Journal: eLife

Article Title: Cytotoxic T-cells mediate exercise-induced reductions in tumor growth

doi: 10.7554/eLife.59996

Figure Lengend Snippet: ( A ) Wild type (WT) and MMTV-PyMT (PyMT) mice on the FVB background were allowed to exercise voluntarily (in running wheels, runners) or left non-exercised (locked running wheels, non-runners) between 4 and 12 weeks of age. ( B ) Running distance (km/day) in WT and PyMT mice. *p<0.05, two-tailed unpaired t test. ( C ) Tumor volume measured twice weekly for PyMT running and non-running mice. Mean and SEM (n = 10–11). ( D ) Survival of PyMT mice, runners, and non-runners. Survival curve ns = not significant, Log-rank (Mantel-Cox) test, (n = 10–11). ( E ) Tumor initiation as age (day) of first indication of a palpable tumor in running (Runners) and non-running (Non-runners) PyMT mice. n = 11, ns = not significant, two-tailed t test. ( F ) Tumor stage from histological scoring (1-4) in mammary glands of running and non-running PyMT mice at 12 (n = 6–10) and 8 (n = 4) weeks of age, ns = not significant, two-tailed t test. ( G ) Individual body weight for WT and PyMT running and non-running mice. n = 7–10, ns = not significant, one-way ANOVA with Tukey’s multiple comparison test. ( H ) Immunohistological characterization of PyMT tumors from non-running and running mice using CD3, F4/80, PCNA, Podocalyxin (PODXL), and Granzyme B (GZMB) antibodies, respectively. n = 8–15, ns = not significant, *p<0.05, Two-tailed unpaired t test. ( I ) Flow-cytometry-based frequency of macrophages within I3TC tumor. Ns = not significant, two-tailed t-test. ( J ) Flow-cytometry-based frequency of neutrofiles within I3TC tumor. Ns = not significant, two-tailed t-test.

Article Snippet: Nonspecific protein interactions were blocked using 20% goat serum in PBS-T. Primary antibodies were used in mammary glands against CD3 (#ab5690, Abcam, UK) to assess lymphocytic infiltration, F4/80 (#MCA497, Abd Serotec, Germany) to assess macrophage density, podocalyxin (#AF1556, R and D Systems, Germany) to assess capillary density, PCNA (#M0879, Dako, Denmark) to identify proliferating cells, and Granzyme B (#ab4059, Abcam, UK) to identify effector lymphocytes.

Techniques: Two Tailed Test, Comparison, Flow Cytometry

Journal: eLife

Article Title: Cytotoxic T-cells mediate exercise-induced reductions in tumor growth

doi: 10.7554/eLife.59996

Figure Lengend Snippet:

Article Snippet: Nonspecific protein interactions were blocked using 20% goat serum in PBS-T. Primary antibodies were used in mammary glands against CD3 (#ab5690, Abcam, UK) to assess lymphocytic infiltration, F4/80 (#MCA497, Abd Serotec, Germany) to assess macrophage density, podocalyxin (#AF1556, R and D Systems, Germany) to assess capillary density, PCNA (#M0879, Dako, Denmark) to identify proliferating cells, and Granzyme B (#ab4059, Abcam, UK) to identify effector lymphocytes.

Techniques: Transgenic Assay, Marker, Derivative Assay, Staining, Software, Plasmid Preparation, Avidin-Biotin Assay

Journal: The Journal of Biological Chemistry

Article Title: Loss of mucin-type O -glycans impairs the integrity of the glomerular filtration barrier in the mouse kidney

doi: 10.1074/jbc.M117.798512

Figure Lengend Snippet: Sialoproteins such as podocalyxin are targets of O-glycosylation in glomerulosclerosis. A, IHC of podocalyxin (PODXL) in WT and iC1galt1−/− adult mice after 4 weeks of tamoxifen-induced deletion. B, Western blotting of podocalyxin and evaluation of O-glycosylation/sialylation status of podocalyxin in WT and iC1galt1−/− mice. VVL is a lectin-recognizing Tn antigen. Sialylation of podocalyxin was probed with lectins: MALII recognizes N-acetylneuraminic acid (Neu5Ac)-linked α2,3-Gal; SNA recognizes α2,6-linked sialic acid. IB, immunoblot; IP, immunoprecipitation. C, podocalyxin–NHERF2 interaction was disrupted after loss of O-glycans. Kidney lysates from adult WT and iC1galt1−/− mice 8 weeks post-induction were immunoprecipitated with polyclonal anti-NHERF2 antibody or anti-podocalyxin, followed by immunoblotting. Arrows indicate podocalyxin proteins with differential degrees of O-glycosylation. Blue arrow indicates hypo-O-glycosylated portion of podocalyxin. D, immunofluorescent staining of pEzrin and podocalyxin in glomeruli from WT and iC1galt1−/− mice. Scale bars = 20 μm.

