Journal: JBMR Plus

Article Title: Osteocytes regulate osteoprotegerin expression via the p38-MAPK-CREB pathway in rheumatoid arthritis

doi: 10.1093/jbmrpl/ziag023

Figure Lengend Snippet: TNF-α-induced changes in Wnt/β-catenin-related transcripts and activation of the p38-MAPK-CREB signaling pathway in osteocytes. (A) Dot plots showing relative mRNA levels of Ctnnb1 (β-catenin), Lef1 , and Axin2 in MLO-Y4 cells treated with or without TNF-α ( n = 3). (B) Representative immunoblotting images showing phosphorylated p38 MAPK (p-p38), total p38, phosphorylated CREB (p-CREB), total CREB, phosphorylated JNK (p-JNK), total JNK, phosphorylated ERK (p-ERK), total ERK, and β-actin in MLO-Y4 cells following TNF-α treatment. Data in (A) are shown as individual values with the mean ± SD and were analyzed by the two-sided Student’s t -test. (C) Representative immunoblot of OPG in TNF-α-stimulated MLO-Y4 cells treated with a p38 inhibitor or a CREB inhibitor. Cells were stimulated with TNF-α in the presence or absence of SB203580 (p38 inhibitor) or 666-15 (CREB inhibitor), and total cell lysates were subjected to immunoblotting for OPG. β-actin serves as the loading control. Numeric values shown beneath each band represent the relative expression levels of OPG/β-actin for the corresponding lane. (D) ChIP-PCR demonstrating enrichment of the OPG promoter in anti-CREB immunoprecipitates from MLO-Y4 cells with or without TNF-α stimulation, with densitometric quantification of the ChIP-PCR bands; input DNA served as the internal control, normal IgG as the negative control, and anti-histone H3 as the positive control.

Article Snippet: Quantitative PCR was performed using TaqMan Gene Expression Assays (Thermo Fisher Scientific) for the following targets: Tnfsf11 (Mm00441906_m1), Tnfrsf11b (Mm00435454_m1), Sost (Mm00470479_m1), Ctnnb1 (Mm00483039_m1), Axin2 (Mm00443610_m1), Lef1 (Mm00550265_m1), and Acp5 (Mm00475-698_m1).

Techniques: Activation Assay, Western Blot, Control, Expressing, Negative Control, Positive Control

Journal: bioRxiv

Article Title: A Wnt-responsive fibrocartilage progenitor system coordinates postnatal mandibular condylar cartilage growth

doi: 10.64898/2026.03.25.714159

Figure Lengend Snippet: (A) Inducible lineage tracing was performed using Axin2 CreERT2 ;Rosa26 ZsGreen mice. Tamoxifen was administered at postnatal day 14 (P14), and ZsGreen-positive (ZsG⁺) cells were analyzed at defined time points after induction (P16, P21, and P42). At 2 days post-induction (P16), ZsG⁺ cells are predominantly localized within the superficial and fibrocartilage zones. At 7 days post-induction (P21), ZsG⁺ cells expand within the fibrocartilage compartment and form small clusters. By 28 days post-induction (P42), ZsG⁺ cells extend vertically into the chondrocartilage zone and form columnar arrays (open arrowheads), while cells expanding laterally within the fibrocartilage layer are indicated by arrows. Following repeated tamoxifen administration, ZsG⁺ cells populate most of the fibrocartilage layer and a large proportion of chondrocytes, indicating both lateral and vertical contributions during postnatal growth. The lower panels show higher-magnification views of the regions outlined by dashed boxes in the upper panels. Closed arrowheads indicate H2B–EGFP–positive cells within the fibrocartilage (fc) zone. Representative images from n = 3 biologically independent mice per time point are shown. Scale bar, 100 μm. (B) To compare canonical Wnt signaling activity with Axin2-lineage labeling in vivo , Axin2 CreERT2 ;Rosa26 Tomato ;R26-WntVis mice were analyzed two days after tamoxifen administration. H2B–EGFP–positive nuclei (green), indicating active canonical Wnt signaling, show substantial spatial overlap with Tomato-labeled Axin2-lineage cells (red) within the superficial (sz) and fibrocartilage (fc) zones. Closed arrowheads indicate H2B–EGFP–positive cells within the fibrocartilage zone, which co-express Tomato, demonstrating concordance between Wnt activity and lineage labeling. Nuclei are counterstained with DAPI (blue). Representative images from three biologically independent mice are shown. Scale bar, 100 μm. (C) To assess proliferative dynamics, EdU label-retention analysis was performed. EdU was administered 28 days prior to tissue harvest, and EdU-retaining cells were detected within the upper fibrocartilage layer. Closed arrowheads indicate EdU-retaining cells that co-express H2B–EGFP, identifying a slow-cycling Wnt-responsive subpopulation. Scale bar, 20 μm. (D) H2B–EGFP–positive cells were isolated from mandibular condylar cartilage of P14 R26-WntVis mice by fluorescence-activated cell sorting (FACS). (E) Clonal colony formation assays were performed following in vitro culture. Isolated cells form colonies, indicating clonogenic potential. (F) Flow cytometry analysis was performed to characterize cell surface marker expression. Isolated cells express mesenchymal stromal cell–associated markers (CD29, CD105, Sca-1, CD44, CD106) and lack hematopoietic markers (CD45 and CD11), indicating a mesenchymal stromal phenotype. (G) Isolated Wnt-responsive cells were cultured under osteogenic, chondrogenic, or adipogenic differentiation conditions. Cells exhibit multilineage differentiation capacity, as evidenced by positive staining with Alizarin Red (osteogenesis), Alcian Blue (chondrogenesis), and Oil Red O (adipogenesis), respectively. Scale bar, 100 μm. Abbreviations: sz, superficial zone; fc, fibrocartilage zone; cc, chondrocartilage zone.

