Journal: Cellular and Molecular Immunology

Article Title: Programming of macrophages by UV-irradiated apoptotic cancer cells inhibits cancer progression and lung metastasis

doi: 10.1038/s41423-019-0209-1

Figure Lengend Snippet: PPARγ-dependent PTEN secretion in exosomes and PTEN uptake by recipient cells. qPCR analysis of PPARγ and PTEN mRNA in RAW cells exposed to apoptotic 344SQ cells (ApoSQ) for the indicated times shown in ( a , b ) or PTEN mRNA in blood MDMs from healthy donors or lung cancer patients exposed to apoptotic (ApoA) or necrotic (NecA) A549 cells for 24 h, as shown in ( c ). d Immunoblot analysis of indicated proteins in RAW cells transfected with PPARγ siRNA before ApoSQ stimulation for 24 h. e Immunoblot analysis of indicated proteins in BMDMs ( left ) or M2-like BMDMs ( right ) pretreated with GW9662 (10 μM) for 1 h before stimulation with ApoSQ for 24 h. f Immunoblot analysis of PTEN with whole-cell lysates from mouse BMDMs stimulated with ApoSQ (WCL) and of the secretion levels of PTEN using affinity pull-down in conditioned medium (CM). PTEN immunoprecipitates were separated by SDS-PAGE in nonreducing conditions. The arrows indicate the immunoglobulin heavy/light chain complex (up) and PTEN (down). g CM from RAW cells pretreated with 10 μM of GW9662 before ApoSQ stimulation for 24 h was fractionated by ultracentrifugation, and the soluble (sol) and insoluble (ins) fractions were immunoblotted with antibodies against PTEN, CD63, CD81, or CD9. h TEM images of exosomes isolated from the CM of ApoSQ-stimulated RAW cells pretreated with or without 20 μM GW4869. Scale bars: 20 μm. i Size distribution analysis of exosomes from the CM of ApoSQ-stimulated RAW cells pretreated with 20 μM GW4869 or vehicle (2% DMSO in saline). The horizontal axis represents particle size (nm), and the vertical axis represents particle concentration (×10 6 particles/ml). The red bars represent the s.e.m. The values represent the mode or mean size ± s.e.m. from three independent experiments. j Immunoblot analysis of GFP-PTEN, CD63, CD81, and CD9 in the soluble and insoluble fractions after ultracentrifugation of CM from a human macrophage cell line (hMϕ) overexpressing GFP-PTEN exposed to ApoA. k Direct fluorescence of 344SQ and A549 cells 24 h after treatment with harvested exosomes containing GFP-PTEN using confocal microcopy. Scale bars: 20 μm. l Lysates from 344SQ and A549 cells after incubation with harvested exosomes from hMϕ overexpressing GFP-PTEN were subjected to western blotting analysis. NS not significant; ** P < 0.01 and *** P < 0.001. Data are from three independent experiments (mean ± s.e.m. in ( a , b , i ) below tables), three donors (mean ± s.e.m. in ( c )), or one experiment representative of three independent experiments with similar results, as shown in ( d – l )

Article Snippet: PPARγ activity was determined in nuclear extracts (8 g) frompharmacological inhibitor-pretreated or siRNA-transfected RAW264.7 or 344SQ cells using a TransAM TM PPARγ Transcription Factor Assay kit (40196, Activ Motif Inc.) according to the manufacturer’s instructions.

Techniques: Western Blot, Transfection, SDS Page, Isolation, Saline, Concentration Assay, Fluorescence, Incubation

