Journal: Signal Transduction and Targeted Therapy

Article Title: Gut dysbiosis conveys psychological stress to activate LRP5/β-catenin pathway promoting cancer stemness

doi: 10.1038/s41392-025-02159-1

Figure Lengend Snippet: Blockage of LRP5-mediated Wnt/β-catenin signaling via butyrate inhibits cancer stemness. a Heatmap showing differentially expressed genes (DEGs) between untreated SK-BR-3 cells and SK-BR-3 cells treated with 4 mM NaBu for 48 hours. The number of significant variant genes (FC > 2) are shown (FC, fold change) ( n = 3 biological replicates). b Venn diagram showing the overlap of downregulated genes from NaBu-treated SK-BR-3 cells (FC > 2, P < 0.05, 1165 genes) and upregulated genes in MDA-MB-231 spheres (FC > 2, P < 0.05, 2571 genes) and the subsequent GO analysis. c Venn diagram showing the overlap of downregulated genes from NaBu-treated SK-BR-3 cells, upregulated genes in MDA-MB-231 spheres and stemness genes. d Expression of mRNA for the indicated genes in SK-BR-3 Ctrl cells and spheres ( n = 3 biological replicates). e Relative LRP5 mRNA expression in SK-BR-3 cells treated with NaBu for 48 h in dose-dependent manner ( n = 3 biological replicates). f Relative mRNA expression of LRP5 , SOX2 , and NANOG in Ctrl and LRP5-forced expression SK-BR-3 cells treated with NaBu (4 mM) for 48 h or vehicle ( n = 3 biological replicates). g ALDH + cells in Ctrl and LRP5-forced expression SK-BR-3 cells treated with NaBu (4 mM) for 48 h or vehicle ( n = 3 biological replicates). h ELDA was performed in Ctrl and LRP5-forced expression SK-BR-3 cells treated with NaBu (4 mM) for 48 h or vehicle, and representative sphere images are shown (Scale bars, 50 μm). Stemness frequency with the upper and lower 95% confidence intervals that indicate the frequency of one stem cell in tumors. Spheres were counted from 24 replicate wells. i Representative images of spheroids formed by single cells in the 4 groups (Scale bars, 100 μm) (up), number of spheres per 1000 cells (d > 50 μm) (middle), and distribution pattern of sphere diameter (down). j Relative LRP5, p-GSK3β, GSK3β and β-catenin protein levels in Ctrl and LRP5-forced expression SK-BR-3 cells treated with NaBu (4 mM) for 48 h or vehicle. k Representative immunofluorescence images of β-catenin protein (green) in the 4 groups, with nuclear staining with DAPI (blue) (Scale bars, 25 μm). l Western blot detects the expression of nuclear and cytoplasmic protein extracts from Ctrl and LRP5 forced expression SK-BR-3 cells treated with NaBu (4 mM) for 48 h or vehicle. Actin was used as a cytoplasmic internal loading control and Lamin B1 as the nuclear internal control. Results are presented as mean ± s.d. ( a, d, e ) by a two-tailed, unpaired Student’s t- test, ( f , g , i ) by one-way ANOVA, h by the likelihood ratio test. For multiple comparisons, p-values were adjusted using the FDR correction. P values are as indicated

Article Snippet: The number of significant variant genes (FC > 2, P < 0.05) are shown. e Relative ZFP36 and pre- ZFP36 mRNA levels of SK-BR-3 and MDA-MB-231 cells treated with NaBu for 48 h in dose-dependent manner ( n = 3 biological replicates). f Relative ZFP36, LRP5, and β-catenin protein levels in SK-BR-3 cells after transfection with the siRNA targeting these three GPRs and treated with NaBu (4 mM). g Relative ZFP36, LRP5, β-catenin, Histone H3-K9, and Histone H3 protein levels in SK-BR-3 cells treated with NaBu (4 mM) and TSA (1 μM; MCE, HY-15144). h Genome browser images showing ChIP-seq signals for the ZFP36 promoter using data from the Cistrome Data Browse (GSM810671). i Relative fold change of the ZFP36 promoter binding motif in SK-BR-3 and MDA-MB-231 cells treated with NaBu (4 mM) for 48 h or vehicle as analyzed by ChIP-qPCR ( n = 3 biological replicates). j Enrichment of endogenous LRP5 3’UTR in SK-BR-3 and MDA-MB-231 cells treated with NaBu (4 mM) for 48 h or vehicle was analyzed by RNA immunoprecipitation (RIP) ( n = 3 biological replicates).

Techniques: Variant Assay, Expressing, Immunofluorescence, Staining, Western Blot, Control, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: Gut dysbiosis conveys psychological stress to activate LRP5/β-catenin pathway promoting cancer stemness

doi: 10.1038/s41392-025-02159-1

Figure Lengend Snippet: Butyrate accelerates LRP5 mRNA decay by transactivating ARE binding protein ZFP36. a Stability of LRP5 mRNA in SK-BR-3 cells treated with NaBu (4 mM) or vehicle ( n = 3 biological replicates). b Stability of LRP5 mRNA in MDA-MB-231 cells treated with 4 mM NaBu or vehicle ( n = 3 biological replicates). c Relative luciferase activity of psiCHECK2-LRP5-3’UTR-WT, Mut1, Mut2, and Mut1 + 2 in HEK293T cells treated with NaBu (4 mM) or vehicle ( n = 3 biological replicates). d Volcano plots displaying DEGs comparing SK-BR-3 cells treated with NaBu (4 mM) or vehicle. The number of significant variant genes (FC > 2, P < 0.05) are shown. e Relative ZFP36 and pre- ZFP36 mRNA levels of SK-BR-3 and MDA-MB-231 cells treated with NaBu for 48 h in dose-dependent manner ( n = 3 biological replicates). f Relative ZFP36, LRP5, and β-catenin protein levels in SK-BR-3 cells after transfection with the siRNA targeting these three GPRs and treated with NaBu (4 mM). g Relative ZFP36, LRP5, β-catenin, Histone H3-K9, and Histone H3 protein levels in SK-BR-3 cells treated with NaBu (4 mM) and TSA (1 μM; MCE, HY-15144). h Genome browser images showing ChIP-seq signals for the ZFP36 promoter using data from the Cistrome Data Browse (GSM810671). i Relative fold change of the ZFP36 promoter binding motif in SK-BR-3 and MDA-MB-231 cells treated with NaBu (4 mM) for 48 h or vehicle as analyzed by ChIP-qPCR ( n = 3 biological replicates). j Enrichment of endogenous LRP5 3’UTR in SK-BR-3 and MDA-MB-231 cells treated with NaBu (4 mM) for 48 h or vehicle was analyzed by RNA immunoprecipitation (RIP) ( n = 3 biological replicates). Relative LRP5 and ZFP36 mRNA expression ( k ) and stability of LRP5 mRNA ( l ) in SK-BR-3 cells treated with knockdown of ZFP36 and NaBu (4 mM), as assessed by RT-qPCR ( n = 3 biological replicates). m Relative luciferase activity of psiCHECK2-LRP5-3’UTR-WT, Mut1, Mut2, and Mut1 + 2 in HEK293T cells treated with ZFP36 knocked down and NaBu (4 mM) ( n = 3 biological replicates). n Relative ZFP36, LRP5, p-GSK3β, GSK3β, and β-catenin protein levels in SK-BR-3 cells treated with ZFP36 knocked down and NaBu (4 mM). o Representative immunofluorescence images of β-catenin protein (green) in SK-BR-3 cells treated with ZFP36 knocked down and NaBu (4 mM). The nucleus was stained with DAPI (blue) (Scale bars, 25 μm). p ALDH + SK-BR-3 cells treated with ZFP36 knocked down and NaBu (4 mM) ( n = 3 biological replicates). Results are presented as mean ± s.d. ( a – e, i , j ) by a two-tailed, unpaired Student’s t- test, ( k , l , m , p ) by one-way ANOVA. For multiple comparisons, p-values were adjusted using the FDR correction. P values are as indicated

