Journal: Cell Communication and Signaling : CCS

Article Title: High expression of AMAP1, an ARF6 effector, is associated with elevated levels of PD-L1 and fibrosis of pancreatic cancer

doi: 10.1186/s12964-020-00608-8

Figure Lengend Snippet: High AMAP1 expression levels statistically correlate with PD-L1 expression in human PDACs and KPC tumors. a Representative IHC images of AMAP1 with IHC scores 0 to 3 and positive and negative staining of PD-L1 in clinical specimens, and their comparison with AMAP1 IHC scores. b IHC images and quantification of the PD-L1 staining of tumors formed by control (Irr) or AMAP1 -silenced ( shAMAP1 #1 and #2) KPC cells in C57BL/6 mice. Error bars represent the mean ± s.e.m. ** P < 0.01. c PD-L1 cell surface expression in IFNγ-treated or non-treated KPC cells, pretreated with shRNAs. MFI, median fluorescence intensity. Error bars represent the mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001. d Kaplan-Meier plots of the overall survival of patients with regard to PD-L1 positivity. P -values were obtained by t -tests ( a , b and c ) and by the log-rank test ( d ). Bars = 100 μm ( a and b )

Article Snippet: Other antibodies were purchased from commercial sources, as follows: rabbit monoclonal antibodies against PD-L1 (Cell Signaling) and phospho-FAK (Thermo Fisher Scientific), rabbit polyclonal antibody against PD-L1 (Novus), rabbit polyclonal antibody against collagen I (Proteintech), rabbit polyclonal antibody against FAK [ ], mouse monoclonal antibody against β-actin (Sigma-Aldrich).

Techniques: Expressing, Negative Staining, Comparison, Staining, Control, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Tumor-Intrinsic PD-L1 Promotes Breast Cancer Proliferation Through Livin and Galectin-1-Mediated Regulation of SKP2 Expression

doi: 10.3390/ijms27062741

Figure Lengend Snippet: Proteomic analysis of nuclear extracts from PD-L1 Cont (Sh-Cont) vs. PD-L1 KD (ShPD-L1(a) and Sh-PDL1(b)) clones of MDA-MB-231 breast cancer cells. Schematic overview of the proteomics-guided workflow used to identify PD-L1-dependent regulators of the SKP2/p21/p27 axis. The heatmap displays the top nuclear differentially expressed proteins (DEPs) in Sh-Cont compared to Sh-PD-L1(a) and Sh-PD-L1(b) (|log 2 FC| ≥ 2 and adjusted p -value ≤ 0.05). DEPs were further filtered based on literature-based relevance to proliferation, the PI3K/AKT pathway, and SKP2, p21, and/or p27 expression (details are in ).

Article Snippet: PD-L1 ORF (RC213071 from OriGene, Rockville, MD, USA) was overexpressed in MDA-MB-468 cells using Lipofectamine LTX (Thermo Fisher Scientific), followed by selection using the eukaryotic antibiotic, G 418 (Thermo Fisher Scientific) at 750 μg/mL.

Techniques: Clone Assay, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Tumor-Intrinsic PD-L1 Promotes Breast Cancer Proliferation Through Livin and Galectin-1-Mediated Regulation of SKP2 Expression

doi: 10.3390/ijms27062741

Figure Lengend Snippet: Knockdown of Livin and Galectin-1 downregulates SKP2 expression and inhibits colony formation. Expression levels of SKP2 ( A ), p21 ( B ), and p27 ( C ) in MDA-MB-231 cells following transient knockdown of six candidate upstream proteins measured using qIF. Data were normalized to negative control siRNA (si-Neg) and are presented as means ± SEMs from four independent experiments. ( D ) Representative IF images (×200 magnification) showing SKP2, p21, and p27 expression after Livin or Galectin-1 knockdown compared with control siRNA. ( E ) Colony-forming ability of MDA-MB-231 cells after transient knockdown of candidate genes. Data were normalized to negative control siRNA–treated cells and are displayed as means ± SEMs from three independent experiments ( n = 3). ( F ) Representative colony-forming assays following knockdown of PD-L1, Livin, Galectin-1, EIF1AX, CALM2, and TCF-3 compared to control siRNA. Statistical significance is indicated as follows: * p < 0.05, ** p < 0.01.

