Journal: Bioactive Materials

Article Title: Phosphorylation inhibition of protein-tyrosine phosphatase 1B tyrosine-152 induces bone regeneration coupled with angiogenesis for bone tissue engineering

doi: 10.1016/j.bioactmat.2020.12.025

Figure Lengend Snippet: 152RM induces the migration of MSCs partly through the FAK/STAT3 signaling pathway. (A) Transwell assays for the migration of MSCs using 152RM (n = 5 each). Representative crystal violet staining images are shown in the left panel. Quantification of cell migration is shown in the right panel. Scale bar, 100 μm. (B) RNA-seq analysis showed the alteration of cell migration-specific gene expression in MSCs cultured with 152RM (n = 3 each). (C) Relative mRNA expression levels of cell migration-specific genes in MSCs cultured with 152RM (n = 5 each). (D) Quantification of the transwell assay and cell wound scratch assay after culture with 152RM, a CXCR4 inhibitor (AMD3100) and an Integrin αvβ3 inhibitor (Cyclo(-RGDfK)) (n = 5 each). (E) Western blot analysis of the expression of E-cadherin in MSCs after the addition of 152RM (n = 5 per group). (F) Gene set enrichment analysis (GSEA) plots showing upregulation of the JAK/STAT signaling pathway in MSCs cultured with 152RM (n = 3 each). (G) RNA-seq analysis showed alterations in JAK/STAT signaling pathway-related gene expression in MSCs cultured with 152RM (n = 3 each). (H) Western blot analysis of the expression of CXCR4, integrin αvβ3, p-Jak2, Jak2, p-FAK, FAK, p-STAT3 and STAT3 in MSCs (pretreated with a CXCR4 inhibitor (AMD3100) and an integrin αvβ3 inhibitor (cyclo(-RGDfK))) after the addition of 152RM (n = 5 per group). Data are shown as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001; ANOVA and Student's t -test were employed. For all panels in this figure, data are representative of three independent experiments.

Article Snippet: Briefly, the bone sections were incubated with individual primary antibodies against mouse CD31 (ab28364; Abcam), endomucin (V.7C7; Santa Cruz), Ki67 (AF7617; R&D), beta-catenin (8480, CST), osterix (bs-1110R; Bioss), osteocalcin (bs-0470R; Bioss), Runx2 (bs-1134R; Bioss), DLL4 (bs-6044R; Bioss), Notch1 (bs-1335R; Bioss), Noggin (bs-2975R; Bioss), CXCR4 D1S7W; Cell Signaling Technology), integrin αvβ3 (bs-1310R; Bioss), CD90 (bs-20640R; Bioss), CD105 (bs-0579R; Bioss), CD271 (bs-0161R; Bioss), OPN (bs-23258R; Bioss), and phospho-VEGFR2(bs-2674R; Bioss) overnight at 4 °C.

Techniques: Migration, Staining, RNA Sequencing Assay, Expressing, Cell Culture, Transwell Assay, Wound Healing Assay, Western Blot

Journal: Bioactive Materials

Article Title: Phosphorylation inhibition of protein-tyrosine phosphatase 1B tyrosine-152 induces bone regeneration coupled with angiogenesis for bone tissue engineering

doi: 10.1016/j.bioactmat.2020.12.025

Figure Lengend Snippet: 152RM induces ECs migration partly through the FAK/ERK signaling pathway. (A) Transwell assay for the migration of ECs using 152RM (n = 5 each). Scale bar, 100 μm. Quantification of cell migration was performed (right). (B) Relative mRNA expression levels of cell migration-specific genes in ECs cultured with 152RM (n = 5 each). (C) Representative immunostaining images of p-VEGFR2 (red) ECs with or without 152RM (n = 5 per group). Scale bar, 100 μm. (D) Quantification of transwell assays after culture with 152RM, a VEGFR2 inhibitor (Ki8751) and an integrin αvβ3 inhibitor cyclo(-RGDfK) (n = 5 each). (E) Western blot analysis of the expression of integrin αvβ3, p-VEGFR2, p-FAK, FAK, p-ERK1/2 and ERK1/2 in ECs (pretreated with a VEGFR2 inhibitor (Ki8751) and an integrin αvβ3 inhibitor (cyclo(-RGDfK))) after the addition of 152RM (n = 5 per group). (F) Schematic illustration of the role of 152RM in promoting ECs migration. Data are shown as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001; ANOVA and Student's t -test were employed. For all panels in this figure, data are representative of three independent experiments.

