ropn  (R&D Systems)

Journal: Frontiers in immunology

Article Title: Increased liver stiffness promotes hepatitis B progression by impairing innate immunity in CCl4-induced fibrotic HBV + transgenic mice.

doi: 10.3389/fimmu.2023.1166171

Figure Lengend Snippet: FIGURE 1 Increased matrix stiffness upregulates HBV infection markers and osteopontin levels in HBV-transfected HepG2.2.15 cells: Cells were plated in both soft and stiff plates for 48hrs. (A) HBV RNA levels were measured by Real-Time PCR. (B) HBV DNA levels were measured by Droplet Digital™PCR (ddPCR). GAPDH was used as an internal control for HBV RNA and HBV DNA. (C) A Sandwich Elisa kit was used to measure HBsAg levels. (D, E) Osteopontin protein and mRNA levels were measured by Elisa and Real-Time PCR. (F) Cleaved caspase 3 protein expression was detected by immunoblotting in cell lysates for apoptosis. Equal (20 ug) amounts of protein were loaded in each lane. b-Actin was used as an internal control. Acetaldehyde-generating system (AGS)-treated HepG2 cell lysates were used as the positive control. Data are from three independent experiments presented as Mean ± SEM. Bars marked with the same letter are not significantly different from each other; bars with different letters are significantly different (p ≤0.05).

Article Snippet: To mimic the overexpression of osteopontin, we used recombinant human osteopontin (rOPN) Protein from R & D Systems, Catalog #: 1433-OP.

Techniques: Infection, Transfection, Real-time Polymerase Chain Reaction, Digital PCR, Control, Sandwich ELISA, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Positive Control

Journal: Frontiers in immunology

Article Title: Increased liver stiffness promotes hepatitis B progression by impairing innate immunity in CCl4-induced fibrotic HBV + transgenic mice.

doi: 10.3389/fimmu.2023.1166171

Figure Lengend Snippet: FIGURE 3 Recombinant OPN treatment increases the expression of HBV marker by suppressing IFNa-inducible anti-viral genes activation in soft gel (2 KPa) attached HBV+ transfected HepG2.2.15 cells: To mimic overexpression, we treated soft (2 kPa) gel-attached HBV transfected HepG2.2.15 cells with rOPN for 24hrs. RT-PCR analysis was done for mRNA expression of IFN-induced genes and HBV RNA. (A) 2′–5′ oligoadenylate synthetase 1 (OAS1); (B) Interferon-stimulated gene 15 (ISG15); (C) Apolipoprotein B Editing Complex (APOBEC3); (D) HBV RNA. GAPDH acted as an internal control. (E) STAT-1 phosphorylation (pSTAT-1) was measured by immunoblotting. Total STAT-1 was used to normalize the data. (F) Quantification of immunoblotting data. Data are from three independent experiments presented as Mean ± SEM. Bars marked with the same letter are not significantly different from each other; bars with different letters are significantly different (P ≤0.05).

Article Snippet: To mimic the overexpression of osteopontin, we used recombinant human osteopontin (rOPN) Protein from R & D Systems, Catalog #: 1433-OP.

Techniques: Recombinant, Expressing, Marker, Activation Assay, Transfection, Over Expression, Reverse Transcription Polymerase Chain Reaction, Control, Phospho-proteomics, Western Blot

Journal: Frontiers in immunology

Article Title: Increased liver stiffness promotes hepatitis B progression by impairing innate immunity in CCl4-induced fibrotic HBV + transgenic mice.

