Journal: Cell death & disease

Article Title: DEF6(differentially exprehomolog) exacerbates pathological cardiac hypertrophy via RAC1.

doi: 10.1038/s41419-023-05948-0

Figure Lengend Snippet: Fig. 1 The expression of DEF6 is increased in hypertrophic hearts and cardiomyocytes. A mRNA levels of DEF6 in the LV myocardium of mice subjected to sham or 4 weeks of TAC surgery (n = 5). B Immunoblot analyses (left) and results of quantification (right) of DEF6 protein expression in the LV myocardium of mice subjected to sham or 4 weeks of TAC surgery (n = 4). C mRNA levels of DEF6 in NRCMs administrated with PBS or 24 h of PE (50 μM) (n = 5). D Immunoblot analyses (left) and results of quantification (right) of DEF6 protein expression in NRCMs administrated with PBS or 24 h of PE (n = 4). *P < 0.05, ***P < 0.001 vs. sham or PBS. Data are displayed as mean ± SD. Statistical analysis were conducted by two-tailed Student’s t test (A, C) or Mann–Whitney U test (B, D).

Article Snippet: The cardiomyocytes were cultured in DMEM/F12 medium (Gibco, C11330) added with 10% fetal bovine serum (FBS), 5-bromodeoxyuridine (0.1 mM), and 1% penicillin/streptomycin for 24 h. The NRCMs were infected with adenoviruses at a multiplicity of infection (MOI) of 100 for 6 h. Subsequently, the medium was replaced with serum-free DMEM/F12, and 12 h later, the cardiomyocytes were stimulated with phosphatebuffered saline (PBS) or PE (50 μM) for 24 h. For rescue experiments, the cardiomyocytes were treated with Rac1 inhibitor NSC23766 (50 μM, 24 h) (S8031, Selleck) before adenovirus infection [31].

Techniques: Expressing, Western Blot, Two Tailed Test, MANN-WHITNEY

Journal: Cell death & disease

Article Title: DEF6(differentially exprehomolog) exacerbates pathological cardiac hypertrophy via RAC1.

doi: 10.1038/s41419-023-05948-0

Figure Lengend Snippet: Fig. 2 Ablation of DEF6 mitigates TAC-induced cardiac hypertrophy. A Strategy to construct KO mice and the sequencing results of WT and KO mice. B Protein levels of cardiac DEF6 in WT and KO mice (n = 5). C Comparisons of HW, HW/BW, LW/BW, and HW/TL in WT and KO mice subjected to sham or 4 weeks of TAC surgery (n = 10). D Left, gross hearts and H&E-stained LV sections of each groups. Scale bars, 0.3 cm and 50 μm, respectively. Right, Comparisons of cardiomyocyte cross-sectional area from groups (n = 6). E RT-PCR analyses of the hypertrophic markers in the indicated groups (n = 4). F–H Comparisons of the LVEDd, LVESd, LVPWd, FS, and EF values in WT and KO mice subjected to sham or 4 weeks of TAC surgery (n = 10). I Left, PSR-stained LV sections in WT and KO mice subjected to sham or 4 weeks of TAC surgery. Scale bars, 50 μm. Right, comparisons of LV collagen volume between groups (n = 6). J RT-PCR analysis of the fibrotic markers in each groups (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001 vs. WT sham, #P < 0.05, ##P < 0.01, ###P < 0.001 vs. WT TAC. Data are displayed as mean ± SD. Statistical analysis were conducted by One-way ANOVA (C, D, F–I) or Kruskal–Wallis test (E, J).

Article Snippet: The cardiomyocytes were cultured in DMEM/F12 medium (Gibco, C11330) added with 10% fetal bovine serum (FBS), 5-bromodeoxyuridine (0.1 mM), and 1% penicillin/streptomycin for 24 h. The NRCMs were infected with adenoviruses at a multiplicity of infection (MOI) of 100 for 6 h. Subsequently, the medium was replaced with serum-free DMEM/F12, and 12 h later, the cardiomyocytes were stimulated with phosphatebuffered saline (PBS) or PE (50 μM) for 24 h. For rescue experiments, the cardiomyocytes were treated with Rac1 inhibitor NSC23766 (50 μM, 24 h) (S8031, Selleck) before adenovirus infection [31].

Techniques: Construct, Sequencing, Staining, Reverse Transcription Polymerase Chain Reaction

Journal: Cell death & disease

Article Title: DEF6(differentially exprehomolog) exacerbates pathological cardiac hypertrophy via RAC1.

doi: 10.1038/s41419-023-05948-0

Figure Lengend Snippet: Fig. 3 Overexpression of DEF6 aggravates TAC-induced cardiac hypertrophy. A mmunoblot analyses (left) and results of quantification (right) of DEF6 protein expression in the hearts of mice injected with AAV9-vector or AAV9-DEF6 (n = 4). B Comparisons of HW, HW/BW, LW/ BW, and HW/TL in AAV9-vector- and AAV9-DEF6-infected mice subjected to sham or 4 weeks of TAC surgery (n = 10). C Left, gross hearts and H&E-stained LV sections of each groups. Scale bars, 0.3 cm and 50 μm, respectively. Right, Comparisons of cardiomyocyte cross-sectional area between groups (n = 6). D RT-PCR analyses of the hypertrophic markers in each groups (n = 4). E–G Comparisons of LVEDd, LVESd, LVPWd, FS, and EF in AAV9-vector- and AAV9-DEF6-infected mice subjected to sham or 4 weeks of TAC surgery (n = 10). H Left, PSR-stained LV sections in AAV9-vector- and AAV9-DEF6-infected mice subjected to sham or 4 weeks of TAC surgery. Scale bars, 50 μm. Right, comparisons of LV collagen volume between groups (n = 6). I RT-PCR analyses of the fibrotic markers in each groups (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001 vs. AAV9- vector or AAV9-vector sham, #P < 0.05, ##P < 0.01, ###P < 0.001 vs. AAV9-vector TAC. Data are displayed as mean ± SD. Statistical analysis were conducted by Mann–Whitney U test (A) or One-way ANOVA (B, C, E–H) or Kruskal–Wallis test (D, I).

Article Snippet: The cardiomyocytes were cultured in DMEM/F12 medium (Gibco, C11330) added with 10% fetal bovine serum (FBS), 5-bromodeoxyuridine (0.1 mM), and 1% penicillin/streptomycin for 24 h. The NRCMs were infected with adenoviruses at a multiplicity of infection (MOI) of 100 for 6 h. Subsequently, the medium was replaced with serum-free DMEM/F12, and 12 h later, the cardiomyocytes were stimulated with phosphatebuffered saline (PBS) or PE (50 μM) for 24 h. For rescue experiments, the cardiomyocytes were treated with Rac1 inhibitor NSC23766 (50 μM, 24 h) (S8031, Selleck) before adenovirus infection [31].

Techniques: Over Expression, Expressing, Injection, Plasmid Preparation, Infection, Staining, Reverse Transcription Polymerase Chain Reaction, MANN-WHITNEY

Journal: Cell death & disease

Article Title: DEF6(differentially exprehomolog) exacerbates pathological cardiac hypertrophy via RAC1.

doi: 10.1038/s41419-023-05948-0

Figure Lengend Snippet: Fig. 4 DEF6 exacerbates PE-induced cardiomyocyte hypertrophy. A Immunoblot analyses (left) and results of quantification (right) of DEF6 protein expression in cultured NRCMs infected with AdshRNA or AdshDEF6 (n = 3). B Left, immunofluorescence staining (α-actinin, red) in cultured NRCMs infected with AdshRNA or AdshDEF6 and administrated with PBS or 24 h of PE. Scale bar, 20 μm. Right, comparisons of the cardiomyocyte surface areas in cultured NRCVs of each groups (n ≥48 cells per group). C RT-PCR analysis of the hypertrophic markers in cultured NRCVs of each groups (n = 3). D Immunoblot analyses (left) and results of quantification (right) of DEF6 protein expression in cultured NRCVs infected with Advector or AdDEF6 (n = 3). E Left, immunofluorescence staining (α-actinin, red) in cultured NRCMs infected with Advector or AdDEF6 and administrated with PBS or 24 h of PE. Scale bar, 20 μm. Right, comparisons of the cardiomyocyte surface areas in cultured NRCVs of each groups (n ≥48 cells per group). F RT-PCR analyses of the hypertrophic markers in cultured NRCVs of each groups (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 vs. AdshRNA or AdshRNA PBS or Advector or Advector PBS, #P < 0.05, ##P < 0.01, ###P < 0.001 vs. AdshRNA PE or Advector PE. Data are displayed as mean ± SD. Statistical analysis were conducted by two-tailed Mann–Whitney U test (A, D) or One-way ANOVA (B, E) or Kruskal–Wallis test (C, F).

Article Snippet: The cardiomyocytes were cultured in DMEM/F12 medium (Gibco, C11330) added with 10% fetal bovine serum (FBS), 5-bromodeoxyuridine (0.1 mM), and 1% penicillin/streptomycin for 24 h. The NRCMs were infected with adenoviruses at a multiplicity of infection (MOI) of 100 for 6 h. Subsequently, the medium was replaced with serum-free DMEM/F12, and 12 h later, the cardiomyocytes were stimulated with phosphatebuffered saline (PBS) or PE (50 μM) for 24 h. For rescue experiments, the cardiomyocytes were treated with Rac1 inhibitor NSC23766 (50 μM, 24 h) (S8031, Selleck) before adenovirus infection [31].