Article Snippet: Briefly, the bone sections were incubated with individual primary antibodies against mouse CD31 (ab28364; Abcam), endomucin (V.7C7; Santa Cruz), Ki67 (AF7617; R&D), beta-catenin (8480, CST), osterix (bs-1110R; Bioss), osteocalcin (bs-0470R; Bioss), Runx2 (bs-1134R; Bioss), DLL4 (bs-6044R; Bioss), Notch1 (bs-1335R; Bioss), Noggin (bs-2975R; Bioss), CXCR4 D1S7W; Cell Signaling Technology), integrin αvβ3 (bs-1310R; Bioss), CD90 (bs-20640R; Bioss), CD105 (bs-0579R; Bioss), CD271 (bs-0161R; Bioss), OPN (bs-23258R; Bioss), and phospho-VEGFR2(bs-2674R; Bioss) overnight at 4 °C.

Techniques: Migration, Transwell Assay, Expressing, Cell Culture, Immunostaining, Western Blot

Journal: Bioactive Materials

Article Title: Phosphorylation inhibition of protein-tyrosine phosphatase 1B tyrosine-152 induces bone regeneration coupled with angiogenesis for bone tissue engineering

doi: 10.1016/j.bioactmat.2020.12.025

Figure Lengend Snippet: DBM-MSN/152RM scaffolds coordinate the recruitment of MSCs and ECs in vivo . (A) HE staining images demonstrating the recruitment of MSCs and ECs 1 week after DBM, DBM-MSN, DBM/152RM and DBM-MSN/152RM scaffold implantation (n = 5 rats per group). Scale bar, 100 μm. (B) Co-immunofluorescence staining of CXCR4, integrin αvβ3 and CD271 in MSCs from 1 week after DBM, DBM-MSN, DBM/152RM and DBM-MSN/152RM scaffold implantation (n = 5 rats per group). Scale bar, 100 μm. (C) Immunofluorescence staining of CD90 + CD105 + MSCs from 1 week after DBM, DBM-MSN, DBM/152RM and DBM-MSN/152RM scaffold implantation (n = 5 rats per group). Scale bar, 100 μm. (D) Co-immunofluorescence staining of emcn and integrin αvβ3 in type H vessels from 4 weeks after DBM, DBM-MSN, DBM/152RM and DBM-MSN/152RM scaffold implantation (n = 5 rats per group). Scale bar, 100 μm. (E) Immunofluorescence staining of CD31 + in EPCs from 4 weeks after DBM, DBM-MSN, DBM/152RM and DBM-MSN/152RM scaffold implantation (n = 5 rats per group). Scale bar, 100 μm. Data are shown as the mean ± SD. ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001; ANOVA was employed. For all panels in this figure, data are representative of three independent experiments.

Article Snippet: Briefly, the bone sections were incubated with individual primary antibodies against mouse CD31 (ab28364; Abcam), endomucin (V.7C7; Santa Cruz), Ki67 (AF7617; R&D), beta-catenin (8480, CST), osterix (bs-1110R; Bioss), osteocalcin (bs-0470R; Bioss), Runx2 (bs-1134R; Bioss), DLL4 (bs-6044R; Bioss), Notch1 (bs-1335R; Bioss), Noggin (bs-2975R; Bioss), CXCR4 D1S7W; Cell Signaling Technology), integrin αvβ3 (bs-1310R; Bioss), CD90 (bs-20640R; Bioss), CD105 (bs-0579R; Bioss), CD271 (bs-0161R; Bioss), OPN (bs-23258R; Bioss), and phospho-VEGFR2(bs-2674R; Bioss) overnight at 4 °C.

Techniques: In Vivo, Staining, Immunofluorescence

Journal: American Journal of Physiology - Renal Physiology

Article Title: NBCe1-A is required for the renal ammonia and K + response to hypokalemia

doi: 10.1152/ajprenal.00481.2019

Figure Lengend Snippet: Antibodies used (alphabetical order)

Article Snippet: ENaCβ , StressMarq Biosciences (SPC-404, Victoria, BC, Canada) , WB, 1:5,000.