Article Snippet: The number of significant variant genes (FC > 2, P < 0.05) are shown. e Relative ZFP36 and pre- ZFP36 mRNA levels of SK-BR-3 and MDA-MB-231 cells treated with NaBu for 48 h in dose-dependent manner ( n = 3 biological replicates). f Relative ZFP36, LRP5, and β-catenin protein levels in SK-BR-3 cells after transfection with the siRNA targeting these three GPRs and treated with NaBu (4 mM). g Relative ZFP36, LRP5, β-catenin, Histone H3-K9, and Histone H3 protein levels in SK-BR-3 cells treated with NaBu (4 mM) and TSA (1 μM; MCE, HY-15144). h Genome browser images showing ChIP-seq signals for the ZFP36 promoter using data from the Cistrome Data Browse (GSM810671). i Relative fold change of the ZFP36 promoter binding motif in SK-BR-3 and MDA-MB-231 cells treated with NaBu (4 mM) for 48 h or vehicle as analyzed by ChIP-qPCR ( n = 3 biological replicates). j Enrichment of endogenous LRP5 3’UTR in SK-BR-3 and MDA-MB-231 cells treated with NaBu (4 mM) for 48 h or vehicle was analyzed by RNA immunoprecipitation (RIP) ( n = 3 biological replicates).

Techniques: Binding Assay, Luciferase, Activity Assay, Variant Assay, Transfection, ChIP-sequencing, ChIP-qPCR, RNA Immunoprecipitation, Expressing, Knockdown, Quantitative RT-PCR, Immunofluorescence, Staining, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: Gut dysbiosis conveys psychological stress to activate LRP5/β-catenin pathway promoting cancer stemness

doi: 10.1038/s41392-025-02159-1

Figure Lengend Snippet: Clinical relevance of A. muciniphila , butyric acid and LRP5/β-catenin expression in breast cancer patients with negative mood. a Alpha diversity of gut microbiota between the HADS low ( n = 24) and high ( n = 33) groups of cohort 1 breast cancer patients, as indicated by Chao1 indices. b PCoA of fecal microbiota from cohort 1 breast cancer patients in the HADS low ( n = 24) and high ( n = 33) groups. c Volcano plots of the species level in the patients’ feces between the HADS low ( n = 24) and high ( n = 33) groups. The number of significant variant genes ( P < 0.05) are shown. Levels of fecal butyric acid ( d ) and serum butyric acid ( e ) in cohort 1 breast cancer patients in the HADS low ( n = 24) and HADS high ( n = 33) groups. f Representative ZFP36, LRP5, β-catenin, and NANOG IHC staining images of tumors in patients in the low and high serum butyric acid groups (Scale bar, 50 μm). Pearson correlation between butyric acid concentrations of feces and ZFP36 ( g ), LRP5 ( h ), β-catenin ( i ) and NANOG ( j ) scores in breast cancer patients ( n = 57). Statistical significance was determined by Pearson’s rank test. Relative ZFP36 ( k ) and LRP5 ( l ) mRNA expression between butyric acid in serum low ( n = 19) and high ( n = 20) groups in cohort 1 breast cancer patients. m Immunoblot analysis of proteins in breast tumor tissues (T) and adjacent normal breast tissues (N) ( n = 5). Kaplan-Meier estimates of overall survival (OS) based on the expression of ZFP36 ( n ), LRP5 ( o ) and CTNNB1 ( p ). Patients were stratified into high-expression and low-expression groups using the median gene expression value as the cutoff in breast cancer patients. Results are presented as mean ± s.d. ( a , c , d , e , k , l ) by a two-tailed, unpaired Student’s t- test, ( b ) by weighted UniFrac ANOSIM analysis, g – j by Pearson’s rank test. For multiple comparisons, p-values were adjusted using the FDR correction. P values are as indicated

Article Snippet: The number of significant variant genes (FC > 2, P < 0.05) are shown. e Relative ZFP36 and pre- ZFP36 mRNA levels of SK-BR-3 and MDA-MB-231 cells treated with NaBu for 48 h in dose-dependent manner ( n = 3 biological replicates). f Relative ZFP36, LRP5, and β-catenin protein levels in SK-BR-3 cells after transfection with the siRNA targeting these three GPRs and treated with NaBu (4 mM). g Relative ZFP36, LRP5, β-catenin, Histone H3-K9, and Histone H3 protein levels in SK-BR-3 cells treated with NaBu (4 mM) and TSA (1 μM; MCE, HY-15144). h Genome browser images showing ChIP-seq signals for the ZFP36 promoter using data from the Cistrome Data Browse (GSM810671). i Relative fold change of the ZFP36 promoter binding motif in SK-BR-3 and MDA-MB-231 cells treated with NaBu (4 mM) for 48 h or vehicle as analyzed by ChIP-qPCR ( n = 3 biological replicates). j Enrichment of endogenous LRP5 3’UTR in SK-BR-3 and MDA-MB-231 cells treated with NaBu (4 mM) for 48 h or vehicle was analyzed by RNA immunoprecipitation (RIP) ( n = 3 biological replicates).

