Journal: Advanced Materials (Deerfield Beach, Fla.)

Article Title: Safe and Localized Lentiviral Gene Delivery via Injectable Mesoporous Scaffolds for Potent Antitumor Immunity

doi: 10.1002/adma.202516661

Figure Lengend Snippet: Alteration of the TME following LVP‐based scaffold vaccination combined with PD‐1 blockade. (a) Experimental design: Mice bearing B16‐OVA tumors were treated with the LVP‐based scaffold vaccine (Vax) on days 5 and 12 post‐tumor inoculation in combination with anti‐PD1 antibody administration ( n = 4 for the untreated group, n = 5 for the Vax+a‐PD1 group). (b) Tumor volumes were measured over time to assess the treatment efficacy. (c) Frequencies of Foxp3 + Tregs within the TME. (d, e) Quantification of tumor‐infiltrating CD4 + (d) and CD8 + (e) T cells per milligram of tumor tissue. (f) M1/M2 macrophage polarization was assessed by the ratio of CD86 to CD206 expression within the F4/80 + macrophage population. (g, h) Concentrations of anti‐OVA IgG1 and IgG2a antibodies in the serum of the mice at the study endpoint. The data in panels (b–h) are presented as the means ± SDs. Statistical analysis was performed using an unpaired Student's t test. p < 0.05 was considered statistically significant.

Article Snippet: The levels of anti‐OVA IgG1 and anti‐OVA IgG2a antibodies were quantified using mouse anti‐OVA IgG1 and mouse anti‐OVA IgG2a antibody assay kits (Chondrex, Inc., Woodinville, WA, USA; Cat# 3013 and 3015) according to the manufacturer's instructions.

Techniques: Expressing

Journal: Journal of Biological Chemistry

Article Title: Amyloid Precursor-like Protein 2 and Sortilin Do Not Regulate the PCSK9 Convertase-mediated Low Density Lipoprotein Receptor Degradation but Interact with Each Other

doi: 10.1074/jbc.m115.647180

Figure Lengend Snippet: FIGURE 1. Exogenous PCSK9 can induce degradation of the LDLR in the absence of APLP2. HepG2 (A) and Huh7 (B) cells were transfected with a control non-target siRNA (Ctrl) or 3 different siRNAs targeting APLP2. Cells wereincubatedovernightwithserum-freeconditionedmedialackingorcon- taining 1 g/ml of PCSK9-V5. HepG2 and Huh7 cell lysates were then sub- jected to Western blotting using LDLR, APLP2, and -actin antibodies. LDLR and APLP2 signals were normalized to that of -actin. C, the input HEK293 conditioned medium was analyzed using mAb-V5 to detect PCSK9-V5. D, duplicate samples of Huh7 cells matching those in panel B were analyzed by FACS to assess the cell surface LDLR levels. Values were normalized to that of the first lane (control non-target siRNA in the absence of PCSK9). Error bars represent S.E. *, p 0.05 (Student’s t test). The data shown here are represen- tative of two to three independent experiments.

Article Snippet: Mouse APLP2 was detected using a rabbit polyclonal antibody kindly provided by Dr. G. Thinakaran (University of Chicago), whereas human APLP2 was detected with either mAb-V5 or a rabbit polyclonal antibody (Novus Biologicals).

Techniques: Transfection, Control, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Amyloid Precursor-like Protein 2 and Sortilin Do Not Regulate the PCSK9 Convertase-mediated Low Density Lipoprotein Receptor Degradation but Interact with Each Other

doi: 10.1074/jbc.m115.647180

Figure Lengend Snippet: FIGURE 4. Sortilin and APLP2 are novel cellular targets of PCSK9. A, overexpressed PCSK9 induces sortilin and APLP2 degradation in HEK293 cells. Triplicate Western blot analyses revealing that both sortilin-Myc and APLP2-V5 expression levels in HEK293 cells were reduced by 90 and 40%, respectively, upon transfection with a PCSK9 plasmid, as compared with a control empty pIRES vector (V). Quantification of sortilin and APLP2 band intensities were normalized against those of -actin. B, HEK293 cells transfected with a cDNA coding for an empty vector control (pIRES; V) or individually with human sortilin or APLP2 tagged at the C terminus with a Myc or V5 epitope, respectively, or together in the absence or presence of a cDNA coding for untagged PCSK9. The following day the cells were washed and then pulsed for 4 h with [35S]Met Cys in the presence or absence of 5 mM NH4Cl. The cells were then extracted and their lysates immunoprecipitated (IP) with a mAb-V5 or mAb-Myc or a polyclonal antibody for PCSK9. The precipitates were separated on an 8% SDS-PAGE. The dried gel was then autoradiographed. Notice the co-precipitation of sortilin and APLP2 in the presence of NH4Cl. These data are representative of at least three independent experiments.

Article Snippet: Mouse APLP2 was detected using a rabbit polyclonal antibody kindly provided by Dr. G. Thinakaran (University of Chicago), whereas human APLP2 was detected with either mAb-V5 or a rabbit polyclonal antibody (Novus Biologicals).

