Journal: Cell reports

Article Title: Human Glial Progenitor Cells Effectively Remyelinate the Demyelinated Adult Brain

doi: 10.1016/j.celrep.2020.107658

Figure Lengend Snippet:

Article Snippet: Mouse anti-A2B5 supernatant, clone 105 , ATCC , Cat#CRL-1520; RRID: CVCL_7946.

Techniques: In Vivo, In Vitro

Journal: Cell reports

Article Title: Human Glial Progenitor Cells Effectively Remyelinate the Demyelinated Adult Brain

doi: 10.1016/j.celrep.2020.107658

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse anti-A2B5 supernatant, clone 105 , ATCC , Cat#CRL-1520; RRID: CVCL_7946.

Techniques: Marker, In Vitro, Recombinant, Clinical Proteomics, Software, Microscopy, Lysis

Journal: Molecular medicine reports

Article Title: Effects of antibodies against human parvovirus B19 on angiogenic signaling.

doi: 10.3892/mmr.2020.10921

Figure Lengend Snippet: Figure 1. Viability of HUVECs. (A) Representative photographs of HUVECs in the presence of different antibodies for 10 min observed under a phase‑contrast microscope (Zeiss Axiovert 200; magnification, x200). (B) Survival ratio of HUVECs in the presence of different antibodies for 10 min. Each experiment was repeated at least three times. HUVEC, human umbilical vein endothelial cell; IgG, immunoglobulin G; NH, normal human; FCS, fetal calf serum; aPL, anti‑phospholipid antibodies; B19V, parvovirus B19; VP1u, VP1 unique region.

Article Snippet: After blocking with 5% non‐fat dry milk for 1 h at 4 ̊C, antibodies against p-mTor (Ser2481; 1:500; cat. no. 09343; eMd Millipore), p-aKT (Ser473; 1:500; cat. no. sc-7985-r), p-aKT (Thr308; 1:1,000; cat. no. sc-135650), p-S6rP (1:500; cat. no. sc-293144), vascular cell adhesion molecule 1 (VcaM-1; 1:250; cat. no. sc-13160), icaM-1 (1:500; cat. no. sc-8439), integrin β1 (1:1,000; cat. no. sc-8978; all Santa cruz Biotechnology, inc.), human inducible factor-1α (HiF-1α; 1:500; cat. no. nB100-105), VeGF (1:500; cat. no. nB100-664; both novus Biologicals, llc) or β-actin (1:5,000; cat. no. MaB1501; eMd Millipore) were diluted in PBS with 2.5% BSa and incubated with the membranes overnight at 4 ̊C with gentle agitation.

Techniques: Microscopy

Journal: Molecular medicine reports

Article Title: Effects of antibodies against human parvovirus B19 on angiogenic signaling.

doi: 10.3892/mmr.2020.10921

Figure Lengend Snippet: Figure 2. Expression of VCAM‑1, ICAM‑1 and integrin β1. (A) Protein expression levels of VCAM‑1, ICAM‑1 and integrin β1 in human umbilical vein endothelial cells treated with different antibodies were detected by immunoblotting. (B‑D) Relative levels of (B) VCAM‑1, (C) ICAM‑1 and (D) integrin β1 are based on β‑actin expression. Similar results were observed in triplicate experiments. *P<0.05 vs. cells treated with NH IgG. VCAM‑1, vascular cell adhesion molecule 1; ICAM‑1, intracellular adhesion molecule 1; NH, normal human; IgG, immunoglobulin G; FCS, fetal calf serum; aPL, anti‑phospholipid antibodies; B19V, parvovirus B19; VP1u, VP1 unique region.

Article Snippet: After blocking with 5% non‐fat dry milk for 1 h at 4 ̊C, antibodies against p-mTor (Ser2481; 1:500; cat. no. 09343; eMd Millipore), p-aKT (Ser473; 1:500; cat. no. sc-7985-r), p-aKT (Thr308; 1:1,000; cat. no. sc-135650), p-S6rP (1:500; cat. no. sc-293144), vascular cell adhesion molecule 1 (VcaM-1; 1:250; cat. no. sc-13160), icaM-1 (1:500; cat. no. sc-8439), integrin β1 (1:1,000; cat. no. sc-8978; all Santa cruz Biotechnology, inc.), human inducible factor-1α (HiF-1α; 1:500; cat. no. nB100-105), VeGF (1:500; cat. no. nB100-664; both novus Biologicals, llc) or β-actin (1:5,000; cat. no. MaB1501; eMd Millipore) were diluted in PBS with 2.5% BSa and incubated with the membranes overnight at 4 ̊C with gentle agitation.