Techniques: Expressing, Variant Assay, Immunohistochemistry, Western Blot, Gene Expression, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: Gut dysbiosis conveys psychological stress to activate LRP5/β-catenin pathway promoting cancer stemness

doi: 10.1038/s41392-025-02159-1

Figure Lengend Snippet: Working model. Psychological stress alters gut microbial community and SCFA profiles during tumor development. Administration of Akkermansia muciniphila , butyrate or high-fiber diet markedly reverses stress-caused cancer stemness and anxiety-like behavior. Butyrate transactivates ZFP36 to accelerate LRP5 mRNA decay, thereby blocking Wnt/β-catenin signaling pathway to attenuate cancer stem-like traits. Part of the elements in this figure generated from BioRender (BioRender.com)

Article Snippet: The number of significant variant genes (FC > 2, P < 0.05) are shown. e Relative ZFP36 and pre- ZFP36 mRNA levels of SK-BR-3 and MDA-MB-231 cells treated with NaBu for 48 h in dose-dependent manner ( n = 3 biological replicates). f Relative ZFP36, LRP5, and β-catenin protein levels in SK-BR-3 cells after transfection with the siRNA targeting these three GPRs and treated with NaBu (4 mM). g Relative ZFP36, LRP5, β-catenin, Histone H3-K9, and Histone H3 protein levels in SK-BR-3 cells treated with NaBu (4 mM) and TSA (1 μM; MCE, HY-15144). h Genome browser images showing ChIP-seq signals for the ZFP36 promoter using data from the Cistrome Data Browse (GSM810671). i Relative fold change of the ZFP36 promoter binding motif in SK-BR-3 and MDA-MB-231 cells treated with NaBu (4 mM) for 48 h or vehicle as analyzed by ChIP-qPCR ( n = 3 biological replicates). j Enrichment of endogenous LRP5 3’UTR in SK-BR-3 and MDA-MB-231 cells treated with NaBu (4 mM) for 48 h or vehicle was analyzed by RNA immunoprecipitation (RIP) ( n = 3 biological replicates).

Techniques: Blocking Assay, Generated

Journal: Nature medicine

Article Title: DKK2 imparts tumor immunity evasion through β-catenin-independent suppression of cytotoxic immune cell activation

doi: 10.1038/nm.4496

Figure Lengend Snippet: (a-c) . Littermates of Apc Min/+ and Apc Min/+ Dkk2 −/− mice were housed for 20 weeks (female) or 22 weeks (male), and their polyps numbers in intestines were counted and measured under a stereomicroscope after staining with methylene-blue (two-tailed Student t-Test for polyp number and two-way Anova for size; n=7). Representative H&E staining images of intestinal sections from an Apc Min/+ mouse and its Apc Min/+ Dkk2 −/− littermates are shown. (d ) Binding of the anti-DKK2 antibody 5F8 to mouse DKK2 and DKK1 proteins in an ELISA assay(n=3). (e) HEK293 cells were transfected with the Wnt reporter gene TOPFlash and treated with WNT3A conditioned medium (CM), DKK2 CM or DKK1 CM, and 5F8 (18 µg/ml) as indicated (one-way Anova; n=3). (f) HEK293 cells were transfected with LacZ (a control) or LRP5 expression plasmid. The binding of DKK2-AP fusion protein to the cells in the presence or absence of 5F8 (18 µg/ml) were determined (Two-way Anova; n=3). (g) Mice (16 weeks old male) were treated with 5F8 and an isotype antibody (IgG3) (8 mg/kg, once a week, i.p.) and their survival was recorded (P=0.004; Two-sided Mantel-Cox Log-Rank test, n=9). Data are presented as means±SEM (a-b) and ±sd (d-f) with P values shown, and experiments were repeated twice (d-f).

Article Snippet: Antibodies to phospho-Stat5 (Tyr694) (CST, 4322s), LAMP1 (sc-19992, Santa Cruz), EEA1 (BD Bioscience, 612006), phospho-AKT(serine 473) (CST, 4060), AKT1 (CST, 9272), phospho-ERK1/2 (Thr202/Tyr204) (CST, 4377), ERK1/2 (CST 9102), perforin (CST, 3693), granzyme B (CST, 4275), β-actin (CST, 3700), FLAG (Sigma Aldrich, F3165), β-catenin (BD Bioscience, 610153), LRP5 (CST, 5731), LRP6 (CST, 3395), mouse CD4-PE (eBioscience, 12-0042-82), mouse NK1.1-APC (BioLegend, 108710), mouse CD8a-PE-Cyanine7 (eBioscience, 25-0081-82), mouse CD69-PE (Biolegend ,104508), human/mouse granzyme B-FITC (BioLegend, 515403), mouse CD314 (NKG2D)-PE-Cyanine7 (eBioscience, 25-5882-81), mouse CD3e-PE (eBioscience, 12-0031-82), mouse IFNγ-PE (eBioscience, 12-7311-81), CTLA-4 / CD152 (1B8)-FITC (Thermo Fisher, HMCD15201), human CD45-eFluor® 450 (eBioscience, 48-0459-41), mouse CD107a-V450 (BD, 560648), mouse CD8a-APC (eBioscience, 17-0081-81), mouse CD25-Alexa Fluor® 488 (eBioscience, 53-0251-82), mouse CD279 (PD-1)-PE (BioLegend, 135205), mouse CD19-PE-Cyanine7 (eBioscience, 25-0193-81), mouse CD3e eFluor® 450 (eBioscience, 48-0031-82), mouse CD11b-PE (eBioscience, 12-0112-82), mouse CD27-FITC (eBioscience, 11-0271-82), Ki67 (Abcam ab,15580), Cleaved Caspase-3 (Asp175) (CST, 9661S), CD31 (Abcam ab, 28364), CD44 antibody (Abcam ab,157107), Fluorescein (FITC)-labeled AffiniPure F(ab') 2 Fragment Donkey Anti-Mouse IgG (H+L) (Jackson lab, 715-096-151), Mouse Integrin alpha 4 beta 7 (LPAM-1) APC (eBioscience, 17-5887-80), Human CD56 (NCAM) APC (eBioscience, 17-0566-41), Human CD16 PE (eBioscience, 12-0167-42), Human CD3 eFluor® 450 (eBioscience, 48-0037-42), and Alexa Fluor® 647-labeled AffiniPure F(ab') 2 Fragment Goat Anti-Rabbit IgG (H+L) (Jackson lab, 111-606-045).

