Journal: eLife

Article Title: Recognition of tumor cells by Dectin-1 orchestrates innate immune cells for anti-tumor responses

doi: 10.7554/eLife.04177

Figure Lengend Snippet: ( A ) Selective contribution of IRF5 in the suppression of lung metastasis of B16F1 cells. Number of metastasized colonies in lungs from wild-type (WT), Irf3 −/− , Irf5 −/− , or Irf7 −/− mice 14 days after intravenous injection of 1 × 10 6 of B16F1 cells. Means are indicated as black bars. *p < 0.05 by Student's t test. ( B ) Representative images of lungs from WT or Irf5 −/− mice 14 days after intravenous injection of 2 × 10 6 of B16F1 cells. ( C ) In vitro killing assay of immune cells from WT or Irf5 −/− mice against B16F1 cells. Whole splenocytes (left panel), purified NK cells (middle panel), or NK-depleted splenocytes (right panel) from WT or Irf5 −/− mice are mixed with 51 Cr-labeled target B16F1 cells at the indicated ratios. 4 hr later, 51 Cr radioactivity released from target cells was monitored. E/T: effector/target cell ratio. ( D ) Purified NK cells (WT; 1 × 10 5 cells) without or with 1 × 10 5 , 2 × 10 5 , or 4 × 10 5 of WT splenic CD11c + , CD11b + , T, or B cells were subjected to in vitro killing assay for B16F1 cells. Target cell lysis was measured by co-culturing target cells and myeloid cells, with (total values) or without NK cells (background values), and background values were subtracted from the total values. Each background lysis was less than 6% of maximum release. The calculated percentage of cytotoxicity was represented as Net lysis (%). In all in vitro killing assays, 1 × 10 4 of 51 Cr-labeled B16F1 cells were used ( C and D ). All in vitro killing assays were performed at least three times with high reproducibility. Represented as means ± SD. DOI: http://dx.doi.org/10.7554/eLife.04177.003

Article Snippet: Splenic CD11b + , CD11c + , B, or T cells were purified using CD11b or CD11c MicroBeads, B cell isolation kit, or pan T cell isolation kit II, respectively (Miltenyi Biotec, Germany).

Techniques: Injection, In Vitro, Purification, Labeling, Radioactivity, Lysis

Journal: eLife

Article Title: Recognition of tumor cells by Dectin-1 orchestrates innate immune cells for anti-tumor responses

doi: 10.7554/eLife.04177

Figure Lengend Snippet: Requirement of IRF5 in myeloid cells for the enhancement of NK cell's in vitro killing activity. In vitro killing activities of purified NK cells (WT; 1 × 10 5 cells) against B16F1 cells were monitored in the absence or presence of 1 × 10 5 , 2 × 10 5 , or 4 × 10 5 of WT or Irf5 −/− splenic CD11b + (left panel) or CD11c + (right panel) cells. The percentage of cytotoxicity was calculated as described in the legend of and represented as Net lysis (%). Each background lysis was less than 6% of maximum release. Represented as means ± SD. *p < 0.05 by Student’s t test. In in vitro killing assays, 1 × 10 4 of 51 Cr-labeled B16F1 cells were used. All in vitro killing assays were performed at least three times and the results were highly reproducible. DOI: http://dx.doi.org/10.7554/eLife.04177.006

Article Snippet: Splenic CD11b + , CD11c + , B, or T cells were purified using CD11b or CD11c MicroBeads, B cell isolation kit, or pan T cell isolation kit II, respectively (Miltenyi Biotec, Germany).

