Journal: Scientific Reports

Article Title: In vivo imaging of kidney glomeruli transplanted into the anterior chamber of the mouse eye

doi: 10.1038/srep03872

Figure Lengend Snippet: (a) and (b) Transmission electron microscopy shows interdigitating podocyte foot processes. (c) Immunogold electron microscopy reveals conserved expression of podocalyxin in podocytes from transplanted glomeruli. Control sections stained with secondary antibody only were completely negative for gold particles.

Article Snippet: Briefly, Ultrathin sections were blocked with 1% ovalbumin in PBS for one hour, followed by incubation with affinity-purified goat antibody to podocalyxin (R&D, AF1556, 1:400) and an anti-goat 10 nm gold conjugate (1:50).

Techniques: Transmission Assay, Electron Microscopy, Expressing, Staining

Journal: Frontiers in Immunology

Article Title: Podocalyxin Expressed in Antigen Presenting Cells Promotes Interaction With T Cells and Alters Centrosome Translocation to the Contact Site

doi: 10.3389/fimmu.2022.835527

Figure Lengend Snippet: PODXL is expressed in human monocyte-derived immature DCs. (A) Determination of PODXL expression in total lysates from human monocytes and monocyte-derived immature DCs from two donors and CD4 + T cells from one donor by Western blot analysis using a specific mouse anti-human PODXL monoclonal antibody. GAPDH was used as loading control. The blot shown is representative of two independent experiments. (B) Flow cytometry gating and overlay histograms depicting the surface expression of PODXL on monocytes (CD14 + CD209 - ) and immature DCs (CD14 - CD209 + ) detected by flow cytometry using a biotinylated goat anti-human PODXL antibody (red line) or an isotype control (grey line) followed by PE-conjugated streptavidin from one representative donor. The graph shows PODXL surface expression ( Δ MFI= PODXL MFI - isotype control MFI) on monocytes and immature DCs from three donors. *p < 0.05, paired t test. (C) Expression of PODXL in monocyte-derived immature DCs by fluorescence microscopy. Monocyte-derived immature DCs were deposited onto glass slides using a cytospin and incubated in complete culture medium for 30 min. Cell were stained with a goat anti-PODXL polyclonal antibody followed by an anti-goat Cy2-labelled secondary antibody (green). Images were acquired with a 63x objective using a confocal fluorescence microscope and are representative of ten rounded and twelve elongated immature DCs photographed from three independent experiments. Example shows maximal intensity z-projections of confocal fluorescence sections. DIC, differential interference contrast. Scale bar corresponds to 5μm. (D) Determination of PODXL expression in cell lysates from different myelomonocytic cell lines by Western blot analysis using a specific anti-PODXL monoclonal antibody. GAPDH was used as loading control. The blot shown is representative of two independent experiments. (E) Overlay histograms showing the surface expression of PODXL on four different myeloid cell lines determined by flow cytometry using a biotinylated goat anti-human PODXL antibody (red line) or an isotype control (grey line) followed by PE-conjugated streptavidin from one representative experiment. Graph shows mean ± SD of PODXL surface expression ( Δ MFI= PODXL MFI - isotype control MFI) from four independent experiments. ***p < 0.001, ****p < 0.0001, one-way ANOVA. (F) Analysis of PODXL mRNA level by RT-PCR in four myelomonocytic cell lines and in T cells, NK cells, monocytes, monocyte-derived immature and mature DCs. Results are depicted relative to THP-1 cells. *p < 0.05, Krustal-Wallis test.

Article Snippet: Then, cells were stained with a mouse anti-human PODXL monoclonal antibody (R&D Systems, cat.n° MAB1658) for 30 min at 4°C followed by a secondary PE-conjugated anti-mouse IgG secondary antibody (R&D Systems) for 20 min at room temperature or with a biotinylated goat anti-human PODXL antibody (R&D Systems, cat.n° BAF1658) for 30 min at 4°C followed by PE-conjugated streptavidin (Biolegend) for 20 min at room temperature, as indicated in figure captions.

