Journal:

Article Title: Functional Evaluation of DC-SIGN Monoclonal Antibodies Reveals DC-SIGN Interactions with ICAM-3 Do Not Promote Human Immunodeficiency Virus Type 1 Transmission

doi: 10.1128/JVI.76.12.5905-5914.2002

Figure Lengend Snippet: HIV-1 transmission mediated by DC is blocked by DC-SIGN MAbs. Transmission of R5-tropic HIV-Luc/JRFL using DC as donor cells and Hut/CCR5 as target cells was performed as described for Fig. ​Fig.3.3. DC cocultured with Hut/CCR5 cells not exposed to HIV-1 were used as a mock-infected control. Mouse IgG was used as a nonspecific antibody control. Anti-DCS(D), cocktail containing the DC-SIGN-specific MAbs 507(D), 516(D), and 531(D) (10 μg/ml combined). Anti-DCS(X): cocktail containing the cross-reactive MAbs 518(X), 526(X), and 612(X) (10 μg/ml combined). The L-SIGN-specific MAb 604(L) was used at 10 μg/ml; mannan was used at 20 μg/ml. DC alone were incubated with the virus, washed to remove unbound virus, and then cultured without Hut/CCR5 target cells. One representative experiment out of two is shown. cps, counts per second.

Article Snippet: To inhibit DC-SIGN and ICAM-3 interactions, fresh DC-SIGN MAb 531(D) and anti-human ICAM-3 MAb (R&D Systems, antibody clone 76205.11) were added daily.

Techniques: Transmission Assay, Infection, Control, Incubation, Virus, Cell Culture

Journal:

Article Title: Functional Evaluation of DC-SIGN Monoclonal Antibodies Reveals DC-SIGN Interactions with ICAM-3 Do Not Promote Human Immunodeficiency Virus Type 1 Transmission

doi: 10.1128/JVI.76.12.5905-5914.2002

Figure Lengend Snippet: Expression of ICAM-3 in GHOST/R5 target cells does not enhance HIV-1 transmission mediated by DC-SIGN. (A) Direct infection of GHOST/R5 and GHOST/R5/ICAM-3 cells. Cells were infected with different amounts of HIV-Luc/JRFL pseudotyped virus as indicated. Luciferase activity was measured 2 days after infection. Cells not exposed to virus were used as a mock-infected control. (B) HIV-1 transmission to ICAM-3-positive or -negative GHOST/R5 cells mediated by DC-SIGN. THP-1 or THP-1/DC-SIGN donor cells were incubated with DC-SIGN-specific MAb 531(D) or mouse IgG control (10 μg/ml) for 30 min at 37°C ([prior]) and then pulsed with HIV-Luc/JRFL for 3 h at 37°C. The washed cells were then added to GHOST/R5 or GHOST/R5/ICAM-3 target cells, respectively. The DC-SIGN-specific MAb 531(D) and anti-ICAM-3 (10 μg/ml) were kept in the cocultivation for 5 h ([post]), and then donor cells were removed and target cells were washed with medium and cultured in 1 ml of fresh medium in the presence of MAb 531(D) or anti-ICAM-3 (refreshed daily) for 2 days before luciferase activity was measured ([post]). Each data set represents the mean of three separate wells of infected cells. One representative experiment out of two is shown. cps, counts per second.

Article Snippet: To inhibit DC-SIGN and ICAM-3 interactions, fresh DC-SIGN MAb 531(D) and anti-human ICAM-3 MAb (R&D Systems, antibody clone 76205.11) were added daily.