Article Snippet: The following mouse lines were used in this study: Axin2 CreERT2 mice (purchased from Jackson laboratory, JAX:018867), Rosa26 ZsGreen reporter mice (purchased from Jackson laboratory, JAX mice 007906), Ctnnb1 fl/fl mice (purchased from Jackson laboratory, JAX:004152), Rosa26 tdTomato reporter mice (purchased from Jackson laboratory, JAX mice 007909), Foxm1 fl/fl mice (kindly provided from Dr. Dragana Kopanja) , Ctnnb1 exon3-flox (kindly provided from Dr. Makoto M. Takeo) ( ) and R26-WntVis reporter mice (purchased from RIKEN BioResource Research Center; CDB0303K https://large.riken.jp/distribution/mutant-list.html ) ( ).

Techniques: Activity Assay, Labeling, In Vivo, Isolation, Fluorescence, FACS, In Vitro, Flow Cytometry, Marker, Expressing, Cell Culture, Staining

Journal: bioRxiv

Article Title: A Wnt-responsive fibrocartilage progenitor system coordinates postnatal mandibular condylar cartilage growth

doi: 10.64898/2026.03.25.714159

Figure Lengend Snippet: (A) Safranin-O/Fast Green staining was performed on sagittal sections of mandibular condylar cartilage from control and Axin2 CreERT2 ;Ctnnb1 fl/fl mice at P42 (28 days after tamoxifen administration). Mutant condyles show marked depletion of the fibrocartilage compartment, whereas the chondrocartilage zone is relatively preserved, indicating impaired maintenance of the fibrocartilage layer upon β-catenin loss. Lower panels show higher magnification views of boxed regions. Scale bar, 100 μm. (B) Immunofluorescence staining for Ki67 was performed in Axin2 CreERT2 ;Ctnnb1 fl/fl ;Rosa26 ZsGreen mice and littermate controls at P21 (7 days post-tamoxifen). ZsGreen marks Axin2-lineage cells, enabling comparison between recombined (ZsGreen-positive) and adjacent non-recombined cells within the same tissue. β-catenin–deficient lineage cells exhibit reduced proliferative activity compared with neighboring cells, indicating a cell-autonomous requirement for β-catenin in fibrocartilage proliferation. Scale bar, 100 μm. (C) Quantification of fibrocartilage thickness, chondrocartilage thickness, and Ki67-positive cells in ZsGreen-positive and ZsGreen-negative populations was performed. β-catenin deficiency significantly reduces fibrocartilage thickness and proliferation within Axin2-lineage cells, without markedly affecting the chondrocartilage compartment. Data represent mean ± SD from biologically independent mice (n = 4–5 per genotype). (D) Safranin-O staining was performed on Axin2 CreERT2 ;Ctnnb1 exon3-flox mice and littermate controls at P42. Constitutive β-catenin activation results in altered cartilage organization, characterized by expansion of the fibrocartilage domain and reduction of the safranin-O–positive chondrocartilage zone, indicating disrupted compartmental balance. Lower panels show higher magnification views of boxed regions. Scale bar, 100 μm. (E) Ki67 immunostaining was performed in Axin2 CreERT2 ;Ctnnb1 exon3-flox mice at P21. Despite expansion of the fibrocartilage compartment, proliferative activity is not increased compared with controls, indicating that β-catenin activation alters tissue organization without proportionally enhancing proliferation. Scale bar, 100 μm. (F) Quantification of fibrocartilage thickness, chondrocartilage thickness, the ratio of fibrocartilage to total cartilage thickness, and Ki67-positive cells in Axin2-lineage cells was performed. β-catenin activation increases fibrocartilage proportion without significantly increasing proliferation. Data represent mean ± SD from biologically independent mice (n = 5 per genotype). Statistical significance was assessed using two-tailed Student’s t-test. n.s., not significant; *P < 0.05; **P < 0.01. Abbreviations: sz, superficial zone; fc, fibrocartilage zone; cc, chondrocartilage zone.

