anti ror2  (Santa Cruz Biotechnology)

Journal: bioRxiv

Article Title: Noncanonical Wnt/Ror2 Signaling Regulates Basal Cell Fidelity and Branching Morphogenesis in the Mammary Gland

doi: 10.1101/2025.02.25.640099

Figure Lengend Snippet: (A) Schematic illustrating the breeding strategy used to generate tamoxifen-inducible p63-specific Ror2 knockout mice carrying a heterozygous ROSA mTmG reporter allele. (B) Experimental timeline showing tamoxifen administration, tissue collection, and phenotype analysis. (C, D) Representative immunofluorescence images of ROSA mTmG -tdTomato (magenta) and ROSA mTmG -eGFP (green) in mammary gland cross-sections from (C) control and (D) p63-Ror2-KO mice 2 days post-tamoxifen injection. Scale bar: 20 μm. (E, F) Representative immunofluorescence images showing Ror2 protein (gray) by immunofluorescence in mammary gland cross-sections from (E) control and (F) p63-Ror2-KO mice at 2 days post-tamoxifen injection. Scale bar: 20 μm. (E’-E”) Magnified inset of E showing Ror2 expression within the p63Cre-eGFP + basal layer (cyan) in controls and (F’-F”) Ror2 –KO glands. Scale bar: 10 μm. (G) Quantitative RT-qPCR analysis of Ror2 mRNA expression in FACS-isolated mammary basal epithelial cells from control and p63-Ror2-KO mice. Expression levels were normalized to GAPDH, with data represented as fold change relative to the control group (p = 0.0076; n = 3 biological replicates per group). (H-H’) Representative whole-mount epifluorescence images of eGFP in mammary gland structures from control mice. (H) Wholemount mammary gland (scale bar: 2 mm) and (H’) a magnified view of ducts (scale bar: 1 mm). (I, I’) Representative whole-mount epifluorescence images of eGFP in mammary gland structures from p63-Ror2-KO mice. (I) Whole mammary gland (scale bar: 2 mm) and (I”) a magnified view of ducts (scale bar: 1 mm). Increased branching in p63-Ror2-KO mammary glands is indicated by white arrowheads. (J, K) Quantification of (J) secondary branching and (K) tertiary branching in mammary glands from control and p63-Ror2-KO mice (p = 0.0184 and p = 0.0019, respectively; n = 6 glands for control and n = 11 glands for p63-Ror2-KO groups). (L, M) Representative differential interference contrast (DIC) bright-field images of branching mammary organoids from control and p63-Ror2-KO groups at 72 hours post-seeding with FGF stimulation. Increased branching morphogenesis in p63-Ror2-KO organoids is indicated by black arrowheads. Scale bar: 50 μm. (N) Quantification of the number of branching projections per organoid in control and p63-Ror2-KO groups (p < 0.0001; n = 18 organoids for control and n = 21 organoids for p63-Ror2-KO).

Article Snippet: The following primary antibodies were used, with their respective dilutions indicated: Ror2 (1:1000; Developmental Studies Hybridoma Bank, Iowa City, IA), RhoA (1:1000; #2117; Cell Signaling Technology [CST], Danvers, MA), ROCK1 (1:1000; #4035; CST), Cdc42 (1:1000; #2466; CST), Rac1/2/3 (1:1000; #2465; CST), YAP/TAZ (1:1000; #8418; CST), phosphorylated YAP (p-YAP, 1:1000; #13008; CST), LATS1 (1:1000; #3477; CST), phosphorylated LATS1 (p-LATS1, 1:1000; #9157; CST), and GAPDH (1:2500; #5174; CST).

