Journal: Cancers

Article Title: Exosome-Mediated Activation of the Prostasin-Matriptase Serine Protease Cascade in B Lymphoma Cells.

doi: 10.3390/cancers15153848

Figure Lengend Snippet: Figure 1. Expression analysis of matriptase, HAI-1, HAI-2, and prostasin in B cancer cells by reverse- transcription/qPCR (a,b), western blotting (c–e), and flow cytometry (f). (a) Bar graph of relative mRNA expression levels of matriptase (Mat), HAI-1, HAI-2, prostasin (Pro) in Daudi (n = 4), Namalwa (n = 5), Ramos (n = 3), Raji (n = 3), JeKo-1 (n = 3), and RS4;11 cells (n = 2) using GAPDH as the reference. The prostasin bars do not appear in the bar graph, as the actual qPCR readouts were registered as “N/A” by the instrument. (b) Bar graph of mRNA quantity ratio of HAI-2 to matriptase after normalization with the GAPDH level in each cell line in (a). (c) Western blotting images of matriptase (Ab: A300-221A), HAI-2, and GAPDH. Twenty micrograms of total protein from the cell lysate of each individual culture (including 2 repeats) were analyzed. Daudi, lanes 1–3; Namalwa, lanes 4–6; Ramos, lanes 7–9. Top panel, matriptase (Mat); middle panel, HAI-2; bottom panel, GAPDH. (d) Densitometry bar graph of relative protein quantities of matriptase and HAI-2 using GAPDH as the reference. (e) The quantitative ratio of HAI-2 to matriptase in each cell line. (f) Flow cytometry histogram of matriptase expression evaluation in Ramos cells. The Ramos cells (4 × 105) were labeled with the matriptase antibody as described in the Materials and Methods section. The matriptase-positive cells are shown in the PE-A subset (blue peak). Cells without the matriptase antibody labeling (red peak) were not detected in the PE-A subset and were used as the gating control.

Article Snippet: The vector control exosomes (Vexo, 10 μg) and prostasin exosomes (Pexo, 10 μg) prepared from the HEK293T cells were tested with four different exosome markers using antibodies against CD63, HSP70, Tsg101, and Alix (all from Santa Cruz Biotechnology).

Techniques: Expressing, Reverse Transcription, Western Blot, Cytometry, Flow Cytometry, Labeling, Antibody Labeling, Control

Journal: Cancers

Article Title: Exosome-Mediated Activation of the Prostasin-Matriptase Serine Protease Cascade in B Lymphoma Cells.

doi: 10.3390/cancers15153848

Figure Lengend Snippet: Figure 2. Prostasin exosomes reduce matriptase quantity in B cancer cells. (a) Western blot images of matriptase (Ab: A300-221A) in samples from the cell lysate (top panel) and the conditioned media (bottom panel) after incubation with prostasin exosomes (Pro) or exosomes without prostasin (KO). The Daudi cells (2 × 105 cells each) were incubated with the exosomes in 50 µL of OPTI-MEM I/2%FBS (lanes 1–4) or RPMI medium (lanes 5–8) overnight. One-half of each cell lysate or 40 µL of each media supernatant were analyzed. (b) Western blot images of GAPDH from (a). (c) Densitometry of relative intensities of matriptase in the cell lysate or media (d). Data presented are the average intensity of lanes 1, 3, 5, 7 versus that of lanes 2, 4, 6, 8 after normalization with GAPDH in (b). (e) Western blot images of matriptase (top panel; Ab: sc-365482) in the Daudi, Namalwa, and Ramos cells treated with exosomes isolated from the HEK293T cells. Cells (2.5 × 105) were co-cultured with prostasin exosomes (Pexo, lanes 3, 6, 9) or vector exosomes (Vexo, lanes 2, 5, 8) in 100 µL of OPTI-MEM I/2%FBS. Cells without exosomes (None, lanes 1, 4, 7) were cultured in the same conditions. Bottom, GAPDH western blot image. (f) Bar graph of (e) expressed as the relative intensities of matriptase

Article Snippet: The vector control exosomes (Vexo, 10 μg) and prostasin exosomes (Pexo, 10 μg) prepared from the HEK293T cells were tested with four different exosome markers using antibodies against CD63, HSP70, Tsg101, and Alix (all from Santa Cruz Biotechnology).