Article Snippet: PD-L1 ORF (RC213071 from OriGene, Rockville, MD, USA) was overexpressed in MDA-MB-468 cells using Lipofectamine LTX (Thermo Fisher Scientific), followed by selection using the eukaryotic antibiotic, G 418 (Thermo Fisher Scientific) at 750 μg/mL.

Techniques: Knockdown, Expressing, Negative Control, Control

Journal: International Journal of Molecular Sciences

Article Title: Tumor-Intrinsic PD-L1 Promotes Breast Cancer Proliferation Through Livin and Galectin-1-Mediated Regulation of SKP2 Expression

doi: 10.3390/ijms27062741

Figure Lengend Snippet: PD-L1 promotes Livin and Galectin-1 expression in TNBC cells. ( A ) Livin and Galectin-1 expression levels in PD-L1 KD MDA-MB-231 clones compared with PD-L1 Cont controls, measured using qIF. Data were normalized to Sh-Cont MFI and are presented as means ± SEMs from four independent experiments. ( B ) Representative IF images of PD-L1 KD clones and controls (×200 magnification). ( C ) Western blot analysis of Livin and Galectin-1 expression following PD-L1 knockdown in MDA-MB-231 cells. ( D ) Densitometric quantification of the Western blots from three independent experiments presented as mean ± SEM ( n = 3). Statistical significance is indicated as follows: * p < 0.05, ** p < 0.01.

Article Snippet: PD-L1 ORF (RC213071 from OriGene, Rockville, MD, USA) was overexpressed in MDA-MB-468 cells using Lipofectamine LTX (Thermo Fisher Scientific), followed by selection using the eukaryotic antibiotic, G 418 (Thermo Fisher Scientific) at 750 μg/mL.

Techniques: Expressing, Clone Assay, Western Blot, Knockdown

Journal: International Journal of Molecular Sciences

Article Title: Tumor-Intrinsic PD-L1 Promotes Breast Cancer Proliferation Through Livin and Galectin-1-Mediated Regulation of SKP2 Expression

doi: 10.3390/ijms27062741

Figure Lengend Snippet: PD-L1 overexpression in MDA-MB-468 breast cancer cells upregulates SKP2 in a Livin/Galectin-1-dependent manner. ( A ) PD-L1 expression measured by flow cytometry following stable overexpression of PD-L1 in MDA-MB-468 cells using a PD-L1 ORF construct (PD-L1-ORF), compared with empty vector-transfected cells (vector control). ( B ) Expression of Livin, Galectin-1, and SKP2 in stable PD-L1 ORF MDA-MB-468 cells, measured using qIF. ( C ) SKP2 expression upon transient knockdown of Livin, Galectin-1, or their combination (combined) in PD-L1-ORF and vector control MDA-MB-468 cells. Data were normalized to empty vector cells and are shown as means ± SEMs from three independent experiments (right) ( n = 3). Representative IF images (×200 magnification) (left). Statistical significance is indicated as follows: * p < 0.05, *** p < 0.001, NS, not significant.

Article Snippet: PD-L1 ORF (RC213071 from OriGene, Rockville, MD, USA) was overexpressed in MDA-MB-468 cells using Lipofectamine LTX (Thermo Fisher Scientific), followed by selection using the eukaryotic antibiotic, G 418 (Thermo Fisher Scientific) at 750 μg/mL.

Techniques: Over Expression, Expressing, Flow Cytometry, Construct, Plasmid Preparation, Transfection, Control, Knockdown

Journal: International Journal of Molecular Sciences

Article Title: Tumor-Intrinsic PD-L1 Promotes Breast Cancer Proliferation Through Livin and Galectin-1-Mediated Regulation of SKP2 Expression

doi: 10.3390/ijms27062741

Figure Lengend Snippet: Knockdown of SKP2, Livin, or Galectin-1 abrogates PD-L1-promoted TNBC cell proliferation. MDA-MB-231 breast cancer cell proliferation was monitored in real time using the RTCA system. Cell index values are shown over 72 h ( upper panel , line graph) and at the 72 h endpoint ( lower panels , bar graphs; n = 3). Cells were transfected with siRNA targeting PD-L1 alone or in combination with Livin ( A ) or Galectin-1 ( B ), with or without SKP2 ( C , D ), and were compared with scrambled siRNA controls. Data are presented as mean ± SEM from at least three independent experiments ( n = 3). Statistical significance is indicated as follows: * p < 0.05, ** p < 0.01; NS, not significant.