Techniques: Knock-Out, Immunofluorescence, Control, Injection, Expressing, Quantitative RT-PCR, Isolation

Journal: bioRxiv

Article Title: Noncanonical Wnt/Ror2 Signaling Regulates Basal Cell Fidelity and Branching Morphogenesis in the Mammary Gland

doi: 10.1101/2025.02.25.640099

Figure Lengend Snippet: (A, B) Confocal Maximum Intensity Projection (MIP) of mammary ducts from ROSA mTmG glands from (A, A’) p63-Cre WT and (B, B’) p63-Cre-Ror2-KO glands optically cleared with CUBIC. Scale bars: 70 μm. (magenta, unrecombined mTomato; orange, recombined mGFP). (C) Schematic of experimental timeline showing tamoxifen administration and tissue collection for RNA sequencing. (D) Heatmap of significantly differentially expressed genes (adjusted p < 0.05 and fold change > 1.5) in control and p63-Ror2-KO basal cells, as identified by RNA sequencing. Expression levels are presented as log 2 (reads/average reads in the control group) and visualized using a gradient from blue (downregulation) to yellow (upregulation). (E, F) Gene ontology analysis performed using the DAVID Bioinformatics Database, highlighting the enrichment of gene expression changes (E) downregulated and (F) upregulated in several biological processes following Ror2 loss. (G, H) Heatmaps displaying RNA sequencing results for differentially expressed genes related to (G) cytokeratin and (H) cell adhesion. Expression changes are represented as fold changes using a gradient from blue (downregulation) to yellow (upregulation). (I) Quantitative RT-qPCR analysis of K14 mRNA expression in FACS-isolated basal epithelial cells from control and p63-Ror2-KO mice. Gene expression levels were normalized to GAPDH , and data are presented as fold change relative to the control group (p = 0.0030; n = 3 biological replicates per group). (J) Quantitative RT-qPCR analysis of K5 mRNA expression in FACS-isolated basal epithelial cells from control and p63-Ror2-KO mice. Gene expression levels were normalized to GAPDH , and data are presented as fold change relative to the control group (p = 0.0016; n = 3 biological replicates per group). (K, L) Representative immunofluorescence images of K14 (green) and K8 (magenta) in mammary gland cross-sections from (K) control and (L) p63-Ror2-KO mice at 4 weeks post-tamoxifen injection. Scale bar: 25 μm. (K’, K”, L’, L”) Magnified inset of single-channel images of K14 and K8 reveal reduced K14 coverage in p63-Ror2-KO mammary ducts. Scale bar 10 μm. (M, N) Representative immunofluorescence images of K5 (cyan) and K8 (magenta) in mammary gland cross-sections from (M) control and (N) p63-Ror2-KO mice at 4 weeks post-tamoxifen injection. Scale bar: 25 μm. (M’, M”, N’, N”) Magnified single-channel images of K5 and K8 reveal reduced K5 coverage in p63-Ror2-KO mammary ducts relative to p63 WT ducts.

Article Snippet: The following primary antibodies were used, with their respective dilutions indicated: Ror2 (1:1000; Developmental Studies Hybridoma Bank, Iowa City, IA), RhoA (1:1000; #2117; Cell Signaling Technology [CST], Danvers, MA), ROCK1 (1:1000; #4035; CST), Cdc42 (1:1000; #2466; CST), Rac1/2/3 (1:1000; #2465; CST), YAP/TAZ (1:1000; #8418; CST), phosphorylated YAP (p-YAP, 1:1000; #13008; CST), LATS1 (1:1000; #3477; CST), phosphorylated LATS1 (p-LATS1, 1:1000; #9157; CST), and GAPDH (1:2500; #5174; CST).

Techniques: RNA Sequencing, Control, Expressing, Gene Expression, Quantitative RT-PCR, Isolation, Immunofluorescence, Injection

Journal: bioRxiv

Article Title: Noncanonical Wnt/Ror2 Signaling Regulates Basal Cell Fidelity and Branching Morphogenesis in the Mammary Gland