Techniques: Western Blot, Expressing, Cell Culture, Infection, Staining, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test, MANN-WHITNEY

Journal: Cell death & disease

Article Title: DEF6(differentially exprehomolog) exacerbates pathological cardiac hypertrophy via RAC1.

doi: 10.1038/s41419-023-05948-0

Figure Lengend Snippet: Fig. 6 Prohypertrophic effect of DEF6 depends on Rac1-MEK-ERK signaling. A Co-IP of DEF6 was performed with anti-Flag and probed by Western blots with anti-HA (left); Co-IP of Rac1 was performed with anti-HA and probed by Western blots with anti-Flag (right). B In vitro GST pulldown assays for the interaction of purified Flag-DEF6 and GST-HA-Rac1 (left), as well as Flag-Rac1 and GST-HA-DEF6 (right). C The activity of Rac1 changes in the same direction as the DEF6 expression. D Immunoblot analyses of total and activated MEK1/2, ERK1/2 in cultured NRCMs infected with Advector or AdDEF6 and treated with PBS or NSC23766 (50 μM, 24 h) under 24 h of PE treatment (50 μM) (n = 3). E Immunofluorescence staining (α-actinin, red) (left) and comparison of cardiomyocyte surface areas (right) of NRCMs infected with Advector and AdDEF6 and treated with PBS or NSC23766 (50 μM, 24 h) under 24 h of PE treatment (50 μM). (n ≥48 cells per group). F RT-PCR analysis of the hypertrophic markers in cultured NRCVs of each groups (n = 3). G Immunoblot analyses of total and activated MEK1/2, ERK1/2 in cultured NRCMs infected with AdshRNA or AdshDEF6 and with Adcontrol or AdRac1(G12V) under PE 24 h of PE treatment (50 μM) (n = 3). H Immunofluorescence staining (α-actinin, red) (left) and comparison of cardiomyocyte surface areas (right) of NRCMs infected with the indicated adenovirus and administrated with 24 h of PE (50 μM) (n ≥48 cells per group). I RT-PCR analysis of the hypertrophic markers in cultured NRCVs of each groups (n = 3). •P < 0.05, P < 0.01,•P < 0.001 vs. Advector PBS PE or AdshRNA Adcontrol PE, *P < 0.05, **P < 0.01, ***P < 0.001 vs. Advector PBS PE or AdshRNA Adcontrol PE, ###P < 0.001 vs. AdDEF6 PBS PE or AdshDEF6 Adcontrol PE, and n.s. indicates no significance. Data are displayed as mean ± SD. Statistical analysis were conducted by Kruskal–Wallis test.

Article Snippet: The cardiomyocytes were cultured in DMEM/F12 medium (Gibco, C11330) added with 10% fetal bovine serum (FBS), 5-bromodeoxyuridine (0.1 mM), and 1% penicillin/streptomycin for 24 h. The NRCMs were infected with adenoviruses at a multiplicity of infection (MOI) of 100 for 6 h. Subsequently, the medium was replaced with serum-free DMEM/F12, and 12 h later, the cardiomyocytes were stimulated with phosphatebuffered saline (PBS) or PE (50 μM) for 24 h. For rescue experiments, the cardiomyocytes were treated with Rac1 inhibitor NSC23766 (50 μM, 24 h) (S8031, Selleck) before adenovirus infection [31].

Techniques: Co-Immunoprecipitation Assay, Western Blot, In Vitro, Activity Assay, Expressing, Cell Culture, Infection, Staining, Comparison, Reverse Transcription Polymerase Chain Reaction

Journal:

Article Title: Persistent and Transient Replication of Full-Length Hepatitis C Virus Genomes in Cell Culture

doi: 10.1128/JVI.76.8.4008-4021.2002

Figure Lengend Snippet: Stability of sfl genomes during continuous cell culture. (A) Six different cell lines were continuously cultured in the presence of G418. At the indicated time points (days [d]), cells were lysed, total RNA was prepared, and 4 μg was analyzed by Northern blotting. For comparison, given amounts of RNA containing subgenomic replicon transcribed in vitro were analyzed in parallel. To allow detection of sequence variants and of smaller HCV RNA pieces, the entire blot was hybridized under low-stringency conditions. (B) Genetic drift of the sfl genome in cell line 21-5. Cells were continuously cultured, and after 57 passages (corresponding to ∼6 months), total RNA was prepared. The coding region of the HCV polyprotein was amplified by long-distance RT-PCR in two overlapping fragments shown as bars below the ORF. For each fragment, two independent clones were sequenced. The boundary of the sequenced clones is indicated by a dashed vertical line. Amino acid substitutions are drawn as vertical lines at their respective positions within the HCV polyprotein. The hypervariable region 1 (HVR1) present at the N terminus of E2 is depicted as a shaded area. Six mutations were conserved between both sequenced clones, and they are indicated (stars).

Article Snippet: For cell lines carrying HCV replicons, various concentrations of G418 (Geneticin; Life Technologies GmbH) were added to the culture medium (100 μg/ml for cell line 20-1; 250 μg/ml for cell lines 21-5 and 21-7; 500 μg/ml for cell lines 22-1, 22-2, 22-6, and 22-16).

Techniques: Cell Culture, Northern Blot, In Vitro, Sequencing, Amplification, Reverse Transcription Polymerase Chain Reaction, Clone Assay

Journal: eLife

Article Title: IL-21/type I interferon interplay regulates neutrophil-dependent innate immune responses to Staphylococcus aureus

doi: 10.7554/eLife.45501

Figure Lengend Snippet: ( A, B ) Intra-tracheal infection with the USA 300 strain of MRSA induced a significant increase in pulmonary Il21 mRNA ( A ) and IL-21 protein ( B ) 24 hr after infection. ( C ) PBS or 2 μg of IL-21 was administered i.t. to WT mice one day prior to MRSA infection, and lung MRSA CFU were quantitated 7 and 24 hr later. ( D, E ) Lung immunopathology was assessed in H and E-stained sections of lung tissue from naïve uninfected mice and mice pre-treated with PBS or IL-21 and then infected for 7 or 24 hr with MRSA (in upper left panel, the bar = 500 μm and inset bar = 10 μm) ( D ), and pathology ( E ) scores were assessed. ( F–K ) animals were infected with MRSA as above. ( F ) Total lung neutrophil cellularity was quantitated by flow cytometry after staining with Ly6G and CD11b. ( G ) RNA-Seq analysis was performed on total lung tissue mRNA (pools of 5 animals) isolated 7 or 24 hr after treatment of WT mice with PBS or IL-21. Boxed regions include genes mentioned in the text. ( H–K ) RT-PCR was used to assess expression of Gzma ( H ) and Gzmb ( I ) mRNA in lungs treated with IL-21 for 7 hr, and ELISA was used to assess the induction of granzyme B ( J ) and IFNγ protein ( K ) in corresponding bronchoalveolar lavage fluid at 7 hr. Data are representative of either three ( A, B, C, F, H–K ) or two ( D, E, G ) independent experiments and validation of RNA-Seq was performed by RT-PCR of mRNA from additional mice.

Article Snippet: Neutrophils were then purified (80–90%) with a mouse neutrophil isolation kit (Miltenyi Biotec) and were used directly in either MRSA killing assays or were in vitro stimulated with IL-21 and then RNA isolated for RT-PCR analysis.

Techniques: Infection, Staining, Flow Cytometry, RNA Sequencing, Isolation, Reverse Transcription Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay, Biomarker Discovery

Journal: eLife

Article Title: IL-21/type I interferon interplay regulates neutrophil-dependent innate immune responses to Staphylococcus aureus

doi: 10.7554/eLife.45501

Figure Lengend Snippet: ( A–B ) Total cellularity of lung was assessed in BAL fluid ( A ) and lung ( B ) 24 hr after IL-21 intratracheal instillation. ( C ) Percentage of neutrophils was assessed by flow cytometry at 24 hr after IL-21 treatment. ( D–E ) Levels of chemokines were measured in BAL fluid at 24 hr after IL-21 treatment.

Article Snippet: Neutrophils were then purified (80–90%) with a mouse neutrophil isolation kit (Miltenyi Biotec) and were used directly in either MRSA killing assays or were in vitro stimulated with IL-21 and then RNA isolated for RT-PCR analysis.

