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

Journal: International journal of molecular sciences

Article Title: L-Type Amino Acid Transporter 1 Regulates Cancer Stemness and the Expression of Programmed Cell Death 1 Ligand 1 in Lung Cancer Cells.

doi: 10.3390/ijms222010955

Figure Lengend Snippet: Figure 1. LAT1 expression increases in pemetrexed-resistant NSCLC cells. (A) The primary or secondary tumorspheres were cultured from A549 and A400 cells according to the protocol described in the Materials and Methods section. Pictures

Article Snippet: The PerCP conjugated mouse monoclonal anti-LAT1 antibody (clone name: BU53) was purchased from Novus Biologicals, LLC. (Centennial, CO, USA).

Techniques: Expressing, Cell Culture

Journal: International journal of molecular sciences

Article Title: L-Type Amino Acid Transporter 1 Regulates Cancer Stemness and the Expression of Programmed Cell Death 1 Ligand 1 in Lung Cancer Cells.

doi: 10.3390/ijms222010955

Figure Lengend Snippet: Figure 2. Knockdown of LAT1 reduces CSC activity and Akt/mTOR activation in NSCLC cells. (A) The knockdown of LAT1 in A400 and H1299 cells was performed by lentiviral delivery of LAT1-specific shRNAs (shSLC7A5#1 or shSLC7A5#2) and 100 nM of LAT1-specific siRNA oligonucleotides, respectively, for 48 h. The CSC activity was determined by tumorsphere cultivation. The inserted scale bars indicate 100 µm in length. *** p< 0.001. (B) The mRNA expressions of cancer stemness genes (BMI1/SOX2/Oct4) and SLC7A5 were determined by real-time RT-PCR. * p < 0.05; ** p < 0.01; *** p < 0.001. (C) The protein expressions of phosphor-mTORser2481, mTOR, phosphor-Aktser473, Akt, and LAT1 were determined by Western blot analysis. GAPDH was used as a protein-loading control. The inserted numbers present relative expression folds in comparison to shLacZ or the control siRNA group.

Article Snippet: The PerCP conjugated mouse monoclonal anti-LAT1 antibody (clone name: BU53) was purchased from Novus Biologicals, LLC. (Centennial, CO, USA).

Techniques: Knockdown, Activity Assay, Activation Assay, Quantitative RT-PCR, Western Blot, Control, Expressing, Comparison

Journal: International journal of molecular sciences

Article Title: L-Type Amino Acid Transporter 1 Regulates Cancer Stemness and the Expression of Programmed Cell Death 1 Ligand 1 in Lung Cancer Cells.

doi: 10.3390/ijms222010955

Figure Lengend Snippet: Figure 3. Inhibition of LAT1 activity by JPH203 suppresses CSC activity and Akt/mTOR activation in NSCLC cells. (A) The CSC activity of A400 and H1299 cells was determined by tumorsphere cultivation, and pictures were presented at 100× magnification. The quantitative data were presented as relative percentages to the tumorsphere number of the 0.1% DMSO treated group (0 µM). The inserted scale bars indicate 100 µm in length * p < 0.05; ** p < 0.01. (B) The protein expressions of phosphor-mTORser2481, mTOR, phosphor-Aktser473, Akt, and LAT1 were determined by Western blot analysis. GAPDH was used as a protein-loading control. The inserted numbers presented are relative expression folds in comparison to the 0.1% DMSO treated group (0 µM) after normalization with the internal control of GAPDH. (C) The protein expression of BMI1 or SOX2 was determined by Western blot. The inserted numbers indicated relative expression levels as compared to DMSO group after normalization to the internal control of GAPDH. (D) The CSC activity of A400 cells under JPH203 treatment with or without 100 µM LLME cotreatment was determined by tumorsphere cultivation and pictures were presented at 200× magnification. The quantitative data were presented as relative percentages of the tumorsphere number of the 0.1% DMSO without LLME treatment group. The inserted scale bars indicate 100 µm in length. * p < 0.05; ** p < 0.01. (E) The protein expressions of phosphor-mTORser2481, mTOR, phosphor-Aktser473, Akt, and LAT1 in A400 and H1299 cells in the presence of LLME were determined by Western blot analysis. GAPDH was used as a protein-loading control. The inserted numbers are presented as relative expression folds in comparison to the cells without LLME treatment group after normalization to GAPDH. (F) The CSC activity of H1299 cells under JPH203 treatment with or without the overexpression of the myr-Akt vector was determined by tumorsphere cultivation, and pictures were presented at 100× magnification. The inserted scale bars indicate 100 µm in length. The quantitative data were presented as relative percentages of the tumorsphere number of the control vector transfected and 0.1% DMSO treatment group. The overexpression of myr-Akt (upper panel) was determined by Western blot analysis. ** p < 0.01. ns, not significant.

