primary antibodies detect pdpn  (OriGene)

Journal: ACS Applied Bio Materials

Article Title: Fibril-Guided Three-Dimensional Assembly of Human Fibroblastic Reticular Cells

doi: 10.1021/acsabm.4c00331

Figure Lengend Snippet: Biological attributes of FRC clusters in EAKbt-av. Live/dead staining of FRCs in (A) 2D monolayer, (B) EAKbt, and (C) EAKbt+av wherein viable cells are observed in clusters [scale = 200 μm]. Immunofluorescent (IF) images with FRCs stained with GP38 (red) and DAPI (blue) [scale = 100 μm] (D) clFRCs, (E) isotype antibody control, (F) 2°Ab control; (G) 3D GP38 slice in clFRCs [scale = 50 μm]; (H&I) image reconstruction from Z-stack IF images demonstrating a spherical 3D assembly of FRCs in clusters (EAKbt+av); (J) ATP assay showing presence of metabolically active FRCs; and (K) GP38 relative expression to isotype and 2°Ab controls in immunofluorescent (IF) images.

Article Snippet: The antihuman GP38 primary antibody and isotype antibody were purchased from Abcam (Waltham, MA).

Techniques: Staining, Control, ATP Assay, Metabolic Labelling, Expressing

Journal: ACS Applied Bio Materials

Article Title: Fibril-Guided Three-Dimensional Assembly of Human Fibroblastic Reticular Cells

doi: 10.1021/acsabm.4c00331

Figure Lengend Snippet: clFRCs retain and engage T cells. Superior retention of human T cell (green-CFSE labeled) in (A) clFRCs (red-GP38) relative to (B) EAKbt-FRCs [scale = 100 μm]; flow cytometric analysis of proliferation of CFSE labeled Jurkat cells at different culture durations of (C) day 3 and (D) day 5; (E–H) RT-qPCR of day 7 clFRCs/allo-PBMCs cocultures; (E) relative exp (GAPDH) of signature FRC genes in the clFRCs fraction; (F) relative exp of tolerance-inducing T cell genes in the lymphocytes fraction recovered from cocultures of clFRCs vs 2D monolayer. (G) Relative expression of Granzyme A (GZMA) in the lymphocytes fraction recovered from cocultures of clFRCs vs 2D monolayer; (H) relative expression of TGFB1 in the lymphocytes fraction recovered from cocultures of clFRCs vs biomaterial control (EAKbt+Av, no FRCs), CFSE-stained aCD3/aCD28 activated human PBMCs (green) cocultured with (I) clFRCs, wherein the PBMCs show infiltration and retention inside the FRC network, (J) 2D monolayer, and (K) EAkbt+Av (no FRC) [scale = 100 μm]. The results were generated from 4 to 5 technical replicates using FRCs from one donor and PBMCs from two donors. Statistical analysis was performed using a one-tailed paired t test where * indicates p < 0.05.

Article Snippet: The antihuman GP38 primary antibody and isotype antibody were purchased from Abcam (Waltham, MA).

Techniques: Labeling, Quantitative RT-PCR, Expressing, Control, Staining, Generated, One-tailed Test

Journal: Human Reproduction (Oxford, England)

Article Title: Single-cell sequencing reveals novel cellular heterogeneity in uterine leiomyomas

doi: 10.1093/humrep/deac183

Figure Lengend Snippet: Lymphatic ECs are present in leiomyomas. (A) Uniform Manifold Approximation and Projection on Principal (UMAP) components showing the clusters of 10 448 endothelial cells. (B, C) UMAP showing the cluster annotation per condition: myometrium and MED12-variant positive leiomyomas. (D) Heatmap of endothelial cell clusters. The colored bar on the top represents cluster number. Columns denote cells; rows denote genes. (E, F) Immunostaining for PDPN (white arrowheads) and NUSAP1 (yellow arrowheads) showing the presence of Lymphatic EC clusters in normal myometrium (E) and MED12-variant positive leiomyomas (F). Scale bar is 100 µm.