Techniques: Derivative Assay, Expressing, Western Blot, Control, Flow Cytometry, Fluorescence, Microscopy, Incubation, Staining, Reverse Transcription Polymerase Chain Reaction

Journal: Frontiers in Immunology

Article Title: Podocalyxin Expressed in Antigen Presenting Cells Promotes Interaction With T Cells and Alters Centrosome Translocation to the Contact Site

doi: 10.3389/fimmu.2022.835527

Figure Lengend Snippet: PODXL expression on monocyte-derived DCs is downregulated upon maturation stimuli. (A) Determination of PODXL expression in monocytes, monocyte-derived immature and mature DCs in total cell lysates from six donors by Western blot using a specific mouse anti-human PODXL monoclonal antibody. Monocytes were incubated with IL-4 and GM-CSF for 5 days to obtained immature DCs, followed by incubation with LPS for 2 days to induce DC maturation. GAPDH was used as loading control. The bar graph represents the mean ± SD of PODXL levels relative to GAPDH from the six donors of three independent experiments. *p < 0.05, ***p < 0.001, RM one-way ANOVA. (B) Expression levels of PODXL on the surface of immature and mature DCs from 15 healthy donors using a mouse anti-human PODXL monoclonal antibody followed by PE-conjugated anti-mouse IgG secondary antibody determined by flow cytometry. Graph shows PODXL surface expression ( Δ MFI= PODXL MFI - isotype control MFI). ***p < 0.001, Wilcoxon matched-pairs signed rank test. (C) Mature DCs were obtained by incubating immature DCs with LPS or the maturation cocktail consisting of TNFα, IL-1, IL-6 and PGE2, and PODXL expression levels determined by flow cytometry as in (B) . Overlay histograms show surface expression of PODXL (red line) and isotype control (grey line) from one representative donor. The graph represents PODXL expression relative to immature DCs from three donors in three independent experiments. **p < 0.01, ***p < 0.001, one-way ANOVA.

Article Snippet: Then, cells were stained with a mouse anti-human PODXL monoclonal antibody (R&D Systems, cat.n° MAB1658) for 30 min at 4°C followed by a secondary PE-conjugated anti-mouse IgG secondary antibody (R&D Systems) for 20 min at room temperature or with a biotinylated goat anti-human PODXL antibody (R&D Systems, cat.n° BAF1658) for 30 min at 4°C followed by PE-conjugated streptavidin (Biolegend) for 20 min at room temperature, as indicated in figure captions.

Techniques: Expressing, Derivative Assay, Western Blot, Incubation, Control, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Podocalyxin Expressed in Antigen Presenting Cells Promotes Interaction With T Cells and Alters Centrosome Translocation to the Contact Site

doi: 10.3389/fimmu.2022.835527

Figure Lengend Snippet: PODXL expression is positively regulated by IL-4 through MEK/ERK and JAK3/STAT6 signaling pathways in myeloid cells. THP-1 cells were stimulated with IL-4 (400 U/ml) and GM-CSF (800 U/ml) cytokines (A) for 96 h or with PMA (100 ng/ml) (B) for 48 h followed by a resting period of 48 (h) Afterwards, total cell lysates were subjected to Western blotting using a specific monoclonal antibody against PODXL. Actin was used as loading control. Bar graphs represent the mean ± SD of PODXL levels relative to those of unstimulated cells of four independent experiments. *p < 0.05, **p < 0.01, one-way ANOVA. (C) PODXL surface expression was analyzed by flow cytometry using a biotinylated goat anti-human PODXL antibody followed by PE-conjugated streptavidin. Overlay histograms show surface expression of PODXL (red line) and isotype control (grey line) from one representative donor. Graph shows PODXL surface expression (ΔMFI= PODXL MFI - isotype control MFI) of four independent experiments. **p < 0.01, ***p < 0.001, one-way ANOVA. (D, E) Effect of signaling pathway inhibitors on IL-4-induced PODXL expression in THP-1 cells. Cells were incubated with 400 U/ml IL-4 in the presence of increasing concentration of MEK/ERK signaling pathway inhibitor (PD98059) (D) or JAK3 signaling pathway inhibitor (PF956980) (E) for 96 (h) Then, cell lysates were subjected to Western blot analysis with a monoclonal anti-PODXL antibody. Actin was used as loading control. Bar graphs represent the mean ± SD of PODXL levels relative to those of unstimulated and untreated cells of three (D) or four (E) independent experiments. A representative blot is shown below each graph. *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA.