Techniques: Staining, Two Tailed Test, Binding Assay, Enzyme-linked Immunosorbent Assay, Transfection, Control, Expressing, Plasmid Preparation

Journal: Nature medicine

Article Title: DKK2 imparts tumor immunity evasion through β-catenin-independent suppression of cytotoxic immune cell activation

doi: 10.1038/nm.4496

Figure Lengend Snippet: (a,b) Primary mouse NK cells were prepared from WT and Lrp5 −/− mice and treated as , followed by flow cytometry (two-way Anova) and Western analysis ( a ) and immunostaining ( b ). Immunostaining was done as . (c) C57BL/6 mice receiving Lrp5 f/f MX1 Cre ( Lrp5 −/− ) or Lrp5 f/f (WT) bone marrows were treated with poly-I:C, followed by s.c. inoculation of MC38 cells. Treatment of 5F8 (10 mg/kg, i.p.) was given at Day 12, 16, and 20 (P<0.01 for tumor growth at Days 20 and 24, IgG+WT vs 5F8+WT; P value for tumor weight is vs 5F8+WT; n=5; two-way Anova). (d) LRP5C and STAT5 coimmunoprecipitate in transfected HEK293 cells. (e-f) HEK293 cells were infected with lentiviruses expressing JAK3, IL2/15Rβ, and the common γ subunit (Rγc). The cells were then transfected with the plasmid for LRP5 intracellular domain (LRP5C), the STAT5-luc reporter gene, and RFP (internal control) for 24 hrs. The cells were stimulated with IL-15 and IL15Rα-Fc for 6 hours before the reporter gene assay ( e , two-way Anova) and Western analysis ( f ). (g-h) HEK293 cells were cotransfected with the STAT5 reporter gene plasmid and plasmids for activated JAK1 (JAK1 CA , V658F) and LRP5C as indicated. After 24 hrs, the cells were analyzed for the reporter gene activity (two-sided Student’s t-test) or by Western ( g ) or immunostaining with a phospho-STAT5 antibody and DAPI ( h ). Immunostained cells were examined by a confocal microscope, and representative images are presented with pseudocolor. Scale bars are 8 µm. (i) HEK293 cells were treated with DKK2 for times indicated. Biotinylated cell surface proteins and cell lysate proteins were analyzed by Western blotting. The LRP5 blots were quantified and normalized against total LRP5. Data are presented as means±sem with P values (a,c,g,e), and experiments were repeated three times (a,b,d-i) or twice (c).

Article Snippet: Antibodies to phospho-Stat5 (Tyr694) (CST, 4322s), LAMP1 (sc-19992, Santa Cruz), EEA1 (BD Bioscience, 612006), phospho-AKT(serine 473) (CST, 4060), AKT1 (CST, 9272), phospho-ERK1/2 (Thr202/Tyr204) (CST, 4377), ERK1/2 (CST 9102), perforin (CST, 3693), granzyme B (CST, 4275), β-actin (CST, 3700), FLAG (Sigma Aldrich, F3165), β-catenin (BD Bioscience, 610153), LRP5 (CST, 5731), LRP6 (CST, 3395), mouse CD4-PE (eBioscience, 12-0042-82), mouse NK1.1-APC (BioLegend, 108710), mouse CD8a-PE-Cyanine7 (eBioscience, 25-0081-82), mouse CD69-PE (Biolegend ,104508), human/mouse granzyme B-FITC (BioLegend, 515403), mouse CD314 (NKG2D)-PE-Cyanine7 (eBioscience, 25-5882-81), mouse CD3e-PE (eBioscience, 12-0031-82), mouse IFNγ-PE (eBioscience, 12-7311-81), CTLA-4 / CD152 (1B8)-FITC (Thermo Fisher, HMCD15201), human CD45-eFluor® 450 (eBioscience, 48-0459-41), mouse CD107a-V450 (BD, 560648), mouse CD8a-APC (eBioscience, 17-0081-81), mouse CD25-Alexa Fluor® 488 (eBioscience, 53-0251-82), mouse CD279 (PD-1)-PE (BioLegend, 135205), mouse CD19-PE-Cyanine7 (eBioscience, 25-0193-81), mouse CD3e eFluor® 450 (eBioscience, 48-0031-82), mouse CD11b-PE (eBioscience, 12-0112-82), mouse CD27-FITC (eBioscience, 11-0271-82), Ki67 (Abcam ab,15580), Cleaved Caspase-3 (Asp175) (CST, 9661S), CD31 (Abcam ab, 28364), CD44 antibody (Abcam ab,157107), Fluorescein (FITC)-labeled AffiniPure F(ab') 2 Fragment Donkey Anti-Mouse IgG (H+L) (Jackson lab, 715-096-151), Mouse Integrin alpha 4 beta 7 (LPAM-1) APC (eBioscience, 17-5887-80), Human CD56 (NCAM) APC (eBioscience, 17-0566-41), Human CD16 PE (eBioscience, 12-0167-42), Human CD3 eFluor® 450 (eBioscience, 48-0037-42), and Alexa Fluor® 647-labeled AffiniPure F(ab') 2 Fragment Goat Anti-Rabbit IgG (H+L) (Jackson lab, 111-606-045).

Techniques: Flow Cytometry, Western Blot, Immunostaining, Transfection, Infection, Expressing, Plasmid Preparation, Control, Reporter Gene Assay, Activity Assay, Microscopy

Journal: Endocrine-Related Cancer

Article Title: The histone methyltransferase EZH2, an oncogene common to benign and malignant parathyroid tumors

doi: 10.1530/erc-13-0497

Figure Lengend Snippet: Figure 4 Wnt/b-catenin signaling on EZH2 knockdown. (A) Transient transfection of siControl and siEZH2 to the sHPT-1 parathyroid tumor cell line and determination of AXIN2 (left panel) and CYCLIN D1 (right panel) expression by real-time quantitative RT-PCR analysis. (B) Western blotting analysis of transiently siRNA-transfected sHPT-1 cells using the anti-active b-catenin antibody. This antibody is specific for transcriptionally active (non- phosphorylated) b-catenin (van Noort et al. 2002). (C) Transient transfec- tion of siControl, siLRP5, and siEZH2 and determination of LRP5 and EZH2 relative mRNA expression by real-time quantitative RT-PCR analysis.

Article Snippet: EZH2 siRNA (HSS103462, Invitrogen), LRP5 siRNA (sc-43900, Santa Cruz), and control nonsilencing siRNA (Qiagen) were transfected using 20 pmol.