doi: 10.1101/2025.02.25.640099

Figure Lengend Snippet: (A, B) Representative immunofluorescence images showing K14 (gray), eGFP (green), and K8 (magenta) in mammary gland cross-sections from (A) control and (B) p63-Ror2-KO mice at 4 weeks post-tamoxifen injection. Scale bars: 25 μm for merged images and 10 μm for magnified insets (A’-B”). (B’, B”) White arrowheads highlight eGFP + cells within the luminal compartment expressing K8 in p63-Ror2-KO mammary ducts. (C, D) Representative immunofluorescence images of p63 (yellow), eGFP (cyan), and K8 (magenta) in mammary gland cross-sections from (C) p63-Ror2-WT and (D) p63-Ror2-KO mice at 4 weeks post-tamoxifen injection. Scale bars: 25 μm for merged images and 10 μm for magnified insets (C’-D”). (D’-D”) White arrowheads indicate “hybrid” cells that are positive for both nuclear p63 and K8 in p63-Ror2-KO mammary ducts. Yellow arrows indicate p63 + basal epithelial cells. (E-F) Flow cytometry analysis (E) eGFP + singlets from a pool of 3 WT (magenta) and 3 Ror2-KO glands (green) depicted by histogram. (F, G) CD24 and CD29 surface markers in eGFP + recombined basal cells from (F) control and (G) p63-Ror2-KO mammary glands. A basal-to-luminal cell fate shift is observed in p63-Ror2-KO glands (10,000 single-cell events analyzed per group; representative of 3 independent experiments). (H, I) Representative immunofluorescence images showing eGFP (cyan) and ERα (orange) in mammary gland cross-sections from (H) control and (I) p63-Ror2-KO mice at 4 weeks post-tamoxifen injection. Scale bar: 20 μm. Nuclear ERα is detected in eGFP + luminal cells within p63-Ror2-KO mammary ducts, while ERα is mutually exclusive from the eGFP + basal cells in controls. (J) Quantification of the percentage of ERα + luminal cells in control and p63-Ror2-KO groups (p = 0.0892; n = 6 and 7 random regions from three mammary glands, respectively). (K) Quantification of the percentage of ERα + cells in eGFP + and eGFP− luminal cells within the p63-Ror2-KO luminal layer (p < 0.0001; n = 7 random regions from three mammary glands). (L, M) Representative immunofluorescence images of eGFP (cyan) and PR (gray) in mammary gland cross-sections from (L) control and (M) p63-Ror2-KO mice at 4 weeks post-tamoxifen injection. Scale bar: 20 μm. Nuclear PR is detected in eGFP + luminal cells within p63-Ror2-KO mammary ducts. (N) Quantification of the percentage of PR + luminal cells in control and p63-Ror2-KO groups (p = 0.1677; n = 6 and 8 random regions from 3 mammary glands, respectively). (O) Quantification of the percentage of PR + cells in eGFP + and eGFP− luminal cells within the p63-Ror2-KO luminal layer (p < 0.0001; n = 8 random regions from 3 mammary glands).

Article Snippet: The following primary antibodies were used, with their respective dilutions indicated: Ror2 (1:1000; Developmental Studies Hybridoma Bank, Iowa City, IA), RhoA (1:1000; #2117; Cell Signaling Technology [CST], Danvers, MA), ROCK1 (1:1000; #4035; CST), Cdc42 (1:1000; #2466; CST), Rac1/2/3 (1:1000; #2465; CST), YAP/TAZ (1:1000; #8418; CST), phosphorylated YAP (p-YAP, 1:1000; #13008; CST), LATS1 (1:1000; #3477; CST), phosphorylated LATS1 (p-LATS1, 1:1000; #9157; CST), and GAPDH (1:2500; #5174; CST).

Techniques: Immunofluorescence, Control, Injection, Expressing, Flow Cytometry

Journal: bioRxiv

Article Title: Noncanonical Wnt/Ror2 Signaling Regulates Basal Cell Fidelity and Branching Morphogenesis in the Mammary Gland