Techniques: Expressing, Transmission Assay, Infection, Virus, Luciferase, Activity Assay, Control, Incubation, Cell Culture

Journal: Stem Cell Research & Therapy

Article Title: Mesenchymal stem cells derived from human induced pluripotent stem cells improve the engraftment of myogenic cells by secreting urokinase-type plasminogen activator receptor (uPAR)

doi: 10.1186/s13287-021-02594-1

Figure Lengend Snippet: Recombinant human uPAR protein promoted engraftment of human myogenic cells in TA muscle of NOD/Scid mice. A Outline of the transplantation experiments. Before transplantation, myogenic cells were cultured in 10% FBS/DMEM supplemented with 20 ng/ml uPAR, BDNF, or IGFBP2 for 3 d. Recombinant uPA was added 24 h before transplantation. The TA muscles of NOD/Scid mice were injured with 1.2% BaCl 2 solution 24 h before cell transplantation and injected with 1.5 × 10 6 cells (Hu5/KD3 cells) or 1.0 × 10 6 cells (hiPSC-derived muscle progenitors) in 50 μl of PBS containing 5 μg/ml uPAR or BDNF. Two weeks after transplantation, the mice were killed, and the TA muscles were dissected for immunohistochemical analysis using an anti-human lamin A/C antibody and human spectrin (green). B Numbers of human lamin A/C-positive nuclei/section of TA muscle injected with Hu5/KD3 cells treated with the indicated cytokines. n = 4–8 mice/group. Data are shown as the means ± SEMs. Dunnett’s analysis. n.s., not significant. *, p < 0.05. C Hu5/KD3 cells were seeded at a density of 1.0 × 10 6 /well in 6-well collagen plates in the absence or presence of uPAR (10 ng/ml) or uPA (10 ng/ml). The next day, a straight scratch was made with a 200 μl pipette tip (0 h). Migration activity in 5 h was evaluated by measuring the width of the wound. Eight points/condition. Data are shown as the means ± SEMs. Dunnett’s analysis. *, p < 0.05. D Representative immunostaining of cross sections of TA muscles injected with Hu5/KD3 cells with uPAR (5 µg/ml) or uPAR + uPA (5 µg/ml, each). Cells were cultured in 10% FBS/DMEM supplemented with 20 ng/ml uPAR or 20 ng/ml uPAR + 20 ng/ml uPA for 3 d. Myofibers formed by Hu5/KD3 cells were identified with anti-human lamin B1 antibody (red) and human spectrin (green). E Quantitative analysis of ( D ). n = 4 mice/group. Unpaired two-tailed Student’s t -test

Article Snippet: To reduce uPAR expression in iMSCs, we transfected 454E2-iMSCs with uPAR shRNA plasmids (human) (Santa Cruz sc-36781-SH) or control shRNA plasmids (human) (sc-108060) using FuGENE HD (Promega).

Techniques: Recombinant, Transplantation Assay, Cell Culture, Muscles, Injection, Derivative Assay, Immunohistochemical staining, Transferring, Migration, Activity Assay, Immunostaining, Two Tailed Test

Journal: Stem Cell Research & Therapy

Article Title: Mesenchymal stem cells derived from human induced pluripotent stem cells improve the engraftment of myogenic cells by secreting urokinase-type plasminogen activator receptor (uPAR)

doi: 10.1186/s13287-021-02594-1

Figure Lengend Snippet: uPAR and BDNF stimulated migration of Hu5/KD3 cells in vitro. A Hu5/KD3 cells were seeded at a density of 1.0 × 10 6 /well in 6-well collagen plates in the presence of uPAR (left), BDNF (middle), or IGFBP2 (light) (0–50 ng/ml). The next day, a straight scratch was made with a 200 μl pipette tip (0 h) and photographed under an inverted microscope. Six hours later, the cells were again photographed (Additional file : Fig. 6). Six views/condition were recorded. Migration activity was evaluated by measuring the width of the wound. B Hu5/KD3 cells were plated onto 24-well collagen plates at 2 × 10 5 cells/well and induced to differentiate in 10% FBS/DMEM medium supplemented with recombinant human uPAR (left), BDNF (middle) or human IGFBP2 protein (right) (0–50 ng/ml). Four (uPAR) or five days (BDNF, IGFBP2) later, the cells were fixed and stained with an anti-muscle myosin heavy chain antibody (MF20) and DAPI (nuclei). Shown are averages of 4 wells/condition. The percentage of MF20-positive area was quantified using ImageJ software. C Hu5/KD3 cells were plated onto 24-well collagen plates at 1 × 10 4 cells/well in 10% FBS/DMEM supplemented with recombinant human uPAR (left), BDNF (middle), or IGFBP2 (right) protein (0–50 ng/ml). Three days later, an MTT assay was performed (4 wells/condition). In ( A – C ), data are shown as the mean ± SEM. Dunnett’s analysis. n.s., not significant. **, p < 0.01. ***, p < 0.001. ****, p < 0.0001