Article Snippet: Then, cells were stained with a mouse anti-human PODXL monoclonal antibody (R&D Systems, cat.n° MAB1658) for 30 min at 4°C followed by a secondary PE-conjugated anti-mouse IgG secondary antibody (R&D Systems) for 20 min at room temperature or with a biotinylated goat anti-human PODXL antibody (R&D Systems, cat.n° BAF1658) for 30 min at 4°C followed by PE-conjugated streptavidin (Biolegend) for 20 min at room temperature, as indicated in figure captions.

Techniques: Expressing, Protein-Protein interactions, Western Blot, Control, Flow Cytometry, Incubation, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Podocalyxin Expressed in Antigen Presenting Cells Promotes Interaction With T Cells and Alters Centrosome Translocation to the Contact Site

doi: 10.3389/fimmu.2022.835527

Figure Lengend Snippet: PODXL expressed in APCs enhances both APC-CD4 + T cell and APC-CD8 + T cell interactions. (A) Raji cells overexpressing PODXL (red) or Raji-Ctrl cells (blue) were pulsed or not with SEA. Afterwards, CD4 + T cells isolated from PBMCs and pre-labelled with CMTMR were mixed with Raji-PODXL or Raji-Ctrl at a ratio of 1:1 and incubated for different time points at 37°C. The formation of conjugates between Raji cells (GFP + ) and CD4 + T cells (CMTMR + ) was detected as CMTMR + and GFP + events by flow cytometry. A representative density plot out of seven independent experiments is shown. Graph shows mean ± SD of the percentage of conjugates (CMTMR + GFP + ) from the total Raji cells (GFP + ) of four independent experiments. *p < 0.05, **p < 0.01, ***p <0.001, ****p < 0.0001, two-way ANOVA. (B) Raji-PODXL or Raji-Ctrl cells were mixed with isolated CD4 + T and CD8 + T at a ratio of 1:1 and incubated for 30 min. CD4 + and CD8 + T cells were detected using anti-CD4-APCH7 and anti-CD8-APCH7 monoclonal antibodies by flow cytometry. The percentage of Raji-CD4 + T cell (GFP + APCH7 + ) or Raji-CD8 + T cell (GFP + APCH7 + ) out of the total Raji cells was determined. Bar graph represents the mean ± SD of five independent experiments. *p < 0.05, RM one-way ANOVA. (C) Raji-PODXL or Raji-Ctrl cells were mixed with CMTMR-labeled Jurkat cells at a ratio of 1:1 and incubated at different time points. The percentage of Raji-Jurkat cell conjugates was determined by flow cytometry as in (A) . Bar graph represents the mean ± SD of five independent experiments. **p < 0.01, two-way ANOVA.

Article Snippet: Then, cells were stained with a mouse anti-human PODXL monoclonal antibody (R&D Systems, cat.n° MAB1658) for 30 min at 4°C followed by a secondary PE-conjugated anti-mouse IgG secondary antibody (R&D Systems) for 20 min at room temperature or with a biotinylated goat anti-human PODXL antibody (R&D Systems, cat.n° BAF1658) for 30 min at 4°C followed by PE-conjugated streptavidin (Biolegend) for 20 min at room temperature, as indicated in figure captions.

Techniques: Isolation, Incubation, Flow Cytometry, Bioprocessing, Labeling

Journal: Frontiers in Immunology

Article Title: Podocalyxin Expressed in Antigen Presenting Cells Promotes Interaction With T Cells and Alters Centrosome Translocation to the Contact Site

doi: 10.3389/fimmu.2022.835527

Figure Lengend Snippet: PODXL decreases the expression of CD86, MHC-I and MHC-II in APCs. (A) Raji-PODXL, Raji-Ctrl cells and CMTMR-labeled Jurkat cells were preincubated separately with a blocking anti-LFA-1 monoclonal antibody for 15 min. Afterwards, Raji-PODXL and Raji-Ctrl were mixed with CMTMR-labeled Jurkat cells at a ratio of 1:1 and incubated for 30 min. The percentage of Raji-Jurkat cell conjugates was determined by flow cytometry as in ( Figure 4A ). Bar graph represents the mean ± SD of nine independent experiments. *p < 0.05, ****p < 0.0001, one-way ANOVA. (B) Raji cells overexpressing PODXL and Raji Ctrl cells were analyzed by flow cytometry for surface expression of CD54, CD80, CD86, MHC-I and MHC-II. Overlay histograms represent the surface expression of the indicated marker in Raji-PODXL cells (red line), Raji-Ctrl cells (blue line) and the isotype control (grey line). Graphs show mean ± SD of surface expression (ΔMFI= Marker MFI - isotype control MFI) from six (CD54, CD80, CD86, MHC-I) or five (MHC-II) independent experiments. *p < 0.05, **p < 0.01, paired t test. 0.0001, ns, not statistically significant; one-way ANOVA.