Techniques: Knockdown, Transfection, Expressing, Quantitative RT-PCR, Western Blot

Journal: Oncogene

Article Title: Hsp90ab1 stabilizes LRP5 to promote epithelial–mesenchymal transition via activating of AKT and Wnt/β-catenin signaling pathways in gastric cancer progression

doi: 10.1038/s41388-018-0532-5

Figure Lengend Snippet: Hsp90ab1 interacted with LRP5 protein in GC. a Immunoprecipitation assays were used to identify proteins associated with Hsp90ab1. The anti-Hsp90ab1 and IgG antibody were incubated with cell extracts, the bands specific were excised and submitted for mass spectrometry, and LRP5 was identified to be a Hsp90ab1-binding protein. b Co-IP of Hsp90ab1 and LRP5 with each other from proteins of BGC823 and MKN45 cells in GC cells. c Confocal immunofluorescence analysis showed the presence and localization of Hsp90ab1 and LRP5 in the cytoplasm of MKN45 cells. d , e Schematics outlines of both Hsp90ab1 and LRP5 structure features. f – h GST pull-down assay results indicated the direct interaction between Hsp90ab1 and LRP5. i Pull-down of LRP5 by GST-tagged Hsp90ab1 fragment (264–609a) and Hsp90ab1 fragment (1–263/610–724a)

Article Snippet: After fixation, cells were permeabilized with 0.25% Triton X-100 for 10 min at room temperature and blocked in 10% normal blocking serum at room temperature for 10 min, then incubated overnight at 4 °C with primary antibodies against Hsp90ab1 (1:200, ab203085, Abcam) and LRP5 (1:100, sc390267, Santa Cruz), and β-catenin (1:200, #8480, CST).

Techniques: Immunoprecipitation, Incubation, Mass Spectrometry, Binding Assay, Co-Immunoprecipitation Assay, Immunofluorescence, Pull Down Assay

Journal: Oncogene

Article Title: Hsp90ab1 stabilizes LRP5 to promote epithelial–mesenchymal transition via activating of AKT and Wnt/β-catenin signaling pathways in gastric cancer progression

doi: 10.1038/s41388-018-0532-5

Figure Lengend Snippet: Hsp90ab1 inhibited the ubiquitin degradation of LRP5 and promoted the proliferation and invasion of GC cells by activating AKT and Wnt/β-catenin signaling pathway. a HEK 293 T cells were transiently transfected with LRP5-wt plasmid or the K142R, K592R, K802R, K1231R, and K1561R mutants, and treated with CHX at 10 μg/ml for the indicated times. Cell lysates were assessed by Western blot analysis. b HEK 293 T cells were transiently transfected with LRP5-wt plasmid or the K142R, K592R, K802R, K1231R and K1561R mutants, and treated with CHX at 10 μg/ml and MG132 at 30 μM for 12 h. Cell lysates were assessed by Western blot analysis. c HEK 293 T cells were transiently co-transfected with LRP5-wt plasmid or the K1561R mutants and Hsp90ab1, and treated with CHX at 10 μg/ml and MG132 at 30 μM for 12 h. Cell lysates were assessed by Western blot analysis. d Hsp90ab1 increased the LRP5 level by inhibiting its ubiquitin-mediated degradation in BGC823 cells. e Western blotting analyses of the levels of Akt, Wnt/β-catenin signaling pathways and classical EMT markers in GC cells treated with Hsp90ab1 or shRNA1. f Western blotting analyses of the levels of Hsp90ab1, LRP5 and classical EMT markers in GC cells treated with co-transfection of Hsp90ab1 and siLRP5 or shHsp90ab1 and LRP5

Article Snippet: After fixation, cells were permeabilized with 0.25% Triton X-100 for 10 min at room temperature and blocked in 10% normal blocking serum at room temperature for 10 min, then incubated overnight at 4 °C with primary antibodies against Hsp90ab1 (1:200, ab203085, Abcam) and LRP5 (1:100, sc390267, Santa Cruz), and β-catenin (1:200, #8480, CST).

Techniques: Ubiquitin Proteomics, Transfection, Plasmid Preparation, Western Blot, Protein-Protein interactions, Cotransfection

Journal: Oncogene

Article Title: Hsp90ab1 stabilizes LRP5 to promote epithelial–mesenchymal transition via activating of AKT and Wnt/β-catenin signaling pathways in gastric cancer progression

doi: 10.1038/s41388-018-0532-5

Figure Lengend Snippet: LRP5 was positively correlated with Hsp90ab1 in GC cells and tissues. a Effects of co-transfection of shRNA1 and LRP5 or Hsp90ab1 and siLRP5-#1 on cell invasion and migration by Matrigel invasion assays and Scratch-wound-healing assays. b Graphical illustration of statistical result of co-transfection of shRNA2 and LRP5 or Hsp90ab1 and siLRP5-#1 on cell invasion and migration. c The Hsp90ab1 expression was positively correlated with LRP5 and β-catenin, but it was meaningless correlation with E-cadherin in 36 GC specimens. Representative figures of two cases were shown. Bottom pictures were local magnification of top pictures. d Graphical illustration of the levels of Hsp90ab1 expression that were associated with the levels of β-catenin, LRP5, and E-cadherin in 36 GC specimens

Article Snippet: After fixation, cells were permeabilized with 0.25% Triton X-100 for 10 min at room temperature and blocked in 10% normal blocking serum at room temperature for 10 min, then incubated overnight at 4 °C with primary antibodies against Hsp90ab1 (1:200, ab203085, Abcam) and LRP5 (1:100, sc390267, Santa Cruz), and β-catenin (1:200, #8480, CST).

Techniques: Cotransfection, Migration, Expressing

Journal: Signal Transduction and Targeted Therapy

Article Title: A novel role of LRP5 in tubulointerstitial fibrosis through activating TGF-β/Smad signaling

doi: 10.1038/s41392-020-0142-x

Figure Lengend Snippet: Levels of LRP5 are upregulated in the renal tubules of CKD. a , c Western blot analyses and ( b , d ) densitometry quantification of LRP5, collagen III, and collagen I in the kidney homogenates from 3-month-old Akita and 6-month-old db/db mice, as well as their respective age- and genetic background-matched, non-diabetic controls ( n = 8–10). e Immunohistochemical staining of LRP5 (brown color) in the kidneys of Akita and db/db mice and their respective non-diabetic controls ( n = 5–6; scale bar = 100 μm). The lower panels are enlarged images of the boxed areas in the upper panels. f Western blot analyses and ( g ) densitometry quantification of LRP5, α-SMA, and CTGF in the kidney homogenates from WT mice with sham surgery or UUO at day 5 and day 10 post-surgery ( n = 5–6). h Immunohistochemical staining of LRP5 (brown color) in the kidneys from WT mice with sham surgery or UUO at day 5 and day 10 ( n = 5; scale bar = 75 μm). The lower panels are enlarged images of the boxed areas in the upper panels. Each lane represents an individual mouse. All values are expressed as mean ± SEM. * p < 0.05; ** p < 0.01, by unpaired Student’s t -test

Article Snippet: HKC-8 cells were co-transfected with a control plasmid pcDNA3 or a plasmid expressing LRP5 together with a plasmid expressing either TβRI or TβRII, and cultured for 48 h. Then cells were incubated with 10 μg/ml primary antibodies for LRP5 (sc-390267, Santa Cruz, Dallas, TX), TβRI (sc-398, Santa Cruz, Dallas, TX), and TβRII (sc-17792, Santa Cruz, Dallas, TX) at 4 °C for 1 h, and washed three times with ice-cold PBS.