Techniques: Flow Cytometry

Journal: eLife

Article Title: IL-21/type I interferon interplay regulates neutrophil-dependent innate immune responses to Staphylococcus aureus

doi: 10.7554/eLife.45501

Figure Lengend Snippet: ( A ) Bone-marrow neutrophils were purified by negative selection using a neutrophil isolation kit (Miltenyi) and either not stimulated or stimulated in vitro with IL-21 (100 ng/ml) for 24 hr in the absence (control) or presence of peptidoglycan (PG) (2 or 5 μg/ml); IL-21R expression on gated Ly6G + CD11b + neutrophils was detected by flow cytometry (blue = isotype control; red = anti-IL-21R). ( B, C ) IL-21 lowers the MRSA CFU in a neutrophil-dependent fashion. Mice were treated i.p. with an isotype control mAb or neutrophil-depleted with anti-Ly6G (1A8 mAb), and the efficacy of neutrophil depletion is shown in BAL and lung ( B ). Mice were then treated with PBS or IL-21 i.t., infected i.t. with MRSA, and CFU quantitated at 24 hr ( C ). CFU quantitation was performed on the left single lobe, whereas immune populations and RNA were determined using the right lobes. ( D, E ) Levels of Gzmb and Gzma mRNAs in lung tissue of untreated or neutrophil-depleted mice (treated as in panel C ) were quantitated by RT-PCR. ( F ) Levels of Gzma and Gzmb mRNAs in purified cell-sorted lung neutrophils (>98% pure Ly6G + CD11b + ) from either untreated or IL-21-treated WT or Il21r KO mice were measured by RT-PCR and normalized to Rpl7 expression. ( G, H ) Lung neutrophils were elicited by i.t treatment with heat-killed S. aureus 24 hr prior to isolation, purified, stimulated in vitro for 4 hr with either PBS or IL-21 (100 ng/ml), and Gzma ( G ) and Gzmb ( H ) mRNAs assayed by RT-PCR and normalized to Rpl7 expression. ( I ) Purified HKSA-elicited lung neutrophils were incubated in vitro with MRSA for 3 hr in the presence of PBS or IL-21 either without or with the granzyme B inhibitor, Z-AAD-CMK. MRSA CFU was quantitated by plating serial dilutions on blood agar plates. Representative experiments are shown; each experiment was performed three times with similar results.

Article Snippet: Neutrophils were then purified (80–90%) with a mouse neutrophil isolation kit (Miltenyi Biotec) and were used directly in either MRSA killing assays or were in vitro stimulated with IL-21 and then RNA isolated for RT-PCR analysis.

Techniques: Purification, Selection, Isolation, In Vitro, Control, Expressing, Flow Cytometry, Infection, Quantitation Assay, Reverse Transcription Polymerase Chain Reaction, Incubation

Journal: eLife

Article Title: IL-21/type I interferon interplay regulates neutrophil-dependent innate immune responses to Staphylococcus aureus

doi: 10.7554/eLife.45501

Figure Lengend Snippet: ( A ) Quantitation of IL-21R MFI in neutrophils treated with or without IL-21 in the presence or absence of peptidoglycan (PG). ( B ) Lung neutrophils from HKSA-stimulated mice were purified via a magnetic neutrophil purification kit (Miltenyi). Shown is CD11b versus Ly6G staining.

Article Snippet: Neutrophils were then purified (80–90%) with a mouse neutrophil isolation kit (Miltenyi Biotec) and were used directly in either MRSA killing assays or were in vitro stimulated with IL-21 and then RNA isolated for RT-PCR analysis.

Techniques: Quantitation Assay, Purification, Staining

Journal: eLife

Article Title: IL-21/type I interferon interplay regulates neutrophil-dependent innate immune responses to Staphylococcus aureus

doi: 10.7554/eLife.45501

Figure Lengend Snippet: ( A ) Human peripheral blood neutrophils were purified by a neutrophil negative selection kit (StemCell Technologies). Shown is CD56 versus CD66b and CD8 versus CD66b staining. ( B ) Western blotting of granzyme B from neutrophils, PBMC, or A549 cells containing 0, 1, or 5% PBMC extracts, confirming that the low level of contamination of purified neutrophils cannot account for the granzyme B expression observed in this population. Also shown is western blotting for actin as a loading control.

Article Snippet: Neutrophils were then purified (80–90%) with a mouse neutrophil isolation kit (Miltenyi Biotec) and were used directly in either MRSA killing assays or were in vitro stimulated with IL-21 and then RNA isolated for RT-PCR analysis.

Techniques: Purification, Selection, Staining, Western Blot, Expressing, Control

Journal: eLife

Article Title: IL-21/type I interferon interplay regulates neutrophil-dependent innate immune responses to Staphylococcus aureus

doi: 10.7554/eLife.45501

Figure Lengend Snippet: ( A, B ) Purified peripheral blood neutrophils were stimulated with PBS (control) or IL-21 (100 ng/ml) for 4 hr, and IL21R mRNA was measured by real-time PCR and normalized to RPL7 expression ( A ), and IL-21R protein levels were measured by flow cytometry ( B ); left panel shows isotype control shaded and anti-IL21R black line, with a summary in the right panel). ( C ) RNA-Seq was performed on neutrophils after 4 or 24 hr incubation with PBS or IL-21 in the absence or presence of heat-killed S. aureus (10 6 /ml). We used HKSA rather than live bacteria in order to allow analysis at 24 hr, as live bacteria would have overgrown the system by then. Genes differentially expressed (fold-change >2.0) are shown. Shown is a representative RNA-Seq analysis. ( D – G ) Human peripheral blood neutrophils were stimulated for 4 hr in vitro in the presence or absence of IL-21 and GZMA ( D ), GZMB ( E ), GNLY ( F ), and IFNG ( G ) mRNA levels were quantitated by RT-PCR and normalized to RPL7 expression. ( H ) Purified human neutrophils were stimulated with IL-21 for 4 hr, fixed, permealized, and stained for intracellular granzyme B protein (gated on CD66b + cells); MFIs are summarized in right panel. ( I ) Granzyme B protein was measured by ELISA in supernatants from human peripheral blood neutrophils cultured for 24 hr in either the absence or presence of IL-21 (100 ng/ml). ( J, K ) Human neutrophils were incubated in vitro with either MRSA ( J ) or S. pyogenes ( K ) for 3 hr with PBS or IL-21 (100 ng/ml). MRSA and S. pyogenes CFU were quantitated by plating serial dilutions on blood agar plates. Results shown are representative of 3 independent experiments, except panel C shows one of two similar independent RNA-Seq experiments, each from a different donor.

Article Snippet: Neutrophils were then purified (80–90%) with a mouse neutrophil isolation kit (Miltenyi Biotec) and were used directly in either MRSA killing assays or were in vitro stimulated with IL-21 and then RNA isolated for RT-PCR analysis.

Techniques: Purification, Control, Real-time Polymerase Chain Reaction, Expressing, Flow Cytometry, RNA Sequencing, Incubation, Bacteria, In Vitro, Reverse Transcription Polymerase Chain Reaction, Staining, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: eLife

Article Title: IL-21/type I interferon interplay regulates neutrophil-dependent innate immune responses to Staphylococcus aureus

doi: 10.7554/eLife.45501

Figure Lengend Snippet: Reactive oxygen species were measured by flow cytometry in CellRox Red loaded peripheral blood neutrophils that were stimulated at 37°C for 30 min with PBS, IL-21, HKSA, or HKSA +IL-21.

Article Snippet: Neutrophils were then purified (80–90%) with a mouse neutrophil isolation kit (Miltenyi Biotec) and were used directly in either MRSA killing assays or were in vitro stimulated with IL-21 and then RNA isolated for RT-PCR analysis.

Techniques: Flow Cytometry

Journal: eLife

Article Title: IL-21/type I interferon interplay regulates neutrophil-dependent innate immune responses to Staphylococcus aureus

doi: 10.7554/eLife.45501

Figure Lengend Snippet: ( A ) WT and Il21r KO mice were infected intratracheally with MRSA (2 × 10 7 ) and CFU in the lung were quantitated at 4 and 24 hr post-infection. ( B ) WT and Il21r KO mice were infected in parallel with WT mice that had been treated with PBS or 2 μg IL-21, and CFU in the lung were quantitated at 7 and 24 hr post infection. ( C–E ) Lung immunopathology was assessed in H and E-stained lung sections (bar in upper left panel = 500 μm; bar in inset = 10 μm) ( C ); lung neutrophil cellularity as assessed by pathology score ( D ) and flow cytometry ( E ) were assessed at 4 and 24 hr post-infection. ( F ) WT or Il21r KO mice were pre-treated with an isotype control antibody or anti-Ly6G to deplete neutrophils, infected with MRSA, and CFU quantitated at 24 hr. Representative experiments are shown; each experiment was performed three times with similar results.

Article Snippet: Neutrophils were then purified (80–90%) with a mouse neutrophil isolation kit (Miltenyi Biotec) and were used directly in either MRSA killing assays or were in vitro stimulated with IL-21 and then RNA isolated for RT-PCR analysis.

Techniques: Infection, Staining, Flow Cytometry, Control

Journal: eLife

Article Title: IL-21/type I interferon interplay regulates neutrophil-dependent innate immune responses to Staphylococcus aureus

doi: 10.7554/eLife.45501

Figure Lengend Snippet: ( A ) WT mice were treated intratracheally with 50 μg of control Fc (from IgG1) or IL-21R-Fc protein for 2 days prior to infection with MRSA and lung CFU were quantitated at 4 and 24 hr post-infection with MRSA. ( B ) Histology of lungs in control Fc or IL-21R-Fc pre-treated mice at 4 and 24 hr post-infection (bar in upper left panel = 500 μm; bar in inset = 10 μm). ( C ) IFNα levels in BAL fluid were measured by ELISA 4 hr after infection in mice pretreated with control Fc or IL-21R-Fc. ( D ) Gzmb mRNA was measured in lungs of WT or Il21r KO mice pre-treated with isotype control or anti-IFNAR1 antibodies prior to MRSA infection, and normalized to Rpl7 mRNA expression. ( E ) Gzmb mRNA was measured in lungs of mice treated with either control Fc or IL-21R-Fc prior to MRSA infection and normalized to Rpl7 expression. ( F ) Like IL-21, IFNβ also induced GZMB mRNA in human peripheral blood neutrophils and normalized to RPL7 expression. ( G ) IFNβ induces increased in vitro killing of MRSA by human peripheral blood neutrophils and this was prevented by a granzyme B inhibitor, Z-AAD-CMK. ( H–J ) Co-cultures of CD4 + T cells and dendritic cells were stimulated with SEB either in the presence of control Fc or IL-21R-Fc, and mRNA was quantitated by RT-PCR after 24 hr and normalized to Rpl7 expression. Representative experiments are shown; each experiment was performed three times with similar results.