Article Snippet: After inactivation of endogenous peroxidase with 0.3% hydrogen peroxide and blocking with normal blocking serum, the sections were incubated with glycosylation-independent goat anti-podocalyxin antibody (R&D Systems) or biotinylated mAb against Tn antigen (mouse IgM, clone Ca3638) overnight at 4 °C.

Techniques: Glycoproteomics, Western Blot, Immunoprecipitation, Staining

Journal: Experimental hematology

Article Title: Novel roles for podocalyxin in regulating stress myelopoiesis, Rap1a, and neutrophil migration.

doi: 10.1016/j.exphem.2017.04.001

Figure Lengend Snippet: Figure 1. Hematopoietic growth factor induction of Podxl, locus targeting, and hematopoietic deletion of Podxl. (A–C) G-CSFand TPO induction of Podxl expres- sion. (A) Upmodulation of Podxl transcript expression in bone marrow GMPs in vivo. Wild-type C57Bl/6 mice were administered G-CSF (125mg/kg/IP) or phosphate-buffered saline. On day 6, GMP populations were isolated from bone marrow via the retrieval of CD11b, Gr-1, Ly6G-positive cells. These HPCs were then analyzed for Podxl expression by reverse transcription quantitative polymerase chain reaction (left) and flow cytometry (right). Values are means 6 SE, n 5 3. (B) Possible effects of GM-CSF on Podxl expression were assessed in isolated bone marrow GMPs as lineage-negative, CD117þ, Sca-1, CD34þ, CD16/ 32þ cells. These HPCs were challenged ex vivo (6GM-CSF), and isolated total RNA was used to determine Podxl levels. (C) In HSCs, possible effects of TPO on Podxl expression were assessed. HPCs were isolated from bone marrow as Linneg cells (using biotinylated antibodies to CD5, CD11b, CD19, CD45R, Ly6G/ C, Ter119). Linneg cells were then labeled with fluorescent antibodies to c-Kit and Sca1, and HSCs were purified via fluorescence-activated cell sorting. HSCs were then challenged with (þ) versus without () TPO. Total RNAwas purified and used to determine Podxl expression levels. (D) Podxl’s structural subdomains are diagrammed, including its signal peptide (S), mucin domain, cysteine-rich region (c-c), stalk plus transmembrane (TM) domains, and cytoplasmic tail (cyto tail). (E) Targeting (floxing) of the Podxl gene. Details are diagrammed for exon floxing and Cre-mediated deletion. (F) Reverse transcription quantitative polymerase chain reaction analysis of Podxl transcripts in HPCs from wild-type and PodxlDHC bone marrow. The HPCs were prepared as Linneg populations. Podxl levels were then determined as normalized to b-Actin. Values are means 6 SE, n 5 3. (G) Representative flow cytometric analysis of Podxl expression in bone marrow cellpreparationsfromwild-typeandPodxlDHC-KOmice(left).Onthe rightare the resultsfor triplicatesamples(meanpercentage positive 6 SE).SE5Standarderror.

Article Snippet: Cells (2 105 in 0.5 mL of PBS, 0.1%BSA, 3 mg/ mL rat IgG) were incubated at 4 C for 15 minutes and were then stained with the following antibodies (at manufacturer’s recommended concentrations): allophycocyanin (APC)-c-Kit (BD Biosciences, cat# 553356), FITCCD11b (BD Biosciences, cat# 553310,), FITC-Gr-1 (BD Biosciences, cat# 553126), PE-CD34 (BD Biosciences, cat# 551387) and APC-Podxl (R&D system, cat# FAB1556A), Alexa fluor-488-CD11b (BD Biosciences, cat # 557672), APC-Ly6G (BD Biosciences, cat # 560599), and BV421-CD16/32 (BD Biosciences, cat # 562896).