Article Snippet: Briefly, tissue sections were incubated with the primary antibodies PDPN (1:200, Cell Signaling) and NUSAP1 (1:1000, Abcam) overnight at 4°C, then incubated with secondary antibodies for 1 h at room temperature.

Techniques: Variant Assay, Immunostaining

Journal: Frontiers in Oncology

Article Title: PDPN marks a subset of aggressive and radiation-resistant glioblastoma cells

doi: 10.3389/fonc.2022.941657

Figure Lengend Snippet: PDPN is expressed in infiltrating gliomas and negatively correlates with survival. (A) Boxplots show increasing PDPN expression across increasing glioma grades in TCGA (n=601, ****p<0.0001). (B) Kaplan–Meier curves show that PDPN mRNA expression inversely correlates with OS across glioma grades in TCGA cohorts. Median OS for patients with PDPN-high and PDPN-low tumors was 14.4 and 94.5 months, respectively (n=601, p<0.0001). (C) Representative IHC examples of PDPN expression in FFPE tumor tissue using the M2A antibody in tumors. Subpanels (A, B) shows intense cytoplasmic staining in nearly all cells, while expression is undetectable in tumors in subpanels (C, D) . (D) Kaplan–Meier curves show that PDPN protein expression inversely correlates with OS among GBM WHO grade IV patients. Median OS for PDPN+ and PDPN− groups was 53.4 and 148.1 weeks, respectively (n=206, p<0.0001). IHC KM curves are shown as solid (PDPN+) and dashed (PDPN−) lines. (E) Kaplan–Meier curves show that PDPN protein expression inversely correlates with PFS among GBM WHO grade IV patients. Median PFS for PDPN+ and PDPN− groups were 21.0 and 34.6 weeks, respectively (n=206, p=0.03). (F) Kaplan–Meier curves show that PDPN protein expression inversely correlates with OS among grade II/III diffuse astrocytoma patients (n=93, p=0.003). (G) Radiation response is correlated with PDPN expression (n=16, *p=0.0326). (H) MRI examples of a radiation responder (A, B) and non-responder (C, D) , pre- and post-radiation (RT).

Article Snippet: Upon blocking, PVDF membranes were incubated overnight with primary monoclonal antibodies targeting PDPN (clone NZ-1, 1:200, AngioBio) and vinculin (1:1,000, Abcam) at 4°C.

Techniques: Expressing, Staining

Journal: Frontiers in Oncology

Article Title: PDPN marks a subset of aggressive and radiation-resistant glioblastoma cells

doi: 10.3389/fonc.2022.941657

Figure Lengend Snippet: Cox multivariate survival analysis for GBM patients.

Article Snippet: Upon blocking, PVDF membranes were incubated overnight with primary monoclonal antibodies targeting PDPN (clone NZ-1, 1:200, AngioBio) and vinculin (1:1,000, Abcam) at 4°C.

Techniques:

Journal: Frontiers in Oncology

Article Title: PDPN marks a subset of aggressive and radiation-resistant glioblastoma cells

doi: 10.3389/fonc.2022.941657

Figure Lengend Snippet: Cox multivariate survival analysis for diffuse astrocytoma patients.

Article Snippet: Upon blocking, PVDF membranes were incubated overnight with primary monoclonal antibodies targeting PDPN (clone NZ-1, 1:200, AngioBio) and vinculin (1:1,000, Abcam) at 4°C.

Techniques:

Journal: Frontiers in Oncology

Article Title: PDPN marks a subset of aggressive and radiation-resistant glioblastoma cells