Techniques: Western Blot, Incubation, Isolation, Cell Culture, Plasmid Preparation

Journal: Cancers

Article Title: Exosome-Mediated Activation of the Prostasin-Matriptase Serine Protease Cascade in B Lymphoma Cells.

doi: 10.3390/cancers15153848

Figure Lengend Snippet: Figure 3. B cell matriptase quantity reduction by wild-type prostasin. (a) Western blot images of matriptase (Ab: sc-365482) and GAPDH in the Daudi (top two panels), Ramos (middle two panels), and Namalwa (bottom two panels) cells treated with exosomes isolated from the Calu-3 cells and sublines with over-expressed prostasin or variants. Calu-3, parent cells; KO, subline with prostasin

Article Snippet: The vector control exosomes (Vexo, 10 μg) and prostasin exosomes (Pexo, 10 μg) prepared from the HEK293T cells were tested with four different exosome markers using antibodies against CD63, HSP70, Tsg101, and Alix (all from Santa Cruz Biotechnology).

Techniques: Western Blot, Isolation

Journal: Cancers

Article Title: Exosome-Mediated Activation of the Prostasin-Matriptase Serine Protease Cascade in B Lymphoma Cells.

doi: 10.3390/cancers15153848

Figure Lengend Snippet: Figure 4. Trypsin-like serine protease activity measurement. (a) Line graphs of trypsin-like serine protease activity expressed as relative fluorescent units (RFU). After mixing 2 × 106 cells with exosomes (Vexo or Pexo) or with RPMI medium alone in a total volume of 500 µL for 24 h, the activity in the conditioned medium was measured continually for 120 min. (b) Activity graph of Vexo or Pexo exosomes from the HEK293T-Vec or Pro cells in RPMI medium without cells, used as background controls. (c) Bar graph of data from (a) after subtracting the background controls of (b). * denotes p < 0.05. (d) Dot plots of trypsin-like serine protease activity in media collected 10 days after cell–exosome co-culturing. a.u., arbitrary units. Data were analyzed in GraphPad Prism 9. ANOVA, p < 0.05 for Daudi, Namalwa, and Ramos, p > 0.05 for RS4.

Article Snippet: The vector control exosomes (Vexo, 10 μg) and prostasin exosomes (Pexo, 10 μg) prepared from the HEK293T cells were tested with four different exosome markers using antibodies against CD63, HSP70, Tsg101, and Alix (all from Santa Cruz Biotechnology).

Techniques: Activity Assay

Journal: Cancers

Article Title: Exosome-Mediated Activation of the Prostasin-Matriptase Serine Protease Cascade in B Lymphoma Cells.