Article Snippet: The following mouse lines were used in this study: Axin2 CreERT2 mice (purchased from Jackson laboratory, JAX:018867), Rosa26 ZsGreen reporter mice (purchased from Jackson laboratory, JAX mice 007906), Ctnnb1 fl/fl mice (purchased from Jackson laboratory, JAX:004152), Rosa26 tdTomato reporter mice (purchased from Jackson laboratory, JAX mice 007909), Foxm1 fl/fl mice (kindly provided from Dr. Dragana Kopanja) , Ctnnb1 exon3-flox (kindly provided from Dr. Makoto M. Takeo) ( ) and R26-WntVis reporter mice (purchased from RIKEN BioResource Research Center; CDB0303K https://large.riken.jp/distribution/mutant-list.html ) ( ).

Techniques: Staining, Control, Mutagenesis, Immunofluorescence, Comparison, Activity Assay, Activation Assay, Immunostaining, Two Tailed Test

Journal: bioRxiv

Article Title: A Wnt-responsive fibrocartilage progenitor system coordinates postnatal mandibular condylar cartilage growth

doi: 10.64898/2026.03.25.714159

Figure Lengend Snippet: (A) Feature plots were generated to examine expression of TGF-β ligands ( Tgfb1 , Tgfb2 , Tgfb3 ) and receptors ( Tgfbr1 , Tgfbr2 ) across mandibular condylar cartilage populations. Tgfb1 , Tgfb2 , and Tgfbr1 show inverse correlation with H2B–EGFP expression within the MC-progenitor population, whereas Tgfb3 and Tgfbr2 show neutral or positive association, indicating selective antagonism between canonical Wnt activity and specific TGF-β signaling components. (B) Quantitative PCR analysis was performed in cultured Wnt-responsive stem/progenitor cells following Ctnnb1 knockdown (KD) or constitutive β-catenin activation (CA). Ctnnb1 KD increases Tgfb1 and Tgfb2 expression, whereas β-catenin CA suppresses their expression, while Tgfb3 remains unchanged, indicating that β-catenin negatively regulates specific TGF-β ligands. (C) Immunofluorescence staining for phosphorylated Smad2/3 (pSmad2/3) and Sox9 was performed in mandibular condyles from control and Axin2 CreERT2 ;Ctnnb1 fl/fl mice. In control mice, pSmad2/3 and Sox9 are largely restricted to the chondrocartilage zone. In contrast, β-catenin–deficient lineage cells show ectopic activation of pSmad2/3 and Sox9 within the fibrocartilage compartment and extension into the chondrocartilage region, indicating enhanced TGF-β–Smad signaling and ectopic activation of chondrogenic programs in vivo . Closed arrowheads indicate cells co-expressing pSmad2/3 and ZsGreen, whereas open arrowheads indicate cells co-expressing Sox9 and ZsGreen. Representative images from n = 3 biologically independent animals per genotype are shown. (D,E) Proliferation assays were performed in Wnt-responsive cells following Ctnnb1 KD or CA. Ctnnb1 KD reduces cell proliferation, whereas β-catenin CA enhances proliferative activity, indicating that β-catenin positively regulates proliferative capacity. (F) Osteogenic differentiation assays were performed and assessed by Alizarin Red staining. Ctnnb1 KD reduces mineralized nodule formation, whereas β-catenin CA enhances osteogenic differentiation, indicating promotion of osteogenic programs by β-catenin. (G) Chondrogenic differentiation assays were performed and assessed by Alcian Blue staining. Ctnnb1 KD increases cartilage matrix deposition, whereas β-catenin CA suppresses chondrogenic differentiation, indicating that β-catenin restrains chondrogenic commitment. (H,I) Quantitative PCR analyses were performed to assess lineage marker expression. Ctnnb1 KD reduces osteogenic markers ( Col1a1 , Alpl ) and increases chondrogenic markers ( Col2a1 , Acan ), whereas β-catenin CA shows the opposite trend, confirming reciprocal regulation of lineage programs. Independent shRNA validation experiments are shown in . Data are presented as mean ± s.d. from n = 3 biologically independent experiments unless otherwise indicated, each performed in technical triplicate. Statistical significance was assessed using two-tailed Student’s t-test. n.s., not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Abbreviations: sz, superficial zone; fc, fibrocartilage zone; cc, chondrocartilage zone. Scale bars, 100 μm.

