rabbit anti human olfm4 antibody  (Novus Biologicals)

Journal: bioRxiv

Article Title: Decreased Colonic Activin Receptor-Like Kinase 1 Disrupts Epithelial Barrier integrity and is associated with a poor clinical outcome in Crohn’s disease

doi: 10.1101/2020.02.21.960070

Figure Lengend Snippet: (A) Proliferation- and stemness-related gene expression in colonic epithelial cells isolated from CD patients (N=15) and NIBD controls (N=12). Each gene expression was normalized to GAPDH . ( B ) Representative immunohistochemical images of OLFM4 expression in the colonic crypts of NIBD controls (left) and CD patients (right). The percentage of OLFM4 staining area in colonic crypts was compared between CD patients and NIBD controls (N=4 per group). *P<0.05, ***P<0.001. P-values were determined by Mann-Whitney test.

Article Snippet: Rabbit anti-human CA1 antibody, Rabbit anti-human OLFM4 antibody, and Mouse/Rabbit IgG VisUCyte HRP polymer antibody were purchased from Novus Biologicals (CO, USA), Cell Signaling Technology (MA, USA), and R&D Systems.

Techniques: Gene Expression, Isolation, Immunohistochemical staining, Expressing, Staining, MANN-WHITNEY

Journal: Nature

Article Title: RANK drives structured intestinal epithelial expansion during pregnancy

doi: 10.1038/s41586-024-08284-1

Figure Lengend Snippet: a , Representative images of anti-RANK immunostaining in mouse jejunal intestinal organoids from Rank WT and Rank Δ Vil mice. DAPI is shown to visualize nuclei. Scale bars, 50 μm. b , Proliferation assay in organoids without (control) or in the presence of recombinant mouse RANKL (rmRANKL; 50 ng/ml) as determined by an MTT assay at the indicated time points. Each plot represents MTT OD, pooled from at least two independent experiments. Group numbers at 2 days: n = 56 (control), n = 56 (rmRANKL); 3 days: n = 50 (control), n = 50 (rmRANKL); 5 days: n = 31 (control), n = 31 (rmRANKL) and 7 days: n = 32 (control), n = 31 (rmRANKL). c , Numbers of buds per intestinal organoid cultured in ENR medium with/without rmRANKL. Data were combined from three independent experiments. Group numbers at 3 days: n = 22 (control), n = 59 (rmRANKL); 4 days: n = 57 (control), n = 102 (rmRANKL) and 5 days: n = 32 (control), n = 66 (rmRANKL) per group. d , Representative images of jejunal organoids at passage 0 and passage 1 cultured in the absence (control) or presence of the indicated concentrations of rmRANKL. Scale bars, 100 μm. e , Ratios of organoid numbers after prolonged passaging in the absence (control) and presence of rmRANKL. Numbers of organoids were counted at each passage. Data from two independent experiments are shown. n = 8 (control), n = 8 (10 ng/ml RANKL), n = 8 (50 ng/ml RANKL), n = 8 (500 ng/ml RANKL). f , Total cell numbers, OLFM4 positive cells per organoid and ratios of OLFM4 positive cell in relation to the total cell number per each jejunal organoid cultured in ENR medium with/without rmRANKL. Data were combined from two independent experiments. 2 days: n = 31 (control), n = 37 (rmRANKL); 5 days: n = 29 (control), n = 29 (rmRANKL) per group. g , Gating strategy for detecting Lgr5-eGFP + cells using fluorescence-activated cell sorting (FACS). Viability was determined using the viability-dye described in the . FSC, forward scatter; SSC, side scatter. h , Representative FACS histograms of Lgr5 high cells and CD44 + cells isolated from Lgr5-eGFPiresCreER/+ jejunal organoids cultured without (control) and with rmRANKL (50 ng/ml) for the indicated times. Numbers of Lgr5 high cells and CD44 + cells among total viable organoid cells are indicated for each group. Data are representative of at least two independent experiments. i , Quantification of Lgr5 high , Lgr5 high CD44 + , and CD44 + cells per well in Lgr5-eGFPiresCreER/+ jejunal organoids cultured without (control) or with rmRANKL (50 ng/ml) for the indicated times. n = 36 (Control, 24hrs), n = 36 (RANKL, 24hrs), n = 21 (Control, 48hrs), n = 24 (RANKL, 48hrs). Data are mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant. One-way analysis of variance (ANOVA) with Tukey’s post hoc test ( b,c,f ); Two-tailed Student’s t-test ( i ). More details on statistics and reproducibility can be found in the .

Article Snippet: Human organoids were stained with anti-human OLFM4 antibodies (1:100; Cell Signaling, 14369) at 4 °C overnight and the TSA Fluorescence System (Alexa Fluor Tyramide SuperBoost Kit) was used according to the manufacturer’s protocol.

Techniques: Immunostaining, Proliferation Assay, Control, Recombinant, MTT Assay, Cell Culture, Passaging, Fluorescence, FACS, Isolation, Two Tailed Test

Journal: Nature

Article Title: RANK drives structured intestinal epithelial expansion during pregnancy

doi: 10.1038/s41586-024-08284-1

Figure Lengend Snippet: a , RANK and EPCAM intestinal staining in Rank WT and Rank Δ vil mice. Scale bars, 25 μm. b , Left, representative images of jejunal organoids cultured without (control) and with recombinant mouse RANKL (rmRANKL; 50 ng ml −1 ) for 3 days. Scale bars, 100 μm. Right, quantification of organoid areas after culture with or without rmRANKL for 3 days. n = 185 (control) and n = 222 (rmRANKL) from three independent experiments. c , Representative 3D images of Lgr5-eGFP;Ires-cre ER/+ organoids. Scale bars, 50 μm. d , Representative images of OLFM4 staining of control and rmRANKL-treated organoids. Scale bars, 50 μm. e , Single-cell log-normalized expression of the indicated anti-apoptotic genes ( y axis) in each cell type ( x axis) (control jejunal organoids versus organoids cultured with 50 ng ml −1 rmRANKL for 12 h). f , Organoids treated with or without rmRANKL (50 ng ml −1 ) were irradiated and cultured in WENR medium with a ROCK inhibitor (Y-27632; 10 μM) (see ) for 7 days. The numbers of surviving organoids from three independent experiments are shown. n = 22 (control and rmRANKL). g , Single-cell log-normalized expression of Bmp2 and the BMP targeted genes Id2 and Id3 ( y axis) in each cell type ( x axis). h , The ratio of organoid numbers cultured in the presence of rmRANKL (50 ng ml −1 ), DMSO (control) or with the BMP inhibitor (BMPi) LDN193189 (0.5 μM). The ratio of organoid numbers in the control + RANKL group was normalized to the control group, whereas the ratio of organoid numbers in the iBMP + RANKL group was normalized to the iBMP group. Data are combined from two experiments. n = 10 for each group shown. i , Representative images of rmRANKL-treated Lgr5-eGFP;Ires-cre ER/+ organoids that were cultured with recombinant mouse NOGGIN. Scale bars, 50 μm. In the indicated images, phalloidin stains actin filaments and DAPI stains nuclei. Data are mean ± s.e.m. Statistical analysis was performed using two-tailed Student’s t -tests ( b and f ), two-sided Wilcoxon rank-sum tests between samples, adjusted using Benjamini–Hochberg correction ( e and g ) and one-way analysis of variance (ANOVA) with Tukey’s post hoc test ( h ); **** P < 0.0001. Further details on statistics and reproducibility are provided in the .

Article Snippet: Human organoids were stained with anti-human OLFM4 antibodies (1:100; Cell Signaling, 14369) at 4 °C overnight and the TSA Fluorescence System (Alexa Fluor Tyramide SuperBoost Kit) was used according to the manufacturer’s protocol.

Techniques: Staining, Cell Culture, Control, Recombinant, Expressing, Irradiation, Two Tailed Test

Journal: Nature

Article Title: RANK drives structured intestinal epithelial expansion during pregnancy

doi: 10.1038/s41586-024-08284-1

Figure Lengend Snippet: a , Left panels, representative images of rmRANKL-treated (50 ng/ml) Lgr5-eGFPiresCre ER/+ organoids cultured without (control, DMSO solvent) or with the BMPi LDN193189 (0,5μM). Phalloidin indicates actin filaments, and DAPI shows nuclei. Scale bars, 50 μm. b , Quantification of the ratio of Lgr5 + cells per well in Lgr5-eGFPiresCreER/+ jejunal organoids cultured in the presence of rmRANKL (50 ng/ml) without (control, DMSO solvent) or with the BMP inhibitor (BMPi) LDN193189 (0,5μM) using FACS. Data points represent individual wells from at least 2 independent experiments. n = 6 (Control), n = 6 (Control+RANKL), n = 6 (BMPi), n = 6 (BMPi+RANKL). c , Quantification of the ratio of Lgr5 + cells per well in Lgr5-eGFPiresCreER/+ jejunal organoids cultured in the presence of rmRANKL (50 ng/ml) together with 1, 100, or 500 ng/ml of rmNOGGIN, determined using FACS. Data points represent individual wells pooled from at least 2 independent experiments. n = 6 (1 ng/ml NOGGIN), n = 6 (1 ng/ml NOGGIN + RANKL), n = 5 (100 ng/ml NOGGIN), n = 5 (100 ng/ml NOGGIN + RANKL), n = 5 (500 ng/ml NOGGIN), n = 5 (500 ng/ml NOGGIN + RANKL). d , Ratios of mouse jejunal organoid numbers cultured in the presence of rmRANKL (50 ng/ml) without (control, DMSO solvent) or with the GSK3 inhibitor CHIR 99021 (20μM). The numbers of organoids were counted at each passage. The ratio of organoid numbers in the Control + RANKL group was normalized to control organoids, whereas the ratio of organoid numbers in the CHIR + RANKL treated group was normalized to CHIR only treated organoids. Data are combined from 2 independent experiments. n = 11 for each group shown. e , Ratios of numbers of jejunal organoids from Rnf43 WT Znrf3 WT and Rnf43 Δ Vil Znrf3 Δ Vil mice after prolonged culture in the presence of rmRANKL (50 ng/ml). The numbers of organoids were counted at each passage. The ratio of organoid numbers in the RANKL treated Rnf43 WT Znrf3 WT organoids was normalized to untreated control Rnf43 WT Znrf3 WT organoids, whereas the ratio of organoid numbers in the RANKL treated Rnf43 ΔVil Znrf3 ΔVil group was normalized to control Rnf43 ΔVil Znrf3 ΔVil organoids. Data are from 2 independent experiments. n = 11 for each group shown. f , Sizes of mouse intestinal organoids generated from the lower small intestine (ileum) cultured in ENR medium (50 ng/ml EGF) with/without rmRANKL (50 ng/ml) for 3 days; n = 63 (control), n = 58 (rmRANKL) per group. Data are from 3 independent experiments. g,h , Sizes of intestinal organoids generated from the ileum cultured in EGF-reduced E low NR medium (50 pg/ml EGF) with/without rmRANKL (50 ng/ml) for 3 days ( n = 99 (control), n = 104 (rmRANKL)) and 5 days ( n = 89 (control), n = 78 (rmRANKL)). Data are from 3 independent experiments. i , Representative images of intestinal organoids generated from the ileum cultured in E low NR medium with/without rmRANKL for 5 days. Scale bars, 100 μm. j , Sizes of intestinal organoids generated from upper small intestine (jejunum) cultured in ENR medium (50 ng/ml EGF) with/without rmRANKL (50 ng/ml) for 3 days; n = 185 (control), n = 222 (rmRANKL). Data are combined from 3 independent experiments. Of note, these are the same data as shown in Fig. , for better comparison. k,l , Sizes of intestinal organoids generated from the jejunum cultured in E low NR medium (50 pg/ml EGF) with/without rmRANKL (50 ng/ml) for 3 days ( n = 74 (control), n = 76 (rmRANKL) per group) and 5 days ( n = 113 (control), n = 105 (rmRANKL) per group). Data are combined from 3 independent experiments. m , Representative images of jejunal organoids cultured in E low NR medium with/without rmRANK for 5 days. Scale bars, 100 μm. n , Representative images of Lgr5eGFPiresCreER/+ jejunal organoids cultured in E low NR medium with/without rmRANK for 5 days. Lgr5 positive stem cells are shown in green. Phalloidin (magenta) and DAPI (blue) are shown to visualize F-actin and nuclei, respectively. Scale bars, 25 μm. o , Representative images of OLFM4 immunostaining of jejunal organoids cultured in E low NR medium with/without rmRANK for 5 days. Phalloidin and DAPI are shown to visualize F-actin and nuclei, respectively. Note secreted OLFM4 in the lumen of the organoids as reported previously . Scale bars, 50 μm. p , Total cell numbers, numbers of OLFM4+ cells and ratios of OLFM4+ cells to total cell numbers per jejunal organoid cultured in E low NR medium with/without rmRANKL for 2 days ( n = 43 (control), n = 44 (rmRANKL)) and 5 days ( n = 44 (control), n = 30 (rmRANKL)). Data were combined from at least 2 independent experiments. q , Proposed mechanism for RANKL-induced cell survival of intestinal epithelial cells via induction of anti-apoptotic genes and NF-κB signalling and intestinal stem cell exhaustion via induction of BMP2. Data are mean ± s.e.m. **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant. One-way analysis of variance (ANOVA) with Tukey’s post hoc test ( b,c, d,e,p ); Two-tailed Student’s t-test ( f-h, j-l ). More details on statistics and reproducibility can be found in the .