doi: 10.3390/cancers15153848

Figure Lengend Snippet: Figure 5. Ectopic expression of prostasin in B cancer cells. (a) Western blot analysis of transient expression of prostasin (P) or vector alone (V) in the Daudi, Namalwa, and Ramos cells. The lysate from 2 × 105 cells of each type was analyzed. Top panel, matriptase (Ab: sc-365482); middle panel, prostasin; bottom panel, GAPDH. (b) Flow cytometry analysis of Namalwa sublines with tetracycline-induced prostasin expression or vector alone. Red peak (vector-alone cells) and sky-blue peak (prostasin-expressing cells) are samples without the prostasin antibody incubation. Orange peak (vector-alone cells) and green peak (prostasin-expressing cells) are samples incubated with the prostasin antibody. All samples were incubated with a secondary antibody conjugated with the fluorophore Cy3, and 10,000 cells of each sample were analyzed in a CytoFLEX S flow cytometer. The data were analyzed with FlowJo™software v10.8.1 and are presented in the histogram. (c) Western blot analysis of NamalwaTR sublines. One hundred thousand cells of each sample were analyzed. Lanes 1 and 4 or V, samples of the vector control subline; lanes 2 and 5 or P, samples of the subline with the wild-type prostasin; lanes 3 and 6 or M, samples of the subline with a serine active-site mutant prostasin. Left panel, cells were grown in OPTI-MEM I/2%FBS with 1 µg/mL tetracycline (with tet); right panel, cells were grown without tetracycline (no tet) for 8 days. Top two panels, matriptase antibody (sc-365482); bottom two panels, prostasin antibody. (d) Western blot analysis of tet-conditioned media from (c). Two hundred milliliters of the conditioned media were precipitated with trichloroacetic acid (TCA) (final 16.7%) at 4 ◦C overnight. The pellet was collected via centrifu- gation and analyzed. The membrane was blotted with the AF3946 human matriptase/ST14 catalytic domain antibody.

Article Snippet: The vector control exosomes (Vexo, 10 μg) and prostasin exosomes (Pexo, 10 μg) prepared from the HEK293T cells were tested with four different exosome markers using antibodies against CD63, HSP70, Tsg101, and Alix (all from Santa Cruz Biotechnology).

Techniques: Expressing, Western Blot, Plasmid Preparation, Flow Cytometry, Incubation, Cytometry, Software, Control, Mutagenesis, Membrane

Journal: Cancers

Article Title: Exosome-Mediated Activation of the Prostasin-Matriptase Serine Protease Cascade in B Lymphoma Cells.

doi: 10.3390/cancers15153848

Figure Lengend Snippet: Figure 6. Impact of prostasin–matriptase cascade activation on B cancer cells. (a) Bar graph of cell count for two consecutive days of B cells treated with exosomes. Namalwa, n = 7; Ramos, n = 6; Raji, n = 5; Jeko-1, n = 6. * denotes p < 0.05. (b) Growth curves of NamalwaTR-Vec and NamalwaTR-Pro cells under tetracycline induction. Left graph, cells were set at 2.5 × 105/mL on day 0 and cultured in the growth medium containing 10%FBS for 4 days. Right graph, on day 4 (reset, indicated by the arrow), the cells were diluted in OPTI-MEM I/2%FBS to 5 × 105/mL and cultured for another 5 days. Tetracycline at 1 µg/mL was added into the culture on day 0 and maintained through culturing. n = 4 for each cell line, and * denotes p < 0.05. (c) Trypsin-like serine protease activity in the conditioned media of NamalwaTR-Vec and NamalwaTR-Pro cells (n = 4). Data were analyzed in Excel with student’s t test. * denotes p < 0.05 between the two sample groups. (d) Bar graph of annexin-V-positive cells analyzed by flow cytometry. Cells under tetracycline induction were cultured for various times (week 1, n = 3; week 2, n = 4; week 3, n = 3) and subjected to direct labeling of annexin V conjugated with fluorophore allophycocyanin (APC). Ten thousand cells for each sample were analyzed on the CytoFLEX S flow cytometer. Propidium iodide staining and FSC/SSC discrimination were used for gating the live singlets, which were further analyzed for annexin V staining. (e) Bar graph of migrated cells treated with exosomes for 24 h. * denotes p < 0.05. (f) Bar graph of migrated Namalwa sublines with the induction of prostasin expression for 2–4 days and reconditioned in RPMI medium for 1 day before seeding in Transwells for migration (n = 7). * denotes p < 0.05. (g) Bar graph of invaded cells treated with exosomes for 24 h. (h) Bar graph of invaded Namalwa sublines (n = 5) treated as in (f). * denotes p < 0.05. (i) Gelatin zymography and western blot analysis. Top panel, the Ramos cells (2 × 106) in 500 µL of RPMI/0.1%BSA were treated with vector exosomes (Vexo), prostasin exosomes (Pexo), or a purified recombinant human matriptase serine protease domain (r-Mat SPD) overnight. One-fifth of the cell lysate (lanes 1–3) or 20 µL of the conditioned medium (lanes 4–6) were analyzed. The Vexo or Pexo exosomes or the r-Mat SPD alone were incubated in RPMI/0.1%BSA and used as controls (lanes 7–9). The clear bands at ~70 kDa marked by a filled arrow are matriptase. These were recognized by matriptase antibodies (middle panel). Unidentified bands with gelatinase activity marked at * locations in lanes 4, 5, 7, 8 are inherited from the exosomes, as shown in the samples with the exosomes alone (lanes 7 and 8). The band marked by the white circle is unknown. Bands at ~28 kDa marked by an unfilled arrow are r-Mat SPD. Bottom panel is GAPDH, which is detected only in the cell lysate, not in media samples or the controls without cells. (j) Gelatin zymography of B cancer cells treated as described in (i). The matriptase gelatinase activity is decreased in the cell lysate (lanes 2, 6, 10, 14) but increased in the corresponding media samples (lanes 4, 8, 12, 16) upon Pexo treatment in comparison to that of the Vexo-treated samples (in lysate, lanes 1, 5, 9, 13; in media, lanes 3, 7, 11, 15), correspondingly.