Techniques: Expressing, Western Blot

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

Article Title: Syk is required for monocyte/macrophage chemotaxis to CX3CL1 (Fractalkine).

doi: 10.4049/jimmunol.175.6.3737

Figure Lengend Snippet: FIGURE 1. RAW cells chemotax toward CX3CL1 (fractalkine). A, Live cells were incubated with rabbit anti-CX3CR1 Ab or control IgG at 4°C before fixation and incubation with anti-rabbit IgG Ab conjugated to Alexa Fluor 488. Immunofluores- cence images are shown as well as the corresponding phase contrast im- ages (see insets). Scale bar 10 m. B, Cell migration in response to in- creasing doses of CX3CL1 was mea- sured as described in Materials and Methods. C, To distinguish between chemotaxis and chemokinesis, cell migration was assessed in response to 50 ng/ml CX3CL1 added in either the bottom chamber or the top chamber of the transmigration apparatus. D, The specificity of CX3CL1-induced chemotaxis was verified by preincu- bating the cells with 10 g/ml CX3CR1 neutralizing Ab or control IgG for 1 h before subjecting the cells to the transmigration assay. E, Cells were preincubated for 5 h with or without 250 ng/ml pertussis toxin (PTX) before measuring cell migra- tion as described above. n 3. , p 0.05 compared with the correspond- ing controls.

Article Snippet: Recombinant mouse CX3CL1 (aa 25–105) and mouse CSF-1 were purchased from R&D Systems.

Techniques: Incubation, Control, Migration, Chemotaxis Assay, Transmigration Assay

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

Article Title: Syk is required for monocyte/macrophage chemotaxis to CX3CL1 (Fractalkine).

doi: 10.4049/jimmunol.175.6.3737

Figure Lengend Snippet: FIGURE 2. CX3CL1 treatment induces a rearrangement of the actin cytoskeleton. A, RAW cells were either untreated (Untr) or treated with 50 ng/ml CX3CL1 for 1 min before fixation and staining of F-actin using phalloidin conjugated to Alexa Fluor 568. Images are representatives of at least five independent experiments. Scale bar 10 m. B, Cells were treated for various times with CX3CL1, then fixed, and total F-actin, nor- malized to the cell number, was quantitatively measured as described in Materials and Methods. n 3–7 independent determinations for each time point.

Article Snippet: Recombinant mouse CX3CL1 (aa 25–105) and mouse CSF-1 were purchased from R&D Systems.

Techniques: Staining

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

Article Title: Syk is required for monocyte/macrophage chemotaxis to CX3CL1 (Fractalkine).

doi: 10.4049/jimmunol.175.6.3737

Figure Lengend Snippet: FIGURE 3. CX3CL1 stimulates tyrosine phosphorylation. A, RAW cells were treated for various times with 50 ng/ml CX3CL1, and total cell lysates were subjected to Western blotting using a phosphotyrosine-spe- cific Ab (PY) or using anti--actin Ab as a verification of equal protein loading. A Western blot representative of three independent experiments is shown. B, RAW cells were either untreated or treated with CX3CL1 for 1 min before fixation and costaining of F-actin and phosphotyrosine-contain- ing proteins. C, Murine bone marrow-derived macrophages, prepared as described in Materials and Methods, were stimulated with CX3CL1 for 1 min before costaining as shown for RAW cells. Unless otherwise noted, all images shown are z-sections collected 2.5 m above the cell-substratum interface (cell midsections) using a confocal microscope. Images noted top represent the same cells as those shown below, but were taken 5 m above the coverslip level to focus on dorsal ruffles. All images are representative of at least three independent experiments. Scale bars 10 m.

Article Snippet: Recombinant mouse CX3CL1 (aa 25–105) and mouse CSF-1 were purchased from R&D Systems.

Techniques: Phospho-proteomics, Western Blot, Derivative Assay, Microscopy

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

Article Title: Syk is required for monocyte/macrophage chemotaxis to CX3CL1 (Fractalkine).

doi: 10.4049/jimmunol.175.6.3737

Figure Lengend Snippet: FIGURE 4. Syk is activated upon CX3CL1 treatment, and piceatannol inhibits CX3CL1-induced cell migration. A, RAW cells were either un- treated () or treated () with 50 ng/ml CX3CL1 for 1 min before lysis and immunoprecipitation of Syk (IP Syk) or phosphotyrosine-containing proteins (IP PY) using specific Abs. Samples were then subjected to West- ern blotting (WB) using the indicated Abs. The phosphospecific Ab used (P-Syk) specifically recognizes activated Syk (phosphorylated on residues Y519/520). Input represents the amount of Syk present in total cell lysates for each condition. Western blots representative of three independent ex- periments are shown. B, Cells were pretreated with 50 m piceatannol (Syk inhibitor) or DMSO (vehicle) and then subjected to a transmigration assay in response to CX3CL1 as described in Materials and Methods. n 3. , p 0.05 compared with CX3CL1-induced stimulation in vehicle- treated cells.

