66-CD18 co-expression after culturing CD34+ cells for 6 days, using anti-human CD66-PE and CD18-FITC antibodies (section i). Isotypes were used for controls (section i, top panel). CD66-CD18 co-expression was quantified in section ii.(D) Fresh peripheral blood (PB) collected from normal human donor. Bactericidal activities of neutrophils induced by Am80-GCSF from PB mononuclear cells were evaluated by bacterial killing assays (section iii).(D) Fresh peripheral blood (PB) collected from normal human donor. Bactericidal activities of neutrophils induced by Am80-GCSF from PB mononuclear cells were evaluated by ROS production (section ii).(D) Fresh peripheral blood (PB) collected from normal human donor. Bacterial killing was assessed in the presence or absence of PBneutrophils (section i).(E) Fresh PBneutrophils collected from normal human donor were tested for ROS production in the presence or absence of specific antibodies. Controls were with or without IgG. Normal IgG, φ; Ab, antibodies; Comb Ab, combined anti-CD18 and -CD11b antibodies.(F) Fresh PB mononuclear cells collected from normal human donors were treated for 3 days. Bacterial killing (sections i) and neutralization of bacterial killing in the presence of IgG or anti-CD18 antibody were assessed at day 3 (section ii).
Figure 2(A) Fresh bone marrow mononuclear cells were collected from acute myeloid leukemia (AML) patient. ROS production (section ii) were analyzed after cells were treated for 4 days.(A) Fresh bone marrow mononuclear cells were collected from acute myeloid leukemia (AML) patient. Proliferation (section i) were analyzed after cells were treated for 4 days.(B) Fresh BM mononuclear cells collected from AML patient were treated for up to 6 days. Proliferation was assessed at day 3 in parallel (section ii).(B) Fresh BM mononuclear cells collected from AML patient were treated for up to 6 days. ROS production were assessed at day 3 in parallel (section iii).(B) Fresh BM mononuclear cells collected from AML patient were treated for up to 6 days. Transcription of RA-target genes was analyzed with qRT-PCR at day 2, 3, and 6 (sections i).
Figure 3(A) Fresh BM mononuclear cells collected from AML patients were treated with 20 or 50 nM Am80 alone or combined with medium dose of GCSF (25 ng/ml) for up to 9 days. Dynamic changes in proliferation (left sections) and live cells on day 9 (right sections) were illustrated for each of specimens. Controls were without treatment.(B) Fresh PB or BM mononuclear cells collected from AML patients were treated with 20, 100, or 150 nM Am80 alone or combined with GCSF for up to 12 days. Dynamic changes in proliferation (left sections) and live cells on day 12 (right sections) were illustrated for each of specimens.(C) Fresh BM mononuclear cells collected from AML patients were treated with 150 nM Am80 alone or combined with GCSF. Proliferation (left sections) and bacterial killing (right sections) were analyzed on day 12 for each of samples.(D) Fresh BM mononuclear cells collected from AML patient were treated with 150 nM Am80 alone or combined with GCSF. Morphologic differentiation was assessed at day 12 (section iv). White arrows indicate neutrophil nuclear segmentation.(D) Fresh BM mononuclear cells collected from AML patient were treated with 150 nM Am80 alone or combined with GCSF. Bacterial killing was assessed at day 12 (sections iii).(D) Fresh BM mononuclear cells collected from AML patient were treated with 150 nM Am80 alone or combined with GCSF. RA-target gene expression was assessed at day 12 (section v).(B) Fresh BM mononuclear cells collected from AML patients were treated with 20, 100, or 150 nM Am80 alone or combined with GCSF for up to 12 days. ROS production was assessed at day 12 (section ii).(D) Fresh BM mononuclear cells collected from AML patient were treated with 150 nM Am80 alone or combined with GCSF. Proliferation was assessed for up to 12 days (section i).
Figure 7(A B) Neutrophil recovery and morphologic differentiation were assessed in BM (panel A) and PB (panel B) of CCIN mice surviving on day 9. White arrows indicate neutrophil nuclear segmentation.(C) PBneutrophil production and nuclear segmentation were assessed in control and Am80-GCSF mice surviving on day 13 (section i).(C) The increased spleen size on day 9 in Am80-GCSF mice dropped to the levels similar to those in control mice on day 13 (section iii).(C) The increased spleen weight on day 9 in Am80-GCSF mice dropped to the levels similar to those in control mice on day 13 (sections iii, iv).(C) PBneutrophil production and nuclear segmentation were assessed in control and Am80-GCSF mice surviving on day 13 (section ii).(D) Significant loss of body weight occurred in GCSFmice on day 9.(E) Ultrastructure analysis of neutrophils collected from mice surviving on day 11. Neutrophil from GCSFmice showed less granules (section i, images 1 vs. 2 or 3), more intracytoplasmic vacuoles (sections i, ii), and inner nuclear membrane dilation (sections i, iii). Pg: primary granules; Sg: secondary granules.