Techniques: In Vitro, Activity Assay, Purification, Lysis, Labeling

Journal: eLife

Article Title: Recognition of tumor cells by Dectin-1 orchestrates innate immune cells for anti-tumor responses

doi: 10.7554/eLife.04177

Figure Lengend Snippet: The effect of various myeloid cells on the enhancement of NK cell killing activities. In vitro killing activities of purified NK cells (WT; 1 × 10 5 cells) were monitored in the absence or presence of 1 × 10 5 , 2 × 10 5 , or 4 × 10 5 of splenic CD8 + CD11c + (left panel), CD8 – CD11c + (middle panel), or CD11c − CD11b + (right panel) cells. CD11c − CD11b + cells were purified from diphtheria toxin-treated CD11c-DTR mice. The percentage of cytotoxicity was calculated as described in the legend of and represented as Net lysis (%). Each background lysis was less than 5% of maximum release. Represented as means ± SD. 1 × 10 4 of 51 Cr-labeled B16F1 cells were used as target cells. All in vitro killing assays were performed at least three times and the results were highly reproducible. DOI: http://dx.doi.org/10.7554/eLife.04177.007

Article Snippet: Splenic CD11b + , CD11c + , B, or T cells were purified using CD11b or CD11c MicroBeads, B cell isolation kit, or pan T cell isolation kit II, respectively (Miltenyi Biotec, Germany).

Techniques: In Vitro, Purification, Lysis, Labeling

Journal: eLife

Article Title: Recognition of tumor cells by Dectin-1 orchestrates innate immune cells for anti-tumor responses

doi: 10.7554/eLife.04177

Figure Lengend Snippet: ( A ) Nuclear translocation of IRF5 in WT or Dectin-1 −/− splenocytes (5 × 10 7 cells) co-incubated with B16F1 cells (5 × 10 5 or 1 × 10 6 cells) or stimulated with curdlan (30 µg/ml) for 6 hr. Nuclear protein fraction from the culture was analyzed by immunoblotting for IRF5 and USF-2. USF-2 was used as a nuclear marker protein. ( B ) In vitro killing activity of whole splenocytes from WT or Dectin-1 −/− mice against B16F1 cells. ( C ) In vitro killing activity of purified NK cells (WT; 1 × 10 5 cells) against B16F1 cells was assessed in the absence or presence of 1 × 10 5 , 2 × 10 5 , or 4 × 10 5 of WT or Dectin-1 −/− splenic CD11b + (left panel) or CD11c + (right panel) cells. The percentage of cytotoxicity was calculated as noted in the legend of and represented as Net lysis (%). Each background lysis was less than 6% of maximum release. In all in vitro killing assays, 1 × 10 4 of 51 Cr-labeled B16F1 cells were used ( B and C ). All in vitro killing assays were performed at least three times with high reproducibility. Represented as means ± SD. *p < 0.05 by Student's t test. Of note, we could not found mRNA induction for typical inflammatory and cytotoxic mediators in the co-culture system, wherein the ratio of B16F1 cells, DCs, and NK cells is 1:30:10 . As such, from these analyses, it seems unlikely that Dectin-1 signaling in DCs affects the expression of these molecules in NK cells. ( D ) Number of metastasized colonies in lungs from WT or Dectin-1 −/− mice intravenously injected with 1 × 10 6 of B16F1 cells. Means are indicated as black bars. *p < 0.05 by Student's t test. ( E ) Representative images of lungs from WT or Dectin-1 −/− mice 14 days after intravenous injection of 2 × 10 6 of B16F1 cells. DOI: http://dx.doi.org/10.7554/eLife.04177.008

Article Snippet: Splenic CD11b + , CD11c + , B, or T cells were purified using CD11b or CD11c MicroBeads, B cell isolation kit, or pan T cell isolation kit II, respectively (Miltenyi Biotec, Germany).