Article Snippet: Recombinant mouse CX3CL1 (aa 25–105) and mouse CSF-1 were purchased from R&D Systems.

Techniques: Migration, Lysis, Immunoprecipitation, Western Blot, Transmigration Assay

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

Article Title: Syk is required for monocyte/macrophage chemotaxis to CX3CL1 (Fractalkine).

doi: 10.4049/jimmunol.175.6.3737

Figure Lengend Snippet: FIGURE 6. Cells with reduced Syk expression show impaired migration toward CX3CL1. A, Syk shRNA- treated cell migration in response to CX3CL1 was de- termined using a Transwell assay as described previ- ously and compared with NI and scr cells. n 4. , p 0.05 compared with CX3CL1-induced migration in NI cells. B, The ability of Syk shRNA and scr cells to mi- grate in response to 20 ng/ml CSF-1 was also evaluated. n 3.

Article Snippet: Recombinant mouse CX3CL1 (aa 25–105) and mouse CSF-1 were purchased from R&D Systems.

Techniques: Expressing, Migration, shRNA, Transwell Assay

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

Article Title: Syk is required for monocyte/macrophage chemotaxis to CX3CL1 (Fractalkine).

doi: 10.4049/jimmunol.175.6.3737

Figure Lengend Snippet: FIGURE 7. CX3CL1 activation of ERK1/2 is not required for RAW cell chemotaxis. A, RAW cells were stimulated with CX3CL1 for the indicated times before Western blotting of the corresponding Triton-soluble whole cell lysates. The activation statuses of ERK1/2 (p42/44MAPK), p38MAPK, and JNK1 were assessed using phosphospecific Abs, and the corresponding total (phosphorylation-independent) levels of MAPK expression are shown below as proof of equal protein loading. B, CX3CL1-induced ERK1/2 activation levels were compared between scrambled and Syk shRNA-treated cells. Results shown were obtained with a clone exhibiting 80% Syk protein reduction. C, RAW cells were preincubated for 1 h with 30 M PD98059 (MEK inhibitor) or with DMSO (vehicle) before being subjected to a transmigration assay. PD98059 efficacy was confirmed through its ability to abolish ERK1/2 phosphorylation (see inset). For all experiments, n 3.

Article Snippet: Recombinant mouse CX3CL1 (aa 25–105) and mouse CSF-1 were purchased from R&D Systems.

Techniques: Activation Assay, Chemotaxis Assay, Western Blot, Phospho-proteomics, Expressing, shRNA, Transmigration Assay

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

Article Title: Syk is required for monocyte/macrophage chemotaxis to CX3CL1 (Fractalkine).

doi: 10.4049/jimmunol.175.6.3737

Figure Lengend Snippet: FIGURE 8. CX3CL1-induced cytoskeletal reorganization is disrupted in cells with reduced Syk expression. A, The ability of cells to increase their F-actin content after a 1-min CX3CL1 stimulation was compared among NI, scr, and Syk shRNA-treated (sh) cells, as previously described. n 4 independent experiments using heterogeneous cell populations. , p 0.05 compared with NI cells. B, Scrambled and Syk shRNA-treated cells were either untreated or treated with CX3CL1 for 1 min, and their ability to exhibit F-actin-rich cell protrusions (ruffles) was compared using F-actin staining. Representative images of three independent experiments are shown. Scale bar 10 m. The extent of CX3CL1-induced ruffles in individual cells was scored as described previously (18); protrusion in- dexes were calculated as the average of at least 50 cells in three different experiments and expressed as a percentage of scr cells. n 3. , p 0.05 compared with scrambled.

Article Snippet: Recombinant mouse CX3CL1 (aa 25–105) and mouse CSF-1 were purchased from R&D Systems.

