recombinant cdh11 ob  (R&D Systems)

Journal: The FASEB Journal

Article Title: Cadherin‐11 binds to PDGFRβ and enhances cell proliferation and tissue regeneration via the PDGFR‐AKT signaling axis

doi: 10.1096/fj.201902613r

Figure Lengend Snippet: FIGURE 1 CDH11 deficiency reduces the proliferation potential of dermal fibroblast in vitro and in vivo. A, Representative trichrome staining of skin tissue from 8-wk old WT and Cdh11−/− mice. Blue color indicates the connective tissue, scale bar: 100 μm. B, Thickness of dermal layer assessed from skin of 8-wk old mice (n = 4). C, Immunostaining for Ki67 in WT or Cdh11−/− mouse skin tissue, scale bar: 50μm. D, Percentage of Ki67 positive cells in WT or Cdh11−/− mouse skin tissue in (C) (n = 4). E, Immunostaining for Ki67 in mouse dermal fibroblasts (passage 5). F, Quantification of percentage of Ki67+ cells (n = 3) in (E). G, Proliferation curve showing the cumulative number of cells as a function of passage. Cells were isolated from the skin of 8-wk old mice. H, Mouse dermal fibroblast doubling time (n = 3). I, qRT-PCR for CMYC, CCND1, CDK4, and CDK6 mRNA in mouse dermal fibroblasts. Values were normalized to cells from WT mice (n=3). The results are presented as mean ± s.d. *denotes P < .05 as compared to WT (unpaired two-tailed Student’s t test)

Article Snippet: Non-tissue culture plates were coated with 50 ng/cm2 recombinant human Fc fused to CDH11 protein (CDH11-Fc) chimera (R&D Systems, Minneapolis, MN) in phosphate-buffered saline (PBS) containing 0.9 mM CaCl2 and 0.9 mM MgCl2 either for 2 hours at 37°C or overnight at 4°C.

Techniques: In Vitro, In Vivo, Staining, Immunostaining, Isolation, Quantitative RT-PCR, Two Tailed Test

Journal: The FASEB Journal

Article Title: Cadherin‐11 binds to PDGFRβ and enhances cell proliferation and tissue regeneration via the PDGFR‐AKT signaling axis

doi: 10.1096/fj.201902613r

Figure Lengend Snippet: FIGURE 2 Loss of CDH11 reduces proliferation of human fibroblasts. A, qRT-PCR measurement of CDH11 mRNA in human dermal fibroblasts (n = 3). B, Western Blot and C, quantification of CDH11 band intensity in human dermal fibroblast (n = 3) (A.U.: arbitrary unit). Immunostaining for D, CDH11 and G, Ki67 of shCDH11 and control human fibroblasts, scale bar =50 µm; and E, quantification of fluorescence intensity (n=3). F, Doubling time of shCDH11 and control human fibroblasts (n = 3). Results are shown as mean ± s.d. *denotes P < .05 as compared to control (unpaired two-tailed Student’s t test)

Article Snippet: Non-tissue culture plates were coated with 50 ng/cm2 recombinant human Fc fused to CDH11 protein (CDH11-Fc) chimera (R&D Systems, Minneapolis, MN) in phosphate-buffered saline (PBS) containing 0.9 mM CaCl2 and 0.9 mM MgCl2 either for 2 hours at 37°C or overnight at 4°C.

Techniques: Quantitative RT-PCR, Western Blot, Immunostaining, Control, Fluorescence, Two Tailed Test

Journal: The FASEB Journal

Article Title: Cadherin‐11 binds to PDGFRβ and enhances cell proliferation and tissue regeneration via the PDGFR‐AKT signaling axis

doi: 10.1096/fj.201902613r

Figure Lengend Snippet: FIGURE 3 Deficiency of CDH11 results in decreased PDGFRβ expression and AKT activity. A, qRT-PCR for PDGFRβ mRNA in mouse (left) and human (right) dermal fibroblasts (n = 3). B, Representative WB for PDGFRβ, p-AKT, and AKT in mouse dermal fibroblasts. C, Band quantitation of WB in (B) (n = 3) (A.U.: arbitrary unit). D, Representative WB for PDGFRβ, CDH11, p-AKT, and AKT in human dermal fibroblasts. E, Band quantitation of WB in (D) (n = 3). F, Immunostaining of PDGFRβ and p-AKT in shCDH11 and control human fibroblasts, scale bar = 50 µm. G, Quantification of fluorescence intensity (F.I.) in (F) (n = 3). Results are shown mean ± s.d. *denotes P < .05 as compared to corresponding control (unpaired two-tailed Student’s t test)

Article Snippet: Non-tissue culture plates were coated with 50 ng/cm2 recombinant human Fc fused to CDH11 protein (CDH11-Fc) chimera (R&D Systems, Minneapolis, MN) in phosphate-buffered saline (PBS) containing 0.9 mM CaCl2 and 0.9 mM MgCl2 either for 2 hours at 37°C or overnight at 4°C.