Article Snippet: Human organoids were stained with anti-human OLFM4 antibodies (1:100; Cell Signaling, 14369) at 4 °C overnight and the TSA Fluorescence System (Alexa Fluor Tyramide SuperBoost Kit) was used according to the manufacturer’s protocol.

Techniques: Cell Culture, Control, Solvent, Generated, Comparison, Immunostaining, Two Tailed Test

Journal: Nature

Article Title: RANK drives structured intestinal epithelial expansion during pregnancy

doi: 10.1038/s41586-024-08284-1

Figure Lengend Snippet: a , Schematic outline of transgenic mice that conditionally express a constitutively active RANK mutant in the ROSA26 locus using gene targeting, termed LSL-caRANK mice. Crossing this line with Villin1Cre mice removes the stop cassette and thereby drives specific expression of the RANK transgene in the intestinal epithelium; these mice are termed caRANK vil-Tg . LSL, LoxP-STOP-LoxP; NeoR, neomycin resistance cassette; eGFP, enhanced green fluorescence protein. b , Representative images of eGFP expression in intestinal epithelial cells of caRANK vil-Tg mice and control mice, using cryo-sections. Phalloidin and DAPI are shown to visualize F-actin and nuclei, respectively. Scale bars, 50 μm. c , Small intestinal villi length, volume and surface areas of 3-week-old control (n = 6 mice, n = 126 villi analysed) and caRANK vil -Tg mice (n = 7/145). Each data point represents the average length, volume, and surface area of villi per mouse. The original data is the same as Fig. . d,e , Reduced cell death of small intestinal epithelial cells in caRANK vil-Tg mice. Representative images (left in d ) and quantification (right in d , e ) of cleaved caspase-3 (CLC3) immunostaining (arrows) in the small intestine are shown for three wks old control mice (n = 7 mice, n = 93 regions analysed) and caRANK vil -Tg mice (n = 8/126). Each data point represents the number of CLC3 positive cells in villi per 2 mm of the intestine ( d ) and the average number per mouse ( e ). Scale bars, 100 μm. f,g , Representative images (left in f ) and quantification of phosphor-Histone H3 (pHH3) immunostaining in the crypts of the upper small intestines from control and caRANK vil -Tg mice (right in f , g ). 3 wks old control mice (n = 3 mice, n = 254 crypts analysed) and caRANK vil -Tg mice (n = 4/395); 4 wks old control (n = 6/379) and caRANK vil -Tg mice (n = 4/351); and 5–8 wks old control (n = 4/556) and caRANK vil -Tg littermates (n = 4/487). Each data point represents the number of pHH3 positive cells per individual crypts ( f ) and average number per mouse ( g ). Scale bars, 20 μm. h , Kaplan–Meier survival curve of control ( n = 31) and caRANK vil -Tg ( n = 37) littermates. i , Numbers of OLFM4 + cells/ crypt in the indicated mice. Each data point represents the average length, volume, and surface area of villi per mouse in the indicated mice described in Fig. . j , Small intestinal villi length, volume and surface areas the indicated mice. Each data point represents the averaged length, volume, and surface area of villi per mouse. The original data is the same as Fig. . k,l , Numbers of villi (left) and crypts (right) per 2 mm were quantified in histological H&E sections of the upper small intestine from control (n = 4 mice, n = 48 regions) and caRANK vil -Tg (n = 4/46) mice at 3 and control (n = 5 mice, n = 63 regions) and caRANK vil -Tg (n = 5/56) mice at 3 and 5–8 wks of age. Each data point represents the number of Numbers of villi (left) and crypts (right) per 2 mm of the intestine ( k ) and the average number per mouse ( l ). m , Representative images of mouse jejunal organoids from 2 weeks old control caRANK vil -Tg mice, cultured in E low NR (50 pg/ml EGF) or ENR (50 ng/ml EGF) media for 3 days after the first passage. Scale bars, 100 μm. n , Representative images (left) of EdU + proliferating cells in control small intestinal organoids (n = 7) and caRANK vil -Tg mice-derived organoids (n = 6). Scale bars, 50 μm. Right; ratios of EdU labelled proliferating cells to all cells presents in each bud. The analysed bud number is n = 27 (control) and n = 24 ( caRANK vil -Tg ). o , Ratios of organoid numbers after prolonged culture of control- and caRANK vil -Tg mice-derived jejunal organoids. Numbers of organoids were counted at each passage. The ratio of organoid numbers in the caRANK vil -Tg mice-derived jejunal organoids was normalized to untreated control jejunal organoids. Data were combined from two independent experiments. n = 6 (control), n = 6 ( caRANK vil -Tg ). p , Representative images of control and caRANK vil -Tg mice-derived small intestinal organoids cultured with/without recombinant human RANKL (rhRANKL; 100, 500 ng/ml) for 4 days. Scale bars, 100 μm. q , Percentages of surviving control and caRANK vil -Tg jejunal organoids after irradiation, as compared to non-irradiated organoids of the same genotypes. Organoids were cultured in ENR medium and irradiated with 4 Gy, followed by subsequent culture for 7 days. Data are from two independent experiments. n = 5 (control), n = 5 ( caRANK vil -Tg ). AVG, average. Data are mean ± s.e.m. *P < 0.05; **P < 0.01; ****P < 0.0001; ns, not significant. Two-tailed Mann–Whitney U -test ( c,e,g,i,j,l,o,q); Kaplan–Meier survival curve with a log-rank test ( h ); Two-tailed Student’s t-test ( d,f,k,n ).

Article Snippet: Human organoids were stained with anti-human OLFM4 antibodies (1:100; Cell Signaling, 14369) at 4 °C overnight and the TSA Fluorescence System (Alexa Fluor Tyramide SuperBoost Kit) was used according to the manufacturer’s protocol.

Techniques: Transgenic Assay, Mutagenesis, Expressing, Fluorescence, Control, Immunostaining, Cell Culture, Derivative Assay, Recombinant, Irradiation, Two Tailed Test, MANN-WHITNEY

Journal: Nature

Article Title: RANK drives structured intestinal epithelial expansion during pregnancy

doi: 10.1038/s41586-024-08284-1

Figure Lengend Snippet: a , Representative 3D reconstruction of small intestine from 3-week-old control and caRANK vil -Tg mice (top). Grid spacing is 200 μm. Bottom, villus length, volume and surface areas of control ( n = 6 mice, n = 126 villi) and caRANK vil -Tg mice ( n = 7 mice, n = 145 villi analysed). b , Representative OLFM4 immunostaining in the small intestines of 3- and 5–8-week-old control and caRANK vil -Tg mice (top). Scale bars, 20 μm. Bottom, the numbers of OLFM4 + cells per crypt in control and caRANK vil -Tg mice were quantified at 3 weeks ( n = 3 mice, n = 234 crypts (control); n = 3 mice, n = 205 crypts ( caRANK vil -Tg )), 4 weeks ( n = 6 mice, n = 162 crypts (control); n = 4 mice and n = 110 crypts ( caRANK vil -Tg )) and at 5–8 weeks ( n = 4 mice, n = 203 crypts (control); n = 4 mice, n = 178 crypts ( caRANK vil -Tg )) of age. c , Representative 3D reconstruction of the small intestines of 5–8-week-old control and caRANK vil -Tg mice (top). Grid spacing is 200 μm. Bottom, the villus length, volume and surface areas of control ( n = 4 mice, n = 47 villi) and caRANK vil -Tg mice ( n = 5 mice, n = 65 villi). d , Representative OLFM4 immunostaining in the small intestines of 8-week-old caRANK vil -Tg;Traf6 fl/+ ( n = 3) and caRANK vil -Tg;Traf6 fl/fl ( n = 5) mice. Scale bars, 100 μm. e , The numbers of OLFM4 + cells per crypt and the villus length in caRANK vil -Tg;Traf6 fl/+ ( n = 3 mice, 172 crypts, 40 villi) and caRANK vil -Tg; Traf6 fl/fl ( n = 5 mice, 299 crypts, 167 villi) mice. f , Representative macroscopic images of the small intestines of Apc min/+ and caRANK vil -Tg;Apc min/+ mice. Scale bars, 7.5 mm. g , The ratio of tumoroid numbers cultured without (control) or with rmRANKL (50 ng ml −1 ) (top). The ratio of organoid numbers in the RANKL-treated group was normalized to the control group. Data were combined from two independent experiments. n = 4 (control) and n = 4 (rmRANKL). Bottom, representative images of tumoroids established from Apc min/+ mice at passage 0, 1 and 2, cultured in the absence (control) or presence of rmRANKL (50 ng ml −1 ). Scale bars, 100 μm. Each point represents the measurement of length, volume and surface area ( a (bottom), c (bottom) and e (top)), and the number of OLFM4 + cells per crypt ( b (bottom) and e (bottom)). Data are mean ± s.e.m. * P < 0.05; NS, not significant. Statistical analysis was performed using two-tailed Student’s t -tests ( a , c , e and g ) and one-way ANOVA with Tukey’s post hoc test ( b ). Further details on statistics and reproducibility are provided in the .

Article Snippet: Human organoids were stained with anti-human OLFM4 antibodies (1:100; Cell Signaling, 14369) at 4 °C overnight and the TSA Fluorescence System (Alexa Fluor Tyramide SuperBoost Kit) was used according to the manufacturer’s protocol.