doi: 10.3389/fimmu.2023.1166171

Figure Lengend Snippet: FIGURE 4 Silencing of OPN decreases expression of HBV marker by increasing IFNa-inducible anti-viral genes activation in soft gel (2 KPa)-attached HBV+ transfected HepG2.2.15 cells, and USP18 is partially responsible for the regulation of HBV infection markers by OPN in fibrotic stiffness gel-attached HBV+ transfected HepG2.2.15 cells: We silenced OPN by specific siRNA transfection, in soft (2 kPa) and stiff (25 kPa) gel-attached HBV transfected HepG2.2.15 cells. RT-PCR analysis was done for mRNA expression of IFN-induced genes and HBV RNA. (A) Transfection efficiency of OPN silencing and OPN mRNA; (B) 2′–5′ oligoadenylate synthetase 1 (OAS1); (C) Interferon-stimulated gene 15 (ISG15); (D) Apolipoprotein B Editing Complex (APOBEC3); (E) HBV RNA. GAPDH acted as an internal control. We silenced OPN by specific siRNA transfection in soft (2 kPa) and stiff (25 kPa) gel-attached HBV-transfected HepG2.2.15 cells, and to mimic overexpression, we treated only soft (2 kPa) gel-attached HBV transfected HepG2.2.15 cells with rOPN for 24hrs. RT-PCR analysis was done for mRNA expression of USP18 (F, G). GAPDH acted as an internal control for RT-PCR analysis. (H) USP18 protein was measured by immunoblotting and b-actin was also used as an internal control. Data are from three independent experiments presented as Mean ± SEM. Bars marked with the same letter are not significantly different from each other; bars with different letters are significantly different (P ≤0.05).

Article Snippet: To mimic the overexpression of osteopontin, we used recombinant human osteopontin (rOPN) Protein from R & D Systems, Catalog #: 1433-OP.

Techniques: Expressing, Marker, Activation Assay, Transfection, Infection, Reverse Transcription Polymerase Chain Reaction, Control, Over Expression, Western Blot

Journal: Frontiers in immunology

Article Title: Increased liver stiffness promotes hepatitis B progression by impairing innate immunity in CCl4-induced fibrotic HBV + transgenic mice.

doi: 10.3389/fimmu.2023.1166171

Figure Lengend Snippet: FIGURE 6 Increased liver tissue stiffness promotes osteopontin which suppresses Interferon-Stimulated Gene (ISG) expression in the livers of CCl4- administered HBVTg mice: Liver fibrosis was induced by injecting CCl4 for 6 weeks in C57Bl/6 parental and HBV+ transgenic (HBVTg) mice. RT-PCR analysis was done for mRNA expression of OPN, IFN-induced genes, and USP18. (A) Osteopontin (OPN); (C) 2’-5’ oligoadenylate synthetase-like 1 (OASL1); (D) Interferon-stimulated gene 15 (ISG15); (E) Apolipoprotein B Editing Complex (APOBEC3); (F) USP18. (B) OPN protein levels were measured by Elisa in serum. Data are expressed as Mean ± SEM (n =4). The data bars marked with the same alphabetical letter are considered not significantly different, while those marked with differing alphabetical letters are considered significantly different from each other (p ≤0.05).

Article Snippet: To mimic the overexpression of osteopontin, we used recombinant human osteopontin (rOPN) Protein from R & D Systems, Catalog #: 1433-OP.

Techniques: Expressing, Transgenic Assay, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: International journal of molecular sciences

Article Title: Osteopontin on the Dental Implant Surface Promotes Direct Osteogenesis in Osseointegration.

doi: 10.3390/ijms23031039

Figure Lengend Snippet: Figure 1. H&E-staining and OPN-immunoreactivity in the tissues surrounding the implants in the WT mice. (a,g,m) H&E-staining in the tissues surrounding the implants at day 5 after implant placement in the control, rOPN, and HABP-OPNpep groups. The infiltration of inflammatory cells and spindle-shaped or flattened cells is observed at the bone-implant interface. (d,j,p) There is a weak OPN positive immunoreaction at the bottom parts of threads and the cement lines of the pre-existing bone (arrowheads). (b,c,h,i,n,o) The formation of direct osteogenesis is clearly observed at week 1 (arrows) and week 2. (e,f,k,l,q,r) OPN-immunoreactivity gradually becomes intense (arrowheads) and elongates along the implant surface at week 2. B, bone. Scale bar = 50 µm.

Article Snippet: We soaked the HAB-implants [11] in different OPN modified protein/peptides solution including rOPN protein (R&D Systems, Minneapolis, MN, USA; catalog no. 441-OP) (rOPN group; 20 μM in phosphate buffer saline [PBS]), Gly-Arg-Gly-Asp-Ser peptide (Peptide Institute Inc, Osaka, Japan; Fibronectin Active Fragment, #4189) (RGDS group; 3.1 mM in PBS), OPN mimic peptide (sequence: GRGDSLAYGLR [OPNpep group], theoretical molecular weight: 1164.27, purity: 97.95% [HPLC method], 3.1 mM in PBS), HABP-OPNpep (sequence: GGGLHAHKKPTQDIRGGGRGDSLAYGLR [HABP-OPNpep group], theo- retical molecular weight: 3101.48, purity: 98.8% [HPLC method] 3.1 mM in PBS) (both peptides were provided from GenScript Japan [Tokyo, Japan] according to our order), and PBS (control) for 2 min in addition to filling the cavity with each solution before implant placement.