Techniques: Expressing, In Vivo, Saline, Isolation, Reverse Transcription, Real-time Polymerase Chain Reaction, Cytometry, Ex Vivo, Labeling, FACS

Journal: Experimental hematology

Article Title: Novel roles for podocalyxin in regulating stress myelopoiesis, Rap1a, and neutrophil migration.

doi: 10.1016/j.exphem.2017.04.001

Figure Lengend Snippet: Figure 2. Governing of stress myelopoiesis by Podxl. (A) Podxlþ/þ and PodxlDHC mice were administered 5-FU (150 mg/kg). On the days indicated, pe- ripheral blood cell populations were assayed. Left and center: Numbers of PB neutrophils and monocytes among Podxlþ/þ and PodxlDHC mice. Right: Total white blood cell, neutrophil, monocyte, and lymphocyte counts in PB from Podxlþ/þ and PodxlDHC mice at day 13 post-5-FU dosing. (B) Podxl deletion dysregulates G-CSF-induced neutrophil and monocyte formation: Podxlf/f and PodxlDHC mice (n 5 4) were dosed on days 1–5 with G-CSF (125 mg/kg) or phosphate-buffered saline. Levels of PB cells then were determined. Top: Neutrophil and monocyte numbers (mean 6 SE) at day 6. Bottom: Overall levels of PB neutrophils, eosinophils, basophils, monocytes, and lymphocytes. (C) After G-CSF administration (125 mg/kg, days 1–5), bone marrow and splenic cells were prepared from Podxlf/f and PodxlDHC mice. HPCs were then prepared as lineage-negative bone marrow populations (depleted using antibodies to CD5, CD11b, CD19, CD45R, Ly6G/C, and Ter119) and were used in CFU assays (means 6 SE, n 5 3). Findings for CFU-GEMM are illustrated. For CFU- GM no significant effects of Podxl-KO were observed (data not shown). (D) For GMPs prepared from Podxlf/f or PodxlDHC mouse bone marrow (at steady- state) populations of FITC-anti-Gr-1- or FITC-anti-CD11b-positive HPCs were assayed by flow cytometry. Here, GMPs were prepared as CD11b-, Gr-1-, Ly6G-positive populations. Data are frequencies of positive cells (mean 6 SE, n 5 3).

Article Snippet: Cells (2 105 in 0.5 mL of PBS, 0.1%BSA, 3 mg/ mL rat IgG) were incubated at 4 C for 15 minutes and were then stained with the following antibodies (at manufacturer’s recommended concentrations): allophycocyanin (APC)-c-Kit (BD Biosciences, cat# 553356), FITCCD11b (BD Biosciences, cat# 553310,), FITC-Gr-1 (BD Biosciences, cat# 553126), PE-CD34 (BD Biosciences, cat# 551387) and APC-Podxl (R&D system, cat# FAB1556A), Alexa fluor-488-CD11b (BD Biosciences, cat # 557672), APC-Ly6G (BD Biosciences, cat # 560599), and BV421-CD16/32 (BD Biosciences, cat # 562896).

Techniques: Saline, Cytometry

Journal: Experimental hematology

Article Title: Novel roles for podocalyxin in regulating stress myelopoiesis, Rap1a, and neutrophil migration.

doi: 10.1016/j.exphem.2017.04.001

Figure Lengend Snippet: Figure 3. Podxl interacts with Rap-1A in HPCs, and regulates Rap1aGTP levels. (A) After G-CSF dosing of wild-type mice, HPCs were isolated from bone marrow cellpreparationsasLinneg populations(depleted for CD5,CD11b,CD19,CD45R, Ly6G/C,and Ter119).Fromcelllysates, Podxl(co-)immunoprecipitates were then prepared,reduced, alkylated,and used togeneratetryptic peptides.Peptideswere analyzedbyLC-MS/MS.For onepredominant Podxl partner,Rap1a,representative time-of-flight mass spectra (MS) are shown (e.g., SKINVNEIFYDLVR). Left: Precursor peptide ion mass 1708.9148, z 5 3, and delta mass 5 0.0001 (99% con- fidence). Right: Collision-induced decay fragment spectra for sequence determination of SKINVNEIFYDLVR. (B) Podxl regulates levels of activated Rap1a-GTP: Hematopoietic progenitor cells were prepared from the bone marrow of Podxlf/f and PodxlDHC mice and plated in IMDM (1 106 cells/mL). Here, Linneg bone marrow HPCs from wild-type and Podxl-KO mice were used. Cells were then exposed to phosphate-buffered saline, G-CSF, or GM-CSF. At 30 min, lysates were generated and analyzed by Western blotting for total Rap-1A levels and levels of GTP-bound Rap-1A (lower panel).(C) For the samples describedin (B),levels of p-MAPK and total MAPK also were assessed. (D) Podxl-KO dysregulates IL3-induced Rap1aGTP formation. HPCs were isolated from Podxlþ/þ and PodxlDHC