Journal: Cellular and Molecular Immunology

Article Title: Programming of macrophages by UV-irradiated apoptotic cancer cells inhibits cancer progression and lung metastasis

doi: 10.1038/s41423-019-0209-1

Figure Lengend Snippet: Macrophage secretion of 15-HETE, lipoxin A4, and 15d-PGJ 2 mediates anti-EMT effects. a – c ELISA of 15-HETE, lipoxin A4 and 15d-PGJ 2 in conditioned medium (CM) from RAW cells alone or from cocultures of RAW/viable 344SQ cells (ViaSQ), RAW/apoptotic 344SQ cells (ApoSQ), or RAW/necrotic 344SQ cells (NecSQ). d Time course of PPARγ activation in 344SQ cells with CM or ApoSQ CM at the indicated times. RAW cells were pretreated with 10 μM PD146176 (PD) for 1 h, as shown in ( e , h , k ), or transfected with two siRNAs against lipocalin-type prostaglandin D synthase (#1 and #2 siL-PGDS) for 48 h, as shown in ( f , g , i , j , l , m ) before stimulation with ApoSQ cells for 24 h. e – m CM was added to 344SQ cells with TGF-β1 (10 ng/ml) for 48 h. PPARγ activation is shown in ( e – g ), and the immunoblot analysis of indicated protein expression in 344SQ cells is shown in ( h – m ). NS not significant; * P < 0.05 and *** P < 0.001. Data are from three independent experiments (mean ± s.e.m.) are shown in ( a – g ), and data from one experiment representative of three independent experiments with similar results are shown in ( h – m )

Article Snippet: PPARγ activity was determined in nuclear extracts (8 g) frompharmacological inhibitor-pretreated or siRNA-transfected RAW264.7 or 344SQ cells using a TransAM TM PPARγ Transcription Factor Assay kit (40196, Activ Motif Inc.) according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Activation Assay, Transfection, Western Blot, Expressing

Journal: Cellular and Molecular Immunology

Article Title: Programming of macrophages by UV-irradiated apoptotic cancer cells inhibits cancer progression and lung metastasis

doi: 10.1038/s41423-019-0209-1

Figure Lengend Snippet: PPARγ and PTEN expression in primary tumors and tumor-infiltrating macrophages. Apoptotic 344SQ cells (ApoSQ) were subcutaneously injected into the skin lesion 2 days after subcutaneous injection of 344SQ cells into syngeneic (129/Sν) mice. Mice were necropsied 6 weeks later. Representative confocal images of primary tumors stained with anti-PPARγ (green) and anti-F4/80 (red) are shown in ( a , d) ; anti-PTEN (green) and anti-F4/80 (red) staining is shown in ( e , g ); and anti-CD36 (red) and anti-F4/80 (green) staining is shown in ( h , j ), as well as staining with the DNA-binding dye DAPI. b , c , f , i Measurements of fluorescence intensity in full-size images. d , g , j ROIs from white squares on the low magnification images of ( a , e , h ), respectively. Arrows indicate the localizations of PPARγ, PTEN, and CD36 in macrophages. NS not significant, * P < 0.05 and ** P < 0.01. Data are representative images from five mice per group in ( a , e , h ) or from independent experiments with five mice per group (mean ± s.e.m. in ( b , c , f , i )). Scale bars: 100 μm in ( a , e , h )

Article Snippet: PPARγ activity was determined in nuclear extracts (8 g) frompharmacological inhibitor-pretreated or siRNA-transfected RAW264.7 or 344SQ cells using a TransAM TM PPARγ Transcription Factor Assay kit (40196, Activ Motif Inc.) according to the manufacturer’s instructions.

Techniques: Expressing, Injection, Staining, Binding Assay, Fluorescence

Journal: Cellular and Molecular Immunology

Article Title: Programming of macrophages by UV-irradiated apoptotic cancer cells inhibits cancer progression and lung metastasis

doi: 10.1038/s41423-019-0209-1

Figure Lengend Snippet: Expression of PPARγ and PTEN and the secretion of PPARγ ligands in isolated TAMs. a qPCR analysis of TAM isolated primary tumors ( n = 8). b – d Representative confocal images of TAMs stained with anti-PPARγ (red), anti-PTEN (red), anti-CD36 (red), and anti-F4/80 (green). e ELISA of 15-HETE, lipoxin A4 and 15d-PGJ 2 in TAM culture ( n = 8). *** P < 0.001 (Student’s t test). Data are from mice with lung metastasis [control; n = 8 in ( a – e )] and without lung metastasis [ApoSQ; n = 8 in ( a – e )] (mean ± s.e.m. in ( a , e ))

Article Snippet: PPARγ activity was determined in nuclear extracts (8 g) frompharmacological inhibitor-pretreated or siRNA-transfected RAW264.7 or 344SQ cells using a TransAM TM PPARγ Transcription Factor Assay kit (40196, Activ Motif Inc.) according to the manufacturer’s instructions.