Article Snippet: PD-L1 ORF (RC213071 from OriGene, Rockville, MD, USA) was overexpressed in MDA-MB-468 cells using Lipofectamine LTX (Thermo Fisher Scientific), followed by selection using the eukaryotic antibiotic, G 418 (Thermo Fisher Scientific) at 750 μg/mL.

Techniques: Knockdown, Transfection

Journal: International Journal of Molecular Sciences

Article Title: Tumor-Intrinsic PD-L1 Promotes Breast Cancer Proliferation Through Livin and Galectin-1-Mediated Regulation of SKP2 Expression

doi: 10.3390/ijms27062741

Figure Lengend Snippet: PD-L1 modulates Livin and Galectin-1 expression in vivo. Expression of Livin and Galectin-1 in PD-L1 Cont and PD-L1 KD MDA-MB-231 xenografts. ( Left ) Representative IF images (×100 magnification). ( Right ) Bar graphs showing expression levels (mean ± SEM), measured using qIF, normalized to Sh-Cont from three different experiments ( n = 3). (*) indicates statistical significance ( p < 0.05).

Article Snippet: PD-L1 ORF (RC213071 from OriGene, Rockville, MD, USA) was overexpressed in MDA-MB-468 cells using Lipofectamine LTX (Thermo Fisher Scientific), followed by selection using the eukaryotic antibiotic, G 418 (Thermo Fisher Scientific) at 750 μg/mL.

Techniques: Expressing, In Vivo

Journal: International Journal of Molecular Sciences

Article Title: Tumor-Intrinsic PD-L1 Promotes Breast Cancer Proliferation Through Livin and Galectin-1-Mediated Regulation of SKP2 Expression

doi: 10.3390/ijms27062741

Figure Lengend Snippet: PD-L1 upregulates Livin/Galectin-1 expression, activating the PI3K/AKT pathway to promote proliferation through SKP2–p21/p27. Schematic illustration of PD-L1-mediated PI3K/AKT activation via Livin and Galectin-1, which sustains PI3K/AKT signaling through a positive feedback loop. This PI3K/AKT activation upregulates SKP-2 and downregulates p21/p27 expression to drive cell cycle progression. Arrows indicate a promoting effect.

Article Snippet: PD-L1 ORF (RC213071 from OriGene, Rockville, MD, USA) was overexpressed in MDA-MB-468 cells using Lipofectamine LTX (Thermo Fisher Scientific), followed by selection using the eukaryotic antibiotic, G 418 (Thermo Fisher Scientific) at 750 μg/mL.

Techniques: Expressing, Activation Assay

Journal: Cancer Immunology Research

Article Title: Programmed Cell Death Ligand 1 Expression in Osteosarcoma

doi: 10.1158/2326-6066.cir-13-0224

Figure Lengend Snippet: Figure 1. PDL1 expression in cancer and noncancer cell lines. A, relative expression of PDL1 from total RNA isolated from cell lines. B, representative PDL1 protein expression levels in corresponding high- and low- expressing cell lines by IHC. C, representative PDL1 protein expression levels in corresponding high- and low- expressing cell lines by flow cytometry.

Article Snippet: To detect PDL1 by flow cytometry and IHC, six antibodies were screened: four noncommercialmousemAbs (CST1, CST2, XW, and LC), a mouse mAb from eBioscience (clone MIH1), and a rabbit polyclonal from ProSci (cat. no. 4059).

Techniques: Expressing, Isolation, Cytometry

Journal: Cancer Immunology Research

Article Title: Programmed Cell Death Ligand 1 Expression in Osteosarcoma

doi: 10.1158/2326-6066.cir-13-0224

Figure Lengend Snippet: Figure 2. Relative expression of PDL1 in 38 osteosarcoma specimens. PDL1 expression was evaluated from total RNA by quantitative real-time RT-PCR and showed that the expression levels range over 4 log (5,000-fold).

Article Snippet: To detect PDL1 by flow cytometry and IHC, six antibodies were screened: four noncommercialmousemAbs (CST1, CST2, XW, and LC), a mouse mAb from eBioscience (clone MIH1), and a rabbit polyclonal from ProSci (cat. no. 4059).

Techniques: Expressing, Quantitative RT-PCR

Journal: Cancer Immunology Research

Article Title: Programmed Cell Death Ligand 1 Expression in Osteosarcoma

doi: 10.1158/2326-6066.cir-13-0224

Figure Lengend Snippet: Figure 3. Overall survival of 37 patients with osteosarcoma in relation to PDL1 gene expression. The median overall survival for PDL1-low patients was 89 months compared with 28 months for PDL1-high patients, which showed a trend but was not statistically significant (P ¼ 0.0544).