Article Snippet: The vector control exosomes (Vexo, 10 μg) and prostasin exosomes (Pexo, 10 μg) prepared from the HEK293T cells were tested with four different exosome markers using antibodies against CD63, HSP70, Tsg101, and Alix (all from Santa Cruz Biotechnology).

Techniques: Activation Assay, Cell Counting, Cell Culture, Activity Assay, Cytometry, Labeling, Staining, Expressing, Migration, Zymography, Western Blot, Plasmid Preparation, Recombinant, Incubation, Comparison

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: HUVEC-derived exosomes alleviate lipopolysaccharide-induced acute lung injury inflammation by restoring the balance of mitochondrial fusion and division

doi: 10.1007/s00018-025-06078-w

Figure Lengend Snippet: HUVEC-derived EVs suppress activation of inflammatory pathways and alleviate lung injury in vivo. (A) Respiratory function parameters, including airway resistance, PaCO₂(mmHg), PaO₂, and pulmonary ventilation, were measured before and after EV administration. ( n = 6). (B) RT-qPCR analysis of inflammatory cytokine mRNA levels (IL-6, IL-1β, and TNF-α) in mouse lung tissues. ( n = 6). (C) H&E staining of lung tissues showing histopathological damage and corresponding ALI scores. ( n = 6). Scale bar = 100 μm (D) ELISA quantification of IL-6, IL-1β, and TNF-α levels in lung tissues, confirming decreased cytokine production following EV administration ( n = 6). (E–F) RNA-seq analysis of mouse lung tissues following EV treatment revealed substantial transcriptional changes, with 1,108 genes upregulated and 1,535 genes downregulated (E). KEGG pathway enrichment analysis indicated that the most significantly affected pathways were NF-κB and MAPK signaling (F). (G) Western blot analysis of key components of the NF-κB and MAPK pathways, including p-IκKα/IκKα, p-IκBα/IκBα, p-NF-κB/NF-κB, p-p38/p38, and p-ERK/ERK. ( n = 6). (H) Depletion of macrophages using clodronate liposomes abolished the therapeutic effects of EVs on LPS-induced lung injury, indicating that HUVEC-derived EVs exert their protective effects primarily via targeting macrophages. (I) Assessment of respiratory function showed no improvement in macrophage-depleted mice following EV treatment, further supporting the notion that EVs act by modulating macrophage activity

Article Snippet: Once HUVECs reached 70–80% confluence (cultured in exosome-depleted serum), the medium was replaced with serum-free medium, and the cells were incubated for an additional 48 h. The supernatant was then collected, and HUVEC-derived exosomes were isolated using density gradient centrifugation as previously described [ , ].