Techniques: Expressing, Isolation, Staining, Enzyme-linked Immunosorbent Assay, Control

Journal: Cellular and Molecular Immunology

Article Title: Programming of macrophages by UV-irradiated apoptotic cancer cells inhibits cancer progression and lung metastasis

doi: 10.1038/s41423-019-0209-1

Figure Lengend Snippet: PPARγ-dependent anti-metastatic effects of UV-irradiated apoptotic lung cancer cell injection in mice. a A schematic of the experimental design. Where indicated, GW9662 (1 mg/kg/day, i.p.) or its vehicle (Veh; 2% DMSO in saline) was administered into the left flank 1 day before the injection of ApoSQ into the right skin lesion ( n = 5 per group). Mice were necropsied 4 weeks after 344SQ cell injections. b Number of mice with lung metastasis/total number of mice examined. The qPCR analysis is shown in ( c ), and the immunoblot analysis of indicated protein expression in primary tumors is shown in ( d ). * P < 0.05, ** P < 0.01, and *** P < 0.001 (Student’s t test). Data are representative images from five mice per group ( d left ) or from independent experiments with five mice per group (mean ± s.e.m. in ( c , d right )

Article Snippet: PPARγ activity was determined in nuclear extracts (8 g) frompharmacological inhibitor-pretreated or siRNA-transfected RAW264.7 or 344SQ cells using a TransAM TM PPARγ Transcription Factor Assay kit (40196, Activ Motif Inc.) according to the manufacturer’s instructions.

Techniques: Irradiation, Injection, Saline, Western Blot, Expressing

Journal: Cellular and Molecular Immunology

Article Title: Programming of macrophages by UV-irradiated apoptotic cancer cells inhibits cancer progression and lung metastasis

doi: 10.1038/s41423-019-0209-1

Figure Lengend Snippet: A schematic diagram summarizing and integrating the effects of enhanced exosomal PTEN secretion and PPARγ ligands from macrophages exposed to UV-irradiated apoptotic cancer cells to prevent cancer cells from undergoing the EMT and metastatic process

Article Snippet: PPARγ activity was determined in nuclear extracts (8 g) frompharmacological inhibitor-pretreated or siRNA-transfected RAW264.7 or 344SQ cells using a TransAM TM PPARγ Transcription Factor Assay kit (40196, Activ Motif Inc.) according to the manufacturer’s instructions.

Techniques: Irradiation

Journal: Clinical and Experimental Otorhinolaryngology

Article Title: Expression of VEGF, HGF, IL-6, IL-8, MMP-9, Telomerase in Peripheral Blood of Patients with Head and Neck Squamous Cell Carcinoma

doi: 10.3342/ceo.2009.2.4.186

Figure Lengend Snippet: Kaplan-Meier overall survival curves. There are significant differences in the Kaplan-Meier overall survival curves related to the telomerase expression and the serum vascular endothelial growth factor (VEGF) level. (A) Telomerase expression ( P =0.045). (B) Serum VEGF level ( P =0.028). (C) Serum hepatocyte growth factor level ( P =0.172). (D) Serum interleukin (IL)-6 level ( P =0.870). (E) Serum IL-8 level ( P =0.675). (F) Serum matrix metalloproteinase-9 level ( P =0.542).

Article Snippet: The measurement and analysis of the telomerase activity of the PBMCs were conducted through the use of the TRAP EZE TM Telomerase Detection Kit (Oncor Co., Gaithersburg, MD, USA), which is a modified the TRAP assay by Kim et al. ( ).

Techniques: Expressing