Article Snippet: The following mouse lines were used in this study: Axin2 CreERT2 mice (purchased from Jackson laboratory, JAX:018867), Rosa26 ZsGreen reporter mice (purchased from Jackson laboratory, JAX mice 007906), Ctnnb1 fl/fl mice (purchased from Jackson laboratory, JAX:004152), Rosa26 tdTomato reporter mice (purchased from Jackson laboratory, JAX mice 007909), Foxm1 fl/fl mice (kindly provided from Dr. Dragana Kopanja) , Ctnnb1 exon3-flox (kindly provided from Dr. Makoto M. Takeo) ( ) and R26-WntVis reporter mice (purchased from RIKEN BioResource Research Center; CDB0303K https://large.riken.jp/distribution/mutant-list.html ) ( ).

Techniques: Generated, Expressing, Activity Assay, Real-time Polymerase Chain Reaction, Cell Culture, Knockdown, Activation Assay, Immunofluorescence, Staining, Control, In Vivo, Marker, shRNA, Biomarker Discovery, Two Tailed Test

Journal: bioRxiv

Article Title: A Wnt-responsive fibrocartilage progenitor system coordinates postnatal mandibular condylar cartilage growth

doi: 10.64898/2026.03.25.714159

Figure Lengend Snippet: (A) Gross morphology of control and Axin2 CreERT2 ;Ctnnb1 fl/fl mice at P42. Representative images from dorsal (top) and lateral (side) views show no overt differences in overall body size or morphology between genotypes. (B) Micro-CT analysis of mandibular condyles from control and Axin2 CreERT2 ;Ctnnb1 fl/fl mice at P42. Three-dimensional reconstructions reveal reduced condylar length in β-catenin–deficient mice compared with littermate controls. Scale bars: 2 mm.

Article Snippet: The following mouse lines were used in this study: Axin2 CreERT2 mice (purchased from Jackson laboratory, JAX:018867), Rosa26 ZsGreen reporter mice (purchased from Jackson laboratory, JAX mice 007906), Ctnnb1 fl/fl mice (purchased from Jackson laboratory, JAX:004152), Rosa26 tdTomato reporter mice (purchased from Jackson laboratory, JAX mice 007909), Foxm1 fl/fl mice (kindly provided from Dr. Dragana Kopanja) , Ctnnb1 exon3-flox (kindly provided from Dr. Makoto M. Takeo) ( ) and R26-WntVis reporter mice (purchased from RIKEN BioResource Research Center; CDB0303K https://large.riken.jp/distribution/mutant-list.html ) ( ).