Article Snippet: To reduce uPAR expression in iMSCs, we transfected 454E2-iMSCs with uPAR shRNA plasmids (human) (Santa Cruz sc-36781-SH) or control shRNA plasmids (human) (sc-108060) using FuGENE HD (Promega).

Techniques: Migration, In Vitro, Transferring, Inverted Microscopy, Activity Assay, Recombinant, Staining, Software, MTT Assay

Journal: Stem Cell Research & Therapy

Article Title: Mesenchymal stem cells derived from human induced pluripotent stem cells improve the engraftment of myogenic cells by secreting urokinase-type plasminogen activator receptor (uPAR)

doi: 10.1186/s13287-021-02594-1

Figure Lengend Snippet: Knockdown of uPAR in iMSCs reduced the efficiency of cell transplantation. A Outline of the experiments. iMSCs were transfected with huPAR shRNA plasmids or control shRNA plasmids. After puromycin treatment for 24 h, iMSCs were co-cultured with Hu5/KD3 cells in 10% FBS/DMEM in a Transwell plate for 3 d. The TA muscles of NOD/Scid mice were injured with 1.2% BaCl 2 solution 24 h before cell transplantation and injected with 5 × 10 myogenic cells (Hu5/KD3 cells). Cells co-cultured with iMSCs transfected with control shRNA plasmids were injected into right TAs (control), and cells co-cultured with iMSCs transfected with uPAR shRNA plasmids were injected left TAs. Ten days after transplantation, the mice were killed, and the TA muscles were dissected for immunohistochemical analysis using an anti-human lamin B1 antibody and human spectrin antibody as described in Fig. . B RT-qPCR analysis performed on RNAs extracted from iMSCs transfected with shRNA plasmids. Prep. 1 and Prep. 2 showed 60% reduction of uPAR mRNA. C Western blotting was performed using proteins extracted from iMSCs transfected with uPAR shRNA plasmids or control shRNA plasmids. Prep. 1 and Prep. 2 showed 40–70% reduction of uPAR protein. D Numbers of human lamin B1-positive, human spectrin-positive myofibers in TA muscles transplanted with Hu5/KD3 cells after co-culture with iMSCs (summary of two independent experiments). n = 5 mice. Paired t -test. E Recombinant PAI-1 (100 ng/ml) was added to the coculture and three days later Hu5/KD3 cells were transplanted into left TA muscles of NOD/Scid mice ( n = 4) or to NSG-mdx 4Cv mice ( n = 4). Right TA muscles were injected with Hu5/KD3 cells that had been co-cultured with iMSCs without PAI-1 (control). Data are shown as the mean ± SEM

Article Snippet: To reduce uPAR expression in iMSCs, we transfected 454E2-iMSCs with uPAR shRNA plasmids (human) (Santa Cruz sc-36781-SH) or control shRNA plasmids (human) (sc-108060) using FuGENE HD (Promega).

Techniques: Knockdown, Transplantation Assay, Transfection, shRNA, Control, Cell Culture, Muscles, Injection, Immunohistochemical staining, Quantitative RT-PCR, Western Blot, Co-Culture Assay, Recombinant

Journal: Stem Cell Research & Therapy

Article Title: Mesenchymal stem cells derived from human induced pluripotent stem cells improve the engraftment of myogenic cells by secreting urokinase-type plasminogen activator receptor (uPAR)

doi: 10.1186/s13287-021-02594-1

Figure Lengend Snippet: Relative levels of cytokines in conditioned medium of BM-MSCs and iMSCs analyzed by cytokine array analysis

Article Snippet: To reduce uPAR expression in iMSCs, we transfected 454E2-iMSCs with uPAR shRNA plasmids (human) (Santa Cruz sc-36781-SH) or control shRNA plasmids (human) (sc-108060) using FuGENE HD (Promega).