Article Snippet: To detect PDL1 by flow cytometry and IHC, six antibodies were screened: four noncommercialmousemAbs (CST1, CST2, XW, and LC), a mouse mAb from eBioscience (clone MIH1), and a rabbit polyclonal from ProSci (cat. no. 4059).

Techniques: Gene Expression

Journal: Cancer Immunology Research

Article Title: Programmed Cell Death Ligand 1 Expression in Osteosarcoma

doi: 10.1158/2326-6066.cir-13-0224

Figure Lengend Snippet: Figure 4. Correlation between PDL1 gene expression and TILs by IHC. A, representative TILs in osteosarcoma tissues (400); score 0, no TILs; 1, rare/few TILs; 2, brisk/prominent TILs. B, significant positive correlation was shown between PDL1 gene expression and TILs in patients with osteosarcoma (P ¼ 0.0117).

Article Snippet: To detect PDL1 by flow cytometry and IHC, six antibodies were screened: four noncommercialmousemAbs (CST1, CST2, XW, and LC), a mouse mAb from eBioscience (clone MIH1), and a rabbit polyclonal from ProSci (cat. no. 4059).

Techniques: Gene Expression

Journal: Cancer Immunology Research

Article Title: Programmed Cell Death Ligand 1 Expression in Osteosarcoma

doi: 10.1158/2326-6066.cir-13-0224

Figure Lengend Snippet: Figure 5. Characterization of the origins of metastases. A, PDL1 expression is significantly higher in metastatic osteosarcoma tumors that originate from lung than from other locations (P ¼ 0.0024). B, TILs also exhibit a positive correlation with pulmonary osteosarcoma metastasis compared with nonpulmonary metastasis (P ¼ 0.0443).

Article Snippet: To detect PDL1 by flow cytometry and IHC, six antibodies were screened: four noncommercialmousemAbs (CST1, CST2, XW, and LC), a mouse mAb from eBioscience (clone MIH1), and a rabbit polyclonal from ProSci (cat. no. 4059).

Techniques: Expressing

Journal: Cellular and Molecular Immunology

Article Title: Selection and characterization of the novel anti-human PD-1 FV78 antibody from a targeted epitope mammalian cell-displayed antibody library

doi: 10.1038/cmi.2016.38

Figure Lengend Snippet: Computer-aided homology modeling. (a) The ribbon structure of the human PD-1 and PD-L1 complex based on the computer-guided modeling and docking methods. (b) The fine structure of the binding mode between human PD-1 and PD-L1. The purple sticks denote the key amino-acid residues in human PD-L1, whereas the green balls and sticks denote the key amino-acid residues in human PD-1. (c) The 3-D theoretical structure of IgHV3-33*01, where the balls and sticks denote H28, H31 and H32. (d) The 3-D theoretical structure of IgHK1-11*01, where the balls and sticks denote L27, L50 and L91.

Article Snippet: The CD274 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_014143","term_id":"1519243726","term_text":"NM_014143"}} NM_014143 ) Human cDNA ORF Clone was purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: Binding Assay

Journal: Cellular and Molecular Immunology

Article Title: Selection and characterization of the novel anti-human PD-1 FV78 antibody from a targeted epitope mammalian cell-displayed antibody library

doi: 10.1038/cmi.2016.38

Figure Lengend Snippet: Preliminary functional assessment of FV78. (a) Binding activity of FV78 to PD-1/Fc. (b) Epitope comparability of FV78 and MIL75 binding to PD-1/Fc by ELISA assay. (c) Analysis of the competitive activity of FV78 and MIL75 against the binding of PD-L1 to PD-1 by ELISA assay. (d) Analysis of the competitive activity of FV78 and MIL75 against the binding of PD-L1 to PD-1 by FACS assay. (e) Analysis of the competitive activity of FV78 and MIL75 against the binding of PD-L2 to PD-1 by ELISA assay. (f) Evaluation of the cross-reactive binding activity of FV78 to other CD28 family members.

Article Snippet: The CD274 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_014143","term_id":"1519243726","term_text":"NM_014143"}} NM_014143 ) Human cDNA ORF Clone was purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: Functional Assay, Binding Assay, Activity Assay, Enzyme-linked Immunosorbent Assay