Techniques: Western Blot, Expressing, Transfection, Plasmid Preparation, Control, Immunoprecipitation, SDS Page

Journal: Journal of Biological Chemistry

Article Title: Amyloid Precursor-like Protein 2 and Sortilin Do Not Regulate the PCSK9 Convertase-mediated Low Density Lipoprotein Receptor Degradation but Interact with Each Other

doi: 10.1074/jbc.m115.647180

Figure Lengend Snippet: FIGURE 5. Sortilin, APLP2, and soluble APLP2 are degraded by both PCSK9 and ER-localized PCSK9-KDEL isoforms. HEK293 cells were transfected with indicated DNA amounts of vectors encoding a control protein 7B2, sortilin (no tag), APLP2-V5, soluble APLP2-V5 (sAPLP2-V5), PCSK9-V5, or PCSK9-V5-KDEL, as indicated. After 48 h, lysates and media were analyzed by Western blotting for the indicated proteins. The data show that overexpressed PCSK9 or PCSK9-KDEL induces degradation of sortilin (A), APLP2 (B), and sAPLP2 (C) in HEK293 cells. Quantification of sortilin and APLP2 band intensities were normalized against those of -actin or GAPDH. These data are representative of two independent experiments.

Article Snippet: Mouse APLP2 was detected using a rabbit polyclonal antibody kindly provided by Dr. G. Thinakaran (University of Chicago), whereas human APLP2 was detected with either mAb-V5 or a rabbit polyclonal antibody (Novus Biologicals).

Techniques: Transfection, Control, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Amyloid Precursor-like Protein 2 and Sortilin Do Not Regulate the PCSK9 Convertase-mediated Low Density Lipoprotein Receptor Degradation but Interact with Each Other

doi: 10.1074/jbc.m115.647180

Figure Lengend Snippet: FIGURE 6. Co-expression of sortilin, APLP2, or both with PCSK9 has no major effect on LDLR degradation. Huh7 cells were transfected with a total of 3 g using 1 g of each vector encoding for either a control protein 7B2 (), sortilin (), APLP2 (), or PCSK9 (), as indicated. After 48 h, lysates were analyzed by Western blotting for expression of the LDLR, sortilin-Myc, APLP2- V5, intracellular pro- and mature-PCSK9-V5, and -actin. Media were ana- lyzed for secreted endogenous and overexpressed PCSK9-V5 using a rabbit polyclonal human PCSK9 antibody. Quantification of LDLR expression was normalized against that of -actin. These data are representative of at least 3 different experiments showing similar results.

Article Snippet: Mouse APLP2 was detected using a rabbit polyclonal antibody kindly provided by Dr. G. Thinakaran (University of Chicago), whereas human APLP2 was detected with either mAb-V5 or a rabbit polyclonal antibody (Novus Biologicals).

Techniques: Expressing, Transfection, Plasmid Preparation, Control, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Amyloid Precursor-like Protein 2 and Sortilin Do Not Regulate the PCSK9 Convertase-mediated Low Density Lipoprotein Receptor Degradation but Interact with Each Other

doi: 10.1074/jbc.m115.647180

Figure Lengend Snippet: FIGURE 7. Sortilin binds APLP2. A, schematic diagram of sortilin and APLP2 fused to the G. princeps luciferase half-domains, Gluc-1 and Gluc-2, respectively. B, heat map generated by G. princeps luciferase complementation assay showing the interaction profile of 36 protein pairs. Normalized luminescence ratio ranging from strong to null interactions is displayed on a light blue to black scale. C, validation of sortilin-Myc and APLP2-V5 interaction was confirmed by co-expression in HEK293 cells and immunoprecipitation with mAb-Myc followed by Western blotting (WB) using a mAb-Myc or mAb-V5.

Article Snippet: Mouse APLP2 was detected using a rabbit polyclonal antibody kindly provided by Dr. G. Thinakaran (University of Chicago), whereas human APLP2 was detected with either mAb-V5 or a rabbit polyclonal antibody (Novus Biologicals).

Techniques: Luciferase, Generated, Biomarker Discovery, Expressing, Immunoprecipitation, Western Blot

Journal: bioRxiv

Article Title: Cell-derived ECM loaded electrospun Polycaprolactone/Chitosan nanofibrous scaffolds for periodontal regeneration

doi: 10.1101/2023.03.30.534964

Figure Lengend Snippet: Characterization of decellularized extracellular matrix (dECM) derived from human periodontal ligament stem cells (PDLSCs). A) Immunofluorescence staining images of fibronectin (FIB, green), collagen I (COL I, red), laminin (LAM, red), asporin (ASP, red), osteopontin (OPN, red) and osteocalcin (OC, red) before and after decellularization DAPI staining (blue) revealed the absence of nuclei after decellularization. Scale bar 100 μm. B) DNA content present in PDLSCs and dECM. C) Quantification of sulphated glycosaminoglycans (sGAGs) in PDLSCs and dECM. D) Content of collagen present in PDLSCs and dECM. Three different samples were used in each quantification assay (N=3) for both conditions; *** p < 0.001.