Techniques: Control, Micro-CT

Journal: bioRxiv

Article Title: A Wnt-responsive fibrocartilage progenitor system coordinates postnatal mandibular condylar cartilage growth

doi: 10.64898/2026.03.25.714159

Figure Lengend Snippet: (A) Differential gene expression analysis was performed comparing H2B–EGFP –positive and H2B–EGFP –negative cells within the MC-progenitor cluster identified by single-cell RNA sequencing. Genes are ranked by statistical significance. Foxm1 is significantly enriched in the H2B–EGFP –positive population, whereas Tgfb1 is enriched in the H2B–EGFP –negative population, indicating divergent transcriptional programs associated with Wnt activity. (B) Feature plots were generated to visualize expression of representative genes across mandibular condylar cartilage populations. Wnt-responsive cells show enriched expression of Foxm1 and IGF signaling–related genes ( Igf1 , Igf1r , Igfbp4 , Igfbp7 ), whereas Wnt-low populations express Tgfb1 and related factors ( Igf2r , Igfbp5 , Igfbp6 ), supporting distinct signaling states. (C) Western blot analysis was performed in isolated Wnt-responsive cells transfected with control vector or constitutively active β-catenin (S33Y). Cells were stimulated with recombinant IGF1 for the indicated time points. β-catenin activation enhances Foxm1 expression and downstream mitogenic signaling, including ERK and IGF1R phosphorylation, indicating that β-catenin promotes proliferative signaling responses. (D) Co-immunoprecipitation was performed to assess interaction between β-catenin and Foxm1. Cell lysates immunoprecipitated with anti–β-catenin antibody show enrichment of Foxm1 compared with control IgG, indicating a physical association between β-catenin and Foxm1. (E,F) Histological and immunofluorescence analyses were performed on mandibular condyles from control and Axin2 CreERT2 ;Ctnnb1 fl/+ ;Foxm1 fl/+ compound heterozygous mice at P42. H&E staining reveals reduced fibrocartilage thickness, and Ki67 staining shows decreased proliferative activity, indicating cooperative effects of β-catenin and Foxm1 in maintaining fibrocartilage proliferation. Scale bar, 100 μm. (G) Quantification of fibrocartilage thickness and Ki67-positive cells was performed. Compound heterozygous mice show reduced fibrocartilage thickness and decreased proliferation compared with controls. Data are presented as mean ± s.d. Each dot represents one biologically independent animal. (H,I) Histological and immunofluorescence analyses were performed on mandibular condyles from control and Axin2 CreERT2 ;Foxm1 fl/fl mice at P42. Foxm1 deletion results in marked condylar hypoplasia and reduced proliferative activity, indicating a critical role for Foxm1 in fibrocartilage growth. Scale bar, 100 μm. (J) Quantification of cartilage thickness and proliferative indices was performed in Foxm1 conditional knockout mice. Foxm1 deficiency significantly reduces cartilage growth and proliferation. Data are presented as mean ± s.d. Each dot represents one biologically independent animal. Statistical significance was assessed using two-tailed Student’s t-test. n.s., not significant; **P < 0.01; ****P < 0.0001. Abbreviations: sz, superficial zone; fc, fibrocartilage zone; cc, chondrocartilage zone.

Article Snippet: The following mouse lines were used in this study: Axin2 CreERT2 mice (purchased from Jackson laboratory, JAX:018867), Rosa26 ZsGreen reporter mice (purchased from Jackson laboratory, JAX mice 007906), Ctnnb1 fl/fl mice (purchased from Jackson laboratory, JAX:004152), Rosa26 tdTomato reporter mice (purchased from Jackson laboratory, JAX mice 007909), Foxm1 fl/fl mice (kindly provided from Dr. Dragana Kopanja) , Ctnnb1 exon3-flox (kindly provided from Dr. Makoto M. Takeo) ( ) and R26-WntVis reporter mice (purchased from RIKEN BioResource Research Center; CDB0303K https://large.riken.jp/distribution/mutant-list.html ) ( ).