doi: 10.3389/fonc.2022.941657

Figure Lengend Snippet: PDPN+ glioma-stem-like cells form aggressive tumors in vivo and mark a stem-like radioresistant subpopulation of cells. (A) PDPN protein is expressed in seven of the nine GSC lines shown. (B) Flow cytometry of GSCs shows PDPN expression is pervasive and more prominent in these GSCs. (C) Kaplan–Meier survival analysis of GSC11 PDPN−/CD133+, PDPN+/CD133+, and PDPN+/CD133− FACS-sorted subpopulations orthotopically injected into the brains of immunocompromised mice reveal that mice harboring tumors from CD133+/PDPN+ cells had a median survival of 99 days (n=5), while mice that received CD133+/PDPN− cells did not succumb to tumor formation (n=3) (p=0.0136, log-rank test). (D) PDPN+ FACS-sorted subpopulations have a higher sphere formation ability (*p<0.005, multiple t-tests, PDPN+ vs . PDPN− groups). (E) PDPN+ FACS-sorted subpopulations have a higher surviving fraction of cells after 2 Gy of radiation (*p<0.005, multiple t-tests, PDPN+ vs . PDPN− groups). (F) Transcriptome analysis revealed distinct gene expression signatures in PDPN+ and PDPN− sorted GSCs (three different cell lines). (G) GSEA of differentially expressed genes (p<0.05) demonstrated significant enrichment of the mesenchymal subtype signature (NES=2.04, q=0.02) in PDPN+ populations.

Article Snippet: Upon blocking, PVDF membranes were incubated overnight with primary monoclonal antibodies targeting PDPN (clone NZ-1, 1:200, AngioBio) and vinculin (1:1,000, Abcam) at 4°C.

Techniques: In Vivo, Flow Cytometry, Expressing, Injection, Gene Expression

Journal: Frontiers in Oncology

Article Title: PDPN marks a subset of aggressive and radiation-resistant glioblastoma cells

doi: 10.3389/fonc.2022.941657

Figure Lengend Snippet: PDPN knockdown sensitizes adherent glioma cell lines to radiation and reduces invasion, migration, and colony formation ability. (A) PDPN expression in adherent glioma cell lines and normal human astrocytes (NHA). (B) Western blot of PDPN knockdown in U87 cell lines. (C) Invasion and migration assays of U87 PDPN knockdown lines (*p<0.005, multiple t-tests, sh-neg vs . sh-PDPN). (D) Clonogenic radiation survival assay with PDPN knockdown lines, PDPN silencing sensitized U87 cells to in vitro radiation compared with controls. (E) Representative crystal violet stains after a colony formation assay with U87 PDPN knockdown lines. (F) Quantification of colony diameter and colony number after colony formation assay of control and PDPN shRNA cell lines (*p<0.005, two tailed t-test).

Article Snippet: Upon blocking, PVDF membranes were incubated overnight with primary monoclonal antibodies targeting PDPN (clone NZ-1, 1:200, AngioBio) and vinculin (1:1,000, Abcam) at 4°C.

Techniques: Knockdown, Migration, Expressing, Western Blot, Clonogenic Cell Survival Assay, In Vitro, Colony Assay, Control, shRNA, Two Tailed Test

Journal: Frontiers in Oncology

Article Title: PDPN marks a subset of aggressive and radiation-resistant glioblastoma cells

doi: 10.3389/fonc.2022.941657

Figure Lengend Snippet: PDPN knockdown in glioma stem-like cells leads to radiosensitization in vitro and slows intracranial tumor growth. (A) Western blot of GSC line 23 (GSC23) showing PDPN expression levels after exposure to doxycycline (doxy), indicating inducible PDPN knockdown. (B) qPCR validation of PDPN knockdown (*p<0.005, two-tailed t-test). (C) In vitro clonogenic survival assays revealed that PDPN silencing by the shRNA MD5 and MD7 was effective in sensitizing GSC23 spheres to radiation in a dose-dependent manner [0 Gy, not significant (ns); 2 Gy, *p<0.0001; 4 Gy, *p<0.0001]. (D) Kaplan–Meier survival analysis (n=5 per condition) of PDPN knockdown, with negative (shLuc and no doxy) controls (p=0.0018, log-rank test). (E) Time course IVIS imaging of tumors from days 1, 30 and 60. (F) Representative tumor histology and PDPN IHC of animal tumors at time of animal death.

Article Snippet: Upon blocking, PVDF membranes were incubated overnight with primary monoclonal antibodies targeting PDPN (clone NZ-1, 1:200, AngioBio) and vinculin (1:1,000, Abcam) at 4°C.

Techniques: Knockdown, In Vitro, Western Blot, Expressing, Biomarker Discovery, Two Tailed Test, shRNA, Imaging