Journal: Oncogene

Article Title: PROX1 promotes hepatocellular carcinoma proliferation and sorafenib resistance by enhancing β-catenin expression and nuclear translocation.

doi: 10.1038/onc.2015.7

Figure Lengend Snippet: Figure 2. Knockdown of PROX1 expression in HCC cells reduces cell proliferation in vitro and tumor growth in vivo. (a) Western blot verification of knockdown of endogenous PROX1 expression in MHCC-97H and Huh7 cells. PROX1 was detected by anti-PROX1 mAb. (b) Cell proliferation and (c) colony formation of MHCC-97H and Huh7 cells upon knockdown of PROX1 expression. Means ± s.d. from three independent experiments were presented. (d) Cell cycle distribution of MHCC-97H cells upon knockdown of PROX1 expression. (e) Knockdown of PROX1 expression reduced HCC growth in tumor xenograft mice. MHCC-97H-SCR and MHCC-97H-si1646 were injected subcutaneously into nude mice respectively (1.0 × 107 cell/mouse, n = 6). Tumor weight was measured after 8 weeks post injection. Significant differences were determined using Student’s-t test, *Po0.05, **Po0.01, ***Po0.001.

Article Snippet: For lentivirus-mediated RNA interference of PROX1 expression, DNA fragments encoding the hairpin precursors for si259 (5′-TTTCCAGGAGCAACCATAATT-3′, corresponding to nt 259–279 of the PROX1 ORF) and si1646 (5′-GGCTCTCCTTGTCGCTCATAA-3′, corresponding to nt 1646–1666 of the PROX1 ORF) were inserted into pLKO.1 TRC (Addgene plasmid 10879) respectively.

Techniques: Knockdown, Expressing, In Vitro, In Vivo, Western Blot, Injection

Journal: Oncogene

Article Title: PROX1 promotes hepatocellular carcinoma proliferation and sorafenib resistance by enhancing β-catenin expression and nuclear translocation.

doi: 10.1038/onc.2015.7

Figure Lengend Snippet: Figure 4. PROX1 activates β-catenin transcription. (a) PROX1 overexpression in HCC cells increased β-catenin mRNA expression. BEL-7402 and Huh7 cells were infected with the vector lentivirus or the PROX1-expressing lentivirus. β-catenin mRNA was measured by using qrtPCR. Means ± s.d. from three independent experiments were presented as relative ratio to the control whose value was taken as 1.0. (b) Knockdown of PROX1 expression in HCC cells reduced β-catenin mRNA expression. Huh7 and MHCC-97H cells were infected with the lentivirus either expressing PROX1 siRNA precursor (si259 and si1646) or scrambled siRNA (SCR). Means ± s.d. from three independent experiments were presented as relative ratio to the control whose value was taken as 1.0. (c) PROX1 dose-dependently activated the full-length β-catenin promoter. HEK293T cells were co-transfected with indicated plasmids. Means ± s.d. of normalized luciferase activity from three independent experiments were presented. (d) Identification of the response region for PROX1 in the β-catenin promoter. HEK293T cells were co-transfected with various deletion constructs of the β-catenin promoter reporter and PROX1 expression construct. Means ± s.d. of normalized luciferase activity from three independent experiments were presented. (e) Endogenous PROX1 associated with the β-catenin promoter. ChIP-PCR was performed with sonicated chromatins immunoprecipitated from Huh7 and MHCC-97H cells by anti-PROX1 mAb or pre-immune IgG (control). β-catenin promoter segments were quantified by using qrtPCR against 5% input. Means ± s.d. from three independent experiments were presented. Significant differences were determined using Student’s-t test, *Po0.05, **Po0.01, ***Po0.001.

Article Snippet: For lentivirus-mediated RNA interference of PROX1 expression, DNA fragments encoding the hairpin precursors for si259 (5′-TTTCCAGGAGCAACCATAATT-3′, corresponding to nt 259–279 of the PROX1 ORF) and si1646 (5′-GGCTCTCCTTGTCGCTCATAA-3′, corresponding to nt 1646–1666 of the PROX1 ORF) were inserted into pLKO.1 TRC (Addgene plasmid 10879) respectively.