Techniques: Control, Immunostaining, Cell Culture, Two Tailed Test

Journal: Nature

Article Title: RANK drives structured intestinal epithelial expansion during pregnancy

doi: 10.1038/s41586-024-08284-1

Figure Lengend Snippet: a-c , Alterations in the NF-κB, anti-apoptotic, and BMP pathways in jejunal organoids from caRANK vil -Tg mice (n = 4) compared to organoids from control mice (n = 4). Upper panels show GSEA enrichment plots. Bottom panels show heatmaps of the top 15 genes upregulated in caRANK vil -Tg organoids. Expression profiles of RANKL stimulated jejunal organoids were compared to non-stimulated (control) jejunal organoids cultured for the same time period. Total RNA was isolated from small intestinal organoids generated from two weeks old control and caRANK vil -Tg littermate (n = 4 for each group) and processed for RNA-seq. d , Differential gene expression analysis of RNA-seq data from mouse jejunal organoids derived from caRANK vil -Tg (n = 4) and control (n = 4) mice. Normalized CPM values of selected transcripts of anti-apoptotic genes, stem cell signature genes and BMP signalling genes are shown. e , Western blotting of phosphorylated IκB-α and total IκB-α in isolated intestinal epithelial cells from 3 wks old control (n = 3) and caRANK vil -Tg (n = 3) mice. β-Actin is shown as a loading control. For gel resource data, see Supplementary Fig. . f , Schematic outline of the proposed role of RANK/RANKL-induced stem cell proliferation and exhaustion. The figure was created with BioRender.com. g , Ratios of the numbers of organoids derived from Traf6 WT and Traf6 ΔVil mice after prolonged culture in the presence of rmRANKL (50 ng/ml). Numbers of organoids were counted at each passage. The ratio of organoid numbers in the RANKL Traf6 WT group was normalized to control Traf6 WT organoids, whereas the ratio of organoid numbers in the RANKL treated Traf6 ΔVil group was normalized to untreated control Traf6 ΔVil organoids. Data were combined from two independent experiments. n = 11 ( Traf6 WT ), n = 11 ( Traf6 WT + RANKL), n = 11 ( Traf6 ΔVil ), n = 11 ( Traf6 ΔVil + RANKL). h , Average length of villi and average number of OLFM4 + cells in each crypt per mouse from the indicated mice in the indicated mice described in Fig. . i , Kaplan–Meier survival curve of control ( n = 14), caRANK vil -Tg ( n = 8), Apc min/+ ( n = 13), and caRANK vil -Tg , Apc min/+ ( n = 11) mice. j , Numbers of macroscopic adenomas and k,l , tumour diameters in the small intestine and colon of 4 months old control Apc min/+ ( n = 5 mice) and caRANK vil -Tg , Apc min/+ ( n = 5) mice. Small intestines were divided equally into 4 parts from the proximal (duodenum, labelled 1) to the distal (ileum, labelled 4) and adenomas assessed for each region. Total tumour numbers in Apc min/+ mice; n = 29 in region 1, n = 53 in region 2, n = 156 in region 3, n = 231 in region 4, n = 469 in total, n = 14 in colon, and in caRANK vil -Tg , Apc min/+ mice ( n = 29/24/29/37/119/23). Each data point in k represents the measurements of the tumour diameter in individual tumours and in l represents the average measurement of the tumour diameters per mouse. Data are mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant. Enrichment-adjusted p-values (p-value), False Discovery Rates (FDR) and Normalized Enrichment Scores (NES) were calculated using two sided-fGSEA ( a-c ). Two-sided DESeq2 Wald tests, adjusted with the Benjamini–Hochberg procedure ( d ). One-way analysis of variance (ANOVA) with Tukey’s post hoc test ( g ); Two-tailed Mann–Whitney U -test ( h , j,l ); Kaplan–Meier survival curve with a log-rank test ( i ); Two-tailed Student’s t-test ( k ).

Article Snippet: Human organoids were stained with anti-human OLFM4 antibodies (1:100; Cell Signaling, 14369) at 4 °C overnight and the TSA Fluorescence System (Alexa Fluor Tyramide SuperBoost Kit) was used according to the manufacturer’s protocol.

Techniques: Control, Expressing, Cell Culture, Isolation, Generated, RNA Sequencing, Gene Expression, Derivative Assay, Western Blot, Two Tailed Test, MANN-WHITNEY

Journal: Nature

Article Title: RANK drives structured intestinal epithelial expansion during pregnancy

doi: 10.1038/s41586-024-08284-1

Figure Lengend Snippet: a , Mating strategy to generate intestinal epithelial cell specific Rank deleted mice (left) and anti-RANK immunostaining on intestinal cryo-sections from nulliparous control Rank WT and Rank Δ Vil mice (right). Scale bars, 50 μm. b , Quantitative RT–PCR analysis of Rank mRNA expression in isolated small intestinal epithelial cells from nulliparous control Rank WT (n = 3) and Rank ΔVil (n = 5) littermates. c , Representative immunostaining to detect CRE expression (blue) in the small intestine (left panel) and mammary gland (right panel) of Villin1-Cre mice. Paraffin sections were counterstained with hematoxylin. Scale bars, 50 μm. d , Representative whole mount images (hematoxylin staining) of mammary glands from nulliparous Rank WT , nulliparous Rank ΔVil , pregnant Rank WT (day 18.5 of pregnancy, P18.5), pregnant Rank ΔVil females, lactating Rank WT (5 days after delivery, L5) and lactating L5 Rank ΔVil females. Scale bars, 500 μm. e , Small intestinal villi length, volume and surface areas in indicated mice. Each data point represents average villi length, volume and surface area per mouse in the indicated mice described in Fig. . f , Quantification of the average number of OLFM4 + cells in each crypt per mouse in the indicated mice described in Fig. . g,h , Cell death of villous epithelial cells from nulliparous Rank WT and Rank ΔVil females and age-matched lactating (L5) Rank WT and Rank ΔVil dams, as determined by immunostaining of cleaved caspase 3 (CLC3). Representative immunostaining. Scale bars, 100 μm (left in g ). Quantification of CLC3 positive cells in villi per 2 mm of the intestine in nulliparous Rank WT (n = 4 mice, n = 49 regions analysed) and Rank ΔVil females (n = 5/59) and age-matched lactating (L5) Rank WT (n = 8/162) and Rank ΔVil dams (n = 8/135) (right in g and h ). Each data point represents the numbers of CLC3 positive cells in villi per each 2 mm of the intestine ( g ) and average number per mouse ( h ). i,j , Quantification of the EdU-labelled intestinal epithelial cell migration along the crypt-villus axis in nulliparous Rank WT (n = 3 mice, n = 29 crypt-villus axis analysed) and Rank ΔVil females (n = 3/37) and age-matched lactating (L5) Rank WT (n = 4/52) and Rank ΔVil dams (n = 4/62). Tissues were harvested 24 h after EdU administration. Each data point represents the measurements of the migration distance in individual crypt-villus axis ( i ) and the average distance per mouse ( j ). k , Representative images of jejunal organoids generated from Rank WT and Rank ΔVil mice. Scale bars, 100 μm. l , Ratios of the number of jejunal organoids derived from Rank WT and Rank ΔVil mice after prolonged culture in the presence of rmRANKL (50 ng/ml). The number of organoids was counted at each passage. The ratio of organoid numbers in the Rank WT + RANKL group was normalized to control Rank WT organoids, whereas the ratio of organoid numbers in the Rank ΔVil + RANKL group was normalized to untreated Rank ΔVil organoids. Data are from two independent experiments. n = 6 ( Rank WT ), n = 6 ( Rank WT + RANKL), n = 6 ( Rank ΔVil ), n = 6 ( Rank ΔVil + RANKL). m,n , Representative hematoxylin and eosin (H&E) stained images of the upper small intestine (left in m ) and quantification of upper small intestinal villus lengths (right in m , n ) in age matched wild type nulliparous females (n = 4 mice, n = 310 villi analysed), pregnant females (P18.5) (n = 4/272), lactating dams (L5, 5 days after delivery) (n = 4/212) and females 6 weeks after weaning of the offspring (n = 4/49). Scale bars, 100 μm. Each data point represents the length of individual villi ( m ) and average length per mouse ( n ). Data are mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant. Two-tailed Mann–Whitney U -test ( e,f,h,j,n); One-way analysis of variance (ANOVA) with Tukey’s post hoc test ( g,i,l,m ).

Article Snippet: Human organoids were stained with anti-human OLFM4 antibodies (1:100; Cell Signaling, 14369) at 4 °C overnight and the TSA Fluorescence System (Alexa Fluor Tyramide SuperBoost Kit) was used according to the manufacturer’s protocol.

Techniques: Immunostaining, Control, Quantitative RT-PCR, Expressing, Isolation, Staining, Migration, Generated, Derivative Assay, Two Tailed Test, MANN-WHITNEY

Journal: Nature

Article Title: RANK drives structured intestinal epithelial expansion during pregnancy

doi: 10.1038/s41586-024-08284-1

Figure Lengend Snippet: a , Representative 3D reconstruction of the upper small intestine from nulliparous and lactating (5 days after delivery, L5) Rank WT and Rank Δvil female mice (top). Grid spacing is 200 μm. Bottom, the small intestinal villus length, volume and surface areas in nulliparous Rank WT ( n = 5 mice, n = 88 villi), nulliparous Rank Δvil ( n = 5 mice, n = 97 villi), P18.5 Rank WT ( n = 6 mice, n = 89 villi), P18.5 Rank Δvil ( n = 6 mice, n = 93 villi), L5 Rank WT ( n = 5 mice, n = 91 villi) and L5 Rank Δvil ( n = 6 mice, n = 141 villi) mice. b , Representative OLFM4 immunostaining in the small intestines of the indicated mice (top). Scale bars, 20 μm. Bottom, quantification of OLFM4 + cells per crypt in nulliparous Rank WT ( n = 3 mice, n = 87 crypts), nulliparous Rank Δvil ( n = 4 mice, n = 130 crypts), L5 Rank WT ( n = 6 mice, n = 160 crypts) and L5 Rank Δvil ( n = 7 mice, n = 181 crypts) mice, and Rank WT ( n = 4 mice, n = 59 crypts) and Rank Δvil ( n = 5 mice, n = 44 crypts) mice 6 weeks after weaning of the offspring. c , Representative 3D images (left) and villi lengths of small intestines from age-matched nulliparous ( n = 3 mice, n = 70 villi analysed) and lactating ( n = 5 mice, n = 94 villi) germ-free C57BL6 mice (right). Grid spacing is 200 μm. d , Left, representative small intestinal sections (haematoxylin and eosin (H&E) staining) of L5 wild-type mice and wild-type mice whose offspring were removed on 1 day after delivery (no lactation). Scale bars, 100 μm. Right, quantification of villus length in L5 dams (with offspring) ( n = 7 mice, n = 170 villi) and female mice with their offspring removed (without offspring) ( n = 5 mice, n = 158 villi). e , Representative small intestinal sections (H&E staining) (left) and quantification of villi lengths (right) of L5 wild-type mice treated with DMSO or cabergoline (5 mg per kg) for 5 consecutive days starting on the day of delivery. Villi were assessed on day 5 after delivery. Scale bars, 100 μm. Right, quantification of villus length in mice treated with DMSO ( n = 4 mice, n = 148 villi) and mice treated with cabergoline ( n = 4 mice, n = 179 villi). f , Representative H&E-stained intestinal sections from nulliparous Traf6 WT and Traf6 Δvil mice, and L5 lactating Traf6 WT and Traf6 Δvil mice (left). Scale bars, 100 μm. Right, quantification of villous length in nulliparous Traf6 WT ( n = 4 mice, n = 76 villi) and Traf6 Δvil ( n = 5 mice, n = 73 villi) mice, and L5 Traf6 WT ( n = 6 mice, n = 198 villi) and Traf6 Δvil ( n = 5 mice, n = 226 villi) mice. Data are mean ± s.e.m. Each point represents the measurement of length, volume and surface area ( a (bottom) and c – f (right)), and the number of OLFM4 + cells per crypt ( b , bottom). Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test ( a , b and f ) and two-tailed Student’s t -tests ( c – e ); ** P < 0.01, *** P < 0.001.

Article Snippet: Human organoids were stained with anti-human OLFM4 antibodies (1:100; Cell Signaling, 14369) at 4 °C overnight and the TSA Fluorescence System (Alexa Fluor Tyramide SuperBoost Kit) was used according to the manufacturer’s protocol.