Techniques: Expressing, shRNA, Staining

Journal: Life science alliance

Article Title: Megakaryocytes possess a STING pathway that is transferred to platelets to potentiate activation.

doi: 10.26508/lsa.202302211

Figure Lengend Snippet: Figure 1. STING stimulation of megakaryocytes induces a type-I interferon response. (A) Brightfield images of megakaryocytes isolated using anti-CD41 magnetic beads at day 4 of in vitro differentiation. Enlargement shows the production of proplatelets (black arrows) by mature megakaryocytes. Scale bar: 30 µm. (B, C) Cytokine production analysis by ELISA of megakaryocytes stimulated with DMXAA for 5 h (B) and 24 h (C) showing a significant increase in IFNB and CCL5 cytokines, n = 4 independent experiments; each dot represents data from one independent experiment. The graph shows data ± STD, and data were analysed by unpaired two-tailed t tests, *P < 0.05. (D, E) Representative immunofluorescence images of megakaryocytes taken by confocal microscopy showing the localisation of cGAS or STING (red), ERp57 or lamin A/C (green), and chromatin (blue), representative of n = 3 independent experiments. Scale bars: 20 µm (D) and 15 µm (E). (F) Representative immunofluorescence images of CD41-positive (magenta for CD41+ and blue for DNA) and CD41-negative (only blue) cells taken by confocal microscopy. Representative images of more than n = 3 independent experiments. Scale bars: 30 µm.

Article Snippet: Cells were then labelled with 10 μl anti-CD41 antibody (coupled to Biotin or APC—Miltenyi Biotec) for 10 min at 4°C, then washed with 2 ml of isolation buffer, and centrifuged at 300g for 10 min at 4°C, twice before the addition of 20 μl of Anti-Biotin/APC MicroBeads (Miltenyi Biotec) and 70 μl of isolation buffer for 15 min at 4°C.

Techniques: Isolation, Magnetic Beads, In Vitro, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Confocal Microscopy

Journal: Molecular Cancer Research

Article Title: The Nuclear Pool of Tetraspanin CD9 Contributes to Mitotic Processes in Human Breast Carcinoma

doi: 10.1158/1541-7786.mcr-14-0242

Figure Lengend Snippet: Figure 1. CD9 is present in BCC nuclei. BCC, fixed and permeabilized, were sequentially incubated with anti-CD9 Ab (Biolegend, clone H19a) and Cy5-conjugated anti-mouse IgG secondary Ab. CD9 positivity was observed in the nuclei of all BCC lines (arrows). Cells were imaged using confocal laser scanning microscopy on a Nikon A1Rþ. Images are representative slices taken from z-stacks using a 0.5 to 0.7 mm step size. Scale bars for images on left represent 50 mm. Scale bars for images on right represent 25 mm. Magenta, CD9. Blue, DAPI.

Article Snippet: © 2014 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from Live cell staining for CD9 partners A total of 10 mg/mL anti-Cep97 Ab (Santa Cruz Biotechnologies) or 5 mg/mL anti-IgSF8 Ab (Santa Cruz Biotechnologies) in 20 mmol/L HEPES buffer (Gibco) was combined with a 1:20 dilution of PE-conjugated anti-goat IgG secondary Ab (Rockland Immunochemicals) in the presence of 40 mg/mL Lipofectamine (Invitrogen) to favor Ab penetration (14) and incubated at room temperature for 15 minutes.

Techniques: Incubation, Confocal Laser Scanning Microscopy

Journal: Molecular Cancer Research

Article Title: The Nuclear Pool of Tetraspanin CD9 Contributes to Mitotic Processes in Human Breast Carcinoma

doi: 10.1158/1541-7786.mcr-14-0242

Figure Lengend Snippet: Figure 2. Treatment of BCC with anti-CD9 mAb results in nuclear CD9 staining. A, non-fixed BCC were sequentially incubated for 90 minutes at 37C with the H19a anti-CD9 Ab and then with Cy5-conjugated anti-mouse IgG secondary Ab, followed by fixation and DAPI staining. CD9 immunopositivity was observed in the nuclei of all BCC lines. Cells were imaged using confocal laser scanning microscopy on a Nikon A1Rþ. Images are representative slices taken from z-stacks using a 0.5 to 0.7 mm step size. Scale bars for images on left represent 50 mm. Scale bars for images on right represent 25 mm. Magenta, CD9. Blue, DAPI. B, exposure of BCC to anti-CD9 mAb causes a shift in nuclear localization of CD9.

Article Snippet: © 2014 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from Live cell staining for CD9 partners A total of 10 mg/mL anti-Cep97 Ab (Santa Cruz Biotechnologies) or 5 mg/mL anti-IgSF8 Ab (Santa Cruz Biotechnologies) in 20 mmol/L HEPES buffer (Gibco) was combined with a 1:20 dilution of PE-conjugated anti-goat IgG secondary Ab (Rockland Immunochemicals) in the presence of 40 mg/mL Lipofectamine (Invitrogen) to favor Ab penetration (14) and incubated at room temperature for 15 minutes.