Article Snippet: Neutrophils were then purified (80–90%) with a mouse neutrophil isolation kit (Miltenyi Biotec) and were used directly in either MRSA killing assays or were in vitro stimulated with IL-21 and then RNA isolated for RT-PCR analysis.

Techniques: Control, Infection, Enzyme-linked Immunosorbent Assay, Expressing, In Vitro, Reverse Transcription Polymerase Chain Reaction

Journal: eLife

Article Title: IL-21/type I interferon interplay regulates neutrophil-dependent innate immune responses to Staphylococcus aureus

doi: 10.7554/eLife.45501

Figure Lengend Snippet: MRSA infection, as detailed in the legend to . ( A, B ) Pathology and neutrophil infiltration scores were assessed from H and E-stained lung sections shown in . ( C ) RNA-Seq was performed on total lung mRNA either before infection (Ctrl) or at 4 hr after MRSA infection of mice pretreated with either Fc protein or an IL-21R-Fc fusion protein. Representative of 2 independent RNA-Seq experiments.

Article Snippet: Neutrophils were then purified (80–90%) with a mouse neutrophil isolation kit (Miltenyi Biotec) and were used directly in either MRSA killing assays or were in vitro stimulated with IL-21 and then RNA isolated for RT-PCR analysis.

Techniques: Infection, Staining, RNA Sequencing

Journal: eLife

Article Title: IL-21/type I interferon interplay regulates neutrophil-dependent innate immune responses to Staphylococcus aureus

doi: 10.7554/eLife.45501

Figure Lengend Snippet: ( A ) Peripheral blood neutrophils from normal donors (NDs) or AD-HIES patients (PTs) were assayed using an in vitro MRSA killing assay in the absence or presence of IL-21 (100 ng/ml). ( B ) RT-PCR was used to quantitate GZMB mRNA in neutrophils 4 hr after in vitro stimulation without or with IL-21. Expression was normalized to RPL7 expression ( C ) RNA-Seq was performed on normal donor and AD-HIES patient neutrophils stimulated for 4 hr with PBS, IL-21, IFNβ, or IL-21 +IFNβ. Genes differentially expressed (fold-change >1.5) in two independent RNA-Seq analyses are shown. ( D ) RT-PCR normalized to RPL7 expression was used to validate the expression pattern of GBP1 and GBP2 in neutrophils from additional normal donors and AD-HIES patients. ( E–G ) Mutant Stat3 transgenic mice were treated i.t. with PBS or 2 μg IL-21, infected i.t. 24 hr later with MRSA, and at 7 hr post-infection lung MRSA CFU quantitated ( E ), and IFNα levels were measured in the serum ( F ) and BAL fluid ( G ).

Article Snippet: Neutrophils were then purified (80–90%) with a mouse neutrophil isolation kit (Miltenyi Biotec) and were used directly in either MRSA killing assays or were in vitro stimulated with IL-21 and then RNA isolated for RT-PCR analysis.

Techniques: In Vitro, Reverse Transcription Polymerase Chain Reaction, Expressing, RNA Sequencing, Mutagenesis, Transgenic Assay, Infection

Journal: eLife

Article Title: IL-21/type I interferon interplay regulates neutrophil-dependent innate immune responses to Staphylococcus aureus

doi: 10.7554/eLife.45501

Figure Lengend Snippet: ( A ) In wild-type (WT) mice, MRSA produces SEB, which bridges between T cells and dendritic cells by interacting with TCR and MHCII, leading to the production of IL-21 by CD4 + T cells. IL-21 stimulates neutrophils to release granzymes, with enhanced killing of MRSA. Type I IFN (e.g., produced by dendritic cells, as shown in the cartoon) can also induce granzyme production to promote killing of MRSA, but IL-21 inhibits dendritic cell production of type I IFN. ( B ) In the absence of either IL-21R or a functional STAT3 signaling response, the IL-21-induced neutrophil granzyme production does not occur. However, IL-21-mediated repression of type I IFN production by dendritic cells also no longer occurs, which leads to enhanced production of type I IFN and hence increased killing of MRSA through this pathway. The functional cross-talk of and relative potency of the IL-21- and type 1 IFN-mediated pathways and levels of each cytokine influence the outcome.

Article Snippet: Neutrophils were then purified (80–90%) with a mouse neutrophil isolation kit (Miltenyi Biotec) and were used directly in either MRSA killing assays or were in vitro stimulated with IL-21 and then RNA isolated for RT-PCR analysis.

Techniques: Produced, Functional Assay

Journal: eLife

Article Title: IL-21/type I interferon interplay regulates neutrophil-dependent innate immune responses to Staphylococcus aureus

doi: 10.7554/eLife.45501

Figure Lengend Snippet:

Article Snippet: Neutrophils were then purified (80–90%) with a mouse neutrophil isolation kit (Miltenyi Biotec) and were used directly in either MRSA killing assays or were in vitro stimulated with IL-21 and then RNA isolated for RT-PCR analysis.

Techniques: In Vivo, Flow Cytometry, Blocking Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Staining, Isolation

Journal: The Science of the Total Environment

Article Title: Sampling methods and assays applied in SARS-CoV-2 exposure assessment

doi: 10.1016/j.scitotenv.2021.145903

Figure Lengend Snippet: Data obtained from the chosen articles.

Article Snippet: , 34. SARS-CoV-2 RNA contamination on surfaces of a COVID-19 ward in a hospital of Northern Italy: what risk of transmission? , Italy , No , Surface samples from ward in University Hospital of Ferrara , Sampling performed with sterile rayon swabs pre-moistened in sterile phosphate-buffered solution , Viral RNA extraction with Patho Gene-spin Extraction kit (Generon) RT-qPCR targeted the RNA-dependent RNA polymerase ( RdRp ) gene (Generon), and the orf1ab , spike ( S ), and nucleocapsid ( N ) genes (ThermoFisher) , • SARS-CoV-2 was only detected in 3 samples of two floors and one-bathroom sink. • Reported to persist for a longer duration on surfaces under controlled laboratory conditions. , ( ) .

Techniques: Sampling, Lysis, RNA Extraction, Environmental Monitoring, Virus, Multiplex Assay, Northern Blot, Marker, RNA Detection, Isolation, Membrane, Control, Environmental Sampling, Amplification, Transmission Assay, Aerosol, Diagnostic Assay, Infection, Sterility, Real-time Polymerase Chain Reaction, Nested PCR, Reverse Transcription, Extraction, Purification, Digital PCR, Preserving, Quantitative RT-PCR, cDNA Synthesis, Magnetic Beads, Incubation, Modification, One Step RT-PCR, Cell Culture, Sequencing

Journal: Genes & development

Article Title: TCF3 alternative splicing controlled by hnRNP H/F regulates E-cadherin expression and hESC pluripotency.

doi: 10.1101/gad.316984.118

Figure Lengend Snippet: Figure 3. hnRNP H/F proteins bind exonic regulatory elements in exon 18b. (A, left) Diagram of wild-type and SWAP constructs in which the positions of exon 18a and exon 18b are switched. (Right) 32P RT–PCR assays of RNAs extracted from transfected HeLa cells using T7 primer and Ex19R. PCR products were digested with PstI. (B, left) Diagram of point-swapping mutant constructs. The details of a series of mutants are described in Supplemental Figure S4. (Right) 32P RT–PCR assays of RNAs extracted from transfected HeLa cells using T7 primer and Ex19R. PCR products were digested with PstI or HaeII. The E12 (exon18a inclusion) percentage was calculated by dividing exon 18a signal by the sum of exon 18a and 18b signals and is indicated below. (C) 32P RT–PCR assays of additional point-swapping mu- tants in sections 3 and 6 in HeLa cells. (D, top) Sequence comparison of sections 3-2 and 6-2. The putative hnRNP H/F-binding sites are underlined. The RNA affinity assay used biotinylated 3-2 and 6-2 short RNA oligonucleotides. Isolated proteins were analyzed by Western blot using the indicated antibodies.

Article Snippet: Western blotting Cells were lysed with SDS sample buffer (50 mM Tris at pH 7.4, 100 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% sodium deoxy- cholate, 1× proteinase inhibitor cocktail [Biotools]), resolved by SDS-PAGE, transferred to nitrocellulose membranes, and incubated with primary antibodies detecting CDH1 (1:1000; Cell Signaling), hnRNP H1 (1:1000; Bethyl Laboratories), hnRNP F (1:1500; Santa Cruz Biotechnology), GAPDH (1:20,000; Sigma), or ACTB (1:30,000; Sigma) overnight at 4°C.