Techniques: Translocation Assay, Incubation, Western Blot, Marker, In Vitro, Activity Assay, Purification, Lysis, Labeling, Co-Culture Assay, Expressing, Injection

Journal: eLife

Article Title: Recognition of tumor cells by Dectin-1 orchestrates innate immune cells for anti-tumor responses

doi: 10.7554/eLife.04177

Figure Lengend Snippet: ( A ) In vitro killing activity of purified NK cells from WT or Dectin-1 −/− mice against B16F1 cells. Represented as means ± SD. E/T: effector/target cell ratio. In in vitro killing assays, 1 × 10 4 of 51 Cr-labeled B16F1 cells were used. In vitro killing assays were performed at least three times, and the results were highly reproducible. ( B ) qRT-PCR analysis of Dectin-1 mRNA in WT splenic CD11b + cells, CD11c + cells and NK cells, and MEFs. Results are presented as relative to the expression of Gapdh mRNA. Represented as means ± SD. ND, none detected. ( C ) Expression levels of Dectin-1 mRNA in myeloid cells from liver and lungs. CD11b + F4/80 + cells and CD11c + cells from liver and lung were isolated by cell sorting. Results are presented as relative to the expression of Gapdh mRNA. Represented as means ± SD. DOI: http://dx.doi.org/10.7554/eLife.04177.011

Article Snippet: Splenic CD11b + , CD11c + , B, or T cells were purified using CD11b or CD11c MicroBeads, B cell isolation kit, or pan T cell isolation kit II, respectively (Miltenyi Biotec, Germany).

Techniques: In Vitro, Activity Assay, Purification, Labeling, Quantitative RT-PCR, Expressing, Isolation, FACS

Journal: eLife

Article Title: Recognition of tumor cells by Dectin-1 orchestrates innate immune cells for anti-tumor responses

doi: 10.7554/eLife.04177

Figure Lengend Snippet: The sDectin-1 binding to splenic CD11b + cells, CD11c + cells, NK cells, liver cells, or lung cells were examined as described in . DOI: http://dx.doi.org/10.7554/eLife.04177.018

Article Snippet: Splenic CD11b + , CD11c + , B, or T cells were purified using CD11b or CD11c MicroBeads, B cell isolation kit, or pan T cell isolation kit II, respectively (Miltenyi Biotec, Germany).

Techniques: Binding Assay

Journal: eLife

Article Title: Recognition of tumor cells by Dectin-1 orchestrates innate immune cells for anti-tumor responses

doi: 10.7554/eLife.04177

Figure Lengend Snippet: ( A ) Effect of the supernatant of myeloid cells after incubation with tumor cells. NK cell killing activity against B16F1 cells was assessed in the presence of supernatants from the co-culture of B16F1 cells with splenic CD11b + cells (left panel) or CD11c + cells (right panel). Represented as means ± SD. ( B ) Effect of antibodies against cytokines on NK cell-mediated killing activity. In vitro killing activity of purified NK cells (WT; 1 × 10 5 cells) against B16F1 cells in the presence of 3 × 10 5 of splenic CD11c + cells was assessed without or with neutralizing antibodies for type I IFNs or IL12p40. ( C ) Induction of mRNAs in DCs co-cultured with B16F1 cells via Dectin-1 signaling. WT or Dectin-1 −/− splenic CD11c + cells (3 × 10 5 cells) were co-cultured with B16F1 cells (1 × 10 4 cells). Total RNA from those cells was then isolated at time zero and 4 hr after the co-culture and then subjected to microarray analysis. We first identified genes for which mRNA is induced more than two-fold in WT DCs co-cultured with B16F1 cells and then, of those, selected the genes whose mRNA levels are increased more than twofold in WT 4 hr sample compared to Dectin-1 −/− 4 hr sample. Those selected genes are listed in the order of fold change (WT 4 hr/ Dectin-1 −/− 4 hr) . Genes encoding a membrane-bound protein are listed. ( D ) Induction of Fam26f ( Inam ) mRNA by the Dectin-1-IRF5 pathway. The expression levels of Inam mRNA were monitored by qRT-PCR analysis of total RNA from splenic CD11c + cells (WT, Irf5 −/− , or Dectin-1 −/− ; 3 × 10 5 cells) co-cultured with B16F1 cells (1 × 10 4 cells) for 4 hr as described in ‘Materials and methods’. Results are presented relative to the expression of Gapdh mRNA. Represented as means ± SD. *p < 0.05 by Student's t test. NS, not significant. Although the IL15 cytokine system is known to promote growth and activity of NK cells, Il15 and Il15ra mRNA expression levels were affected only marginally in the Dectin-1 −/− DCs . The results suggest that this cytokine system will not be involved in this particular experimental setting. ( E ) Effect of INAM expression in DCs on the enhancement of NK cell killing activity. Purified NK cells (WT; 1 × 10 5 cells) were mixed with increasing amounts (1 × 10 4 , 3 × 10 4 , and 1 × 10 5 cells) of INAM-transduced WT BMDCs (BMDC-INAM) or mock-transduced WT BMDCs (control BMDC) and killing activities against B16F1 cells were monitored. Represented as means ± SD. In all in vitro killing assays, 1 × 10 4 of 51 Cr-labeled B16F1 cells were used. 51 Cr radioactivity released from target cells was measured. The percentage of cytotoxicity was calculated as described in the legend of and represented as Net lysis (%). DOI: http://dx.doi.org/10.7554/eLife.04177.024