Techniques: Immunostaining, Staining, Two Tailed Test

Journal: Nature

Article Title: RANK drives structured intestinal epithelial expansion during pregnancy

doi: 10.1038/s41586-024-08284-1

Figure Lengend Snippet: a , Uniform manifold approximation and projection (UMAP) of 2,265 human intestinal epithelial cells. Data were taken from Fujii et al. . Cells are colour coded according to epithelial cell-type annotation based on unsupervised clustering. b , Violin plots show single cell log-normalized expression of RANK and RANKL in each intestinal cell-type. Each dot represents an individual cell. c , Quantitative RT–PCR analyses to compare expression levels of anti-apoptotic genes, stem cell signature genes, and BMP signalling genes in human duodenal organoids. Data represent the relative expression of the indicated genes in rhRANKL (500 ng/ml) stimulated duodenal organoids compared to control (no RANKL) organoids (set at 1). rhRANKL stimulation was for 12 and 96 h. n = 4 (control), n = 4 (RANKL). d , Representative images (left) and quantified areas (right) of human duodenal organoids cultured for 2 days without (control; n = 83) and with recombinant human RANKL (rhRANKL; 500 ng/ml; n = 54) in growth-factor-reduced medium (GFR medium) lacking EGF, IGF-1, and FGF-2. Scale bars, 100 μm. Each dot represents an organoid, assessed in three independent experiments. e , RANKL-induced proliferation of duodenal organoids. Organoids were left untreated (control) or treated with rhRANKL (500 ng/ml) and proliferation determined using the MTT assay. Each plot represents an MTT OD, pooled from two independent experiments. n = 20 (control), n = 20 (RANKL). f , Representative cell cycle FACS plots (left) and quantification of S-phase and subG1 + G1 entry (right) assessing EdU labelled human duodenal organoids cultured in GFR medium and stimulated with rhRANKL for 24 and 72 h. Dots represent individual organoids, assessed in three independent experiments. n = 14 (control, 24hrs), n = 16 (control, 72hrs), n = 18 (rmRANKL, 24hrs), n = 18 (rmRANKL, 72hrs). g , Representative images of anti-OLFM4 immunostaining (green) of human duodenal organoids cultured in GFR medium in the absence (control) and presence of rhRANKL (500 ng/ml) for 24 and 96 h. Organoids were counterstained with DAPI (blue) to detect nuclei and Phalloidin (magenta) to detect filamentous actin. Scale bars, 50 μm. h , Representative images of OLFM4 + stem cells in human intestinal organoids cultured in the presence of rhRANKL (500 ng/ml) without (control, DMSO solvent) or with the BMP inhibitor (BMPi) LDN193189 (1.6μM) for 96 hrs. Organoids were stained with DAPI (blue) to detect nuclei and Phalloidin to detect filamentous actin. Scale bars, 50 μm. i , Proposed function of RANK–RANKL in the small intestine during pregnancy and lactation. During pregnancy and lactation, RANK–RANKL signalling promotes intestinal stem cell proliferation and differentiation as well as intestinal cell survival, ultimately resulting in massive villous expansion. Villous expansion facilitates nutritional uptake, which is important for nourishment of offspring as well as their transgenerational metabolic health. The figure was created with BioRender.com. Data are mean ± s.e.m. **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant. Two-tailed Student’s t-test ( c-f) . More details on statistics and reproducibility can be found in the .

Article Snippet: Human organoids were stained with anti-human OLFM4 antibodies (1:100; Cell Signaling, 14369) at 4 °C overnight and the TSA Fluorescence System (Alexa Fluor Tyramide SuperBoost Kit) was used according to the manufacturer’s protocol.

Techniques: Expressing, Quantitative RT-PCR, Control, Cell Culture, Recombinant, MTT Assay, Immunostaining, Solvent, Staining, Two Tailed Test

Journal: Nature

Article Title: RANK drives structured intestinal epithelial expansion during pregnancy

doi: 10.1038/s41586-024-08284-1

Figure Lengend Snippet: a , Human duodenal organoids were cultured without (control) and with recombinant human RANKL (rhRANKL; 500 ng ml −1 ) for 2 days. Left, representative images. Scale bars, 100 μm. Right, quantification of organoid sizes. n = 83 (control) and n = 83 (rhRANKL). Data were combined from three independent experiments. b , Organoids were cultured with or without rhRANKL (500 ng ml −1 ) for 2 days and then irradiated and cultured for additional 7 days. Data show the number of surviving organoids combined from three independent experiments. n = 12 (control) and n = 12 (rhRANKL). c , Cell cycle analyses of RANK-stimulated human duodenal organoids. Left, representative fluorescence-activated cell sorting (FACS) plots of the EdU cell cycle analysis assessed at 24 h and 72 h of rhRANKL stimulation. Right, quantification of S phase and subG1 + G1 entry. Data were combined from two independent experiments. n = 12 for each condition. d , The ratio of the numbers of wild-type (WT) and BMPR1A -knockout ( BMPR1A -KO) human small intestinal organoids in the absence or presence of rhRANKL (500 ng ml −1 ). The ratio of organoid numbers in the WT + RANKL group was normalized to untreated WT organoids; and the ratio of organoid numbers in the BMPR1A- KO + RANKL organoids was normalized to the untreated BMPR1A- KO organoids. n = 3 for each condition. e , Cell cycle analyses of RANK-stimulated duodenal organoids in the absence (control) and presence of the BMPi LDN193189 (1.6 μM). Left, representative FACS plots depicting EdU cell cycle analysis after 72 h rhRANKL stimulation with or without BMPi. Right, quantification of S phase and sbG1 + G1 entry. n = 11 (control), n = 12 (control + RANKL), n = 12 (BMPi) and n = 12 (BMPi + RANKL). Data were combined from two independent experiments. f , Representative images of OLFM4 + stem cells in WT and BMPR1A -KO human small intestinal organoids with or without rhRANKL (500 ng ml −1 ). Scale bars, 50 μm. Dots represent individual datapoints. Data are mean ± s.e.m. Statistical analysis was performed using two-tailed Student’s t -tests ( a – c ) and one-way ANOVA with Tukey’s post hoc test ( d and e ). Further details on statistics and reproducibility are provided in the .

Article Snippet: Human organoids were stained with anti-human OLFM4 antibodies (1:100; Cell Signaling, 14369) at 4 °C overnight and the TSA Fluorescence System (Alexa Fluor Tyramide SuperBoost Kit) was used according to the manufacturer’s protocol.

Techniques: Cell Culture, Control, Recombinant, Irradiation, Fluorescence, FACS, Cell Cycle Assay, Knock-Out, Two Tailed Test

Journal: Nature

Article Title: RANK drives structured intestinal epithelial expansion during pregnancy

doi: 10.1038/s41586-024-08284-1

Figure Lengend Snippet: a , Representative images of anti-RANK immunostaining in mouse jejunal intestinal organoids from Rank WT and Rank Δ Vil mice. DAPI is shown to visualize nuclei. Scale bars, 50 μm. b , Proliferation assay in organoids without (control) or in the presence of recombinant mouse RANKL (rmRANKL; 50 ng/ml) as determined by an MTT assay at the indicated time points. Each plot represents MTT OD, pooled from at least two independent experiments. Group numbers at 2 days: n = 56 (control), n = 56 (rmRANKL); 3 days: n = 50 (control), n = 50 (rmRANKL); 5 days: n = 31 (control), n = 31 (rmRANKL) and 7 days: n = 32 (control), n = 31 (rmRANKL). c , Numbers of buds per intestinal organoid cultured in ENR medium with/without rmRANKL. Data were combined from three independent experiments. Group numbers at 3 days: n = 22 (control), n = 59 (rmRANKL); 4 days: n = 57 (control), n = 102 (rmRANKL) and 5 days: n = 32 (control), n = 66 (rmRANKL) per group. d , Representative images of jejunal organoids at passage 0 and passage 1 cultured in the absence (control) or presence of the indicated concentrations of rmRANKL. Scale bars, 100 μm. e , Ratios of organoid numbers after prolonged passaging in the absence (control) and presence of rmRANKL. Numbers of organoids were counted at each passage. Data from two independent experiments are shown. n = 8 (control), n = 8 (10 ng/ml RANKL), n = 8 (50 ng/ml RANKL), n = 8 (500 ng/ml RANKL). f , Total cell numbers, OLFM4 positive cells per organoid and ratios of OLFM4 positive cell in relation to the total cell number per each jejunal organoid cultured in ENR medium with/without rmRANKL. Data were combined from two independent experiments. 2 days: n = 31 (control), n = 37 (rmRANKL); 5 days: n = 29 (control), n = 29 (rmRANKL) per group. g , Gating strategy for detecting Lgr5-eGFP + cells using fluorescence-activated cell sorting (FACS). Viability was determined using the viability-dye described in the . FSC, forward scatter; SSC, side scatter. h , Representative FACS histograms of Lgr5 high cells and CD44 + cells isolated from Lgr5-eGFPiresCreER/+ jejunal organoids cultured without (control) and with rmRANKL (50 ng/ml) for the indicated times. Numbers of Lgr5 high cells and CD44 + cells among total viable organoid cells are indicated for each group. Data are representative of at least two independent experiments. i , Quantification of Lgr5 high , Lgr5 high CD44 + , and CD44 + cells per well in Lgr5-eGFPiresCreER/+ jejunal organoids cultured without (control) or with rmRANKL (50 ng/ml) for the indicated times. n = 36 (Control, 24hrs), n = 36 (RANKL, 24hrs), n = 21 (Control, 48hrs), n = 24 (RANKL, 48hrs). Data are mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant. One-way analysis of variance (ANOVA) with Tukey’s post hoc test ( b,c,f ); Two-tailed Student’s t-test ( i ). More details on statistics and reproducibility can be found in the .

Article Snippet: The sections were blocked for 1 h in 5% BSA (VWR Life Science) and 10% goat serum (Sigma-Aldrich, 9023) and incubated with primary antibodies against phospho-histone H3 (1:100; CellPath, PBC-ACI3130C), mouse OLFM4 (1:800; Cell Signaling Technology, 39141), human OLFM4 (1:100; Cell Signaling Technology, 14369), cleaved caspase-3 (1:100; Cell Signaling Technology, 9661) or CRE (1:100, Cell Signaling Technology, 15036), all diluted in blocking solution.

Techniques: Immunostaining, Proliferation Assay, Control, Recombinant, MTT Assay, Cell Culture, Passaging, Fluorescence, FACS, Isolation, Two Tailed Test

Journal: Nature

Article Title: RANK drives structured intestinal epithelial expansion during pregnancy

doi: 10.1038/s41586-024-08284-1

Figure Lengend Snippet: a , RANK and EPCAM intestinal staining in Rank WT and Rank Δ vil mice. Scale bars, 25 μm. b , Left, representative images of jejunal organoids cultured without (control) and with recombinant mouse RANKL (rmRANKL; 50 ng ml −1 ) for 3 days. Scale bars, 100 μm. Right, quantification of organoid areas after culture with or without rmRANKL for 3 days. n = 185 (control) and n = 222 (rmRANKL) from three independent experiments. c , Representative 3D images of Lgr5-eGFP;Ires-cre ER/+ organoids. Scale bars, 50 μm. d , Representative images of OLFM4 staining of control and rmRANKL-treated organoids. Scale bars, 50 μm. e , Single-cell log-normalized expression of the indicated anti-apoptotic genes ( y axis) in each cell type ( x axis) (control jejunal organoids versus organoids cultured with 50 ng ml −1 rmRANKL for 12 h). f , Organoids treated with or without rmRANKL (50 ng ml −1 ) were irradiated and cultured in WENR medium with a ROCK inhibitor (Y-27632; 10 μM) (see ) for 7 days. The numbers of surviving organoids from three independent experiments are shown. n = 22 (control and rmRANKL). g , Single-cell log-normalized expression of Bmp2 and the BMP targeted genes Id2 and Id3 ( y axis) in each cell type ( x axis). h , The ratio of organoid numbers cultured in the presence of rmRANKL (50 ng ml −1 ), DMSO (control) or with the BMP inhibitor (BMPi) LDN193189 (0.5 μM). The ratio of organoid numbers in the control + RANKL group was normalized to the control group, whereas the ratio of organoid numbers in the iBMP + RANKL group was normalized to the iBMP group. Data are combined from two experiments. n = 10 for each group shown. i , Representative images of rmRANKL-treated Lgr5-eGFP;Ires-cre ER/+ organoids that were cultured with recombinant mouse NOGGIN. Scale bars, 50 μm. In the indicated images, phalloidin stains actin filaments and DAPI stains nuclei. Data are mean ± s.e.m. Statistical analysis was performed using two-tailed Student’s t -tests ( b and f ), two-sided Wilcoxon rank-sum tests between samples, adjusted using Benjamini–Hochberg correction ( e and g ) and one-way analysis of variance (ANOVA) with Tukey’s post hoc test ( h ); **** P < 0.0001. Further details on statistics and reproducibility are provided in the .