Techniques: Gene Expression, Single Cell, RNA Sequencing, Activity Assay, Generated, Expressing, Western Blot, Isolation, Transfection, Control, Plasmid Preparation, Recombinant, Activation Assay, Phospho-proteomics, Immunoprecipitation, Immunofluorescence, Staining, Knock-Out, Two Tailed Test

Journal: Scientific Reports

Article Title: Wnt/β -catenin signalling shapes macrophage behaviour during injury and repair in the mouse submandibular gland

doi: 10.1038/s41598-026-38873-1

Figure Lengend Snippet: Histological changes in the SMG 8 days after duct ligation. Sirius Red trichrome staining of Axin2 CreERT2/+ ; R26 mTmG/+ SMGs shows extensive acinar cell loss, ductal dilation, and capsule thickening with increased collagen deposition ( B , D ) compared with non-operated controls ( A , C ). Alcian Blue labels acinar cells (white arrows), while ducts appear pale yellow (black arrows). Double-headed arrows in ( C ) and ( D ) indicate SMG capsule thickness. ( E ) Schematic of the ligation procedure of the MED, with the sublingual duct left intact. ( F , G ) Experimental timeline indicating ligation, non-operated controls, and tamoxifen administration. TAM: tamoxifen; CO: Corn Oil; MED: Main Excretory Duct. Scale bar: 100 μm.

Article Snippet: pCagCre ERT2/+ , Axin2 CreERT2/+ , R26 mTmG/+ , and Wntless flox−flox (fl/fl) mice were obtained from The Jackson Laboratory.

Techniques: Ligation, Staining

Journal: Scientific Reports

Article Title: Wnt/β -catenin signalling shapes macrophage behaviour during injury and repair in the mouse submandibular gland

doi: 10.1038/s41598-026-38873-1

Figure Lengend Snippet: Increased Axin2⁺ cells within the stromal compartment of the SMG after injury. GFP immunostaining of Axin2 CreERT2/ + ; R26 mTmG/+ SMGs shows a marked expansion of Axin2⁺ (GFP⁺) cells within the capsule, septa, and interstitial regions between acini and ducts 8 days after ligation ( D – F ) compared with non-operated controls (48 h) ( A – C ). Double immunostaining for GFP and the ductal marker K7 reveals few K7⁺ ductal cells with active Wnt/β-catenin signalling ( C , white arrows). At baseline, only occasional elongated Axin2⁺ cells were detected in septa ( B , inset). Nuclei were counterstained with Hoechst (blue). GFP: Green; Tomato/K7: Red. ED: Excretory duct. Scale bars: 250 μm ( A , B , D – F ); 25 μm ( C ).

Article Snippet: pCagCre ERT2/+ , Axin2 CreERT2/+ , R26 mTmG/+ , and Wntless flox−flox (fl/fl) mice were obtained from The Jackson Laboratory.

Techniques: Immunostaining, Ligation, Double Immunostaining, Marker

Journal: Scientific Reports

Article Title: Wnt/β -catenin signalling shapes macrophage behaviour during injury and repair in the mouse submandibular gland

doi: 10.1038/s41598-026-38873-1

Figure Lengend Snippet: Inflammatory and macrophage populations exhibit active Wnt/β-catenin signalling after SMG injury. ( A , B ) Double immunostaining of ligated Axin2 CreERT2/+ ; R26 mTmG/+ SMGs shows colocalisation of GFP⁺ cells with CD45⁺ immune cells ( A ) and F4/80⁺ macrophages ( B ) within the capsule and septa 8 days after injury, indicating activation of canonical Wnt signalling. Nuclei were counterstained with Hoechst (blue). GFP: Green; CD45/F4/80: Red. ( C – F ) Flow cytometry analysis reveals that 18% of total cells were CD45⁺, of which 37% were F4/80⁺; 29% of CD45⁺ cells and 34.3% of macrophages exhibited GFP-associated Wnt/β-catenin activity. Scale bar: 250 μm.

Article Snippet: pCagCre ERT2/+ , Axin2 CreERT2/+ , R26 mTmG/+ , and Wntless flox−flox (fl/fl) mice were obtained from The Jackson Laboratory.

Techniques: Double Immunostaining, Activation Assay, Flow Cytometry, Activity Assay

Journal: Scientific Reports

Article Title: Wnt/β -catenin signalling shapes macrophage behaviour during injury and repair in the mouse submandibular gland

doi: 10.1038/s41598-026-38873-1

Figure Lengend Snippet: Concomitant upregulation of Axin2 and F4/80 expression following SMG injury. Quantitative PCR analysis of wild-type SMGs shows dynamic changes in gene expression after duct ligation. ( A ) Axin2 expression peaks significantly at days 3 and 6 post-injury. ( B ) F4/80 expression follows a similar pattern, with significant upregulation at the same time points, indicating a temporal correlation between macrophage recruitment and Wnt/β-catenin activation. Data are normalised to Gapdh expression. * P < 0.05, ** P < 0.01 (Student’s t -test).