Article Snippet: Then, cells were stained with a mouse anti-human PODXL monoclonal antibody (R&D Systems, cat.n° MAB1658) for 30 min at 4°C followed by a secondary PE-conjugated anti-mouse IgG secondary antibody (R&D Systems) for 20 min at room temperature or with a biotinylated goat anti-human PODXL antibody (R&D Systems, cat.n° BAF1658) for 30 min at 4°C followed by PE-conjugated streptavidin (Biolegend) for 20 min at room temperature, as indicated in figure captions.

Techniques: Expressing, Labeling, Blocking Assay, Incubation, Flow Cytometry, Marker, Control

Journal: Frontiers in Immunology

Article Title: Podocalyxin Expressed in Antigen Presenting Cells Promotes Interaction With T Cells and Alters Centrosome Translocation to the Contact Site

doi: 10.3389/fimmu.2022.835527

Figure Lengend Snippet: PODXL expressed in APCs localizes to the T-cell contact side and partially colocalizes with actin. (A) Raji cells overexpressing PODXL-GFP and pulsed with SEA were incubated with CD4 + T cells at a ratio of 1:2 for 30 min. Images of conjugates comprising one Raji cell and one CD4 + T cell were obtained using confocal fluorescence microscopy with a 63x objective and are representative of three independent experiments. The example shows maximal intensity z-projections of confocal fluorescence sections. The position of PODXL in Raji-CD4 + T cell conjugates was determined using images obtained with a fluorescence microscope and a 20x objective and scored as 1 when located in the quadrant close to the contact area, 2 and 3 when located at the intermediate quadrants, and 4 when present at the distal quadrant. Bar graph represents the percentage of conjugates with PODXL in each indicated position from three independent experiments and a total of 426 Raji-CD4 + T cell conjugates. The results are shown as mean ± SD. ****p < 0.0001, one-way ANOVA. (B) Immature DC were incubated with CD4 + T cells for 30 min and conjugates were analyzed with a 63x objective using a confocal fluorescence microscope. Cells were stained with a goat polyclonal anti-PODXL antibody followed by an anti-goat Cy2-labelled secondary antibody (green), and Hoechst 33342 for nuclei staining (blue). Images show a representative immature DC-CD4 + T cell conjugate of 15 conjugates photographed from three independent experiments. Example shows maximal intensity z-projections of confocal fluorescence sections. (C) Raji cells overexpressing PODXL-GFP (green) and pulsed with SEA were incubated with CD4 + T cells and stained for actin with Alexa 555-phalloidin (red) and for nuclei with Hoechst 33342 (blue). A confocal single z-section from a representative conjugate is depicted. An enlarged image of the region corresponding to the cell-to-cell contact zone is shown. Histogram depicts intensity profiles of PODXL (green) and actin (red) obtained using ImageJ-Fiji software, along an ideal line (white dashed line on the left image) crossing the contact zone of a representative conjugate. Bar graph represent the Pearson´s correlation coefficient of PODXL and actin colocalization at cell-to-cell contact site in Raji-PODXL cells conjugated with CD4 + T cells. A representative image showing the ROI selected for the quantification of colocalization is depicted (right image). Twenty two Raji-PODXL- CD4 + T cell conjugates from five independent experiments were analyzed. Error bar displays the standard error. Scale bars in confocalimages correspond to 5 um.

Article Snippet: Then, cells were stained with a mouse anti-human PODXL monoclonal antibody (R&D Systems, cat.n° MAB1658) for 30 min at 4°C followed by a secondary PE-conjugated anti-mouse IgG secondary antibody (R&D Systems) for 20 min at room temperature or with a biotinylated goat anti-human PODXL antibody (R&D Systems, cat.n° BAF1658) for 30 min at 4°C followed by PE-conjugated streptavidin (Biolegend) for 20 min at room temperature, as indicated in figure captions.