Article Snippet: The sections were blocked for 1 h in 5% BSA (VWR Life Science) and 10% goat serum (Sigma-Aldrich, 9023) and incubated with primary antibodies against phospho-histone H3 (1:100; CellPath, PBC-ACI3130C), mouse OLFM4 (1:800; Cell Signaling Technology, 39141), human OLFM4 (1:100; Cell Signaling Technology, 14369), cleaved caspase-3 (1:100; Cell Signaling Technology, 9661) or CRE (1:100, Cell Signaling Technology, 15036), all diluted in blocking solution.

Techniques: Staining, Cell Culture, Control, Recombinant, Expressing, Irradiation, Two Tailed Test

Journal: Nature

Article Title: RANK drives structured intestinal epithelial expansion during pregnancy

doi: 10.1038/s41586-024-08284-1

Figure Lengend Snippet: a , Left panels, representative images of rmRANKL-treated (50 ng/ml) Lgr5-eGFPiresCre ER/+ organoids cultured without (control, DMSO solvent) or with the BMPi LDN193189 (0,5μM). Phalloidin indicates actin filaments, and DAPI shows nuclei. Scale bars, 50 μm. b , Quantification of the ratio of Lgr5 + cells per well in Lgr5-eGFPiresCreER/+ jejunal organoids cultured in the presence of rmRANKL (50 ng/ml) without (control, DMSO solvent) or with the BMP inhibitor (BMPi) LDN193189 (0,5μM) using FACS. Data points represent individual wells from at least 2 independent experiments. n = 6 (Control), n = 6 (Control+RANKL), n = 6 (BMPi), n = 6 (BMPi+RANKL). c , Quantification of the ratio of Lgr5 + cells per well in Lgr5-eGFPiresCreER/+ jejunal organoids cultured in the presence of rmRANKL (50 ng/ml) together with 1, 100, or 500 ng/ml of rmNOGGIN, determined using FACS. Data points represent individual wells pooled from at least 2 independent experiments. n = 6 (1 ng/ml NOGGIN), n = 6 (1 ng/ml NOGGIN + RANKL), n = 5 (100 ng/ml NOGGIN), n = 5 (100 ng/ml NOGGIN + RANKL), n = 5 (500 ng/ml NOGGIN), n = 5 (500 ng/ml NOGGIN + RANKL). d , Ratios of mouse jejunal organoid numbers cultured in the presence of rmRANKL (50 ng/ml) without (control, DMSO solvent) or with the GSK3 inhibitor CHIR 99021 (20μM). The numbers of organoids were counted at each passage. The ratio of organoid numbers in the Control + RANKL group was normalized to control organoids, whereas the ratio of organoid numbers in the CHIR + RANKL treated group was normalized to CHIR only treated organoids. Data are combined from 2 independent experiments. n = 11 for each group shown. e , Ratios of numbers of jejunal organoids from Rnf43 WT Znrf3 WT and Rnf43 Δ Vil Znrf3 Δ Vil mice after prolonged culture in the presence of rmRANKL (50 ng/ml). The numbers of organoids were counted at each passage. The ratio of organoid numbers in the RANKL treated Rnf43 WT Znrf3 WT organoids was normalized to untreated control Rnf43 WT Znrf3 WT organoids, whereas the ratio of organoid numbers in the RANKL treated Rnf43 ΔVil Znrf3 ΔVil group was normalized to control Rnf43 ΔVil Znrf3 ΔVil organoids. Data are from 2 independent experiments. n = 11 for each group shown. f , Sizes of mouse intestinal organoids generated from the lower small intestine (ileum) cultured in ENR medium (50 ng/ml EGF) with/without rmRANKL (50 ng/ml) for 3 days; n = 63 (control), n = 58 (rmRANKL) per group. Data are from 3 independent experiments. g,h , Sizes of intestinal organoids generated from the ileum cultured in EGF-reduced E low NR medium (50 pg/ml EGF) with/without rmRANKL (50 ng/ml) for 3 days ( n = 99 (control), n = 104 (rmRANKL)) and 5 days ( n = 89 (control), n = 78 (rmRANKL)). Data are from 3 independent experiments. i , Representative images of intestinal organoids generated from the ileum cultured in E low NR medium with/without rmRANKL for 5 days. Scale bars, 100 μm. j , Sizes of intestinal organoids generated from upper small intestine (jejunum) cultured in ENR medium (50 ng/ml EGF) with/without rmRANKL (50 ng/ml) for 3 days; n = 185 (control), n = 222 (rmRANKL). Data are combined from 3 independent experiments. Of note, these are the same data as shown in Fig. , for better comparison. k,l , Sizes of intestinal organoids generated from the jejunum cultured in E low NR medium (50 pg/ml EGF) with/without rmRANKL (50 ng/ml) for 3 days ( n = 74 (control), n = 76 (rmRANKL) per group) and 5 days ( n = 113 (control), n = 105 (rmRANKL) per group). Data are combined from 3 independent experiments. m , Representative images of jejunal organoids cultured in E low NR medium with/without rmRANK for 5 days. Scale bars, 100 μm. n , Representative images of Lgr5eGFPiresCreER/+ jejunal organoids cultured in E low NR medium with/without rmRANK for 5 days. Lgr5 positive stem cells are shown in green. Phalloidin (magenta) and DAPI (blue) are shown to visualize F-actin and nuclei, respectively. Scale bars, 25 μm. o , Representative images of OLFM4 immunostaining of jejunal organoids cultured in E low NR medium with/without rmRANK for 5 days. Phalloidin and DAPI are shown to visualize F-actin and nuclei, respectively. Note secreted OLFM4 in the lumen of the organoids as reported previously . Scale bars, 50 μm. p , Total cell numbers, numbers of OLFM4+ cells and ratios of OLFM4+ cells to total cell numbers per jejunal organoid cultured in E low NR medium with/without rmRANKL for 2 days ( n = 43 (control), n = 44 (rmRANKL)) and 5 days ( n = 44 (control), n = 30 (rmRANKL)). Data were combined from at least 2 independent experiments. q , Proposed mechanism for RANKL-induced cell survival of intestinal epithelial cells via induction of anti-apoptotic genes and NF-κB signalling and intestinal stem cell exhaustion via induction of BMP2. Data are mean ± s.e.m. **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant. One-way analysis of variance (ANOVA) with Tukey’s post hoc test ( b,c, d,e,p ); Two-tailed Student’s t-test ( f-h, j-l ). More details on statistics and reproducibility can be found in the .

Article Snippet: The sections were blocked for 1 h in 5% BSA (VWR Life Science) and 10% goat serum (Sigma-Aldrich, 9023) and incubated with primary antibodies against phospho-histone H3 (1:100; CellPath, PBC-ACI3130C), mouse OLFM4 (1:800; Cell Signaling Technology, 39141), human OLFM4 (1:100; Cell Signaling Technology, 14369), cleaved caspase-3 (1:100; Cell Signaling Technology, 9661) or CRE (1:100, Cell Signaling Technology, 15036), all diluted in blocking solution.

Techniques: Cell Culture, Control, Solvent, Generated, Comparison, Immunostaining, Two Tailed Test

Journal: Nature

Article Title: RANK drives structured intestinal epithelial expansion during pregnancy

doi: 10.1038/s41586-024-08284-1

Figure Lengend Snippet: a , Schematic outline of transgenic mice that conditionally express a constitutively active RANK mutant in the ROSA26 locus using gene targeting, termed LSL-caRANK mice. Crossing this line with Villin1Cre mice removes the stop cassette and thereby drives specific expression of the RANK transgene in the intestinal epithelium; these mice are termed caRANK vil-Tg . LSL, LoxP-STOP-LoxP; NeoR, neomycin resistance cassette; eGFP, enhanced green fluorescence protein. b , Representative images of eGFP expression in intestinal epithelial cells of caRANK vil-Tg mice and control mice, using cryo-sections. Phalloidin and DAPI are shown to visualize F-actin and nuclei, respectively. Scale bars, 50 μm. c , Small intestinal villi length, volume and surface areas of 3-week-old control (n = 6 mice, n = 126 villi analysed) and caRANK vil -Tg mice (n = 7/145). Each data point represents the average length, volume, and surface area of villi per mouse. The original data is the same as Fig. . d,e , Reduced cell death of small intestinal epithelial cells in caRANK vil-Tg mice. Representative images (left in d ) and quantification (right in d , e ) of cleaved caspase-3 (CLC3) immunostaining (arrows) in the small intestine are shown for three wks old control mice (n = 7 mice, n = 93 regions analysed) and caRANK vil -Tg mice (n = 8/126). Each data point represents the number of CLC3 positive cells in villi per 2 mm of the intestine ( d ) and the average number per mouse ( e ). Scale bars, 100 μm. f,g , Representative images (left in f ) and quantification of phosphor-Histone H3 (pHH3) immunostaining in the crypts of the upper small intestines from control and caRANK vil -Tg mice (right in f , g ). 3 wks old control mice (n = 3 mice, n = 254 crypts analysed) and caRANK vil -Tg mice (n = 4/395); 4 wks old control (n = 6/379) and caRANK vil -Tg mice (n = 4/351); and 5–8 wks old control (n = 4/556) and caRANK vil -Tg littermates (n = 4/487). Each data point represents the number of pHH3 positive cells per individual crypts ( f ) and average number per mouse ( g ). Scale bars, 20 μm. h , Kaplan–Meier survival curve of control ( n = 31) and caRANK vil -Tg ( n = 37) littermates. i , Numbers of OLFM4 + cells/ crypt in the indicated mice. Each data point represents the average length, volume, and surface area of villi per mouse in the indicated mice described in Fig. . j , Small intestinal villi length, volume and surface areas the indicated mice. Each data point represents the averaged length, volume, and surface area of villi per mouse. The original data is the same as Fig. . k,l , Numbers of villi (left) and crypts (right) per 2 mm were quantified in histological H&E sections of the upper small intestine from control (n = 4 mice, n = 48 regions) and caRANK vil -Tg (n = 4/46) mice at 3 and control (n = 5 mice, n = 63 regions) and caRANK vil -Tg (n = 5/56) mice at 3 and 5–8 wks of age. Each data point represents the number of Numbers of villi (left) and crypts (right) per 2 mm of the intestine ( k ) and the average number per mouse ( l ). m , Representative images of mouse jejunal organoids from 2 weeks old control caRANK vil -Tg mice, cultured in E low NR (50 pg/ml EGF) or ENR (50 ng/ml EGF) media for 3 days after the first passage. Scale bars, 100 μm. n , Representative images (left) of EdU + proliferating cells in control small intestinal organoids (n = 7) and caRANK vil -Tg mice-derived organoids (n = 6). Scale bars, 50 μm. Right; ratios of EdU labelled proliferating cells to all cells presents in each bud. The analysed bud number is n = 27 (control) and n = 24 ( caRANK vil -Tg ). o , Ratios of organoid numbers after prolonged culture of control- and caRANK vil -Tg mice-derived jejunal organoids. Numbers of organoids were counted at each passage. The ratio of organoid numbers in the caRANK vil -Tg mice-derived jejunal organoids was normalized to untreated control jejunal organoids. Data were combined from two independent experiments. n = 6 (control), n = 6 ( caRANK vil -Tg ). p , Representative images of control and caRANK vil -Tg mice-derived small intestinal organoids cultured with/without recombinant human RANKL (rhRANKL; 100, 500 ng/ml) for 4 days. Scale bars, 100 μm. q , Percentages of surviving control and caRANK vil -Tg jejunal organoids after irradiation, as compared to non-irradiated organoids of the same genotypes. Organoids were cultured in ENR medium and irradiated with 4 Gy, followed by subsequent culture for 7 days. Data are from two independent experiments. n = 5 (control), n = 5 ( caRANK vil -Tg ). AVG, average. Data are mean ± s.e.m. *P < 0.05; **P < 0.01; ****P < 0.0001; ns, not significant. Two-tailed Mann–Whitney U -test ( c,e,g,i,j,l,o,q); Kaplan–Meier survival curve with a log-rank test ( h ); Two-tailed Student’s t-test ( d,f,k,n ).