Article Snippet: pCagCre ERT2/+ , Axin2 CreERT2/+ , R26 mTmG/+ , and Wntless flox−flox (fl/fl) mice were obtained from The Jackson Laboratory.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Gene Expression, Ligation, Activation Assay

Journal: Scientific Reports

Article Title: Wnt/β -catenin signalling shapes macrophage behaviour during injury and repair in the mouse submandibular gland

doi: 10.1038/s41598-026-38873-1

Figure Lengend Snippet: Axin2⁺ cells are predominantly recruited to the SMG 6 days after injury. Immunofluorescence staining for GFP in ligated Axin2 CreERT2/+ ; R26 mTmG/+ SMGs reveals few Axin2⁺ cells within the capsule and septa at day 3 post-injury ( A , B ), but a marked increase by day 6 ( C , D ). Schematics illustrate tamoxifen administration and collection timelines for 3-day ( E ) and 6-day ( F ) injury protocols. Nuclei were counterstained with Hoechst (blue). GFP: Green; Tomato: Red. TAM: tamoxifen. Scale bar: 250 μm.

Article Snippet: pCagCre ERT2/+ , Axin2 CreERT2/+ , R26 mTmG/+ , and Wntless flox−flox (fl/fl) mice were obtained from The Jackson Laboratory.

Techniques: Immunofluorescence, Staining

Journal: Scientific Reports

Article Title: Wnt/β -catenin signalling shapes macrophage behaviour during injury and repair in the mouse submandibular gland

doi: 10.1038/s41598-026-38873-1

Figure Lengend Snippet: Axin2⁺ cells arise predominantly from recruited Axin2⁻ cells following SMG injury. Immunofluorescence staining for GFP in Axin2 CreERT2/+ ; R26 mTmG/+ SMGs shows few Axin2⁺ cells when tamoxifen was administered before ligation ( B , E , H ) compared with a marked increase when given after injury ( C , F , I ). Recruited Axin2⁺ cells were abundant in the interstitial space, gland capsule, and around excretory ducts relative to non-operated controls ( A , D , G ). Schematics illustrate tamoxifen administration and tissue collection timelines for the non-operated control (J), tamoxifen-before (K), and tamoxifen-after (L) protocols. Nuclei were counterstained with Hoechst (blue). GFP: Green; Tomato: Red. Scale bar: 250 μm.

Article Snippet: pCagCre ERT2/+ , Axin2 CreERT2/+ , R26 mTmG/+ , and Wntless flox−flox (fl/fl) mice were obtained from The Jackson Laboratory.

Techniques: Immunofluorescence, Staining, Ligation, Control

Journal: Scientific Reports

Article Title: Wnt/β -catenin signalling shapes macrophage behaviour during injury and repair in the mouse submandibular gland

doi: 10.1038/s41598-026-38873-1

Figure Lengend Snippet: Wls depletion increases CD206⁺ macrophages and reduces fibrosis following SMG injury. Sirius red staining and immunofluorescence of ligated Axin2 CreERT2/+ ; Wls fl/fl and pCAG CreERT2/+ ; Wls fl/fl SMGs show fewer dilated ducts and a thinner gland capsule following Wls depletion compared with controls ( A – H ). Immunostaining for CD206 (pro-reparative macrophages) and CD45 reveals a marked increase in CD206⁺ macrophages within the interstitial space of Wls -depleted glands ( J , L ) relative to controls ( I , K ). Schematics illustrate the timeline of ligation, tamoxifen administration, and tissue collection for the Wls (M) and Axin2Wls (N) protocols. Nuclei were counterstained with Hoechst (blue). TAM: tamoxifen; CO: Corn Oil. Scale bars: 100 μm (I, J), 250 μm ( K , L ).

Article Snippet: pCagCre ERT2/+ , Axin2 CreERT2/+ , R26 mTmG/+ , and Wntless flox−flox (fl/fl) mice were obtained from The Jackson Laboratory.

Techniques: Staining, Immunofluorescence, Immunostaining, Ligation