Techniques: Over Expression, Expressing, Infection, Plasmid Preparation, Control, Knockdown, Transfection, Luciferase, Activity Assay, Construct, Sonication, Immunoprecipitation

Journal: bioRxiv

Article Title: Using peptide-exchange systems to interrogate peptide-specific KIR binding to HLA Class I

doi: 10.64898/2026.03.03.708729

Figure Lengend Snippet: (A) Display of ULBP1 and CD155 on CombiCells detected by mAbs and NKG2D-Fc, and DNAM-1-Fc, respectively. (B&C) KIR2DL1+ and KIR2DL1-NK-cell degranulation (CD107a upregulation) in response to ULBP1, CD155, and HLA-C*05:01 displayed on CombiCells. (C) Three types of HLA-C*05:01 were tested, all containing P2 (IIDKSGSTV); wild-type, open and dipeptide exchanged. Data from three independent experiments with NK cells from separate donors are shown.

Article Snippet: For binding assays, APC-conjugated Fc fusion protein (NKG2D-Fc (R&D Systems, Cat. No: 1299-NK) or DNAM-1-Fc (R&D Systems, Cat. No: # 666-DN) or KIR-Fc (R&D Systems, KIR2DL1-Fc (Cat. No: 1844-KR-050), KIR2DL3-Fc (Cat. No: 2014-KR-050), KIR2DS4-Fc (Cat. No: 1847-KR-050) (3.6 μg mL -1 ) was added at 25 μL per well and incubated for 30 min at 4 °C in the dark.

Techniques:

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: Differential expression of the T-cell inhibitor TIGIT in glioblastoma and MS

doi: 10.1212/NXI.0000000000000712

Figure Lengend Snippet: (A) TIGIT + , CD226 + , and CD155 + infiltrates in tumor tissue from patients with GBM and chronic active lesions from patients with MS. Arrows and insets indicate representative immunoreactivities in lymphocytes. (B) Area corresponding to TIGIT-stained GBM tissue in (A), immunolabeled with antibody against CD3. CD155 expression in tumor cells and cortical neurons. (C) Quantification of TIGIT + , CD226 + , and CD155 + infiltrates in GBM tumor tissue and MS lesions. Statistical significance was assessed by unpaired Student t tests with a p value threshold of 0.05. ns = not significant. High magnification (scale bar = 40 μm). Low magnification (scale bar = 10 μm). GBM = glioblastoma multiforme.

Article Snippet: Serial sections were stained with primary antibodies against CD68 (Cell Signaling #76437, 1:500), MBP (Millipore Sigma MAB386, 1:500), CD3 (Dako A 40452, 1:200), TIGIT (Santa Cruz sc-103349, 1:800), CD226 (Novus Biologicals NBP1-85001, 1:50), CD155 (Bioss Inc. bs-2525R, 1:750), PD-1 (Acris Antibodies AP23805PU-M, 1:100), and PD-L1 (Spring Bioscience M4420, 1:25) and processed with the appropriate biotinylated secondary antibodies (Vector Laboratories, Burlingame CA) and avidin-biotin staining kit with diaminobenzidine as chromogen.

Techniques: Staining, Immunolabeling, Expressing

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: Differential expression of the T-cell inhibitor TIGIT in glioblastoma and MS

doi: 10.1212/NXI.0000000000000712

Figure Lengend Snippet: (A) PD-1 + and PD-L1 + infiltrates in tumor tissue from patients with GBM and chronic active lesions from patients with MS. Isotype control for PD-1 and PD-L1 antibody in perivascular infiltrates of MS tissue. Arrows and insets indicate representative immunoreactivities in lymphocytes. (B) Quantification of PD-1 + and PD-L1 + infiltrates in GBM tumor tissue and MS lesions. (C) Frequency of TIGIT + and CD226 + lymphocytes in the perivascular space and deep parenchyma, as well as the number of CD3 + lymphocytes counted in the perivascular space and deep parenchyma per sample. Statistical significance was assessed by unpaired or paired Student t tests with a p value threshold of 0.05. ns = not significant. High magnification (scale bar = 40 μm). Low magnification (scale bar = 10 μm). GBM = glioblastoma multiforme.

Article Snippet: Serial sections were stained with primary antibodies against CD68 (Cell Signaling #76437, 1:500), MBP (Millipore Sigma MAB386, 1:500), CD3 (Dako A 40452, 1:200), TIGIT (Santa Cruz sc-103349, 1:800), CD226 (Novus Biologicals NBP1-85001, 1:50), CD155 (Bioss Inc. bs-2525R, 1:750), PD-1 (Acris Antibodies AP23805PU-M, 1:100), and PD-L1 (Spring Bioscience M4420, 1:25) and processed with the appropriate biotinylated secondary antibodies (Vector Laboratories, Burlingame CA) and avidin-biotin staining kit with diaminobenzidine as chromogen.