Techniques: Construct, Reverse Transcription Polymerase Chain Reaction, Transfection, Mutagenesis, Sequencing, Comparison, Binding Assay, Isolation, Western Blot

Journal: Genes & development

Article Title: TCF3 alternative splicing controlled by hnRNP H/F regulates E-cadherin expression and hESC pluripotency.

doi: 10.1101/gad.316984.118

Figure Lengend Snippet: Figure 4. hnRNP H/F expression and recruitment to exonic regulatory elements decrease during differentiation. (A) E12/E47 ratio was examined by 32P RT–PCR. PCR products were digested by PstI. (B) Expression of pluripotency and differentiation markers as determined by quantitative PCR (qPCR) after 8 d of differentiation. The P-value was determined by two-tailed Student’s t-test. (∗) P < 0.05. (C) CLIP analysis to measure the hnRNP H protein enrichment on exon 18b of TCF3 mRNA by qPCR after 8 d of differentiation. Data were nor- malized to hnRNP H immunoprecipitation signal in undifferentiated H9 cells. All results represent the means ± SD from at least three independent experiments. (∗) P < 0.05. (D) Expression of hnRNP H and hnRNP F during 8 d of differentiation of H9 cells. mRNA levels were determined by qPCR. (E) Protein levels were analyzed by Western blot using the indicated antibodies (left), and the bar graph shows its quantification (right).

Article Snippet: Western blotting Cells were lysed with SDS sample buffer (50 mM Tris at pH 7.4, 100 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% sodium deoxy- cholate, 1× proteinase inhibitor cocktail [Biotools]), resolved by SDS-PAGE, transferred to nitrocellulose membranes, and incubated with primary antibodies detecting CDH1 (1:1000; Cell Signaling), hnRNP H1 (1:1000; Bethyl Laboratories), hnRNP F (1:1500; Santa Cruz Biotechnology), GAPDH (1:20,000; Sigma), or ACTB (1:30,000; Sigma) overnight at 4°C.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Two Tailed Test, Protein Enrichment, Immunoprecipitation, Western Blot

Journal: Genes & development

Article Title: TCF3 alternative splicing controlled by hnRNP H/F regulates E-cadherin expression and hESC pluripotency.

doi: 10.1101/gad.316984.118

Figure Lengend Snippet: Figure 5. hnRNP H1 and hnRNP F regulate TCF3 splicing and hESC morphology. (A) E12/E47 ratio in hESCs after the indicated siRNA treatment analyzed by 32P RT–PCR. RT–PCR products were digested by PstI and resolved by 5% PAGE. (Top) The E12 percentage is in- dicated below. (Bottom) Immunoblots showing protein levels after the indicated siRNA treatment. (B) E12/E47 ratio in 293T cells after the indicated siRNA treatment analyzed by 32P RT–PCR. (C) Splicing assays analyzed by 32P RT–PCR using T7 primer and Ex19R. The TCF3 minigene and the indicated expression plasmids were transfected into 293T cells. Total RNA from the transfected cells was used for 32P RT–PCR. (D) Cell morphology of H9 hESCs transfected by the indicated siRNAs. (E) Expression of pluripotency markers in hnRNP H/F knockdown cells, determined by qPCR. (F) Expression of differentiation markers in H9 hESCs after the indicated siRNA treatment, determined by qPCR.

Article Snippet: Western blotting Cells were lysed with SDS sample buffer (50 mM Tris at pH 7.4, 100 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% sodium deoxy- cholate, 1× proteinase inhibitor cocktail [Biotools]), resolved by SDS-PAGE, transferred to nitrocellulose membranes, and incubated with primary antibodies detecting CDH1 (1:1000; Cell Signaling), hnRNP H1 (1:1000; Bethyl Laboratories), hnRNP F (1:1500; Santa Cruz Biotechnology), GAPDH (1:20,000; Sigma), or ACTB (1:30,000; Sigma) overnight at 4°C.

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Transfection, Knockdown

Journal: Journal of Cancer Prevention

Article Title: Nuclear Localization of Fibroblast Growth Factor Receptor 1 in Breast Cancer Cells Interacting with Cancer Associated Fibroblasts

doi: 10.15430/jcp.2022.27.1.68

Figure Lengend Snippet: Figure 1. Involvement of FGF2-FGFR1 axisin Akt activation. (A) The effect of CAF-CM on proliferation of breast cancer (MCF-7, MDA-MB-231, and MDA-MB-468) cells was determined by the MTT assay. Cells were incubated with or without CAF-CM for 72 hours. ***Significantly different be- tween the groups compared (P < 0.001). (B) MDA-MB-231 cells were incubated with CAF-CM for the indicated time periods. Phosphorylation of Akt and STAT3 were detected by Western blot analysis. (C) MDA-MB-231 cells were exposed to CAF-CM with or without FGF-2-neutralizing antibody for 3 hours. Phosphorylation of Akt was detected by Western blot analysis. *,***Significantly different between the groups compared (*P < 0.05; ***P < 0.001). (D) MDA-MB-231 cells were treated with 20 ng/mL of FGF2 for the indicated time periods. The phosphorylation of FRS2α as well as Akt was analyzed by Western blot. (E) RNA-seq data set of TCGA breast invasive carcinoma was downloaded from XenaBrower (https://xenabrowser.net). mRNA expression levels of total 1,097 samples (Illumina HiSeq log [normalized counts + 1]) were prepared by quantile normalization. Pearson cor- relation coefficient was calculated to assess the relationship between FGF2 and FGFR1. (F, G) Correlation of FGFR1 protein expression with FGF2 (F) and Akt (G), based on 105 breast invasive carcinoma protein specimens (TCGA, Pan-Cancer Atlas) from the cBioportal database (www.cbiopor- tal.org). FGF2, fibroblast growth factor 2; FGFR1, FGF receptor 1; CAFs, cancer-associated fibroblasts; NFs, normal fibroblasts; CM, conditioned medium; ns, not significantly different; FRS2, FGFR substrate 2; TCGA, The Cancer Genome Atlas; CPTAC, the Clinical Proteomic Tumor Analysis Consortium.

Article Snippet: For neutralization of FGF2 in the CM of CAFs, CM was pre-incubated with 25 μg/mL of human FGF2 antibody or its IgG control (R&D Systems, Inc., Minneapolis, MN, USA) for 1 hour at room temperature prior to use.

Techniques: Activation Assay, MTT Assay, Incubation, Phospho-proteomics, Western Blot, RNA Sequencing, Expressing

Journal: Journal of Cancer Prevention

Article Title: Nuclear Localization of Fibroblast Growth Factor Receptor 1 in Breast Cancer Cells Interacting with Cancer Associated Fibroblasts

doi: 10.15430/jcp.2022.27.1.68

Figure Lengend Snippet: Figure 2. Role of FGFR1 in Akt phosphorylation and breast cancer cell growth and progression. (A) MDA-MB-231 cells were transfected with scrambled or FGFR1 si-RNA for 24 hours. Cells were then incubated with 20 ng/mL of FGF2 for 15 minutes to measure phosphorylated FRS2α. (B) Mice were subjected to xenograft co-injecting with fibroblasts and MDA-MB-231 breast cancer cells. A complex collagen network was detected in H&E-stained tumors by an intense pink and in Masson’s trichrome stain by a blue stain (arrows). Stromal compartment was also detected by α-SMA immunostaining. Magnification, x100. Bars, 100 μm. (C) Phosphorylated Akt in the xenograft tumors was determined by Western blot analysis. *Sig- nificantly different between the groups compared (P < 0.05). (D) Enrichment plots of hallmark gene sets in the high FGFR1-expressing group. FGF2, fibroblast growth factor 2; FGFR1, FGF receptor 1; FRS2, FGFR substrate 2; α-SMA, alpha-smooth muscle actin; CONT, control; EMT, epithelial- mesenchymal transition.

Article Snippet: For neutralization of FGF2 in the CM of CAFs, CM was pre-incubated with 25 μg/mL of human FGF2 antibody or its IgG control (R&D Systems, Inc., Minneapolis, MN, USA) for 1 hour at room temperature prior to use.