Techniques: Staining, Incubation, Confocal Laser Scanning Microscopy

Journal: Molecular Cancer Research

Article Title: The Nuclear Pool of Tetraspanin CD9 Contributes to Mitotic Processes in Human Breast Carcinoma

doi: 10.1158/1541-7786.mcr-14-0242

Figure Lengend Snippet: Figure 4. CD9 is associated with Cep97 and IgSF8 in MDA nuclei. A, immunoblotting of nuclear (N) and cytoplasmic (C) lysates from MDA-CD9-GFP and MA-11-CD9- GFP before and after exposure of the cells to anti-CD9 mAb for 90 minutes. Single protein bands of about 50 and 100 kDa were observed for CD9-GFP and Cep97, respectively; 2 bands of about 55 and 70 kDa for IgSF8, and one band of about 65 kDa for ADAM10. B, co-immunoprecipitation of Cep97 and IgSF8 with CD9-GFP at the nuclear (N), but not at the cytoplasmic (C) level. C–F, colocalization experiments were performed by combining 10 mg/mL anti-Cep97 Ab or 5 mg/mL anti- IgSF8 Ab with a 1:20 dilution of PE-conjugated anti-mouse IgG secondary Ab in 20 mmol/L HEPES buffer in the presence of 40 mg/mL Lipofectamine 2000. After incubating for 4 hours at 37C, cells were imaged using confocal laser scanning microscopy. D and F, anti-Cep97 and anti-IgSF8 were preceded by 90-minute incubation with anti- CD9 Ab to enhance nuclear translocation of CD9. White arrows indicate areas of colocalization. Scale bars represent 50 mm.

Article Snippet: © 2014 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from Live cell staining for CD9 partners A total of 10 mg/mL anti-Cep97 Ab (Santa Cruz Biotechnologies) or 5 mg/mL anti-IgSF8 Ab (Santa Cruz Biotechnologies) in 20 mmol/L HEPES buffer (Gibco) was combined with a 1:20 dilution of PE-conjugated anti-goat IgG secondary Ab (Rockland Immunochemicals) in the presence of 40 mg/mL Lipofectamine (Invitrogen) to favor Ab penetration (14) and incubated at room temperature for 15 minutes.

Techniques: Western Blot, Immunoprecipitation, Confocal Laser Scanning Microscopy, Incubation, Translocation Assay

Journal: Molecular Cancer Research

Article Title: The Nuclear Pool of Tetraspanin CD9 Contributes to Mitotic Processes in Human Breast Carcinoma

doi: 10.1158/1541-7786.mcr-14-0242

Figure Lengend Snippet: Figure 5. Colocalization and FRET analysis of CD9 and Cep-97 or CD9 and IgSF8. A and B, colocalization of CD9-GFP and Cep97 or CD9-GFP and IgSF8. MDA-CD9-GFP cells were fixed, permeabilized, and stained with anti-Cep97 Ab (A) or anti-IgSF8 (B) followed by TRITC-conjugated anti-goat IgG secondary Ab. Images are representative confocal slices from z-stacks collected before FRET analysis. From left to right: GFP channel, TRITC channel, and both channels merged onto DIC image. Colocalization is observed in and around the nucleus for Cep97 and CD9 and on the plasma membrane for IgSF8 (arrows). C and D, average relative fluorescence values for GFP and TRITC over time during sequential acceptor photobleaching FRET in multiple ROIs for the samples described in A and B. The donor (TRITC) was sequentially bleached for 8 seconds, followed by donor (GFP) and acceptor acquisition for 576 seconds total. TRITC fluorescence decreased over time, but there was no increase in GFP fluorescence in either Cep97- or IgSF8-stained dishes.

Article Snippet: © 2014 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from Live cell staining for CD9 partners A total of 10 mg/mL anti-Cep97 Ab (Santa Cruz Biotechnologies) or 5 mg/mL anti-IgSF8 Ab (Santa Cruz Biotechnologies) in 20 mmol/L HEPES buffer (Gibco) was combined with a 1:20 dilution of PE-conjugated anti-goat IgG secondary Ab (Rockland Immunochemicals) in the presence of 40 mg/mL Lipofectamine (Invitrogen) to favor Ab penetration (14) and incubated at room temperature for 15 minutes.

Techniques: Staining, Clinical Proteomics, Membrane