Techniques: Control

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: Differential expression of the T-cell inhibitor TIGIT in glioblastoma and MS

doi: 10.1212/NXI.0000000000000712

Figure Lengend Snippet: (A) Expression of TIGIT and CD226 measured by flow cytometry on CD4 and CD8 T cells from tumor infiltrates. (B) Percent of CD4 and CD8 T cells expressing TIGIT and CD226 and percent of CD226 + , CD4, and CD8 T cells coexpressing TIGIT (C). Histograms represent mean ± SEM. (D) Quantification of the frequency of TIGIT + , CD226 + , and TIGIT + among CD226 + for CD4 and CD8 circulating and tumor-infiltrating T cells. Frequencies were assessed by flow cytometry. Dots connected by a line represent the same patient. TIL = tumor-infiltrating lymphocytes. Statistical significance was assessed by the paired Student t test with a p value threshold of 0.05; ns = not significant. GBM = glioblastoma multiforme.

Article Snippet: Serial sections were stained with primary antibodies against CD68 (Cell Signaling #76437, 1:500), MBP (Millipore Sigma MAB386, 1:500), CD3 (Dako A 40452, 1:200), TIGIT (Santa Cruz sc-103349, 1:800), CD226 (Novus Biologicals NBP1-85001, 1:50), CD155 (Bioss Inc. bs-2525R, 1:750), PD-1 (Acris Antibodies AP23805PU-M, 1:100), and PD-L1 (Spring Bioscience M4420, 1:25) and processed with the appropriate biotinylated secondary antibodies (Vector Laboratories, Burlingame CA) and avidin-biotin staining kit with diaminobenzidine as chromogen.

Techniques: Expressing, Flow Cytometry

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: Differential expression of the T-cell inhibitor TIGIT in glioblastoma and MS

doi: 10.1212/NXI.0000000000000712

Figure Lengend Snippet: Expression of TIGIT and CD226 measured by flow cytometry on circulating CD4 (A) and CD8 (B) T cells from healthy donors (HDs) and patients with GBM. Quantification of the frequency of TIGIT + , CD226 + , and TIGIT + among CD226 + and PD-1 + for CD4 (C) and CD8 (D) circulating T cells. The values for GBM are the same as depicted in in the “Blood” group. Histograms represent mean ± SEM. Statistical significance was assessed by the unpaired Student t test with a p value threshold of 0.05; ns = not significant. GBM = glioblastoma multiforme.

Article Snippet: Serial sections were stained with primary antibodies against CD68 (Cell Signaling #76437, 1:500), MBP (Millipore Sigma MAB386, 1:500), CD3 (Dako A 40452, 1:200), TIGIT (Santa Cruz sc-103349, 1:800), CD226 (Novus Biologicals NBP1-85001, 1:50), CD155 (Bioss Inc. bs-2525R, 1:750), PD-1 (Acris Antibodies AP23805PU-M, 1:100), and PD-L1 (Spring Bioscience M4420, 1:25) and processed with the appropriate biotinylated secondary antibodies (Vector Laboratories, Burlingame CA) and avidin-biotin staining kit with diaminobenzidine as chromogen.

Techniques: Expressing, Flow Cytometry

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: Differential expression of the T-cell inhibitor TIGIT in glioblastoma and MS

doi: 10.1212/NXI.0000000000000712

Figure Lengend Snippet: Proliferation as measure by CellTrace™ dilution vs CD226 expression in TIGIT + CD4 and CD8 T cells at the end of a 5-day in vitro stimulation with αCD3+αCD28. Representative stainings from 2 patients presenting different baseline degrees of proliferation (A) and quantifications from 6 patients (B). Statistical significance was assessed by the paired Student t test with a p value threshold of 0.05. GBM = glioblastoma multiforme.

Article Snippet: Serial sections were stained with primary antibodies against CD68 (Cell Signaling #76437, 1:500), MBP (Millipore Sigma MAB386, 1:500), CD3 (Dako A 40452, 1:200), TIGIT (Santa Cruz sc-103349, 1:800), CD226 (Novus Biologicals NBP1-85001, 1:50), CD155 (Bioss Inc. bs-2525R, 1:750), PD-1 (Acris Antibodies AP23805PU-M, 1:100), and PD-L1 (Spring Bioscience M4420, 1:25) and processed with the appropriate biotinylated secondary antibodies (Vector Laboratories, Burlingame CA) and avidin-biotin staining kit with diaminobenzidine as chromogen.

Techniques: Expressing, In Vitro