Techniques: Expressing, Activity Assay, Quantitative RT-PCR, Quantitation Assay, Immunostaining, Control, Fluorescence, Two Tailed Test

Journal: The FASEB Journal

Article Title: Cadherin‐11 binds to PDGFRβ and enhances cell proliferation and tissue regeneration via the PDGFR‐AKT signaling axis

doi: 10.1096/fj.201902613r

Figure Lengend Snippet: FIGURE 4 CDH11 engagement promotes proliferation. A, Cumulative number of human fibroblasts plated on CDH11-Fc coated or TC plates in medium containing 2% FBS at the indicated times (n = 3). B, Doubling time of human fibroblasts cultured on CDH11-Fc coated or TC plates in medium containing 2% FBS (n = 3). C, Ki67 immunostaining of human fibroblasts cultured on CDH11-Fc coated or TC plates for 2 days (n = 3), scale bar: 50 µm. D, Quantification of fluorescence intensity in (C) (A.U.: arbitrary unit, n = 3). E, qRT-PCR for CDK4, CDK6, CYCLIND1, CMYC, E2F1, PDGFR-β, and CDH11 mRNA at 16 hours after plating on CDH11-Fc coated or TC plates (n = 3). Data was normalized to TC. *denotes P < .05 as compared to cells seeding on TC plates (unpaired two-tailed Student’s t test)

Article Snippet: Non-tissue culture plates were coated with 50 ng/cm2 recombinant human Fc fused to CDH11 protein (CDH11-Fc) chimera (R&D Systems, Minneapolis, MN) in phosphate-buffered saline (PBS) containing 0.9 mM CaCl2 and 0.9 mM MgCl2 either for 2 hours at 37°C or overnight at 4°C.

Techniques: Cell Culture, Immunostaining, Fluorescence, Quantitative RT-PCR, Two Tailed Test

Journal: The FASEB Journal

Article Title: Cadherin‐11 binds to PDGFRβ and enhances cell proliferation and tissue regeneration via the PDGFR‐AKT signaling axis

doi: 10.1096/fj.201902613r

Figure Lengend Snippet: FIGURE 5 CDH11 engagement activates the AKT pathway. A, WB for p-AKT and AKT at the indicated times after plating human dermal fibroblasts on CDH11-Fc coated or TC plates. B, Band quantitation of WB in (A) (n = 3, A.U.: arbitrary unit; N.S.: not significant). C, WB for p-AKT and AKT from cells seeded on plates coated with the indicated CDH11-Fc concentrations for 2 hours in the presence of 2% serum. D, Band quantitation of p-AKT normalized to total AKT (n = 3). E, WB for p-PDGFRβ and PDGFRβ at the indicated times after plating human dermal fibroblast on CDH11-Fc or TC plates for 2 hours in the presence of 2% serum. F, Band quantitation of WB in (E) (n = 3). G, WB for p-AKT and AKT after plating human dermal fibroblast on CDH11-Fc or TC surface for 2 hours in the presence of 2% serum; anti-PDGFRβ is a PDGFRβ functional blocking antibody. H, Band quantitation of p-AKT normalized to total AKT (n = 3). I, WB for p-AKT and AKT after plating human dermal fibroblast on CDH11-Fc or TC plates for 2 hours in the presence of 2% serum plus the indicated chemical inhibitors: PF573228 (PF, 10μM) is EGFR inhibitor; PD173074 (PD, 10 µM) is FGFR and VEGFR inhibitor; PPPsc204008 (PPP, 10 µM) is IGFR inhibitor; LY294002 (LY, 10 µM) is PI3K inhibitor; and JNJ10198409 (JNJ, 50 µM) is PDGFR inhibitor. J, Band quantitation of p-AKT normalized to total AKT (n = 3). K, Cell number as a function of time on CDH11-Fc or TC plates in the presence of 2% serum and the indicated inhibitors, LY294002 (10 µM) or JNJ10198409 (50 µM). Results are shown mean ± s.d. *denotes P < .05 as compared to corresponding control (unpaired two-tailed Student’s t test)

Article Snippet: Non-tissue culture plates were coated with 50 ng/cm2 recombinant human Fc fused to CDH11 protein (CDH11-Fc) chimera (R&D Systems, Minneapolis, MN) in phosphate-buffered saline (PBS) containing 0.9 mM CaCl2 and 0.9 mM MgCl2 either for 2 hours at 37°C or overnight at 4°C.