Techniques: Phospho-proteomics, Transfection, Incubation, Staining, Immunostaining, Western Blot, Expressing, Control

Journal: Journal of Cancer Prevention

Article Title: Nuclear Localization of Fibroblast Growth Factor Receptor 1 in Breast Cancer Cells Interacting with Cancer Associated Fibroblasts

doi: 10.15430/jcp.2022.27.1.68

Figure Lengend Snippet: Figure 3. The involvement of FGF2-induced ROS generation in nuclear localization of FGFR1. (A) MDA-MB-231 cells were co-cultured with NFs or CAFs for 24 hours. MDA-MB-231 (5 x 10 3 cells) and NFs or CAFs (5 x 10 3 cells) were mixed prior to seeding and incubated for 24 hours. Immunocytochemical analysis was performed using anti-FGFR1 antibody. Cells were then stained with DAPI for detection of nuclei. Magnification, x100. Bars, 200 μm. (B) MDA-MB-231 cells were incubated with FGF2 for 1 hour. Immunocytochemical analysis was performed using anti-FGFR1 antibody. Cells were then stained with PI for detection of nuclei. Magnification, x100. Bars, 200 μm. (C) MDA-MB-231 cells were treated with 20 ng/ mL of FGF2 for 1 hour, followed by Western blot analysis of FGFR1 in cytosolic and nuclear extracts. Lamin B was used as a nuclear marker. *Sig- nificantly different between the groups compared (P < 0.05). (D, E) MDA-MD-231 cells were incubated with CAF-CM or FGF2 for 3 hours and 1 hour, respectively. After staining with DCF-DA for 30 minutes, fluorescent microscopic (D) or flow cytometric (E) analysis was performed to detect intracellu- lar ROS accumulation. Magnification, x40. (F) After pretreatment with NAC for 3 hours, cells were exposed to FGF2 for additional 1 hour. Nuclear ex- tracts were subjected to Western blot analysis to detect the presence of FGFR1 and Nrf2 in the nucleus. **Significantly different between the groups compared (P < 0.01). (G) MDA-MB-231 cells were exposed to FGF2 (20 ng/mL) for 1 hour. Cell lysates were subjected to immunoprecipitation using CBP antibody for 16 hours followed by immunoblotting with. FGFR1 or Nrf2 antibody. FGF2, fibroblast growth factor 2; FGFR1, FGF receptor 1; ROS, reactive oxygen species; CAFs, cancer-associated fibroblasts; CM, conditioned medium; NFs, normal fibroblasts; DAPI, 4′,6-diamidino-2-phenylindole; PI, propidium iodide; CONT, cotrol; DCF-DA, 2’,7’-dichlorodihydrofluorescein diacetate; NAC, N-acetylcysteine; CBP, CREB-binding protein.

Article Snippet: For neutralization of FGF2 in the CM of CAFs, CM was pre-incubated with 25 μg/mL of human FGF2 antibody or its IgG control (R&D Systems, Inc., Minneapolis, MN, USA) for 1 hour at room temperature prior to use.

Techniques: Cell Culture, Incubation, Staining, Western Blot, Marker, Immunoprecipitation, Binding Assay

Journal: Journal of Cancer Prevention

Article Title: Nuclear Localization of Fibroblast Growth Factor Receptor 1 in Breast Cancer Cells Interacting with Cancer Associated Fibroblasts

doi: 10.15430/jcp.2022.27.1.68

Figure Lengend Snippet: Figure 4. Possible association between nuclear FGFR1 and Nrf2. (A) TNBC patient cohorts were validated based on the mean expression value of the indicated single genes (FGFR1 or NFE2L2) or as a signature of two genes together and patient survival was analyzed (n = 255). (B, C) MDA- MB-231 cells were transfected with scrambled or Nrf2 si-RNA for 24 hours. Cells were then incubated with 20 ng/mL of FGF2 for 3 hours. The mRNA (B) and protein (C) expression of cyclin D1 was assessed by RT-PCR and Western blot analyses, respectively. The expression of cyclin D1 was mea- sured by RT-PCR (B) and Western blot (C) analyses. (D) In tumor microenvironment, fibroblasts are activated to form CAFs, which secrete FGF2. CAF-derived FGF2 could induces nuclear translocation as well as de novo synthesis of FGFR1, ultimately contributing to cancer cell proliferation, mi- gration and tumor growth. While membrane bound FGFR1 may translocate to nucleus as a complex with FGF2 which has nuclear localization signal (NLS), the complex is likely rather to stimulate the intracellular signaling via FRS2α, which induces transcription of FGFR-1 gene. On the other hand, newly synthesized FGFR-1 is speculated to enter the nucleus as a complex with a cargo protein harboring NLS. FGFR-1 is translocated to the inner nuclear membrane through the nuclear pore complexes (NPCs), which is regulated by importin β. FGF2, fibroblast growth factor 2; FGFR1, FGF receptor 1; TNBC, triple negative breast cancer; HR, hazard ratio; CAFs, cancer-associated fibroblasts; ER, endoplasmic reticulum; FRS2, FGFR substrate 2; CBP, CREB-binding protein.

Article Snippet: For neutralization of FGF2 in the CM of CAFs, CM was pre-incubated with 25 μg/mL of human FGF2 antibody or its IgG control (R&D Systems, Inc., Minneapolis, MN, USA) for 1 hour at room temperature prior to use.

Techniques: Expressing, Transfection, Incubation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Derivative Assay, Translocation Assay, Membrane, Synthesized, Binding Assay

Journal: Hepatology (Baltimore, Md.)

Article Title: CD151 modulates expression of matrix metalloproteinase 9 and promotes neoangiogenesis and progression of hepatocellular carcinoma.

doi: 10.1002/hep.23661

Figure Lengend Snippet: Fig. 1. Expression of CD151 and MMP9 in HCC cell lines. The expression of CD151 and MMP9 in the HCC cell lines HCCLM3, MHCC97-L, PLC/PRF/5, Hep3B, and HepG2 (A) at the mRNA level via qRT-PCR and (B) at the protein level. The results showed that CD151high HCC cell lines possessed a high level of expression of MMP9. (A,D) The aforementioned results were validated by shRNA-mediated repression of CD151. (B,C) The bioactivity and concentration of MMP9 in the supernatant were assayed by gelatin zymography (lower panel) and ELISA, respectively. The results were the same as those presented previously. (D,E) The bioactivity and concentration of MMP9 and MMP2 in the supernatants from HCCLM3-mock (HCCLM3-pGCSIL-GFP) and shRNA-CD151-HCCLM3 cells precoated or not precoated with laminin 5 (10 lg/mL) were revealed by gelatin zymography. Up-regulation of MMP9 in the supernatant in HCCLM3-mock cells treated with laminin 5 was compared to that in HCCLM3- mock cells. However, no difference in the secretion of MMP9 was found between shRNA-CD151-HCCLM3 cells and shRNA-CD151-HCCLM3 cells treated with laminin 5. Similarly, no difference in the concentration of MMP2 was noted between HCCLM3-mock and shRNA-CD151-HCCLM3 cells treated with laminin 5 or not treated. Abbreviation: GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: The type IV collagenase activity and the concentrations of MMP9 in a conditioned medium were determined by gelatin zymography6 with a human MMP9 ELISA kit (R&D Systems); the concentrations of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) were determined with human VEGF and bFGF ELISA kits (R&D Systems), respectively, essentially as described previously.23 All experiments were performed in triplicate.

Techniques: Expressing, Quantitative RT-PCR, shRNA, Concentration Assay, Zymography, Enzyme-linked Immunosorbent Assay

Journal: Hepatology (Baltimore, Md.)

Article Title: CD151 modulates expression of matrix metalloproteinase 9 and promotes neoangiogenesis and progression of hepatocellular carcinoma.

doi: 10.1002/hep.23661

Figure Lengend Snippet: Fig. 2. Role of the PI3K/Akt/GSK-3b/Snail signal in the up-regulation of MMP9 mediated by CD151 in HCC cells. (A) The phosphorylation level of AktSer473 and GSK-3bSer9 and the expression of Snail and MMP9 were reduced significantly in shRNA-CD151-HCCLM3 cells versus HCCLM3-mock (HCCLM3-pGCSIL-GFP). (B) In comparison with Hep3B-mock (pcDNA3-Hep3B), when the expression of CD151 in Hep3B cells was up-regulated through the transfection of pcDNA3/CD151 plasmids, the phosphorylation level of AktSer473 and GSK-3bSer9 and the expres- sion of Snail and MMP9 were enhanced correspondingly. (C) When HCCLM3 cells were supplemented with 50 lM LY294002 (a specific PI3K inhibitor), the phosphorylation level of GSK-3bSer9 and the expression of Snail and MMP9 were reduced, whereas the expression of GSK-3b was enhanced. (D) This was similar to the situation for shRNA-Akt-HCCLM3 cells. (E) When U0126 (25 lmol/L) was used to block ERK1/2-MAPK in HCCLM3 cells, the expression of MMP9 was slightly reduced. However, the expression of GSK-3b and Snail remained unchanged. (F) After the expression of GSK-3 in HCCLM3 cells was down-regulated by siRNA, the expression of CD151 and Akt remained untouched, but the expression of Snail and MMP9 and the phosphorylation level of AktSer473 were slightly up-regulated in comparison with HCCLM3-control (a negative control mismatch sequence). (G) Similar results were also found in HCCLM3 cells blocked with GSK-3 inhibitor XV (0.6 nM). (H) After constitutively active GSK-3bS9A plasmids were transfected into HCCLM3 cells, the expression of Snail and MMP9 and the phosphorylation level of AktSer473

Article Snippet: The type IV collagenase activity and the concentrations of MMP9 in a conditioned medium were determined by gelatin zymography6 with a human MMP9 ELISA kit (R&D Systems); the concentrations of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) were determined with human VEGF and bFGF ELISA kits (R&D Systems), respectively, essentially as described previously.23 All experiments were performed in triplicate.

Techniques: Phospho-proteomics, Expressing, shRNA, Comparison, Transfection, Blocking Assay, Control, Negative Control, Sequencing

Journal: Hepatology (Baltimore, Md.)