Techniques: Generated

Journal: Tissue Engineering Part A

Article Title: Bone Marrow-Derived Cells Implanted into Radiation-Injured Urinary Bladders Reconstruct Functional Bladder Tissues in Rats

doi: 10.1089/ten.tea.2012.0061

Figure Lengend Snippet: FIG. 5. Differentiation of bone marrow-derived cells into nerve- like cells. Some GFP-positive im- planted cells (A–C, green, arrows) were positive for S100 (D), tubu- lin beta 3 (E), or calcitonin gene- related peptide (F), all of which are differentiation markers for nerve cell (red, arrows). These double-positive cells showed that the implanted cells differentiated into nerve-like cells (G–I, yellow, arrows). Blue: nuclei.

Article Snippet: Alternatively, they were incubated with antibodies for S100 (1:50, mouse monoclonal; Abcom, Cambridge, United Kingdom), tubulin beta 3 (1:50, rabbit monoclonal; Novus Biologicals, Inc., Littleton, CO), or calcitonin gene-related peptide (CGRP, 1:500, guinea pig polyclonal; Progen Biotechnik GmbH) as markers for nerve cells.

Techniques: Derivative Assay

Journal: The Journal of cell biology

Article Title: Proteolytic regulation of a galectin-3/Lrp1 axis controls osteoclast-mediated bone resorption.

doi: 10.1083/jcb.202206121

Figure Lengend Snippet: Figure 4. Mmp9/Mmp14 regulates galectin-3 lattice remodeling in osteoclasts. (A and B) Galectin-3 expression and cleavage as assessed with anti–galectin-3 monoclonal antibody (ab2785; Abcam; epitopes mapped against N-terminal region) by Western blot (A) and quantification (B) in BMDMs and osteoclasts (OC) generated from wild-type or DKO mice. Data are presented as mean ± SEM (n = 3 biological replicates). (C and D) Galectin-3 (green) im- munofluorescence of non-permeabilized wild-type or DKO osteoclasts stained with an anti–galectin-3 monoclonal antibody (#125401; Biolegend; clone M3/38, epitopes mapped against N-terminal region; C), and surface galectin-3 level quantified (D). Scale bar, 20 μm. Data are presented as mean ± SEM (n = 3 biological replicates with two technical replicates each). (E) Following cell surface biotinylation and capture with streptavidin magnetic beads, Mmp9, Mmp14, galectin-3, and β3 integrin expression in the membrane fraction of wild-type and DKO osteoclasts as assessed by Western blot. Results are representative of three independent experiments. (F) Measurements of surface galectin-3 in wild-type and DKO osteoclasts with eFluor 660–conjugated anti–galectin-3 monoclonal antibody (#50-5301-82; Thermo Fisher Scientific; clone M3/38, epitopes mapped within the N-terminal domain) by flow cytometry. Results are representative of three independent experiments. (G) Measurements of surface galectin-1 in wild-type and DKO osteoclasts by flow cytometry. Results are representative of three independent experiments. (H) DKO BMDMs were transduced with lentiviral vectors expressing full-length MMP9, an MMP9E/A mutant, or an empty control, and differentiated into osteoclasts. Cell lysates were collected for MMP9 and galectin-3 expression and cleavage as assessed by Western blot. Results are representative of three independent experiments. (I) DKO BMDMs were transduced with lentiviral vectors expressing full-length MMP14, MMP14E/A, or an empty control and differentiated into osteoclasts. Cell lysates were collected for MMP14 and galectin-3 expression and cleavage as assessed by Western blot. Results are representative of three independent experiments. **P < 0.01. Statistical significance was assessed using two-way ANOVA with Bonferroni cor- rection (B) and unpaired two-sided Student’s t test (D). Source data are available for this figure: SourceData F4.