Techniques: Western Blot, Immunohistochemical staining, Staining

Journal: Signal Transduction and Targeted Therapy

Article Title: A novel role of LRP5 in tubulointerstitial fibrosis through activating TGF-β/Smad signaling

doi: 10.1038/s41392-020-0142-x

Figure Lengend Snippet: Knockout of Lrp5 alleviates the renal tubulointerstitial fibrosis in CKD. a , c Western blot analyses and ( b , d ) densitometry quantification of ( a , b ) CTGF, fibronectin, collagen I, and collagen III in the kidneys from WT and Lrp5 −/− mice at day 10 post-surgery ( n = 5–7), and ( c , d ) E-cadherin and α-SMA in the kidneys from WT and Lrp5 −/− mice at day 5 post-surgery ( n = 5–6). e Staining of collagen III (brown color; scale bar = 200 μm) and picro-sirius red (red color; scale bar = 200 μm) in the kidneys from WT and Lrp5 −/− mice with sham surgery or UUO at day 10 post-surgery, and staining of α-SMA (green color; scale bar = 75 μm) and E-cadherin (green color; scale bar = 100 μm) in the kidneys from WT and Lrp5 −/− mice with sham surgery or UUO at day 5 post-surgery. The lower panels are enlarged images of the boxed areas in the upper panels. White arrows indicate polarized distribution of E-cadherin in the basolateral membrane of tubules; red arrows indicate re-distribution of E-cadherin to the apical membrane of tubules. n = 5–6. Each lane represents an individual mouse. All values are expressed as mean ± SEM. ** p < 0.01, by two-way ANOVA with pair-wise multiple comparisons

Article Snippet: HKC-8 cells were co-transfected with a control plasmid pcDNA3 or a plasmid expressing LRP5 together with a plasmid expressing either TβRI or TβRII, and cultured for 48 h. Then cells were incubated with 10 μg/ml primary antibodies for LRP5 (sc-390267, Santa Cruz, Dallas, TX), TβRI (sc-398, Santa Cruz, Dallas, TX), and TβRII (sc-17792, Santa Cruz, Dallas, TX) at 4 °C for 1 h, and washed three times with ice-cold PBS.

Techniques: Knock-Out, Western Blot, Staining, Membrane

Journal: Signal Transduction and Targeted Therapy

Article Title: A novel role of LRP5 in tubulointerstitial fibrosis through activating TGF-β/Smad signaling

doi: 10.1038/s41392-020-0142-x

Figure Lengend Snippet: Knockout of Lrp5 attenuates TGF-β/Smad signaling activation in renal tubules. a Western blot analyses, ( b ) densitometry quantification and ( c ) ELISA of total TGF-β1 in the kidneys from WT and Lrp5 −/− mice at day 10 post-surgery ( n = 6–8). d Western blot analyses and ( e ) densitometry quantification of p-Smad2/3, total Smad2/3, TβRI and TβRII in the kidneys from WT and Lrp5 −/− mice at day 10 post-surgery ( n = 5–7). f Immunostaining of TβRI (red color; scale bar = 100 μm) and α-SMA (green color; scale bar = 100 μm) in the kidney sections from WT and Lrp5 −/− mice at day 5 post-surgery ( n = 5–7), and of Smad2/3 (red color; scale bar = 25 μm), and ( g ) quantification of nuclear Smad2/3 in the kidney sections from WT and Lrp5 −/− mice at day 10 post-surgery ( n = 5–10). h Luciferase activity measurement in the kidney homogenates from WT/SBE-Luc and Lrp5 −/− / SBE-Luc mice at day 10 post-surgery ( n = 5–6). Each lane represents an individual mouse. All values are expressed as mean ± SEM. ** p < 0.01; n.s., not significant, by two-way ANOVA with pair-wise multiple comparisons

Article Snippet: HKC-8 cells were co-transfected with a control plasmid pcDNA3 or a plasmid expressing LRP5 together with a plasmid expressing either TβRI or TβRII, and cultured for 48 h. Then cells were incubated with 10 μg/ml primary antibodies for LRP5 (sc-390267, Santa Cruz, Dallas, TX), TβRI (sc-398, Santa Cruz, Dallas, TX), and TβRII (sc-17792, Santa Cruz, Dallas, TX) at 4 °C for 1 h, and washed three times with ice-cold PBS.

Techniques: Knock-Out, Activation Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Immunostaining, Luciferase, Activity Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: A novel role of LRP5 in tubulointerstitial fibrosis through activating TGF-β/Smad signaling

doi: 10.1038/s41392-020-0142-x

Figure Lengend Snippet: LRP5 promotes TGF-β/Smad signaling in renal tubule epithelial cells. a , b Western blot analyses of p-Smad2/3 and total Smad2/3 ( a ) in WT PTECs and Lrp5 −/− PTECs treated with 2 ng/ml TGF-β1 for the indicated times, and ( b ) in HKC-8 cells after transfection of a control plasmid (pcDNA3) or a plasmid expressing LRP5 for 48 h, followed by the treatment with/without 2 ng/ml TGF-β1 for the indicated times. c Immunostaining of Smad2/3 (red color; scale bar = 25 μm) in HKC-8 cells after transfection of a pcDNA3 plasmid or a plasmid expressing LRP5 for 48 h, followed by the treatment with/without 2 ng/ml TGF-β1 for 60 min. d Western blot analyses and ( e ) densitometry quantification of nuclear levels of p-Smad2/3 and Smad2/3 in the fractions extracted from HKC-8 cells after transfection of a pcDNA3 plasmid or a plasmid expressing LRP5 for 48 h, followed by the treatment with/without 2 ng/ml TGF-β1 for 60 min ( n = 6). f 3TP-Lux luciferase activity assay in HKC-8 cells after transfection of the 3TP-Lux plasmid, a renilla plasmid, and a pcDNA3 plasmid or a plasmid expressing LRP5 for 48 hr, followed by the treatment with/without 4 ng/ml TGF-β1 for 16 h. Relative luciferase activity was presented as folds of that in the cells with transfection of pcDNA3 control ( n = 3). g , h Western blot analyses of fibronectin, CTGF, and α-SMA ( g ) in primary PTECs from WT and Lrp5 −/− mice exposed to the indicated concentrations of TGF-β1 for 24 h, and ( h ) in HKC-8 cells after transfection of a pcDNA3 plasmid or a plasmid expressing LRP5 for 48 h, followed by the treatment with/without 2 ng/ml TGF-β1 for 24 h. Representative images are shown from at least 3 independent experiments. All values are expressed as mean ± SEM. ** p < 0.01, by two-way ANOVA with pair-wise multiple comparisons