Techniques: Staining, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Osteopontin Promotes Protective Antigenic Tolerance against Experimental Allergic Airway Disease

doi: 10.4049/jimmunol.1701345

Figure Lengend Snippet: (A) Mice received three doses of endotoxin-free OVA for tolerance induction, along with rOpn or PBS. Controls (without tolerance) were PBS-treated mice (white bars). (B) Percentages and numbers of CD3+CD4+Foxp3+ T cells quantified in dLNs by flow cytometry at day 7. (C) Relative expression to Hprt of immuno-regulatory genes in dLNs. cDNAs were pooled from three separate experiments. Data are mean 6 SEM (n = 6 mice per group), one representative of three independent experiments. *p # 0.0332, **p # 0.0021, ***p # 0.0002.

Article Snippet: In vivo experimental protocols For tolerance induction, mice received 200 mg of EndoGrade OVA (Hyglos) i.n. in the presence of 2.5 mg recombinant Opn protein (rOpn) (R&D Systems), 72 ng of synthetic secreted Opn 134–153 fragments (frOpn; IVPTVDVPNGRGDSLAYGLR), or PBS (control).

Techniques: Flow Cytometry, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Osteopontin Promotes Protective Antigenic Tolerance against Experimental Allergic Airway Disease

doi: 10.4049/jimmunol.1701345

Figure Lengend Snippet: (A) [3H]Thymidine incorporation in co-cultures of DO11.10 T cells preconditioned with OVA323-339 and rOpn-treated pDCs with responder DO11.10 T cells. The same numbers of cells were cultured in five different wells per group. Data are mean 6 SEM, onerepresentative of three independent experiments. (B) Sorted pDCs pre-conditioned in vitro with PBS or OVA or OVA/rOpn were adoptively transferred to recipient mice before the induction of allergic airway inflammation. Total and eosinophil cell count in BAL were evaluated (day 20). Histological assessment of lung inflammation (H&E scoring) and lungmucus production (PAS score) (C) and levels of IL-4 and IL-13 (D) in supernatants of OVA-stimulated mLN cells. Different wells of pooled mLN cells. Data are mean 6 SEM (n = 6–8 mice per group), one representative of three independent experiments. *p # 0.0332, **p # 0.0021, ***p # 0.0002, ****p, 0.0001.

Article Snippet: In vivo experimental protocols For tolerance induction, mice received 200 mg of EndoGrade OVA (Hyglos) i.n. in the presence of 2.5 mg recombinant Opn protein (rOpn) (R&D Systems), 72 ng of synthetic secreted Opn 134–153 fragments (frOpn; IVPTVDVPNGRGDSLAYGLR), or PBS (control).

Techniques: Cell Culture, In Vitro, Cell Counting

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Osteopontin Promotes Protective Antigenic Tolerance against Experimental Allergic Airway Disease

doi: 10.4049/jimmunol.1701345

Figure Lengend Snippet: Treatment of mice with endotoxin-free OVA (tolerance induction), together with endotoxin-free rOpn (A) or treatment of micewith PBS without OVA for control (B–D). Controls (without tolerance) were PBS-treated mice (white bars). (B–D) 7AAD2 CD32CD192CD11c+PDCA-1+CCR7+ pDCs and 7AAD2CD32CD192CD11c+CD11b+ CCR7+ cDCs quantified in dLNs by flow cytometry. (B) Representative percentages inflow cytometric plots and numbers of dLNpDCs (upper panels) and cDCs (lower pan-els). (C) Representative flow cytometric plotsand percentages of peripheral blood CCR7+pDCs and cDCs among total pDCs andcDCs, respectively. (D) Relative expression to Hprt of Ccl19 and Ccl21 in dLNs of tolerized mice. Data are mean 6 SEM (n = 10 mice per group; cDNAs were pooled from three separate experiments). (E) Sorted pDCs fromLNs and spleens of naive BALB/c mice werepulsed with vehicle or rOpn for 24 h and thensubjected to transmigrate in response toCCL19. Numbers of migrating PBS-treatedpDCs (white bars) or rOpn-treated pDCs (black bars). Triplicate wells of pooled pDCssorted from spleens and LNs (n = 8). Data aremean 6 SEM (n = 5-8 mice per group), one representative of five independent experiments (A-C) and from three independent experiments (E). *p # 0.0332, **p # 0.0021, ***p # 0.0002, ****p, 0.0001.