Techniques: Incubation, Fluorescence, Microscopy, Staining, Software

Journal: Frontiers in Immunology

Article Title: Podocalyxin Expressed in Antigen Presenting Cells Promotes Interaction With T Cells and Alters Centrosome Translocation to the Contact Site

doi: 10.3389/fimmu.2022.835527

Figure Lengend Snippet: PODXL expressed in APCs perturbs CD4 + T cell-centrosome polarization to the contact site. (A) Raji PODXL or Raji control cells pulsed with SEA were incubated with CD4 + T cells at a ratio of 1:1 for 30 min or 60 min. Then, cells were stained with a monoclonal antibody against γ-tubulin to visualize the centrosome (red) and Hoechst 33342 for nuclei staining (blue). Confocal fluorescence microscopy images show separate and merged channels of maximal intensity z-projections of z-sections from a representative experiment performed at 60 min of incubation. Arrows point to T-cell centrosome in each T-cell. DIC: differential interference contrast. Scale bars correspond to 5 μm. (B) Quantification of T-cell centrosome polarization in Raji-CD4 + T cells conjugates. Scatter graph depicts the distance of CD4 + T cell centrosome to Raji-cell contact site in each conjugate for a total of 41 (Raji Ctrl, 30 min), 37 (Raji PODXL, 30 min), 51 (Raji Ctrl, 60 min), and 45 (Raji PODXL, 60 min) conjugates from three independent experiments. The results are shown as mean ± SD. *p < 0.05, two-way ANOVA followed by Holm-Šídák multiple comparisons test. Bar graph represents the percentage of conjugates in which T-cell centrosome localizes at a distal ( ≥ 3 μm), intermediate (1-3 μm) or proximal ( ≤ 1 μm) distance from Raji-cell contact site for the indicated conditions.

Article Snippet: Then, cells were stained with a mouse anti-human PODXL monoclonal antibody (R&D Systems, cat.n° MAB1658) for 30 min at 4°C followed by a secondary PE-conjugated anti-mouse IgG secondary antibody (R&D Systems) for 20 min at room temperature or with a biotinylated goat anti-human PODXL antibody (R&D Systems, cat.n° BAF1658) for 30 min at 4°C followed by PE-conjugated streptavidin (Biolegend) for 20 min at room temperature, as indicated in figure captions.

Techniques: Control, Incubation, Staining, Fluorescence, Microscopy

Journal: Cancers

Article Title: An Anti-VEGF-B Antibody Reduces Abnormal Tumor Vasculature and Enhances the Effects of Chemotherapy

doi: 10.3390/cancers16101902

Figure Lengend Snippet: Immunohistochemical analysis of HT29 xenografts from anti-VEGF-treated mice. ( A ) Representative IHC images of matched HT29 tumor sections from PBS, 2H10- and bevacizumab-treated mice (from ), stained with anti-Podocalyxin for blood vessels, or anti-Ki-67 for proliferation, versus IgG controls. ( B , C ) Graphs show the average number of Podocalyxin-positive vessels ( B ) and vessel diameter ( C ) determined in ten regions/section. ( D ) Average number of Ki-67-positive nuclei. Graphs show means +/− SEM (ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: Podocalyxin: sections were incubated with 1ug goat anti-human podocalyxin (R&D, AF1556) antibody for 2 h at room temperature.

Techniques: Immunohistochemical staining, Staining

Journal: Cancers

Article Title: An Anti-VEGF-B Antibody Reduces Abnormal Tumor Vasculature and Enhances the Effects of Chemotherapy

doi: 10.3390/cancers16101902

Figure Lengend Snippet: Combination of 2H10 with chemotherapy 5-FU. ( A , B ) Mice with HT29 tumors were treated with 2H10 (1 mg), bevacizumab (0.4 mg) or 5FU (20 mg/kg), alone and in combination as indicated. Tumor growth was monitored and growth curves plotted ( A ). ( B ) All data point at day 23. ( C – E ) Quantification of tumor IHC staining of markers for blood vessels (Podocalyxin) and proliferation (Ki-67). Graphs show vessel numbers ( C ) and diameter ( D ), and numbers of Ki-67-positive cells ( E ), in matched control and treated tumors (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: Podocalyxin: sections were incubated with 1ug goat anti-human podocalyxin (R&D, AF1556) antibody for 2 h at room temperature.

Techniques: Immunohistochemistry