Article Snippet: Cells (2 105 in 0.5 mL of PBS, 0.1%BSA, 3 mg/ mL rat IgG) were incubated at 4 C for 15 minutes and were then stained with the following antibodies (at manufacturer’s recommended concentrations): allophycocyanin (APC)-c-Kit (BD Biosciences, cat# 553356), FITCCD11b (BD Biosciences, cat# 553310,), FITC-Gr-1 (BD Biosciences, cat# 553126), PE-CD34 (BD Biosciences, cat# 551387) and APC-Podxl (R&D system, cat# FAB1556A), Alexa fluor-488-CD11b (BD Biosciences, cat # 557672), APC-Ly6G (BD Biosciences, cat # 560599), and BV421-CD16/32 (BD Biosciences, cat # 562896).

Techniques: Isolation, Tandem Mass Spectroscopy, Sequencing, Saline, Generated, Western Blot

Journal: Experimental hematology

Article Title: Novel roles for podocalyxin in regulating stress myelopoiesis, Rap1a, and neutrophil migration.

doi: 10.1016/j.exphem.2017.04.001

Figure Lengend Snippet: Figure 3. (continued) (E) Podxl deletion enhances neutrophil migration. Peripheral blood neutrophils were prepared via density gradient centrifugation using Histopaque-1077 and Histopaque-1119. Trans-well migration of peripheral blood neutrophils from wild-type and Podxl-KO mice was then assessed 6 the Rap1a inhibitor GGTI-2147. Graphed results are mean values 6 SE (n 5 3) for trans-membrane migrating cells. (F) Initial model for Podxl governing of stress myelopoiesis. Left: HGF effects on Podxl expression are outlined (top), together with Vav1-conditional Podxl-KO phenotypes. Right: A working model is outlined at a cellular level for proposed podocalyxin governing of peripheral blood neutrophil (e)migrations.

Article Snippet: Cells (2 105 in 0.5 mL of PBS, 0.1%BSA, 3 mg/ mL rat IgG) were incubated at 4 C for 15 minutes and were then stained with the following antibodies (at manufacturer’s recommended concentrations): allophycocyanin (APC)-c-Kit (BD Biosciences, cat# 553356), FITCCD11b (BD Biosciences, cat# 553310,), FITC-Gr-1 (BD Biosciences, cat# 553126), PE-CD34 (BD Biosciences, cat# 551387) and APC-Podxl (R&D system, cat# FAB1556A), Alexa fluor-488-CD11b (BD Biosciences, cat # 557672), APC-Ly6G (BD Biosciences, cat # 560599), and BV421-CD16/32 (BD Biosciences, cat # 562896).

Techniques: Migration, Gradient Centrifugation, Membrane, Expressing

Journal: The Journal of Experimental Medicine

Article Title: Upregulated LRRC55 promotes BK channel activation and aggravates cell injury in podocytes

doi: 10.1084/jem.20192373

Figure Lengend Snippet: Knockout of Lrrc55 ameliorates podocyte injury in Ang II–induced mice. (A and B) RT-PCR and Western blot analysis of LRRC55 in podocytes of mice treated with Ang II ( n = 6). Mice glomeruli were isolated by using the magnetic bead–based isolation technique, single-cell suspensions were obtained with enzymatic disaggregation, and then podocytes were enriched by using anti-podocalyxin beads for RT-PCR and Western blot analysis. (C) IHC analysis of LRRC55 in glomerular tissues of mice treated with Ang II ( n = 6). Black arrow indicates positive staining for LRRC55. (D) The BK current measured at +80 mV in podocytes of freshly isolated glomeruli from mice treated with Ang II ( n = 6). To analyze the whole-cell recording of the BK current in situ, the isolated glomeruli were attached to poly-L-lysine–coated coverslips, and then the coverslips were placed into a patch-clamp chamber and perfused with bath solution to conduct a patch-clamp experiment. (E) Urinary albumin excretion of mice treated with Ang II ( n = 6). (F) Periodic acid–Schiff staining and electron microscopy analysis of renal sections. (G) Mean width of podocyte foot processes ( n = 6). (H) The number of apoptotic podocytes in glomerular tissues of mice treated with Ang II ( n = 6). (I) Western blot analysis of cleaved caspase-3 (casp-3) in glomerular tissues of mice treated with Ang II. Data shown are representative of three experiments. For statistical analysis, a two-tailed Student’s t test was used for A, and one-way ANOVA with Tukey’s post hoc test was used for D, E, G, and H. ***, P < 0.001. Scale bar = 20 µm, unless otherwise indicated.