Article Snippet: The sections were blocked for 1 h in 5% BSA (VWR Life Science) and 10% goat serum (Sigma-Aldrich, 9023) and incubated with primary antibodies against phospho-histone H3 (1:100; CellPath, PBC-ACI3130C), mouse OLFM4 (1:800; Cell Signaling Technology, 39141), human OLFM4 (1:100; Cell Signaling Technology, 14369), cleaved caspase-3 (1:100; Cell Signaling Technology, 9661) or CRE (1:100, Cell Signaling Technology, 15036), all diluted in blocking solution.

Techniques: Transgenic Assay, Mutagenesis, Expressing, Fluorescence, Control, Immunostaining, Cell Culture, Derivative Assay, Recombinant, Irradiation, Two Tailed Test, MANN-WHITNEY

Journal: Nature

Article Title: RANK drives structured intestinal epithelial expansion during pregnancy

doi: 10.1038/s41586-024-08284-1

Figure Lengend Snippet: a , Representative 3D reconstruction of small intestine from 3-week-old control and caRANK vil -Tg mice (top). Grid spacing is 200 μm. Bottom, villus length, volume and surface areas of control ( n = 6 mice, n = 126 villi) and caRANK vil -Tg mice ( n = 7 mice, n = 145 villi analysed). b , Representative OLFM4 immunostaining in the small intestines of 3- and 5–8-week-old control and caRANK vil -Tg mice (top). Scale bars, 20 μm. Bottom, the numbers of OLFM4 + cells per crypt in control and caRANK vil -Tg mice were quantified at 3 weeks ( n = 3 mice, n = 234 crypts (control); n = 3 mice, n = 205 crypts ( caRANK vil -Tg )), 4 weeks ( n = 6 mice, n = 162 crypts (control); n = 4 mice and n = 110 crypts ( caRANK vil -Tg )) and at 5–8 weeks ( n = 4 mice, n = 203 crypts (control); n = 4 mice, n = 178 crypts ( caRANK vil -Tg )) of age. c , Representative 3D reconstruction of the small intestines of 5–8-week-old control and caRANK vil -Tg mice (top). Grid spacing is 200 μm. Bottom, the villus length, volume and surface areas of control ( n = 4 mice, n = 47 villi) and caRANK vil -Tg mice ( n = 5 mice, n = 65 villi). d , Representative OLFM4 immunostaining in the small intestines of 8-week-old caRANK vil -Tg;Traf6 fl/+ ( n = 3) and caRANK vil -Tg;Traf6 fl/fl ( n = 5) mice. Scale bars, 100 μm. e , The numbers of OLFM4 + cells per crypt and the villus length in caRANK vil -Tg;Traf6 fl/+ ( n = 3 mice, 172 crypts, 40 villi) and caRANK vil -Tg; Traf6 fl/fl ( n = 5 mice, 299 crypts, 167 villi) mice. f , Representative macroscopic images of the small intestines of Apc min/+ and caRANK vil -Tg;Apc min/+ mice. Scale bars, 7.5 mm. g , The ratio of tumoroid numbers cultured without (control) or with rmRANKL (50 ng ml −1 ) (top). The ratio of organoid numbers in the RANKL-treated group was normalized to the control group. Data were combined from two independent experiments. n = 4 (control) and n = 4 (rmRANKL). Bottom, representative images of tumoroids established from Apc min/+ mice at passage 0, 1 and 2, cultured in the absence (control) or presence of rmRANKL (50 ng ml −1 ). Scale bars, 100 μm. Each point represents the measurement of length, volume and surface area ( a (bottom), c (bottom) and e (top)), and the number of OLFM4 + cells per crypt ( b (bottom) and e (bottom)). Data are mean ± s.e.m. * P < 0.05; NS, not significant. Statistical analysis was performed using two-tailed Student’s t -tests ( a , c , e and g ) and one-way ANOVA with Tukey’s post hoc test ( b ). Further details on statistics and reproducibility are provided in the .

Article Snippet: The sections were blocked for 1 h in 5% BSA (VWR Life Science) and 10% goat serum (Sigma-Aldrich, 9023) and incubated with primary antibodies against phospho-histone H3 (1:100; CellPath, PBC-ACI3130C), mouse OLFM4 (1:800; Cell Signaling Technology, 39141), human OLFM4 (1:100; Cell Signaling Technology, 14369), cleaved caspase-3 (1:100; Cell Signaling Technology, 9661) or CRE (1:100, Cell Signaling Technology, 15036), all diluted in blocking solution.

Techniques: Control, Immunostaining, Cell Culture, Two Tailed Test

Journal: Nature

Article Title: RANK drives structured intestinal epithelial expansion during pregnancy

doi: 10.1038/s41586-024-08284-1

Figure Lengend Snippet: a-c , Alterations in the NF-κB, anti-apoptotic, and BMP pathways in jejunal organoids from caRANK vil -Tg mice (n = 4) compared to organoids from control mice (n = 4). Upper panels show GSEA enrichment plots. Bottom panels show heatmaps of the top 15 genes upregulated in caRANK vil -Tg organoids. Expression profiles of RANKL stimulated jejunal organoids were compared to non-stimulated (control) jejunal organoids cultured for the same time period. Total RNA was isolated from small intestinal organoids generated from two weeks old control and caRANK vil -Tg littermate (n = 4 for each group) and processed for RNA-seq. d , Differential gene expression analysis of RNA-seq data from mouse jejunal organoids derived from caRANK vil -Tg (n = 4) and control (n = 4) mice. Normalized CPM values of selected transcripts of anti-apoptotic genes, stem cell signature genes and BMP signalling genes are shown. e , Western blotting of phosphorylated IκB-α and total IκB-α in isolated intestinal epithelial cells from 3 wks old control (n = 3) and caRANK vil -Tg (n = 3) mice. β-Actin is shown as a loading control. For gel resource data, see Supplementary Fig. . f , Schematic outline of the proposed role of RANK/RANKL-induced stem cell proliferation and exhaustion. The figure was created with BioRender.com. g , Ratios of the numbers of organoids derived from Traf6 WT and Traf6 ΔVil mice after prolonged culture in the presence of rmRANKL (50 ng/ml). Numbers of organoids were counted at each passage. The ratio of organoid numbers in the RANKL Traf6 WT group was normalized to control Traf6 WT organoids, whereas the ratio of organoid numbers in the RANKL treated Traf6 ΔVil group was normalized to untreated control Traf6 ΔVil organoids. Data were combined from two independent experiments. n = 11 ( Traf6 WT ), n = 11 ( Traf6 WT + RANKL), n = 11 ( Traf6 ΔVil ), n = 11 ( Traf6 ΔVil + RANKL). h , Average length of villi and average number of OLFM4 + cells in each crypt per mouse from the indicated mice in the indicated mice described in Fig. . i , Kaplan–Meier survival curve of control ( n = 14), caRANK vil -Tg ( n = 8), Apc min/+ ( n = 13), and caRANK vil -Tg , Apc min/+ ( n = 11) mice. j , Numbers of macroscopic adenomas and k,l , tumour diameters in the small intestine and colon of 4 months old control Apc min/+ ( n = 5 mice) and caRANK vil -Tg , Apc min/+ ( n = 5) mice. Small intestines were divided equally into 4 parts from the proximal (duodenum, labelled 1) to the distal (ileum, labelled 4) and adenomas assessed for each region. Total tumour numbers in Apc min/+ mice; n = 29 in region 1, n = 53 in region 2, n = 156 in region 3, n = 231 in region 4, n = 469 in total, n = 14 in colon, and in caRANK vil -Tg , Apc min/+ mice ( n = 29/24/29/37/119/23). Each data point in k represents the measurements of the tumour diameter in individual tumours and in l represents the average measurement of the tumour diameters per mouse. Data are mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant. Enrichment-adjusted p-values (p-value), False Discovery Rates (FDR) and Normalized Enrichment Scores (NES) were calculated using two sided-fGSEA ( a-c ). Two-sided DESeq2 Wald tests, adjusted with the Benjamini–Hochberg procedure ( d ). One-way analysis of variance (ANOVA) with Tukey’s post hoc test ( g ); Two-tailed Mann–Whitney U -test ( h , j,l ); Kaplan–Meier survival curve with a log-rank test ( i ); Two-tailed Student’s t-test ( k ).

Article Snippet: The sections were blocked for 1 h in 5% BSA (VWR Life Science) and 10% goat serum (Sigma-Aldrich, 9023) and incubated with primary antibodies against phospho-histone H3 (1:100; CellPath, PBC-ACI3130C), mouse OLFM4 (1:800; Cell Signaling Technology, 39141), human OLFM4 (1:100; Cell Signaling Technology, 14369), cleaved caspase-3 (1:100; Cell Signaling Technology, 9661) or CRE (1:100, Cell Signaling Technology, 15036), all diluted in blocking solution.

Techniques: Control, Expressing, Cell Culture, Isolation, Generated, RNA Sequencing, Gene Expression, Derivative Assay, Western Blot, Two Tailed Test, MANN-WHITNEY

Journal: Nature

Article Title: RANK drives structured intestinal epithelial expansion during pregnancy

doi: 10.1038/s41586-024-08284-1

Figure Lengend Snippet: a , Mating strategy to generate intestinal epithelial cell specific Rank deleted mice (left) and anti-RANK immunostaining on intestinal cryo-sections from nulliparous control Rank WT and Rank Δ Vil mice (right). Scale bars, 50 μm. b , Quantitative RT–PCR analysis of Rank mRNA expression in isolated small intestinal epithelial cells from nulliparous control Rank WT (n = 3) and Rank ΔVil (n = 5) littermates. c , Representative immunostaining to detect CRE expression (blue) in the small intestine (left panel) and mammary gland (right panel) of Villin1-Cre mice. Paraffin sections were counterstained with hematoxylin. Scale bars, 50 μm. d , Representative whole mount images (hematoxylin staining) of mammary glands from nulliparous Rank WT , nulliparous Rank ΔVil , pregnant Rank WT (day 18.5 of pregnancy, P18.5), pregnant Rank ΔVil females, lactating Rank WT (5 days after delivery, L5) and lactating L5 Rank ΔVil females. Scale bars, 500 μm. e , Small intestinal villi length, volume and surface areas in indicated mice. Each data point represents average villi length, volume and surface area per mouse in the indicated mice described in Fig. . f , Quantification of the average number of OLFM4 + cells in each crypt per mouse in the indicated mice described in Fig. . g,h , Cell death of villous epithelial cells from nulliparous Rank WT and Rank ΔVil females and age-matched lactating (L5) Rank WT and Rank ΔVil dams, as determined by immunostaining of cleaved caspase 3 (CLC3). Representative immunostaining. Scale bars, 100 μm (left in g ). Quantification of CLC3 positive cells in villi per 2 mm of the intestine in nulliparous Rank WT (n = 4 mice, n = 49 regions analysed) and Rank ΔVil females (n = 5/59) and age-matched lactating (L5) Rank WT (n = 8/162) and Rank ΔVil dams (n = 8/135) (right in g and h ). Each data point represents the numbers of CLC3 positive cells in villi per each 2 mm of the intestine ( g ) and average number per mouse ( h ). i,j , Quantification of the EdU-labelled intestinal epithelial cell migration along the crypt-villus axis in nulliparous Rank WT (n = 3 mice, n = 29 crypt-villus axis analysed) and Rank ΔVil females (n = 3/37) and age-matched lactating (L5) Rank WT (n = 4/52) and Rank ΔVil dams (n = 4/62). Tissues were harvested 24 h after EdU administration. Each data point represents the measurements of the migration distance in individual crypt-villus axis ( i ) and the average distance per mouse ( j ). k , Representative images of jejunal organoids generated from Rank WT and Rank ΔVil mice. Scale bars, 100 μm. l , Ratios of the number of jejunal organoids derived from Rank WT and Rank ΔVil mice after prolonged culture in the presence of rmRANKL (50 ng/ml). The number of organoids was counted at each passage. The ratio of organoid numbers in the Rank WT + RANKL group was normalized to control Rank WT organoids, whereas the ratio of organoid numbers in the Rank ΔVil + RANKL group was normalized to untreated Rank ΔVil organoids. Data are from two independent experiments. n = 6 ( Rank WT ), n = 6 ( Rank WT + RANKL), n = 6 ( Rank ΔVil ), n = 6 ( Rank ΔVil + RANKL). m,n , Representative hematoxylin and eosin (H&E) stained images of the upper small intestine (left in m ) and quantification of upper small intestinal villus lengths (right in m , n ) in age matched wild type nulliparous females (n = 4 mice, n = 310 villi analysed), pregnant females (P18.5) (n = 4/272), lactating dams (L5, 5 days after delivery) (n = 4/212) and females 6 weeks after weaning of the offspring (n = 4/49). Scale bars, 100 μm. Each data point represents the length of individual villi ( m ) and average length per mouse ( n ). Data are mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant. Two-tailed Mann–Whitney U -test ( e,f,h,j,n); One-way analysis of variance (ANOVA) with Tukey’s post hoc test ( g,i,l,m ).