Article Snippet: HKC-8 cells were co-transfected with a control plasmid pcDNA3 or a plasmid expressing LRP5 together with a plasmid expressing either TβRI or TβRII, and cultured for 48 h. Then cells were incubated with 10 μg/ml primary antibodies for LRP5 (sc-390267, Santa Cruz, Dallas, TX), TβRI (sc-398, Santa Cruz, Dallas, TX), and TβRII (sc-17792, Santa Cruz, Dallas, TX) at 4 °C for 1 h, and washed three times with ice-cold PBS.

Techniques: Western Blot, Transfection, Control, Plasmid Preparation, Expressing, Immunostaining, Luciferase, Activity Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: A novel role of LRP5 in tubulointerstitial fibrosis through activating TGF-β/Smad signaling

doi: 10.1038/s41392-020-0142-x

Figure Lengend Snippet: LRP5 interacts with TβRs and stimulates the formation of TβRI/TβRII heterodimers. a Immunostaining of LRP5 (green color; scale bar = 25 μm) and TβRI (red color; scale bar = 25 μm) in the kidney sections from WT mice at day 5 post-surgery showing co-localization of LRP5 and TβRI in the injured tubules of UUO kidneys ( n = 5–7). b IP of endogenous LRP5 in HKC-8 cells. LRP5 was precipitated with its specific antibody. The precipitated proteins were immunoblotted for TβRI, TβRII, and LRP5. c , d IP of ( c ) TβRI and ( d ) LRP5 in HEK293A cells co-transfected with TβRI-His and LRP5-Flag for 48 h, followed by the treatment with/without 2 ng/ml TGF-β1 for 15 min. TβRI-His was precipitated by Ni-NTA resin and LRP5-Flag by anti-Flag resin. The precipitated proteins were immunoblotted for His and Flag. e , f IP of ( e ) LRP5 or ( f ) TβRII in HKC-8 cells co-transfected with TβRII and LRP5 for 48 h, followed by the treatment with/without 2 ng/ml TGF-β for 15 min. TβRII and LRP5 were precipitated using their specific antibodies. The precipitated proteins were immunoblotted for TβRII and LRP5. g IP of TβRI in HEK293A cells co-transfected with TβRI-His and LRP5FL-Flag, LRP5∆N-Flag or LRP5∆C-Flag for 48 h. TβRI-His was precipitated by Ni-NTA resin. The precipitated proteins were immunoblotted for His and Flag. h Measurement of 3TP-Lux luciferase activity in HKC-8 cells after transfection of the 3TP-Lux plasmid, a renilla plasmid and a control plasmid or plasmid expressing LRP5FL, LRP5∆N, or LRP5∆C for 48 h, followed by the treatment with/without 4 ng/ml TGF-β1 for 16 h. Relative luciferase activity was presented as folds of that in the cells with transfection of pcDNA3 control ( n = 3). i IP of TβRI in HKC-8 cells co-transfected with TβRI-His and LRP5-Flag or LDLR-Flag for 48 h, followed by the treatment with/without 2 ng/ml TGF-β for 15 min. TβRI-His was precipitated by Ni-NTA resin. The precipitated proteins were immunoblotted for His, Flag, and TβRII. Arrowheads indicate non-specific bands; arrows indicate target proteins. All values are expressed as mean ± SEM. ** p < 0.01; n.s., not significant, by two-way ANOVA with pair-wise multiple comparisons

Article Snippet: HKC-8 cells were co-transfected with a control plasmid pcDNA3 or a plasmid expressing LRP5 together with a plasmid expressing either TβRI or TβRII, and cultured for 48 h. Then cells were incubated with 10 μg/ml primary antibodies for LRP5 (sc-390267, Santa Cruz, Dallas, TX), TβRI (sc-398, Santa Cruz, Dallas, TX), and TβRII (sc-17792, Santa Cruz, Dallas, TX) at 4 °C for 1 h, and washed three times with ice-cold PBS.

Techniques: Immunostaining, Transfection, Luciferase, Activity Assay, Plasmid Preparation, Control, Expressing

Journal: Signal Transduction and Targeted Therapy

Article Title: A novel role of LRP5 in tubulointerstitial fibrosis through activating TGF-β/Smad signaling

doi: 10.1038/s41392-020-0142-x

Figure Lengend Snippet: LRP5 promotes the basal membrane presentation and ligand-induced internalization of TβRs. a , c , f , h Western blot analyses and ( b , d , g , i ) densitometry quantification of TβRI, TβRII, and LRP5 in the cell factions from HKC-8 cells after transfection with ( a , b , f , g ) a control plasmid (pcDNA3) or a plasmid expressing LRP5, and ( c , d , h , i ) a control plasmid (sh-scramble) or a plasmid expressing shRNA for LRP5 (shLRP5) for 48 h, followed by the treatment of 2 ng/ml TGF-β1 for the indicated times. Cells were fractionated and immunoblotted for TβRI, TβRII, and LRP5, with pan-cadherin and α-tubulin as loading controls ( n = 3). e Immunostaining of internalized TβRI, TβRII, and LRP5 in HKC-8 cells after transfection of pcDNA3 or a plasmid expressing LRP5 together with either a plasmid expressing TβRI or TβRII for 48 hr, followed by the treatment of 2 ng/ml TGF-β1 for 7.5 min (green color; scale bar = 10 μm). Representative images are shown from at least three independent experiments. All values are expressed as mean ± SEM. * p < 0.05; ** p < 0.01, by unpaired Student’s t -test

Article Snippet: HKC-8 cells were co-transfected with a control plasmid pcDNA3 or a plasmid expressing LRP5 together with a plasmid expressing either TβRI or TβRII, and cultured for 48 h. Then cells were incubated with 10 μg/ml primary antibodies for LRP5 (sc-390267, Santa Cruz, Dallas, TX), TβRI (sc-398, Santa Cruz, Dallas, TX), and TβRII (sc-17792, Santa Cruz, Dallas, TX) at 4 °C for 1 h, and washed three times with ice-cold PBS.