Article Snippet: Antigen detection was performed using antibodies directed against c-Src (rabbit antimouse/human antibody; #2109; Cell Signaling), Ctsk (mouse anti-mouse/human antibody; sc-48353; Santa Cruz), Rho (mouse anti-mouse/human antibody; #05-778; Millipore), galectin-3 (mouse anti-mouse/human antibody; ab2785; Abcam; epitopes mapped within the N-terminal region), Lrp1 (mouse anti-mouse antibody; MABN1796; Millipore), Mmp9 (rabbit anti-mouse antibody; ab38898; Abcam), Mmp14 (rabbit anti-mouse antibody; ab53712; Abcam), OXPHOS (rabbit anti-mouse antibody; ab110413; Abcam), vinculin (mouse anti-mouse antibody; V9131; Sigma-Aldrich), β3 integrin (rabbit anti-mouse antibody; #4702; Cell Signaling), or β-actin (rabbit anti-mouse antibody; #4970; Cell Signaling).

Techniques: Expressing, Western Blot, Generated, Staining, Magnetic Beads, Membrane, Flow Cytometry, Transduction, Mutagenesis, Control

Journal: The Journal of cell biology

Article Title: Proteolytic regulation of a galectin-3/Lrp1 axis controls osteoclast-mediated bone resorption.

doi: 10.1083/jcb.202206121

Figure Lengend Snippet: Figure 7. Mmp9/Mmp14 co-dependent regulation of osteoclast function through the galectin-3–centric control of Lrp1 activation. (A–C) Pre- osteoclasts were incubated with either human GALECTIN-3 or GALECTIN-3-biotin (hGAL3-biotin; 1.5 µM) for 30 min at 4°C. Biotinylated GALECTIN-3 was precipitated with streptavidin magnetic beads, separated by SDS-PAGE (A), and analyzed by mass spectrometry (B). Hits were classified according to the peptides spectral matches and the number of peptides per protein. Red columns: the top hit of GALECTIN-3 binding partner. The numbering on the x axis relates to Table S3. Pie chart depicts GALECTIN-3 interactors based on the GO annotation “cellular component” in FunRich (C). (D) Lrp1 and galectin-3 ex- pression from the above streptavidin magnetic beads–precipitated lysates as assessed by Western blot. Results are representative of three independent experiments. (E) Protein lysates from wild-type osteoclasts were immunoprecipitated with the galectin-3 antibody, and Lrp1 and galectin-3 levels assessed by Western blot. Results are representative of three independent experiments. (F and G) Cell-free binding assay demonstrating direct binding between 0.1 and 3.2 μg GALECTIN-3 and 0.5 μg LRP1 for 4 h at 30°C (F), and the competitive inhibition of binding between GALECTIN-3 and LRP1 (1.6 versus 0.5 μg) by 50 mM sucrose, 50 mM lactose, or 10 mg/ml GCS-100 (G). Data are presented as mean ± SEM (n = 3 biological replicates). (H) Following cell surface biotinylation and capture with streptavidin magnetic beads, Lrp1 and β3 integrin expression in the membrane fraction of wild-type and DKO osteoclasts as assessed by Western blot. Results are representative of three independent experiments. **P < 0.01. Statistical significance was assessed using one-way ANOVA with Bonferroni correction. Source data are available for this figure: SourceData F7.

Article Snippet: Antigen detection was performed using antibodies directed against c-Src (rabbit antimouse/human antibody; #2109; Cell Signaling), Ctsk (mouse anti-mouse/human antibody; sc-48353; Santa Cruz), Rho (mouse anti-mouse/human antibody; #05-778; Millipore), galectin-3 (mouse anti-mouse/human antibody; ab2785; Abcam; epitopes mapped within the N-terminal region), Lrp1 (mouse anti-mouse antibody; MABN1796; Millipore), Mmp9 (rabbit anti-mouse antibody; ab38898; Abcam), Mmp14 (rabbit anti-mouse antibody; ab53712; Abcam), OXPHOS (rabbit anti-mouse antibody; ab110413; Abcam), vinculin (mouse anti-mouse antibody; V9131; Sigma-Aldrich), β3 integrin (rabbit anti-mouse antibody; #4702; Cell Signaling), or β-actin (rabbit anti-mouse antibody; #4970; Cell Signaling).

Techniques: Control, Activation Assay, Incubation, Magnetic Beads, SDS Page, Mass Spectrometry, Binding Assay, Western Blot, Immunoprecipitation, Inhibition, Expressing, Membrane