Article Snippet: The sections were blocked for 1 h in 5% BSA (VWR Life Science) and 10% goat serum (Sigma-Aldrich, 9023) and incubated with primary antibodies against phospho-histone H3 (1:100; CellPath, PBC-ACI3130C), mouse OLFM4 (1:800; Cell Signaling Technology, 39141), human OLFM4 (1:100; Cell Signaling Technology, 14369), cleaved caspase-3 (1:100; Cell Signaling Technology, 9661) or CRE (1:100, Cell Signaling Technology, 15036), all diluted in blocking solution.

Techniques: Immunostaining, Control, Quantitative RT-PCR, Expressing, Isolation, Staining, Migration, Generated, Derivative Assay, Two Tailed Test, MANN-WHITNEY

Journal: Nature

Article Title: RANK drives structured intestinal epithelial expansion during pregnancy

doi: 10.1038/s41586-024-08284-1

Figure Lengend Snippet: a , Representative 3D reconstruction of the upper small intestine from nulliparous and lactating (5 days after delivery, L5) Rank WT and Rank Δvil female mice (top). Grid spacing is 200 μm. Bottom, the small intestinal villus length, volume and surface areas in nulliparous Rank WT ( n = 5 mice, n = 88 villi), nulliparous Rank Δvil ( n = 5 mice, n = 97 villi), P18.5 Rank WT ( n = 6 mice, n = 89 villi), P18.5 Rank Δvil ( n = 6 mice, n = 93 villi), L5 Rank WT ( n = 5 mice, n = 91 villi) and L5 Rank Δvil ( n = 6 mice, n = 141 villi) mice. b , Representative OLFM4 immunostaining in the small intestines of the indicated mice (top). Scale bars, 20 μm. Bottom, quantification of OLFM4 + cells per crypt in nulliparous Rank WT ( n = 3 mice, n = 87 crypts), nulliparous Rank Δvil ( n = 4 mice, n = 130 crypts), L5 Rank WT ( n = 6 mice, n = 160 crypts) and L5 Rank Δvil ( n = 7 mice, n = 181 crypts) mice, and Rank WT ( n = 4 mice, n = 59 crypts) and Rank Δvil ( n = 5 mice, n = 44 crypts) mice 6 weeks after weaning of the offspring. c , Representative 3D images (left) and villi lengths of small intestines from age-matched nulliparous ( n = 3 mice, n = 70 villi analysed) and lactating ( n = 5 mice, n = 94 villi) germ-free C57BL6 mice (right). Grid spacing is 200 μm. d , Left, representative small intestinal sections (haematoxylin and eosin (H&E) staining) of L5 wild-type mice and wild-type mice whose offspring were removed on 1 day after delivery (no lactation). Scale bars, 100 μm. Right, quantification of villus length in L5 dams (with offspring) ( n = 7 mice, n = 170 villi) and female mice with their offspring removed (without offspring) ( n = 5 mice, n = 158 villi). e , Representative small intestinal sections (H&E staining) (left) and quantification of villi lengths (right) of L5 wild-type mice treated with DMSO or cabergoline (5 mg per kg) for 5 consecutive days starting on the day of delivery. Villi were assessed on day 5 after delivery. Scale bars, 100 μm. Right, quantification of villus length in mice treated with DMSO ( n = 4 mice, n = 148 villi) and mice treated with cabergoline ( n = 4 mice, n = 179 villi). f , Representative H&E-stained intestinal sections from nulliparous Traf6 WT and Traf6 Δvil mice, and L5 lactating Traf6 WT and Traf6 Δvil mice (left). Scale bars, 100 μm. Right, quantification of villous length in nulliparous Traf6 WT ( n = 4 mice, n = 76 villi) and Traf6 Δvil ( n = 5 mice, n = 73 villi) mice, and L5 Traf6 WT ( n = 6 mice, n = 198 villi) and Traf6 Δvil ( n = 5 mice, n = 226 villi) mice. Data are mean ± s.e.m. Each point represents the measurement of length, volume and surface area ( a (bottom) and c – f (right)), and the number of OLFM4 + cells per crypt ( b , bottom). Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test ( a , b and f ) and two-tailed Student’s t -tests ( c – e ); ** P < 0.01, *** P < 0.001.

Article Snippet: The sections were blocked for 1 h in 5% BSA (VWR Life Science) and 10% goat serum (Sigma-Aldrich, 9023) and incubated with primary antibodies against phospho-histone H3 (1:100; CellPath, PBC-ACI3130C), mouse OLFM4 (1:800; Cell Signaling Technology, 39141), human OLFM4 (1:100; Cell Signaling Technology, 14369), cleaved caspase-3 (1:100; Cell Signaling Technology, 9661) or CRE (1:100, Cell Signaling Technology, 15036), all diluted in blocking solution.

Techniques: Immunostaining, Staining, Two Tailed Test

Journal: Nature

Article Title: RANK drives structured intestinal epithelial expansion during pregnancy

doi: 10.1038/s41586-024-08284-1

Figure Lengend Snippet: a , Uniform manifold approximation and projection (UMAP) of 2,265 human intestinal epithelial cells. Data were taken from Fujii et al. . Cells are colour coded according to epithelial cell-type annotation based on unsupervised clustering. b , Violin plots show single cell log-normalized expression of RANK and RANKL in each intestinal cell-type. Each dot represents an individual cell. c , Quantitative RT–PCR analyses to compare expression levels of anti-apoptotic genes, stem cell signature genes, and BMP signalling genes in human duodenal organoids. Data represent the relative expression of the indicated genes in rhRANKL (500 ng/ml) stimulated duodenal organoids compared to control (no RANKL) organoids (set at 1). rhRANKL stimulation was for 12 and 96 h. n = 4 (control), n = 4 (RANKL). d , Representative images (left) and quantified areas (right) of human duodenal organoids cultured for 2 days without (control; n = 83) and with recombinant human RANKL (rhRANKL; 500 ng/ml; n = 54) in growth-factor-reduced medium (GFR medium) lacking EGF, IGF-1, and FGF-2. Scale bars, 100 μm. Each dot represents an organoid, assessed in three independent experiments. e , RANKL-induced proliferation of duodenal organoids. Organoids were left untreated (control) or treated with rhRANKL (500 ng/ml) and proliferation determined using the MTT assay. Each plot represents an MTT OD, pooled from two independent experiments. n = 20 (control), n = 20 (RANKL). f , Representative cell cycle FACS plots (left) and quantification of S-phase and subG1 + G1 entry (right) assessing EdU labelled human duodenal organoids cultured in GFR medium and stimulated with rhRANKL for 24 and 72 h. Dots represent individual organoids, assessed in three independent experiments. n = 14 (control, 24hrs), n = 16 (control, 72hrs), n = 18 (rmRANKL, 24hrs), n = 18 (rmRANKL, 72hrs). g , Representative images of anti-OLFM4 immunostaining (green) of human duodenal organoids cultured in GFR medium in the absence (control) and presence of rhRANKL (500 ng/ml) for 24 and 96 h. Organoids were counterstained with DAPI (blue) to detect nuclei and Phalloidin (magenta) to detect filamentous actin. Scale bars, 50 μm. h , Representative images of OLFM4 + stem cells in human intestinal organoids cultured in the presence of rhRANKL (500 ng/ml) without (control, DMSO solvent) or with the BMP inhibitor (BMPi) LDN193189 (1.6μM) for 96 hrs. Organoids were stained with DAPI (blue) to detect nuclei and Phalloidin to detect filamentous actin. Scale bars, 50 μm. i , Proposed function of RANK–RANKL in the small intestine during pregnancy and lactation. During pregnancy and lactation, RANK–RANKL signalling promotes intestinal stem cell proliferation and differentiation as well as intestinal cell survival, ultimately resulting in massive villous expansion. Villous expansion facilitates nutritional uptake, which is important for nourishment of offspring as well as their transgenerational metabolic health. The figure was created with BioRender.com. Data are mean ± s.e.m. **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant. Two-tailed Student’s t-test ( c-f) . More details on statistics and reproducibility can be found in the .

Article Snippet: The sections were blocked for 1 h in 5% BSA (VWR Life Science) and 10% goat serum (Sigma-Aldrich, 9023) and incubated with primary antibodies against phospho-histone H3 (1:100; CellPath, PBC-ACI3130C), mouse OLFM4 (1:800; Cell Signaling Technology, 39141), human OLFM4 (1:100; Cell Signaling Technology, 14369), cleaved caspase-3 (1:100; Cell Signaling Technology, 9661) or CRE (1:100, Cell Signaling Technology, 15036), all diluted in blocking solution.

Techniques: Expressing, Quantitative RT-PCR, Control, Cell Culture, Recombinant, MTT Assay, Immunostaining, Solvent, Staining, Two Tailed Test

Journal: Nature

Article Title: RANK drives structured intestinal epithelial expansion during pregnancy

doi: 10.1038/s41586-024-08284-1

Figure Lengend Snippet: a , Human duodenal organoids were cultured without (control) and with recombinant human RANKL (rhRANKL; 500 ng ml −1 ) for 2 days. Left, representative images. Scale bars, 100 μm. Right, quantification of organoid sizes. n = 83 (control) and n = 83 (rhRANKL). Data were combined from three independent experiments. b , Organoids were cultured with or without rhRANKL (500 ng ml −1 ) for 2 days and then irradiated and cultured for additional 7 days. Data show the number of surviving organoids combined from three independent experiments. n = 12 (control) and n = 12 (rhRANKL). c , Cell cycle analyses of RANK-stimulated human duodenal organoids. Left, representative fluorescence-activated cell sorting (FACS) plots of the EdU cell cycle analysis assessed at 24 h and 72 h of rhRANKL stimulation. Right, quantification of S phase and subG1 + G1 entry. Data were combined from two independent experiments. n = 12 for each condition. d , The ratio of the numbers of wild-type (WT) and BMPR1A -knockout ( BMPR1A -KO) human small intestinal organoids in the absence or presence of rhRANKL (500 ng ml −1 ). The ratio of organoid numbers in the WT + RANKL group was normalized to untreated WT organoids; and the ratio of organoid numbers in the BMPR1A- KO + RANKL organoids was normalized to the untreated BMPR1A- KO organoids. n = 3 for each condition. e , Cell cycle analyses of RANK-stimulated duodenal organoids in the absence (control) and presence of the BMPi LDN193189 (1.6 μM). Left, representative FACS plots depicting EdU cell cycle analysis after 72 h rhRANKL stimulation with or without BMPi. Right, quantification of S phase and sbG1 + G1 entry. n = 11 (control), n = 12 (control + RANKL), n = 12 (BMPi) and n = 12 (BMPi + RANKL). Data were combined from two independent experiments. f , Representative images of OLFM4 + stem cells in WT and BMPR1A -KO human small intestinal organoids with or without rhRANKL (500 ng ml −1 ). Scale bars, 50 μm. Dots represent individual datapoints. Data are mean ± s.e.m. Statistical analysis was performed using two-tailed Student’s t -tests ( a – c ) and one-way ANOVA with Tukey’s post hoc test ( d and e ). Further details on statistics and reproducibility are provided in the .