Article Snippet: In vivo experimental protocols For tolerance induction, mice received 200 mg of EndoGrade OVA (Hyglos) i.n. in the presence of 2.5 mg recombinant Opn protein (rOpn) (R&D Systems), 72 ng of synthetic secreted Opn 134–153 fragments (frOpn; IVPTVDVPNGRGDSLAYGLR), or PBS (control).

Techniques: Control, Flow Cytometry, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Osteopontin Promotes Protective Antigenic Tolerance against Experimental Allergic Airway Disease

doi: 10.4049/jimmunol.1701345

Figure Lengend Snippet: (A) Mice received four doses of 120G8 pDC-depleting Ab or Ig control i.p. on days 26, 25, 24, and 23, followed by tolerance induction for Treg cell generation, with or without rOpn administration. (B and C) Also, allergic airway disease was subsequently induced on day 10. Control mice were pretreated with PBS (A) or sensitized with PBS (B and C). (A) Representative percentages in flowcytometric plots (left panel) and numbers of Treg cells (CD3+CD4+Foxp3+) accumulated in dLNs (right panel). (B) Eosinophil counts in BAL of allergic mice. (C) [3H]Thymidine incorporation in OVA-stimulated dLN cells. Quadruplicate wells of pooled mLN cells. Data are mean 6 SEM (n = 5 mice per group), one representative of three independent experiments. *p # 0.0332, **p # 0.0021, ***p # 0.0002, ****p, 0.0001.

Article Snippet: In vivo experimental protocols For tolerance induction, mice received 200 mg of EndoGrade OVA (Hyglos) i.n. in the presence of 2.5 mg recombinant Opn protein (rOpn) (R&D Systems), 72 ng of synthetic secreted Opn 134–153 fragments (frOpn; IVPTVDVPNGRGDSLAYGLR), or PBS (control).

Techniques: Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Osteopontin Promotes Protective Antigenic Tolerance against Experimental Allergic Airway Disease

doi: 10.4049/jimmunol.1701345

Figure Lengend Snippet: (A) Numbers of dLN 7AAD*CD11c+B220+PDCA1+Siglec-H+ pDCs osinophil cell count in BAL, histological assessment of lung inflammation (H&E scoring) and lung mucus production (PAS score), and numbers of T1ST2+CD4+ TH2 cells in mLNs. after 40 h of tolerance induction with EndoGrade OVA i.n., along with rOpn, frOpn1, frOpn2, frOpn3, or PBS (as in Fig. 3A). (B) Mice were treated with OVA/frOpn during tolerance induction before allergic airway disease induction in (C)–(E). Control mice were pretreated with PBS, and allergy was subsequently induced (white bars). (C) AHR (depicted as Penh) at day 19. (D) Eosinophil cell count in BAL, histological assessment of lung inflammation (H&E scoring) and lung mucus production (PAS score), and numbers of T1ST2+CD4+ TH2 cells in mLNs. (E) Levels of IL-4, IL-5, and IL-13 in supernatants of OVA-stimulated mLN cells. The same numbers of pooled mLN cells were cultured in four to six different wells per group. Data are mean 6 SEM (n = 5, mice per group), one representative of three independent experiments. *p # 0.0332, **p # 0.0021, ***p # 0.0002, ****p, 0.0001.

Article Snippet: In vivo experimental protocols For tolerance induction, mice received 200 mg of EndoGrade OVA (Hyglos) i.n. in the presence of 2.5 mg recombinant Opn protein (rOpn) (R&D Systems), 72 ng of synthetic secreted Opn 134–153 fragments (frOpn; IVPTVDVPNGRGDSLAYGLR), or PBS (control).

Techniques: Cell Counting, Control, Cell Culture