Article Snippet: The PerCP conjugated mouse monoclonal anti-LAT1 antibody (clone name: BU53) was purchased from Novus Biologicals, LLC. (Centennial, CO, USA).

Techniques: Inhibition, Activity Assay, Activation Assay, Western Blot, Control, Expressing, Comparison, Over Expression, Plasmid Preparation, Transfection

Journal: International journal of molecular sciences

Article Title: L-Type Amino Acid Transporter 1 Regulates Cancer Stemness and the Expression of Programmed Cell Death 1 Ligand 1 in Lung Cancer Cells.

doi: 10.3390/ijms222010955

Figure Lengend Snippet: Figure 4. LAT1 activity participates in PD-L1 expression on NSCLC cells. (A,B). The knockdown of LAT1 by lentiviral delivery of LAT1-specific shRNAs (shSLC7A5#1 or shSLC7A5#2) for A400 cells or by transfection of LAT1-specific siRNA oligonucleotides (si-SLC7A5) for H1299 cells was confirmed by Western blot analysis (A). The cell surface expressions of PD-L1 were determined by Western blot (A) or FACS analysis (B), respectively. The FACS data was analyzed by FlowJo software. The inserted numbers in (A) represent the relative expression levels of PD-L1 or LAT1 in comparison to the sh-LacZ or si-NC (indicated as 0 nM) control group, respectively, after normalization with GAPDH. Blue peaks and red peaks in (B) represent an isotype control and an anti-PD-L1 antibody, respectively. siNC, negative control siRNA. (C,D) The PD-L1 total protein and cell surface expressions of A400 and H1299 cells under JPH203 treatment were determined by Western blot (C) and FACS analysis (D), respectively. The inserted numbers in (A) present the relative expression levels of PD-L1 or LAT1 in comparison to the 0.1% DMSO control group (0 µM) after normalization with GAPDH. Blue peaks and red peaks in (D) represent an isotype control and an anti-PD-L1 antibody, respectively.

Article Snippet: The PerCP conjugated mouse monoclonal anti-LAT1 antibody (clone name: BU53) was purchased from Novus Biologicals, LLC. (Centennial, CO, USA).

Techniques: Activity Assay, Expressing, Knockdown, Transfection, Western Blot, Software, Comparison, Control, Negative Control

Journal: International journal of molecular sciences

Article Title: L-Type Amino Acid Transporter 1 Regulates Cancer Stemness and the Expression of Programmed Cell Death 1 Ligand 1 in Lung Cancer Cells.

doi: 10.3390/ijms222010955

Figure Lengend Snippet: Figure 6. LAT1+/PD-L1+ enriches CSC activity of NSCLC cells. (A) The cell surface expressions of LAT1 and PD-L1 on A549 and H1299 cells were determined by FACS with fluorescence-labeled antibodies. (B) Three populations of NSCLC cells (PD-L1−/LAT1−, PD-L1+/LAT1−, PD-L1+/LAT1+) were isolated by FACS cell sorting, and their CSC activities were measured by tumorsphere cultivation. Pictures were presented at 100× magnification and the inserted scale bars indicate 100 µm in length. The data were presented as relative percentages of the tumorsphere number of PD-L1−/LAT1−cells. * p < 0.05. (C) The expressions of cancer stemness proteins (BMI1, c-MYC, EZH2, OCT4), phosphor-mTORser2481, mTOR, phosphor-Aktser473, and Akt were determined by Western blot. The inserted numbers indicated relative expression levels in comparison to PD-L1−/LAT1−cells after normalization to GAPDH. (D) The overall survival curves of NSCLC patients in the TCGA database regarding expression levels of CD274 and the two-gene signature of CD274 and SLC7A5 using median level as the cutoff criteria were analyzed using the GEPIA2 webtool [18].

Article Snippet: The PerCP conjugated mouse monoclonal anti-LAT1 antibody (clone name: BU53) was purchased from Novus Biologicals, LLC. (Centennial, CO, USA).

Techniques: Activity Assay, Labeling, Isolation, FACS, Western Blot, Expressing, Comparison