Article Snippet: The sections were blocked for 1 h in 5% BSA (VWR Life Science) and 10% goat serum (Sigma-Aldrich, 9023) and incubated with primary antibodies against phospho-histone H3 (1:100; CellPath, PBC-ACI3130C), mouse OLFM4 (1:800; Cell Signaling Technology, 39141), human OLFM4 (1:100; Cell Signaling Technology, 14369), cleaved caspase-3 (1:100; Cell Signaling Technology, 9661) or CRE (1:100, Cell Signaling Technology, 15036), all diluted in blocking solution.

Techniques: Cell Culture, Control, Recombinant, Irradiation, Fluorescence, FACS, Cell Cycle Assay, Knock-Out, Two Tailed Test

Journal: Journal of innate immunity

Article Title: The Olfactomedin-4-Defined Human Neutrophil Subsets Differ in Proteomic Profile in Healthy Individuals and Patients with Septic Shock.

doi: 10.1159/000527649

Figure Lengend Snippet: Fig. 1. Proteomic analysis of Olfm4-defined neutrophil subsets in healthy blood donors. Olfm4-H and Olfm4-L neutrophils isolated from healthy blood donors (n = 8) were stained, sorted by FACS, and analysed by LC-MS/MS. a Box-and-whisker plots showing the number of proteins and peptide groups identified in Olfm4-H and Olfm4-L neutrophil samples. b Box-and-whisker plot showing protein abundance log2 ratios for the 20 most abundant proteins between paired Olfm4-H/L neutrophil samples. Common con- taminants and histones are not shown.

Article Snippet: AntiOlfm4 serum as described above or commercial rabbit anti-human Olfm4 (HPA077718, Atlas antibodies, Stockholm, Sweden) at 2 μg/mL were used.

Techniques: Isolation, Staining, Liquid Chromatography with Mass Spectroscopy, Whisker Assay, Quantitative Proteomics

Journal: Journal of innate immunity

Article Title: The Olfactomedin-4-Defined Human Neutrophil Subsets Differ in Proteomic Profile in Healthy Individuals and Patients with Septic Shock.

doi: 10.1159/000527649

Figure Lengend Snippet: Fig. 2. Differential proteomic profiles of Olfm4-H and Olfm4-L neutrophil subsets in healthy blood donors. Olfm4-H and Olfm4- L neutrophils isolated from healthy blood donors (n = 8) were sort- ed by FACS and analysed by LC-MS/MS. a Volcano plot showing median log2 abundance ratios between the identified proteins in Olfm4-H and Olfm4-L neutrophils and their p values. b Left box- and-whisker plot showing proteins with a statistically significant positive median log2 abundance ratio, indicating increased abun- dance in the Olfm4-H neutrophils. Right box-and-whisker plot showing proteins with a statistically significant negative median log2 abundance ratio, indicating increased abundance in the Olfm4-L neutrophils. c Neutrophils from healthy blood donors (n = 7) were isolated and stained for proteins of interest and analysed by live imaging flow cytometry. Box-and-whisker plot comparing median fluorescence intensity of the immunofluorescently stained proteins Rab3d, S100A7, Rab3a, and CXCR1 between Olfm4-H and Olfm4-L neutrophils. Wilcoxon signed rank tests were used to test for statistical significance. Common contaminants and his- tones are not shown.

Article Snippet: AntiOlfm4 serum as described above or commercial rabbit anti-human Olfm4 (HPA077718, Atlas antibodies, Stockholm, Sweden) at 2 μg/mL were used.

Techniques: Isolation, Liquid Chromatography with Mass Spectroscopy, Whisker Assay, Staining, Imaging, Flow Cytometry, Fluorescence

Journal: Journal of innate immunity

Article Title: The Olfactomedin-4-Defined Human Neutrophil Subsets Differ in Proteomic Profile in Healthy Individuals and Patients with Septic Shock.

doi: 10.1159/000527649

Figure Lengend Snippet: Fig. 3. In vitro functional comparison of Olfm4-H and Olfm4-L neutrophils. a For the ROS assay, neutrophils isolated from healthy donors (n = 4) were loaded with CellROX and stimulated with PMA or buf- fer for 5 min before fixation and Olfm4 staining. Diagram showing CellROX in- tensity in Olfm4-H and Olfm4-L subsets, as analysed by imaging flow cytometry. Statistical significance was tested by mul- tiple Wilcoxon signed rank tests. Represen- tative images are shown. b For the bacte- rial infection assay, S. aureus at MOI 10 was allowed to adhere on ice to neutrophils iso- lated from healthy donors (n = 6), followed by incubation at 37°C for the indicated times, fixation, Olfm4 staining, and analy- sis by imaging flow cytometry. Top dia- gram showing the mean (error bars show SD) number of bacteria per Olfm4-H or Olfm4-L neutrophil over time. Lower dia- gram showing the mean number of bacte- ria (error bars show 95% confidence inter- vals) per neutrophil over time in a control experiment (n > 150 neutrophils) where samples were treated with DPI or solvent control during infection. Statistical signifi- cance was tested by 2-way ANOVA. Repre- sentative images are shown. c The propor- tion of Olfm4-H neutrophils was analysed by imaging flow cytometry in LDNs (local- izing to the PBMC layer during density gra- dient separation) and NDN (localizing to the granulocyte layer) of the same donors (n = 8), upon staining of cell surface CD15, to identify neutrophils, in combination with intracellular Olfm4. Diagram show- ing the percentage of Olfm4-H neutrophils in each neutrophil phenotype in each do- nor. Wilcoxon signed rank test was used to test for statistical significance. Representa- tive images are shown.

Article Snippet: AntiOlfm4 serum as described above or commercial rabbit anti-human Olfm4 (HPA077718, Atlas antibodies, Stockholm, Sweden) at 2 μg/mL were used.

Techniques: In Vitro, Functional Assay, Comparison, ROS Assay, Isolation, Staining, Imaging, Flow Cytometry, Infection, Incubation, Bacteria, Control, Solvent

Journal: Journal of innate immunity

Article Title: The Olfactomedin-4-Defined Human Neutrophil Subsets Differ in Proteomic Profile in Healthy Individuals and Patients with Septic Shock.

doi: 10.1159/000527649

Figure Lengend Snippet: Fig. 4. Proteomic analysis of Olfm4-defined neutrophil subsets in patients with septic shock. Olfm4-H and Olfm4-L neutrophils iso- lated from patients with septic shock (n = 3) were sorted by FACS and analysed by LC-MS/MS. a Box-and-whisker plots showing the number of proteins and peptide groups identified in Olfm4-H and Olfm4-L neutrophil samples. b Box-and-whisker plot showing protein abundance log2 ratios for the 20 most abundant proteins between paired Olfm4-H/L neutrophil samples. Common con- taminants and histones are not shown.

Article Snippet: AntiOlfm4 serum as described above or commercial rabbit anti-human Olfm4 (HPA077718, Atlas antibodies, Stockholm, Sweden) at 2 μg/mL were used.

Techniques: Liquid Chromatography with Mass Spectroscopy, Whisker Assay, Quantitative Proteomics

Journal: Journal of innate immunity

Article Title: The Olfactomedin-4-Defined Human Neutrophil Subsets Differ in Proteomic Profile in Healthy Individuals and Patients with Septic Shock.

doi: 10.1159/000527649

Figure Lengend Snippet: Fig. 5. Differential proteomic profiles of Olfm4-H and Olfm4-L neutrophil subsets in patients with septic shock. Olfm4-H and Olfm4-L neutrophils isolated from patients with septic shock (n = 3) were sorted by FACS and analysed by LC-MS/MS. a Vol- cano plot showing median log2 abundance ratios between the identified proteins in Olfm4-H and Olfm4-L neutrophils and their p values. b Left box-and-whisker plot showing proteins with a statistically signif- icant positive median log2 abundance ra- tio, indicating increased abundance in the Olfm4-H neutrophils. Right box-and- whisker plot showing proteins with a statis- tically significant negative median log2 abundance ratio, indicating increased abundance in the Olfm4-L neutrophils. Common contaminants are not shown.

Article Snippet: AntiOlfm4 serum as described above or commercial rabbit anti-human Olfm4 (HPA077718, Atlas antibodies, Stockholm, Sweden) at 2 μg/mL were used.

Techniques: Isolation, Liquid Chromatography with Mass Spectroscopy, Whisker Assay

Journal: Journal of innate immunity

Article Title: The Olfactomedin-4-Defined Human Neutrophil Subsets Differ in Proteomic Profile in Healthy Individuals and Patients with Septic Shock.

doi: 10.1159/000527649

Figure Lengend Snippet: Fig. 6. Analysis of Olfm4-H proportion and plasma Olfm4 concen- tration in patients with septic shock. Peripheral blood was col- lected from patients with septic shock (n = 20) and healthy blood donors (n = 20). The proportion of Olfm4-H neutrophils was ana- lysed by imaging flow cytometry after fixation of leukocytes, stain- ing of CD15 for neutrophil identification, permeabilization and antibody staining of Olfm4. a Box-and-whisker plot showing the proportion of Olfm4-H neutrophils in sepsis patients and healthy controls. b Scatterplots showing the correlation between the pro- portion of Olfm4-H neutrophils and SOFA score (left panel) or OSFD (right panel). c Box-and-whisker plots showing SOFA score (left panel) or OSFD (right panel) of sepsis patients with percent- age of Olfm4-H neutrophils above 37.6% (n = 3) or below 37.6% (n = 17). d Olfm4 concentrations in plasma were quantified by ELISA. Box-and-whisker plots showing plasma Olfm4 concentra- tions in plasma obtained from patients with septic shock and healthy donors.

Article Snippet: AntiOlfm4 serum as described above or commercial rabbit anti-human Olfm4 (HPA077718, Atlas antibodies, Stockholm, Sweden) at 2 μg/mL were used.

Techniques: Clinical Proteomics, Imaging, Flow Cytometry, Staining, Whisker Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of gastroenterology

Article Title: Single cell analysis of Crohn's disease patient-derived small intestinal organoids reveals disease activity-dependent modification of stem cell properties.

doi: 10.1007/s00535-018-1437-3

Figure Lengend Snippet: Fig. 1 Immunostaining of small intestinal tissues for stem cell- specific genes, OLFM4 and SLC12A2. Small intestinal surgical specimens obtained from CD patients were subjected to immunos- taining of OLFM4 and SLC12A2 (green). Results of small intestinal tissues obtained from active lesions of a CD patient (CD active) and those from CD patients in remission (CD remission) are shown. The same regions of the ileum were analyzed

Article Snippet: The primary antibodies used are as follows: Rabbit-anti-human-OLFM4 mAb (1:1000, Cell Signalling Technology, Danvers, MA, USA); Mouse-anti-human-SLC12A2 Ab (1:1000, LS-Bio, Seattle, WA, USA); Mouse-anti-human-E-cadherin (1:500, Takara Bio, Kusatsu, Shiga, Japan).

Techniques: Immunostaining