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: Primary antibodies used including a biotinylated anti-mouse PODXL antibody (cat# BAF1658, R&D Systems), anti-Actin-B (Sigma-Aldrich, cat# A5441), anti-Rap1a (Cell Signaling Technology, cat# 4938), phosphorylated MAPK1, 2 (Erk1/2) (Cell Signaling Technology, cat# 4370) and p44/42 MAPK (Cell Signaling Technology, cat# 4695).

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: Primary antibodies used including a biotinylated anti-mouse PODXL antibody (cat# BAF1658, R&D Systems), anti-Actin-B (Sigma-Aldrich, cat# A5441), anti-Rap1a (Cell Signaling Technology, cat# 4938), phosphorylated MAPK1, 2 (Erk1/2) (Cell Signaling Technology, cat# 4370) and p44/42 MAPK (Cell Signaling Technology, cat# 4695).

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: Primary antibodies used including a biotinylated anti-mouse PODXL antibody (cat# BAF1658, R&D Systems), anti-Actin-B (Sigma-Aldrich, cat# A5441), anti-Rap1a (Cell Signaling Technology, cat# 4938), phosphorylated MAPK1, 2 (Erk1/2) (Cell Signaling Technology, cat# 4370) and p44/42 MAPK (Cell Signaling Technology, cat# 4695).

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: Primary antibodies used including a biotinylated anti-mouse PODXL antibody (cat# BAF1658, R&D Systems), anti-Actin-B (Sigma-Aldrich, cat# A5441), anti-Rap1a (Cell Signaling Technology, cat# 4938), phosphorylated MAPK1, 2 (Erk1/2) (Cell Signaling Technology, cat# 4370) and p44/42 MAPK (Cell Signaling Technology, cat# 4695).

Techniques: Migration, Gradient Centrifugation, Membrane, Expressing