Techniques:

Journal: Cancer Research

Article Title: FAP Promotes Immunosuppression by Cancer-Associated Fibroblasts in the Tumor Microenvironment via STAT3–CCL2 Signaling

doi: 10.1158/0008-5472.can-15-2973

Figure Lengend Snippet: Figure 2. FAP is responsible for inflammatory phenotype of CAFs by activating the uPAR–FAK–c-Src–JAK2–STAT3 pathway. A, representative flow cytometric analysis of uPAR expression. B, CoIP assays to analyze the interaction between FAP and uPAR in CAFs. Normal rabbit/goat IgG antibodies were served as a negative control. C, representative Western blots showing the total and phosphorylated FAK, and densitometry quantification. D, representative Western blots showing the effect of different inhibitors of downstream molecules of FAK on STAT3 activation in CAFs. E, ELISA analysis of CCL2 concentrations in the culture of CAFs treated with DMSO or different inhibitors. , P < 0.01; , P < 0.001 versus CAFs treated with DMSO. ns, not significant. F, representative Western blots showing the phosphorylation of c-Src, JAK2, and STAT3 in CAFs and FAPnegCAFs, and the effect of inhibitors of FAK, c-Src, and JAK2 on the phosphorylation of c-Src, JAK2, and STAT3 in CAFs. DMSO was used as a vehicle control. In C and E, data represented as mean SEM. Results are representative of at least three independent experiments.

Article Snippet: The following antibodies were used: fluorochrome-labeled anti-mouse CD45 (30-F11), CD31 (390), PDGFR (53-6.7), CD4 (GK1.5), CD8 (APA5), CD11b (M1/70), F4/80 (BM8), IFNg (AN-18), Gr-1(RB6-8C5; all from eBioscience); Ly6G (1A8) and Ly6C (HK1.4; both from BD), CCR2 (475301) and uPAR (MAB531; both from R&D Systems), FAP (Abgent).

Techniques: Expressing, Negative Control, Western Blot, Activation Assay, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Control

Journal: Cancer Research

Article Title: FAP Promotes Immunosuppression by Cancer-Associated Fibroblasts in the Tumor Microenvironment via STAT3–CCL2 Signaling

doi: 10.1158/0008-5472.can-15-2973

Figure Lengend Snippet: Figure 3. FAP activates fibroblastic STAT3 in a uPAR-dependent way. A, representative Western blots showing the phosphorylation of FAK, c-Src, JAK2, and STAT3. B, luciferase assays using the STAT3 reporter vector. C, qRT-PCR analysis of inflammatory gene expression. Insets, Western blots showing the effect of uPAR siRNA. D and E, representative Western blots showing the total and phosphorylated STAT3 (D) and CCL2 concentrations (E). CAFs and FAP-Fbs were treated with or without PT-100 (5 nmol/L), the inhibitor of DDPIV. Data, mean SEM. Results are representative of at least three independent experiments. ns, not significant.

Article Snippet: The following antibodies were used: fluorochrome-labeled anti-mouse CD45 (30-F11), CD31 (390), PDGFR (53-6.7), CD4 (GK1.5), CD8 (APA5), CD11b (M1/70), F4/80 (BM8), IFNg (AN-18), Gr-1(RB6-8C5; all from eBioscience); Ly6G (1A8) and Ly6C (HK1.4; both from BD), CCR2 (475301) and uPAR (MAB531; both from R&D Systems), FAP (Abgent).

Techniques: Western Blot, Phospho-proteomics, Luciferase, Plasmid Preparation, Quantitative RT-PCR, Gene Expression