Figure 1(A) Survival graphs of newborn pups from breeding of Rab11aFl/+;Rab11b+/-;Villin-Cre and Rab11aFl/+;Rab11b+/- (or Rab11aFl/+;Rab11b-/-) mice. The total number of pups for each genotype was labeled next to the corresponding curve. The graph represents over 6 independent litters from three different mating pairs.(B) Western blots for Rab11a and Rab11b using small intestinal lysates prepared from adult wild type (WT, lane 1), Rab11aFl/Fl;Villin-CreER (aKO, lane 2) 2 days after tamoxifen treatment, Rab11b-/- (bKO, lane 3), and Rab11aFl/Fl;Rab11b-/-;Villin-CreER (DKO) mice 1-day (lane 4), 2-day (lane 5), and 3-day (lane 6) after tamoxifen treatment. All mice were given a single tamoxifen injection. β-actin was used as loading control. Results represent more than 3 independent experiments.(C) H. & E. staining of adult Rab11aFl/Fl;Rab11b-/-;Villin-CreER mice, before and after tamoxifen injection. Images represent jejunum tissues collected 1, 2, and 3 days following tamoxifen injection. Experiments were repeated over 5 times using independent litters (n>10). Scale bars, 50 μ(D) Alkaline phosphatase (AP) staining was performed on intestinal tissue sections of adult Rab11aFl/Fl;Rab11b-/-;Villin-CreER mice that were treated with corn oil or with tamoxifen. Results represent at least 3 independent experiments. Scale bars, 50 μm.(F) Alcian blue staining was performed on adult Rab11aFl/Fl;Rab11b-/-;Villin-CreER mice before or 1 to 3 days after tamoxifen injection. Results represent at least 3 independent experiments. Scale bars, 100 μm. (G) Quantification of the number of alcian blue positive cells per crypt-villus axis in mice of various genotypes. Data represent average values of approximately 10-15 different microscopic fields taken from 3 mice per condition.(H) Immunofluorescent staining for lysozyme and E-Cad was performed on adult Rab11aFl/Fl;Rab11b-/-;Villin-CreER mice before and after tamoxifen injection. Experiments represent 3 independent replicates. Scale bars, 50 μm. (I) Quantification of lysozyme fluorescent signal abundance per crypt in mice of various genotypes. Data represent approximately 10-15 different microscopic fields taken from 3 mice per condition.
Figure 2(B) Immunohistochemistry for Ki67 staining was performed on intestinal sections of adult Rab11aFl/Fl;Rab11b-/-;Villin-CreER mice before and after tamoxifen injection. Experiments represent 3 independent replicates. Scale bars, 50 μm. (C) Quantification of ratio of Ki67+ cells over total epithelial cells per crypt-villus axis in DKO mice before and after tamoxifen treatment. Data represent approximately 10-15 different microscopic fields taken from 3 mice per condition.(D) Immunofluorescent staining for pHH3 and E-Cad was performed on intestinal sections of adult Rab11aFl/Fl;Rab11b-/-;Villin-CreER mice before and after tamoxifen injection. Experiments represent 3 independent replicates. Scale bars, 50 μm. (E) Quantification of ratio of pHH3+ cells over total IECs per crypt in DKO mice before and after tamoxifen treatment. Data represent approximately 10-15 different microscopic fields taken from 3 mice per condition.(G) Representative Western blots for Olfm4 and pHH3 in intestinal tissue lysates of DKO mice before and after tamoxifen injection.(H) Immunohistochemistry for Olfm4 was performed on intestinal sections of adult Rab11aFl/Fl;Rab11b-/-;Villin-CreER mice before and after tamoxifen injection. Experiments represent 3 independent replicates. Scale bars, 50 μm. (I) Quantification of Olfm4+ cell number per crypt in DKO mice before and after tamoxifen treatment. Data represent approximately 10-15 different microscopic fields taken from 3 mice per condition.(J) Immunofluorescent staining for Sox9 and E-Cad in DKO mice before and after tamoxifen treatment. Scale bars, 50 μm. (K) Quantification of Sox9+ cell number per crypt in DKO mice before and after tamoxifen treatment. Data represent approximately 10-15 different microscopic fields taken from 3 mice per condition.(L) Immunohistochemistry for c-Myc was performed on intestinal sections of adult Rab11aFl/Fl;Rab11b-/-;Villin-CreER mice before and after tamoxifen injection. Experiments represent 3 independent replicates. Scale bars, 50 μm. (M) Quantification of c-Myc+ cell number per crypt-villus axis in DKO mice before and after tamoxifen treatment. Data represent approximately 10-15 different microscopic fields taken from 3 mice per condition.
Figure 3(F) High magnification images were taken from CIdU/IdU labeled WT and DKO mouse crypt regions to visualize double positive cells. White arrows point to shedding CIdU cells. Scale bars, 50 μm. (G-H) Quantification of IdU+ cells, and shedding CIdU+ cells per crypt-villus axis in WT and DKO mice. Data represent 10-15 different microscopic fields taken from 3 mice per condition.