Techniques: Derivative Assay, Activation Assay, In Vivo, Quantitative RT-PCR, Staining, Enzyme-linked Immunosorbent Assay, RNA Sequencing, Western Blot, Liposomes, Activity Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: HUVEC-derived exosomes alleviate lipopolysaccharide-induced acute lung injury inflammation by restoring the balance of mitochondrial fusion and division

doi: 10.1007/s00018-025-06078-w

Figure Lengend Snippet: Encapsulation of IPR into HUVEC-derived EVs as a novel therapeutic strategy for ALI. (A) Molecular docking showing a strong binding interaction between MARCH5 and IPR. (B) Thermal stability assay demonstrating that IPR significantly enhances the thermal stability of MARCH5, while GAPDH remains unaffected ( n = 6). (C) Measurement of respiratory function changes in mice before and after IPR treatment, including airway resistance, CO₂ partial pressure, O₂ partial pressure, and pulmonary ventilation ( n = 6). (D) Toluidine blue staining to assess lung epithelial barrier damage in mice, with darker blue indicating more severe epithelial barrier disruption ( n = 6). (E) HE staining to evaluate pathological changes in mouse lung tissue before and after IPR treatment, with corresponding lung injury scores ( n = 6). Scale bar = 100 μm (F) Examination of NF-κB and MAPK pathway activation before and after IPR treatment. The results show that IPR does not affect the activation of NF-κB and MAPK pathways ( n = 6). (G) RT-qPCR analysis of IL-6, IL-1β, and TNF-α mRNA levels in mice after IPR treatment. (H) TEM analysis of EV morphology after encapsulation of IPR, showing vesicles filled with content and more robust shapes ( n = 6). Scale bar = 200 μm (I) HE staining to evaluate the potential effects of exosome@IPR on LPS-induced acute lung injury in mice ( n = 6). Scale bar = 100 μm (J) Assessment of the protective effect of exosome@IPR on the lung epithelial barrier through alveolar lavage. (K) RT-qPCR analysis of inflammatory cytokines IL-6 mRNA levels after exosome@IPR treatment ( n = 6). (L) Effects of engineered vesicles (exosome@IPR) on pulmonary function in a mouse model of acute lung injury. (M) Western blot (WB) analysis of NF-κB pathway activation before and after exosome@IPR treatment ( n = 6). (N) Immunofluorescence detection of NF-κB nuclear translocation, with results showing a significant reduction in NF-κB nuclear fluorescence intensity following exosome@IPR treatment. Scale bar = 10 μm

Article Snippet: Once HUVECs reached 70–80% confluence (cultured in exosome-depleted serum), the medium was replaced with serum-free medium, and the cells were incubated for an additional 48 h. The supernatant was then collected, and HUVEC-derived exosomes were isolated using density gradient centrifugation as previously described [ , ].

Techniques: Encapsulation, Derivative Assay, Binding Assay, Stability Assay, Staining, Disruption, Activation Assay, Quantitative RT-PCR, Western Blot, Immunofluorescence, Translocation 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: 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: Western blotting analysis was performed for the identification of exosomal PTEN in CM with anti-CD63 (ab216130, Abcam) or anti-PTEN (9559, Cell Signaling Technology).