doi: 10.1101/2025.02.25.640099

Figure Lengend Snippet: (A) Uniform manifold approximation and projection (UMAP) visualization of single-cell ATAC-seq data, showing 8,823 control (blue) and 9,500 p63-Ror2-KO (yellow) eGFP + mammary epithelial cells. (B, C) Heatmaps displaying Z-scores of transcription factor motif enrichment for (B) TP63 and (C) ESR1 across single cells in the UMAP including both control and p63-Ror2-KO eGFP + cells. Enrichment scores are represented by a gradient from blue (low score) to maroon (high score). (D) Volcano plot illustrating significantly upregulated (orange; adjusted p < 0.05 and log₂FC > 0.25) and downregulated (blue; adjusted p < 0.05 and log₂FC < –0.25) motifs in p63-Ror2-KO cells compared to p63-WT control cells. Motifs not significantly altered are shown in gray. (E) UMAP visualization of scATAC-seq data colored by cell type. Control cells primarily cluster as basal cells (blue), while p63-Ror2-KO cells distribute into basal (red), luminal progenitor (green), and ER + luminal (orange) cell types. (F) Heatmap showing significantly differentially expressed genes (adjusted p < 0.05) identified through promoter enrichment analysis among four cell types. Expression levels are represented as log 2 FC using a gradient from blue (downregulation) to maroon (upregulation). Genes within the two most significantly altered clusters are listed. (G) Peak tracks depicting accessible genomic regions for basal marker genes, including Keratin 14 , Keratin 5 , and s mooth muscle actin . These genomic elements show greater accessibility in basal cell types in both control and p63-Ror2-KO groups. (H) Peak tracks showing accessible genomic elements for luminal marker genes, including Keratin 18 , Keratin 8 , and Cdh1 ( E-cadherin ). These regions exhibit increased accessibility in luminal and luminal progenitor cell types in the p63-Ror2-KO eGFP + cells. (I) Peak tracks highlighting accessible genomic regions for luminal progenitor marker genes, including c-Kit, Aldh1a3 , and Elf5 . These regions are more accessible in luminal progenitor cell types in the p63-Ror2-KO eGFP + cells.

Article Snippet: The following primary antibodies were used, with their respective dilutions indicated: Ror2 (1:1000; Developmental Studies Hybridoma Bank, Iowa City, IA), RhoA (1:1000; #2117; Cell Signaling Technology [CST], Danvers, MA), ROCK1 (1:1000; #4035; CST), Cdc42 (1:1000; #2466; CST), Rac1/2/3 (1:1000; #2465; CST), YAP/TAZ (1:1000; #8418; CST), phosphorylated YAP (p-YAP, 1:1000; #13008; CST), LATS1 (1:1000; #3477; CST), phosphorylated LATS1 (p-LATS1, 1:1000; #9157; CST), and GAPDH (1:2500; #5174; CST).

Techniques: Control, Expressing, Marker

Journal: bioRxiv

Article Title: Noncanonical Wnt/Ror2 Signaling Regulates Basal Cell Fidelity and Branching Morphogenesis in the Mammary Gland

doi: 10.1101/2025.02.25.640099

Figure Lengend Snippet: (A) Bubble plot showing Reactome Pathway Analysis enrichment based on genes with significantly altered promoter accessibility in scATAC-seq data from control and p63-Ror2-KO cells. Pathways are ranked by the number of genes with altered promoter accessibility, represented by bubble size and red color gradients. (B) Quantitative RT-qPCR analysis of Rho GTPase-activating proteins Arhgap24 and Arhgap32 in FACS-isolated basal epithelial cells from control and p63-Ror2-KO mice. Gene expression levels were normalized to GAPDH, and fold changes were plotted relative to the control group ( p = 0.008 for Arhgap24 ; p < 0.001 for Arhgap32 ; n = 3 biological replicates per group). (C) Western blot for Ror2 showing efficient Ror2 knockdown with LeGO-shRor2 cells relative to LeGO-shLUC control cells. (D) Western blot analysis of GTP-bound RhoA, total RhoA, ROCK1, Cdc42, Rac1/2/3, and GAPDH in shLUC and shRor2 mammary basal epithelial cells with or without Wnt5a treatment. (E) Peak tracks illustrating accessible genomic elements for YAP1 downstream target genes Vgll3, Ctgf, and Ankrd1. These regions are less accessible in luminal cell types in p63-Ror2-KO groups. (F) Venn diagram showing the overlap between significantly altered transcription factors identified in scATAC-seq motif enrichment analysis and YAP1-binding transcription factors from previous ChIP sequencing data . (G) Western blot analysis of phospho-YAP1, total YAP1, TAZ, phospho-LATS1, LATS1, and GAPDH in shLUC and shRor2 mammary basal epithelial cells with or without Wnt5a treatment. (H) Quantitative RT-qPCR analysis of Hippo/YAP1 signaling genes in FACS-isolated basal epithelial cells from control and p63-Ror2-KO mice. Gene expression levels were normalized to GAPDH , and fold changes were plotted relative to the control group ( p = 0.015 for LATS2; p < 0.001 for other genes; n = 3 biological replicates per group). (I, J) Representative immunofluorescence images showing eGFP (green) and YAP1 (gray) in mammary gland cross-sections from (I) control and (J) p63-Ror2-KO mice at 4 weeks post-tamoxifen injection. Scale bars: 20 μm for merged images and 10 μm for magnified insets (I’, I”, J’, J”). White arrowheads indicate nuclear YAP1 in eGFP + basal cells, while yellow arrowheads indicate the absence of nuclear YAP1 in eGFP + luminal cells within p63-Ror2-KO mammary ducts.