Techniques: Membrane, Western Blot, Transfection, Control, Plasmid Preparation, Expressing, shRNA, Immunostaining

Journal: Nature communications

Article Title: Senescent cells perturb intestinal stem cell differentiation through Ptk7 induced noncanonical Wnt and YAP signaling.

doi: 10.1038/s41467-022-35487-9

Figure Lengend Snippet: Fig. 4 | Ptk7 acts through noncanonical Wnt signaling. a Schematic repre- sentation of canonical and noncanonical Wnt signaling pathways. While canonical and noncanonical Wnt signaling pathways share components such as FZD and Dvl, other components such as LRP5/6 and β-Catenin are specific for canonical Wnt signaling. The figure was created with BioRender.com. b Representative images of organoids cultured in corresponding conditioned media with DMSO or inhibitors. Scale bar, 200 μm. c Quantification of data from (b). The percentage of cystic organoids in organoids cultured in conditioned media is shown. d Representative images of organoids treated with DMSO (carrier), Wnt3a or Wnt5a. Scale bar, 200 μm. e Quantification of data from (d). The percentage of cystic organoids in organoids cultured in conditioned media is shown. f Representative images of organoids cultured in Wnt5a with DMSO or inhibitors. Scale bar, 200 μm. g Quantification of data from (f). mean ± s.d. percentage of cystic organoids in organoids cultured in conditioned media. h Representative images of organoids

Article Snippet: A dilution series of the extracellular domain of recombinant mouse LRP5 (R&D Systems, #7344-LR) was injected.

Techniques: Protein-Protein interactions, Cell Culture

Journal: Cartilage

Article Title: The Effect of Overexpression of Lrp5 on the Temporomandibular Joint.

doi: 10.1177/1947603520968875

Figure Lengend Snippet: Figure 1. Temporomandibular joint (TMJ) subchondral bone changes in Lrp5-high bone mass (HBM) mice. Representative micro- computed tomography (CT) scans of the mandibular condyle from (A) experimental (EXP) and (B) control (CTRL) animals. The dotted lines denote the region of interest (ROI) in the condylar head that was used to quantify the following subchondral bone micro-CT parameters: (C) bone volume (BV), (D) total volume (TV), (E) percent bone volume (BV/TV), (F) trabecular thickness (Tb.Th), (G) trabecular spacing (Tb.Sp), and (H) trabecular number (Tb.N). Each bar on a graph represents mean ± standard error of the mean (SEM) for n = 10 per group. Statistically significant differences between groups are denoted by * (P < 0.05).

Article Snippet: Serial sagittal sections were incubated with Lrp5 and Sost primary antibodies (Aviva Systems Biology, San Diego, CA).

Techniques: Micro-CT, Control

Journal: Cartilage

Article Title: The Effect of Overexpression of Lrp5 on the Temporomandibular Joint.

doi: 10.1177/1947603520968875

Figure Lengend Snippet: Figure 2. Histopathology grade assessment of the MCC in Lrp5-HBM mice. Safranin-O-stained sagittal histologic sections of the TMJ from (A) EXP-A, (B) EXP-G, and (C) CTRL groups. The black rectangle denotes the middle third of the MCC in the (D) EXP-A, (E) EXP-G, and (F) CTRL animals. (G) The Osteoarthritis Research Society International (OARSI) score was calculated from the images in panels D to F. Each bar on the graph represents mean ± standard error of the mean (SEM) for n = 6 per group. HBM, high bone mass; TMJ, temporomandibular joint; EXP, experimental group; CTRL, control group; TB, temporal bone; AD, articular disc; MCC, mandibular condylar cartilage.

Article Snippet: Serial sagittal sections were incubated with Lrp5 and Sost primary antibodies (Aviva Systems Biology, San Diego, CA).

Techniques: Histopathology, Staining, Control

Journal: Cartilage

Article Title: The Effect of Overexpression of Lrp5 on the Temporomandibular Joint.

doi: 10.1177/1947603520968875

Figure Lengend Snippet: Figure 3. Increased cartilage thickness and extracellular matrix accumulation in Lrp5-HBM mice. Toluidine blue–stained sagittal histologic sections of the TMJ from (A) EXP-A, (B) EXP-G, and (C) CTRL groups. The black rectangle denotes the middle third of the MCC in the (D) EXP-A, (E) EXP-G, and (F) CTRL animals. (G) The boundary of the MCC was outlined as the ROI in ImageJ from the images in panels A to C. (H) Total cartilage area and (I) area fraction of the toluidine blue positive area were calculated in the ROIs. Each bar on the graph represents mean ± standard error of the mean (SEM) for n = 6 per group. HBM, high bone mass; TMJ, temporomandibular joint; EXP, experimental group; CTRL, control group; ROI, region of interest; TB, temporal bone; AD, articular disc; MCC, mandibular condylar cartilage.

Article Snippet: Serial sagittal sections were incubated with Lrp5 and Sost primary antibodies (Aviva Systems Biology, San Diego, CA).

Techniques: Staining, Control

Journal: Cartilage

Article Title: The Effect of Overexpression of Lrp5 on the Temporomandibular Joint.

doi: 10.1177/1947603520968875

Figure Lengend Snippet: Figure 4. Increased expression of Lrp5 and sclerostin (Sost) in the MCC in Lrp5-HBM mice. Immunohistochemistry for Lrp5 (A, B) and Sost (C, D) in the middle third of serial sagittal sections of the MCC. HBM, high bone mass; MCC, mandibular condylar cartilage.

Article Snippet: Serial sagittal sections were incubated with Lrp5 and Sost primary antibodies (Aviva Systems Biology, San Diego, CA).

Techniques: Expressing, Immunohistochemistry

Journal: Cartilage

Article Title: The Effect of Overexpression of Lrp5 on the Temporomandibular Joint.

doi: 10.1177/1947603520968875

Figure Lengend Snippet: Figure 5. Subchondral bone mineralization in Lrp5-HBM mice. (A) Expression of fluorescent bone labels calcein (Cal; green), alizarin complexone (AC; red), and demeclocycline (Dem; yellow) in an experimental animal. (B) Interlabel width (iL.Wi) and (C) mineral apposition rate (MAR) were calculated. Each bar on the graph represents mean ± standard error of the mean (SEM) for n = 4 per group. HBM, high bone mass; TB, temporal bone; AD, articular disc; MCC, mandibular condylar cartilage; AC-Cal, separation between alizarin complexone and calcein; Dem-AC, separation between demeclocycline and alizarin complexone.

Article Snippet: Serial sagittal sections were incubated with Lrp5 and Sost primary antibodies (Aviva Systems Biology, San Diego, CA).

Techniques: Expressing