Article Snippet: The primary antibodies, including rabbit polyclonal collagen I (1:200, Abcam), rabbit polyclonal asporin (1:100, Thermo Fisher Scientific), mouse monoclonal osteopontin (1:100, Thermo Fisher Scientific), mouse monoclonal osteocalcin (1:50, R&D Systems), rabbit polyclonal periostin (1:100, Abcam) and rabbit polyclonal cementum protein 1 (1:100, Thermo Fisher Scientific) prepared in a solution of 0.3% Triton X-100, 1% BSA, 10% FBS (in PBS) were added into the samples and incubated overnight at 4°C.

Techniques: Derivative Assay, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Cell-derived ECM loaded electrospun Polycaprolactone/Chitosan nanofibrous scaffolds for periodontal regeneration

doi: 10.1101/2023.03.30.534964

Figure Lengend Snippet: Immunofluorescent staining images of collagen I (COL I, red), asporin (ASP, red), osteopontin (OPN, red), osteocalcin (OC, red), periostin (POSTN, red) and cementum protein 1 (CMP, red) expressed by PDLSCs cultured on PCL, PCL-CTS and PCL-CTS-ECM electrospun scaffolds for 21 days under osteogenic differentiation conditions. Nuclei were counterstained with DAPI (blue). Scale bar 100 μm.

Article Snippet: The primary antibodies, including rabbit polyclonal collagen I (1:200, Abcam), rabbit polyclonal asporin (1:100, Thermo Fisher Scientific), mouse monoclonal osteopontin (1:100, Thermo Fisher Scientific), mouse monoclonal osteocalcin (1:50, R&D Systems), rabbit polyclonal periostin (1:100, Abcam) and rabbit polyclonal cementum protein 1 (1:100, Thermo Fisher Scientific) prepared in a solution of 0.3% Triton X-100, 1% BSA, 10% FBS (in PBS) were added into the samples and incubated overnight at 4°C.

Techniques: Staining, Cell Culture

Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology

Article Title: Flow Cytometric Detection of Circulating Osteosarcoma Cells in Dogs.

doi: 10.1002/cyto.a.23847

Figure Lengend Snippet: Figure 1. Canine OSA cell lines OVC-cOSA-31 (A) and OVC-cOSA-75 (B), gated as indicated, were highly positive for intracellular Col I (center panel) and variably positive for OC (right panel). Blue = anti-rat CD4 control antibody; red = Col I or OC antibody. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: The following isotype- and fluorochrome-matched antibodies were used as negative controls in all assays: (1) Mouse antirat CD4 monoclonal antibody OX-35 (IgG2a) conjugated to fluorescein isothiocyanate (FITC); (2) Mouse anti-rat CD71 monoclonal antibody OX-26 (IgG2a) conjugated to phycoerythrin (PE), both from BD (Mississauga, ON); (3) mouse monoclonal antibody to bovine retinal pigment epithelium (RPE) 65 (401.8B11.3D9, IgG1) conjugated to FITC (Novus Biologicals, Oakville, ON); and (4) mouse monoclonal antibody to human CD146 (P1H12, IgG1) conjugated to PE from EMD Millipore (Etobicoke, ON).

Techniques: Control

Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology

Article Title: Flow Cytometric Detection of Circulating Osteosarcoma Cells in Dogs.

doi: 10.1002/cyto.a.23847

Figure Lengend Snippet: Figure 8. Cytomorphology of OSA cells. Cytocentrifuge preparations of (A) cultured OVC-cOSA-31 cells (400×); (B) normal dog blood leukocytes spiked with primary OSA tumor aspirate cells, fixed, and permeabilized (arrow on OSA cell, 400×); (C) CTC sorted based on Col I positivity from blood of dog with a primary OSA (1,000×); (D) cell directly aspirated from a primary OSA (1,000×) with neutrophil (arrow); (E) primary OSA cells aspirated from tumor and sorted based on Col I positivity (1,000×); (F) OVC-cOSA-31 cells sorted from spiked normal dog blood (1,000×). All slides were Wright stained. Immunofluorescence of Col I-FITC positive; (G) OVC-cOSA-31 cells; and (H) CTC cells sorted from blood of a dog with OSA. Nuclei are stained with DAPI (1,000×). [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: The following isotype- and fluorochrome-matched antibodies were used as negative controls in all assays: (1) Mouse antirat CD4 monoclonal antibody OX-35 (IgG2a) conjugated to fluorescein isothiocyanate (FITC); (2) Mouse anti-rat CD71 monoclonal antibody OX-26 (IgG2a) conjugated to phycoerythrin (PE), both from BD (Mississauga, ON); (3) mouse monoclonal antibody to bovine retinal pigment epithelium (RPE) 65 (401.8B11.3D9, IgG1) conjugated to FITC (Novus Biologicals, Oakville, ON); and (4) mouse monoclonal antibody to human CD146 (P1H12, IgG1) conjugated to PE from EMD Millipore (Etobicoke, ON).

Techniques: Cell Culture, Staining