Article Title: CD151 modulates expression of matrix metalloproteinase 9 and promotes neoangiogenesis and progression of hepatocellular carcinoma.

doi: 10.1002/hep.23661

Figure Lengend Snippet: Fig. 3. Role of CD151 in vascular remodeling and neoangiogenesis via the modulation of MMP9. (A) The Matrigel assay was used to determine the ability of HUVECs to form capillaries when cultured with the supernatant collected from different cells. A closely knit network of capillary-like structures was found in the HCCLM3 group and in the HCCLM3-mock group. However, the ability of HUVECs to form capillaries was significantly impaired when they were cultured with the supernatant collected from shRNA-CD151-HCCLM3, Hep3B, and shRNA-MMP9-HCCLM3. (B) Thoracic aortic rings were embedded in Matrigel and incubated for 4 days. Phase-contrast images are shown of branching vessel-like structures in the HCCLM3, HCCLM3-mock, shRNA-CD151-HCCLM3, Hep3B, and shRNA-MMP9-HCCLM3 groups. (D) The lengths of the vessel-like structures were measured, and the results showed that more neoangiogenesis occurred in HCCLM3 and HCCLM3-mock cells than in Hep3B, shRNA-CD151- HCCLM3, and shRNA-MMP9-HCCLM3. (C) The mouse cornea micropocket angiogenesis model was developed, and this was followed by the estab- lishment of subcutaneous xenografts of HCCLM3, HCCLM3-mock, shRNA-CD151-HCCLM3, Hep3B, and shRNA-MMP9-HCCLM3. (E) The results also supported the consistency between neoangiogenesis and the expression of CD151 and MMP9. The scale bar represents 100 lm.

Article Snippet: The type IV collagenase activity and the concentrations of MMP9 in a conditioned medium were determined by gelatin zymography6 with a human MMP9 ELISA kit (R&D Systems); the concentrations of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) were determined with human VEGF and bFGF ELISA kits (R&D Systems), respectively, essentially as described previously.23 All experiments were performed in triplicate.

Techniques: Matrigel Assay, Cell Culture, shRNA, Incubation, Expressing

Journal: Hepatology (Baltimore, Md.)

Article Title: CD151 modulates expression of matrix metalloproteinase 9 and promotes neoangiogenesis and progression of hepatocellular carcinoma.

doi: 10.1002/hep.23661

Figure Lengend Snippet: Fig. 4. CD151-dependent neoangiogenesis in a spontaneous metastasis assay. Samples of HCCLM3, HCCLM3-mock, shRNA-CD151-HCCLM3, Hep3B, and shRNA-MMP9-HCCLM3 cells (8 106) were inoculated into nude mice subcutaneously. (A) All groups formed tumors successfully. (B) After identification by H&E staining, (C) neoangiogenesis in xenografts was assayed with CD34 staining. HCCLM3-derived and HCCLM3- mock–derived xenografts presented with more neoangiogenesis than the shRNA-CD151-HCCLM3, Hep3B, and shRNA-MMP9-HCCLM3 groups. (D- F) Metastasis assays revealed that the pulmonary metastasis rates and metastatic tumor clusters per mouse were 100% (5/5) and 132.8 6 4.0 in the HCCLM3 group and 100% (5/5) and 134.0 6 8.0 in the HCCLM3-mock group, whereas they were 20% (1/5) and 33.6 6 19.6 in the shRNA-CD151-HCCLM3 group, 20% (1/5) and 5.6 6 12.5 in the Hep3B group, and 20% (1/5) and 24.0 6 22.8 in the shRNA-MMP9- HCCLM3 group, with statistically significant differences (P < 0.01). (D) Fluorescence stereomicroscopy showed that the numbers of lung meta- static loci in HCCLM3-mock (arrow) were much greater than those in HCCLM3-shRNA. (E) Serial sections also showed the aforementioned results. (F) Magnification of the circle in panel E. The scale bar represents 100 lm. Abbreviation: H&E, hematoxylin and eosin.

Article Snippet: The type IV collagenase activity and the concentrations of MMP9 in a conditioned medium were determined by gelatin zymography6 with a human MMP9 ELISA kit (R&D Systems); the concentrations of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) were determined with human VEGF and bFGF ELISA kits (R&D Systems), respectively, essentially as described previously.23 All experiments were performed in triplicate.

Techniques: shRNA, Staining, Derivative Assay, Fluorescence

Journal: Hepatology (Baltimore, Md.)

Article Title: CD151 modulates expression of matrix metalloproteinase 9 and promotes neoangiogenesis and progression of hepatocellular carcinoma.

doi: 10.1002/hep.23661

Figure Lengend Snippet: Fig. 5. Coexpression of CD151, MMP9, and MVD in HCC samples. (A) The diagnosis of primary HCC in a tissue microassay was performed with H&E staining (left panel). Immunohistochemical double staining was used for CD151 and MVD-CD34 and for MMP9 and MVD-CD34. The immuno- reactivity of CD151 and CD34 was located in the plasma membrane of the tumor cells (brown) and capillary-like vessels (red; middle panel), whereas MMP9 was located in HCC cells and stroma fibroblasts (brown; right panel). Representative HCC tumor samples showed that the expression of CD151, MMP9, and MVD-CD34 was diverse. (A,B) High expression of CD151, MMP9, and MVD-CD34 together. (C,D) Low-level expression of CD151, MMP9, and MVD-CD34 together. (E) Significantly positive correlations were found between CD151 and MMP9 in cancerous tissues (r ¼ 0.663, P < 0.001; left panel) and between CD151 and MVD-CD34 expression (r ¼ 0.610, P < 0.001; right panel) with a scatter plot. (F) RT-PCR (top panel) and Immunoblotting (bottom panel) validated the consistency between overexpression of CD151 and high-level expression of MMP9. The scale bar represents 100 lm. Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; H&E, hematoxylin and eosin.

Article Snippet: The type IV collagenase activity and the concentrations of MMP9 in a conditioned medium were determined by gelatin zymography6 with a human MMP9 ELISA kit (R&D Systems); the concentrations of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) were determined with human VEGF and bFGF ELISA kits (R&D Systems), respectively, essentially as described previously.23 All experiments were performed in triplicate.

Techniques: Biomarker Discovery, Staining, Immunohistochemical staining, Double Staining, Clinical Proteomics, Membrane, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Over Expression

Journal: Hepatology (Baltimore, Md.)

Article Title: CD151 modulates expression of matrix metalloproteinase 9 and promotes neoangiogenesis and progression of hepatocellular carcinoma.

doi: 10.1002/hep.23661

Figure Lengend Snippet: Fig. 6. Prognostic roles by Kaplan-Meier analysis. Patients were classified into three groups according to their CD151, MMP9, and MVD-CD34 density: group I, high expression of all three markers; group II, high expression of one or two of the three markers; and group III, low expression of all three markers. Kaplan-Meier analysis revealed that HCC patients with overexpression of CD151, MMP9, and MVD-CD34 together presented with (A) the worst OS and (B) the highest cumulative recurrence rate.

Article Snippet: The type IV collagenase activity and the concentrations of MMP9 in a conditioned medium were determined by gelatin zymography6 with a human MMP9 ELISA kit (R&D Systems); the concentrations of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) were determined with human VEGF and bFGF ELISA kits (R&D Systems), respectively, essentially as described previously.23 All experiments were performed in triplicate.

Techniques: Expressing, Over Expression

Journal: Hepatology (Baltimore, Md.)

Article Title: CD151 modulates expression of matrix metalloproteinase 9 and promotes neoangiogenesis and progression of hepatocellular carcinoma.

doi: 10.1002/hep.23661

Figure Lengend Snippet: Fig. 7. Working model: CD151 overexpression facilitates the expres- sion of MMP9. Cooperation between CD151 overexpression, laminin 5, and integrin results in the enhancement of phosphorylated AKTser473

Article Snippet: The type IV collagenase activity and the concentrations of MMP9 in a conditioned medium were determined by gelatin zymography6 with a human MMP9 ELISA kit (R&D Systems); the concentrations of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) were determined with human VEGF and bFGF ELISA kits (R&D Systems), respectively, essentially as described previously.23 All experiments were performed in triplicate.

Techniques: Over Expression

Journal: Cancer Immunology Research

Article Title: Granulocyte–Macrophage Colony-Stimulating Factor Influence on Soluble and Membrane-Bound ICOS in Combination with Immune Checkpoint Blockade

doi: 10.1158/2326-6066.CIR-22-0702

Figure Lengend Snippet: Comparison between human ICOS full length and splice variant. A, Schematic representation of full length and spliced variant of human ICOS. The full length of ICOS consists of 5 exons. The gray boxes denote untranslated regions, and the black boxes denote coding sequences. The transmembrane domain (TM) is located in exon 3 and labeled with a green bar. The splicing region is shown in red. B, Comparison of cDNA nucleotide sequences, amino acid sequences, and 5′UTR sequences between ICOS-FL and the splice variant. Nucleotide sequences of cDNA for ICOS-FL (b1) and the splice variant of ICOS (b4) are represented; spliced region of ICOS is shown in red and delineated by red brackets; the nucleotides and amino acids of TM are shown in green. Asterisk indicates the stop codon. Amino acid sequences of full length of ICOS (b2) and the splice variant of ICOS (b5) are shown. The underline depicts additional differences in amino acid sequence compared with ICOS-FL. “Stop” represents a stop codon. 5′UTR sequences of ICOS-FL (b3) and the splice variant of ICOS (b6) are shown. The 5′UTR of the ICOS-SV lacks 32 nucleotides compared with the 5′UTR of ICOS-FL shown in red and delineated by red brackets. C, Schematic diagram of ICOS-FL and the ICOS-SV protein. Thirty-two out of 38 amino acids in the intracellular domain are spliced, as shown in red.