Article Snippet: The following primary antibodies were used, with their respective dilutions indicated: Ror2 (1:1000; Developmental Studies Hybridoma Bank, Iowa City, IA), RhoA (1:1000; #2117; Cell Signaling Technology [CST], Danvers, MA), ROCK1 (1:1000; #4035; CST), Cdc42 (1:1000; #2466; CST), Rac1/2/3 (1:1000; #2465; CST), YAP/TAZ (1:1000; #8418; CST), phosphorylated YAP (p-YAP, 1:1000; #13008; CST), LATS1 (1:1000; #3477; CST), phosphorylated LATS1 (p-LATS1, 1:1000; #9157; CST), and GAPDH (1:2500; #5174; CST).

Techniques: Control, Quantitative RT-PCR, Isolation, Gene Expression, Western Blot, Knockdown, Binding Assay, ChIP-sequencing, Immunofluorescence, Injection

Journal: bioRxiv

Article Title: Noncanonical Wnt/Ror2 Signaling Regulates Basal Cell Fidelity and Branching Morphogenesis in the Mammary Gland

doi: 10.1101/2025.02.25.640099

Figure Lengend Snippet: (A-C) Representative immunofluorescence images showing eGFP (cyan), ERα (orange), K8 (magenta), and nuclei (gray) in cross-sections of mammary gland organoids cultured in Matrigel for 4 days under three conditions: (A) control WT, (B) p63-Ror2-KO, and (C) WT treated with the ROCK inhibitor Y-27632. (A’-C“) Insets within A-C showing eGFP (cyan) with ERα (orange) or eGFP (cyan) with K8 (magenta). Vehicle controls exhibit mutual exclusivity between eGFP and ERα, as well as eGFP and K8, unlike Y-27632 or Ror2-KO organoids. Scale bar: 20 μm. White arrowheads indicate eGFP + cells expressing ERα. (D) Quantification of the percentage of ERα+ cells among eGFP + cells across the 3 groups. Statistical comparisons: p = 0.0003 for WT-Veh vs. KO-Veh; p < 0.0001 for WT-Veh vs. WT-ROCKi; p = 0.06 for KO-Veh vs. WT-ROCKi ( n = 11, 17, and 12 random organoids from 3 independent wells, respectively). (E) Quantification of the percentage of K8 + cells among eGFP + cells in the three groups. Statistical comparisons: p < 0.0001 for WT-Veh vs. KO-Veh; p < 0.0001 for WT-Veh vs. WT-ROCKi Y-27632; p = 0.10 for KO-Veh vs. WT-ROCKi Y-27632 ( n = 11, 17, and 12 random organoids from three independent wells, respectively).

Article Snippet: The following primary antibodies were used, with their respective dilutions indicated: Ror2 (1:1000; Developmental Studies Hybridoma Bank, Iowa City, IA), RhoA (1:1000; #2117; Cell Signaling Technology [CST], Danvers, MA), ROCK1 (1:1000; #4035; CST), Cdc42 (1:1000; #2466; CST), Rac1/2/3 (1:1000; #2465; CST), YAP/TAZ (1:1000; #8418; CST), phosphorylated YAP (p-YAP, 1:1000; #13008; CST), LATS1 (1:1000; #3477; CST), phosphorylated LATS1 (p-LATS1, 1:1000; #9157; CST), and GAPDH (1:2500; #5174; CST).

Techniques: Immunofluorescence, Cell Culture, Control, Expressing