Techniques: Quantitation Assay, Functional Assay, Blocking Assay, Control, Two Tailed Test

Journal: The FASEB Journal

Article Title: Cadherin‐11 binds to PDGFRβ and enhances cell proliferation and tissue regeneration via the PDGFR‐AKT signaling axis

doi: 10.1096/fj.201902613r

Figure Lengend Snippet: FIGURE 6 CDH11 engagement leads to stronger and sustained PDGFR-Akt signaling. A, WB for p-AKT and AKT after plating human dermal fibroblast on CDH11-Fc coated or TC plates for 2 hours at the indicated serum concentration. B, Band quantitation of p-AKT normalized to total AKT in (A) (n = 3, A.U.: arbitrary unit; N.S.: not significant). C, Human fibroblasts were plated on CDH11-Fc coated or TC in DMEM without serum at 37°C for 2 hours to allow for cell spreading and then treated with PDGF-BB (10 ng/mL) for 2 hours at 4°C to prevent receptor internalization. Then the cells were returned to 37°C and at the indicated times they were lysed and processed for WB. D, Band quantitation p-PDGFRβ(Y751) normalized to total PDGFRβ in (C) (n = 3). E, Human fibroblasts were plated on CDH11-Fc coated or TC plates for 2 h in serum free medium and then treated with PDGF-BB (10 ng/mL) and at the indicated times they were lysed and processed for WB for p-AKT and AKT. F, Band quantitation of p-AKT normalized to total AKT in (E) (n = 3). G, WB for p-PDGFRβ (Y751), PDGFRβ, p-AKT, and AKT after plating human dermal fibroblast on CDH11-Fc coated or TC plates for 2 hours in the absence of serum and in the presence of the indicated concentrations of PDGF-BB for 10 minutes. Band quantitation of H, p-PDGFRβ (Y751)/PDGFRβ, and I, p-AKT/AKT in (G). Results are shown mean ± s.d. *denotes P < .05 as compared to corresponding control (unpaired two-tailed Student’s t test)

Article Snippet: Non-tissue culture plates were coated with 50 ng/cm2 recombinant human Fc fused to CDH11 protein (CDH11-Fc) chimera (R&D Systems, Minneapolis, MN) in phosphate-buffered saline (PBS) containing 0.9 mM CaCl2 and 0.9 mM MgCl2 either for 2 hours at 37°C or overnight at 4°C.

Techniques: Concentration Assay, Quantitation Assay, Control, Two Tailed Test

Journal: The FASEB Journal

Article Title: Cadherin‐11 binds to PDGFRβ and enhances cell proliferation and tissue regeneration via the PDGFR‐AKT signaling axis

doi: 10.1096/fj.201902613r

Figure Lengend Snippet: FIGURE 7 CDH-11 forms a complex with PDGFR. A, Human dermal fibroblasts were plated on CDH11-Fc surface for 6 hours and lysates were co-immunoprecipitated with antibodies against CDH11 or PDGFRβ, followed by WB for CDH11 and PDGFRβ. B, Schematics for CDH11 mutants; SP, EC, and TM denote signal peptide, extracellular domain and transmembrane domain, respectively. C-E, L-cells were transfected with CDH11 constructs with deletions of various ECs as indicated. After 2 days the cells were lysed. C, Cells lysates were processed for WB using an antibody targeting the intracellular domain of CDH11. D, E, Cell lysates were co-immunoprecipitated with antibody targeting the intracellular domain of CDH11 followed by WB for PDGFRβ. *denotes P < .05 as compared to cells seeding on TC plates (unpaired two-tailed Student’s t test)

Article Snippet: Non-tissue culture plates were coated with 50 ng/cm2 recombinant human Fc fused to CDH11 protein (CDH11-Fc) chimera (R&D Systems, Minneapolis, MN) in phosphate-buffered saline (PBS) containing 0.9 mM CaCl2 and 0.9 mM MgCl2 either for 2 hours at 37°C or overnight at 4°C.

Techniques: Immunoprecipitation, Transfection, Construct, Two Tailed Test

Journal: The FASEB Journal

Article Title: Cadherin‐11 binds to PDGFRβ and enhances cell proliferation and tissue regeneration via the PDGFR‐AKT signaling axis

doi: 10.1096/fj.201902613r

Figure Lengend Snippet: FIGURE 8 CDH11 promotes tissue regeneration in mouse cutaneous wound healing assay. A, Representative images of the wounds of Cdh11−/− and WT mice (n = 6 for each condition) over 14 days after wounding. B, Measurements of the area of each wound over time normalized to the initial wound area. C, Representative images of the wounds of Cdh11−/− and WT stained with trichrome, or immunostained for vimentin, Ki67, and p-AKT; dashed line indicates the dermo-epidermal junction. D, Collagen deposition was quantified by measuring the intensity of blue color in the trichrome staining (n = 3). WT-W and Cdh11−/− W denote the wound bed; WT-N and Cdh11−/−N denote unwounded skin tissue. E, Quantitation of total number of vimentin positive cells/mm2 in the wound bed (n = 3). F, Quantitation of the percentage of Ki67 positive cells in the wound bed (n = 3). G, Quantitation of fluorescence intensity of p-AKT per cell in the wound bed (n = 3). *denotes P < .05 as compared to cells on TC plate (unpaired two-tailed Student’s t test)

Article Snippet: Non-tissue culture plates were coated with 50 ng/cm2 recombinant human Fc fused to CDH11 protein (CDH11-Fc) chimera (R&D Systems, Minneapolis, MN) in phosphate-buffered saline (PBS) containing 0.9 mM CaCl2 and 0.9 mM MgCl2 either for 2 hours at 37°C or overnight at 4°C.

Techniques: Wound Healing Assay, Staining, Quantitation Assay, Fluorescence, Two Tailed Test