Article Snippet: Isolated glomeruli were incubated with 10 μM PBFI-AM, 0.04% Pluronic F-127, and PE-conjugated podocalyxin antibody (1:200; FAB1556P; R&D Systems) for 40 min at room temperature.

Techniques: Knock-Out, Reverse Transcription Polymerase Chain Reaction, Western Blot, Isolation, Staining, In Situ, Patch Clamp, Electron Microscopy, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Upregulated LRRC55 promotes BK channel activation and aggravates cell injury in podocytes

doi: 10.1084/jem.20192373

Figure Lengend Snippet: Effect of BK channel knockout on podocyte injury in Ang II–treated mice. (A) The BK current measured at +80 mV in podocytes of freshly isolated glomeruli from WT and Kcnma1 −/− mice treated with Ang II ( n = 6). (B) The level of intracellular potassium in podocytes of freshly isolated glomeruli from mice ( n = 6). To analyze intracellular potassium in podocytes of mouse glomeruli in situ, isolated glomeruli were incubated with 10 µM PBFI-AM, 0.04% Pluronic F-127, and PE-conjugated podocalyxin antibody (1:200) for 40 min at room temperature. The glomeruli were then attached to poly-L-lysine–coated glass coverslips. Fluorescence images were acquired by using a Zeiss LSM710 confocal microscope. PBFI fluorescence intensities in podocalyxin-positive cells were analyzed to determine the intracellular potassium level in podocytes of mouse glomeruli. (C) Western blot analysis of cleaved caspase-3 (casp-3) in glomerular tissues of mice ( n = 6). (D) Apoptotic podocytes in mouse glomeruli ( n = 6). (E) Urinary albumin excretion in WT and Kcnma1 −/− mice treated with Ang II ( n = 6). (F) Periodic acid–Schiff staining and electron microscopy analysis of renal sections. (G) Mean width of podocyte foot processes ( n = 6). Data shown are representative of three experiments. For statistical analysis, one-way ANOVA with Tukey’s post hoc test was used for A, B, D, E, and G. ***, P < 0.001. Scale bar = 20 µm, unless otherwise indicated.

Article Snippet: Isolated glomeruli were incubated with 10 μM PBFI-AM, 0.04% Pluronic F-127, and PE-conjugated podocalyxin antibody (1:200; FAB1556P; R&D Systems) for 40 min at room temperature.

Techniques: Knock-Out, Isolation, In Situ, Incubation, Fluorescence, Microscopy, Western Blot, Staining, Electron Microscopy

Journal: The Journal of Experimental Medicine

Article Title: Upregulated LRRC55 promotes BK channel activation and aggravates cell injury in podocytes

doi: 10.1084/jem.20192373

Figure Lengend Snippet: Intracellular potassium assay in the podocytes of isolated rodent glomeruli in situ. (A) Image of the PBFI-AM and a PE-conjugated podocalyxin antibody-loaded glomerulus. (B) The podocyte area selected according to the podocalyxin fluorescence signal. (C) Image used to determinate the PBFI fluorescence intensities in podocytes. (D) Calculation of the PBFI fluorescence ratio. Scale bar = 20 µm.

Article Snippet: Isolated glomeruli were incubated with 10 μM PBFI-AM, 0.04% Pluronic F-127, and PE-conjugated podocalyxin antibody (1:200; FAB1556P; R&D Systems) for 40 min at room temperature.

Techniques: Isolation, In Situ, Fluorescence

Journal: American Journal of Physiology - Cell Physiology

Article Title: Nucleolin mediates the binding of cancer cells to L-selectin under conditions of lymphodynamic shear stress

doi: 10.1152/ajpcell.00035.2019

Figure Lengend Snippet: Flow cytometry screening for cell surface expression of other potential L-selectin ligands

Article Snippet: Mouse anti-human podocalyxin (PCLP, clone 53D11; cat. no. M084-3) antibody was purchased from MBL International (Woburn, MA).

Techniques: Flow Cytometry, Expressing, Fluorescence