Article Snippet: To evaluate soluble ICOS-SVs in the sera of melanoma patients or cell culture supernatants, the Human ICOS DuoSet ELISA kit (DY169, R&D Systems) was used according to the manufacturer's protocol.

Techniques: Comparison, Variant Assay, Labeling, Sequencing

Journal: Cancer Immunology Research

Article Title: Granulocyte–Macrophage Colony-Stimulating Factor Influence on Soluble and Membrane-Bound ICOS in Combination with Immune Checkpoint Blockade

doi: 10.1158/2326-6066.CIR-22-0702

Figure Lengend Snippet: Secretion of the ICOS-SV from ICOS-SV–overexpressing cells. A, RT-PCR to detect the ICOS-SV in human T cells. Total RNA was isolated from purified T cells in PBMCs of a healthy donor. Expression of the ICOS variant was examined by RT-PCR with specific primers. RT-PCR of ICOS-FL is shown as a control. B, DNA sequencing of ICOS-SV cDNA from human T cells. The cDNA of the PCR product was cloned into a TOPO TA vector. The ICOS-SV was confirmed by DNA sequencing using an M13 forward primer. Splicing point is shown by the red arrow. C, DNA sequencing of ICOS-FL cDNA is shown as a control. No splicing occurs in the region corresponding to the position in ( B ), as shown in blue. D, Expression of ICOS-SV and ICOS-FL in 293T cells determined by immunoblot. 293T cells were transduced with recombinant GFP retroviral vector encoding either ICOS-SV or ICOS-FL as a positive control. The parental cells and the GFP-empty vector–transduced cells were used as negative controls. Sample loading was normalized to actin. E, Cell culture supernatants were collected from the cells described in ( A ). Secreted ICOS-SV was examined by ELISA. Two-tailed unpaired t test was used to compare ICOS secretion between the two groups. ***, P < 0.001. Standard deviation of the mean (SD) is shown. F, To validate the secreted ICOS variant protein in the cell supernatant, immunoprecipitation, SDS-PAGE, and immunoblotting assays were performed. Sample loading was normalized to cell numbers.

Article Snippet: To evaluate soluble ICOS-SVs in the sera of melanoma patients or cell culture supernatants, the Human ICOS DuoSet ELISA kit (DY169, R&D Systems) was used according to the manufacturer's protocol.

Techniques: Reverse Transcription Polymerase Chain Reaction, Isolation, Purification, Expressing, Variant Assay, Control, DNA Sequencing, Clone Assay, Plasmid Preparation, Western Blot, Transduction, Recombinant, Retroviral, Positive Control, Cell Culture, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Standard Deviation, Immunoprecipitation, SDS Page

Journal: Cancer Immunology Research

Article Title: Granulocyte–Macrophage Colony-Stimulating Factor Influence on Soluble and Membrane-Bound ICOS in Combination with Immune Checkpoint Blockade

doi: 10.1158/2326-6066.CIR-22-0702

Figure Lengend Snippet: Suppressive effects of sICOS-SV on the costimulation of T cells. A, CHO-K1 cells were transduced with either ICOSL-expressing lentiviral vector or GFP-empty vector (CHO-ICOSL − ) as a negative control. Expression of ICOSL on the cell surface of CHO-K1 was examined by flow cytometry using anti-ICOSL (open histogram with solid line) or lentivirus GFP-empty vector (open histogram with dotted line). Isotype antibody was used as a negative control (filled histogram). B, ICOSL-expressing CHO-K1 cells were stained with ICOS-SV Ig (red line), control IgG Ig (blue line), or PD-1 Ig (orange line) and analyzed by flow cytometry. Both soluble control Ig and PD-1 Ig were used as negative controls. GFP-empty vector-expression CHO-K1 cells stained with ICOS-SV Ig (green line) were used as another negative control for the binding assay. Isotype antibody control was used as a negative control (gray filled histogram). C, CD154 expression was determined by FACS analysis. Isotype control antibody is shown in gray. Activated T cells treated with a suboptimal dose of anti-CD3 were incubated with control Ig + CHO-ICOSL − cells (CHO cells transduced with GFP-empty vector, blue), control Ig + CHO-ICOSL + cells (green), PD-1 Ig + CHO-ICOSL + cells (red), or ICOS-SV Ig + CHO-ICOSL + cells (yellow). Both control Ig and PD-1 Ig were used as negative controls. D, Two-tailed unpaired t test was used to compare expression of CD154 between two groups. **, P < 0.01; ***, P < 0.001. Standard deviation of the mean (SD) is shown. E, CD69 expression was determined by FACS analysis. Isotype control antibody is shown in gray. Activated T cells treated with a suboptimal dose of anti-CD3 were incubated with control Ig + CHO-ICOSL − cells (CHO cells transduced with GFP-empty vector, blue), control Ig + CHO-ICOSL + cells (green), PD-1 Ig + CHO-ICOSL + cells (red), or ICOS-SV Ig + CHO-ICOSL + cells (yellow). Both control Ig and PD-1 Ig were used as negative controls. F, Two-tailed unpaired t test was used to compare expression of CD69 between the two groups. **, P < 0.01; ***, P < 0.001. Standard deviation of the mean (SD) is shown. G, CHO cells transduced with GFP-empty vector (CHO-ICOSL − ) were used as negative controls. Sample loading was normalized to total pan-Akt. H, T-cell proliferation was determined by a [ 3 H]-TdR thymidine incorporation assay. CHO cells transduced with empty vector (CHO-ICOSL − ) were used as negative controls. Two-tailed unpaired t test was used to compare 3 H uptake between the two groups, respectively. *, P < 0.1. Standard deviation of the mean (SD) is shown. I, Schematic diagram of ICOS/ICOSL costimulatory T-cell proliferation, as well as the blocking function of the sICOS-SV. Depicted are the cytoplasmic tail sequences of ICOS-FL and sICOS-SV isoforms. The YMFM Src Homology 2 (SH2) binding motif in the cytoplasmic tail of the ICOS-FL is highlighted in pink. Upon ICOS engagement by the ICOSL on CHO cells, the unique YMFM motif recruits a p85a and a p50a subunits of PI3K, resulting in the elevated phosphorylation of Akt, thereby inducing PI3K activity. In contrast, the ICOS-SV, a truncated isoform lacking the YMFM motif in its cytoplasmic tail, cannot elicit phosphorylation of Akt. Consequently, it fails to promote T-cell proliferation. The secreted ICOS-SV (red) competes with membrane-bound ICOS for binding to ICOSL, thereby blocking the interaction between ICOSL and membrane ICOS. As a result, the sICOS-SV suppresses phosphorylation of Akt and T-cell proliferation, leading to the inhibition of T-cell immunity. The diagram was created with BioRender.com. All data shown are representative of at least 2 independent experiments.

Article Snippet: To evaluate soluble ICOS-SVs in the sera of melanoma patients or cell culture supernatants, the Human ICOS DuoSet ELISA kit (DY169, R&D Systems) was used according to the manufacturer's protocol.

Techniques: Transduction, Expressing, Plasmid Preparation, Negative Control, Flow Cytometry, Staining, Control, Binding Assay, Incubation, Two Tailed Test, Standard Deviation, Thymidine Incorporation Assay, Blocking Assay, Phospho-proteomics, Activity Assay, Membrane, Inhibition

Journal: Cancer Immunology Research

Article Title: Granulocyte–Macrophage Colony-Stimulating Factor Influence on Soluble and Membrane-Bound ICOS in Combination with Immune Checkpoint Blockade

doi: 10.1158/2326-6066.CIR-22-0702

Figure Lengend Snippet: sICOS from melanoma patients inhibits T-cell activation and proliferation induced by GM-CSF–driven DCs in MLRs. A, CD4 + T cells (responders) were stimulated by allogeneic GM-CSF–driven MoDCs (stimulators: negative control DCs, DCs generated by GM-CSF/IL4 + anti-IgG, or DCs generated by GM-CSF/IL4 + anti-CD116) in MLRs. T cells were assessed for activation via flow cytometry using anti-ICOS (blue), anti-GITR (green), or anti-CD25 (gray). Isotype control antibodies were used as negative controls. B, Statistical analysis of the percentage of CD4 + ICOS + , CD4 + GITR + , and CD4 + CD25 + T-cell populations from different groups as described above. C, Soluble ICOS levels were determined by ELISA using supernatants from the MLRs described above. D – E, Serum from sICOS-high and sICOS-negative patients were added to the MLRs, and T-cell proliferation ( D ) and CD69 expression ( E ) were evaluated. Additionally, sICOS was depleted from sICOS-high serum to assess its effects on T cells. F, Statistical analysis of the percentage of CD4 + CD69 + T-cell populations from different groups as described above. All data shown are representative of at least 2 independent experiments. Two-tailed unpaired t test was used between the two groups. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant ( P > 0.05). Standard deviation of the mean (SD) is shown.

Article Snippet: To evaluate soluble ICOS-SVs in the sera of melanoma patients or cell culture supernatants, the Human ICOS DuoSet ELISA kit (DY169, R&D Systems) was used according to the manufacturer's protocol.

Techniques: Activation Assay, Negative Control, Generated, Flow Cytometry, Control, Enzyme-linked Immunosorbent Assay, Expressing, Two Tailed Test, Standard Deviation