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: Internalized PTEN alters signaling, retains polarity, and inhibits the EMT and invasion of 344SQ cells. a Immunoblot analysis of indicated proteins in 344SQ cell lysates after incubation with CM for the indicated times. b Immunoblot analysis of PTEN in 344SQ cells after incubation with ApoSQ-exposed CM at various dilutions (from 1/5 to 1) with control CM for 12 h. The qPCR analysis of PTEN mRNA is shown in ( c ). The immunoblot analysis of indicated proteins in 344SQ cells after the addition of TGF-β1 (10 ng/ml) is shown in ( d ) and that after the addition of CM is shown in (e) . f – l RAW cells were transfected with PTEN siRNA (#1 siPTEN) before ApoSQ cell stimulation for 24 h. f Immunoblot analysis of PTEN in RAW cells. g – l CM was added to 344SQ cells with or without TGF-β1 (10 ng/ml) for the indicated times. g , h , j Immunoblot analysis of indicated proteins in 344SQ cells. i Confocal microscopic images of 3D acini at 12 h after treatment with TGF-β1 (10 ng/ml). Normal acini of 344SQ cells were grown in 3D Matrigel containing CM and stained with anti-β-catenin (green) and DAPI. Scale bars: 20 μm. k qPCR analysis of Snai1 and Zeb1 mRNAs in 344SQ cells. l The numbers of invaded cells were analyzed to assess their invasive ability using Matrigel-coated Transwells. NS not significant; *** P < 0.001. Data are from three independent experiments (mean ± s.e.m. in ( c , k , l )) or one experiment representative of three independent experiments with similar results, as shown in ( a , b , d – j )

Article Snippet: Western blotting analysis was performed for the identification of exosomal PTEN in CM with anti-CD63 (ab216130, Abcam) or anti-PTEN (9559, Cell Signaling Technology).

Techniques: Western Blot, Incubation, Control, Transfection, Cell Stimulation, Staining

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: Downregulation of EMT, the Akt/p38 signaling cascades, and cancer cell invasion by purified exosomes and the exosomal PTEN level in recipient cancer cells. 344SQ cells were treated with exosomes isolated from RAW conditioned medium with (Exo ApoSQ CM ) or without (Exo CM ) apoptotic 344SQ cells, in the presence of TGF-β1 (10 ng/ml) for the indicated times, as shown in ( a – d ). a – c Immunoblot analysis of indicated proteins in 344SQ cell lysates after incubation with exosomes. d Confocal microscopic images of 3D acini were taken 10 days after cell seeding. The normal acini of 344SQ cells were grown in 3D Matrigel containing exosomes and stained with anti-β-catenin (green) and DAPI. Scale bars: 20 μm. 344SQ cells were treated with purified exosomes from the culture medium of apoptotic cancer cell-stimulated RAW 264.7 cells transfected with or without two siRNAs for PTEN (#1 and #2 siPTEN) for the indicated times, as shown in ( e , f ). The immunoblot analysis of indicated proteins in purified exosomes is shown in ( e ), that in 344SQ cell lysates after treatment with purified exosomes is shown in ( f ). g qPCR analysis of PTEN mRNA in 344SQ cells 12 h after treatment with exosomes isolated from RAW CM with (Exo ApoSQ CM ) or without (Exo CM ) apoptotic 344SQ cells. NS not significant; ** P < 0.01. Data are from one experiment representative of three independent experiments with similar results are shown in ( a – e , f upper panel), or data from three independent experiments (mean ± s.e.m.) are shown in ( f lower panel) and ( g )

Article Snippet: Western blotting analysis was performed for the identification of exosomal PTEN in CM with anti-CD63 (ab216130, Abcam) or anti-PTEN (9559, Cell Signaling Technology).

Techniques: Purification, Isolation, Western Blot, Incubation, Staining, Transfection

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: Western blotting analysis was performed for the identification of exosomal PTEN in CM with anti-CD63 (ab216130, Abcam) or anti-PTEN (9559, Cell Signaling Technology).

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: Western blotting analysis was performed for the identification of exosomal PTEN in CM with anti-CD63 (ab216130, Abcam) or anti-PTEN (9559, Cell Signaling Technology).

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: 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: Western blotting analysis was performed for the identification of exosomal PTEN in CM with anti-CD63 (ab216130, Abcam) or anti-PTEN (9559, Cell Signaling Technology).

Techniques: Irradiation