Article Snippet: Splenic CD11b + , CD11c + , B, or T cells were purified using CD11b or CD11c MicroBeads, B cell isolation kit, or pan T cell isolation kit II, respectively (Miltenyi Biotec, Germany).

Techniques: Incubation, Activity Assay, Co-Culture Assay, In Vitro, Purification, Cell Culture, Isolation, Microarray, Membrane, Expressing, Quantitative RT-PCR, Control, Labeling, Radioactivity, Lysis

Journal: Oncotarget

Article Title: Role of BCL9L in transforming growth factor-β (TGF-β)-induced epithelial-to-mesenchymal-transition (EMT) and metastasis of pancreatic cancer

doi: 10.18632/oncotarget.12455

Figure Lengend Snippet: E-cadherin, α-catenin, BCL9L and α-tubulin (loading control) protein levels were determined in stably shRNA-transduced Panc-1 cells ( A ) as well as S2-007, S2-028 and Panc-1 cells transiently transfected with siRNA ( B ). Knockdown of BCL9L by both shRNA (shBCL9L) and siRNA (siRNA1, siRNA2, siRNA pool) induced an up-regulation of E-cadherin protein in all cell lines compared to controls (Vector control, siCTR). Shown are representative images of 3 western blot experiments. qRT-PCR analyses of BCL9L and E-cadherin mRNA expression levels in Panc-1 ( C ), S2-007 ( D ), and S2-028 ( E ) cells transfected with control siRNA or BCL9L-targeting siRNA showed a significant up-regulation of E-cadherin expression in response to knockdown of BCL9L. Data represent mean ± s.e.m. of three independent experiments. ** p ≤ 0.01, *** p ≤ 0.001 (Student's t-test ). ( F ) Untransduced (UNT), Vector control and BCL9L-shRNA transduced Panc-1 cells were treated or not with 10 μM CHIR and subjected to immunofluorescence co-staining of E-cadherin (left panel; visualized by Alexa 555-tagged secondary antibody) and β-catenin (middle panel; visualized by Alexa 488-tagged secondary antibody), respectively. Irrespective of CHIR treatment BCL9L-knockdown cells show increased retention of E-cadherin and β-catenin staining at the plasma membrane compared to controls (see arrows, bottom right panel). ( G ) Quantification of CHIR-induced nuclear translocation of β-Catenin in samples from (F). Data shown represent mean and standard deviation of mean grey values from nuclear ROIs of each 20 cells quantified as described in Materials and Methods. ( H ) β-catenin was immunoprecipitated from total cell lysates of Panc-1 BCL9L-knockdown and control cells treated or not with CHIR inhibitor. Western Blot analysis of β-catenin, E-cadherin and GAPDH (control) protein levels in input (IP) and bound (B) fractions of the immunoprecipitation revealed increased binding of E-cadherin to β-catenin protein in presence and absence of CHIR in BCL9L knockdown cells compared to controls. Shown is a representative image of 2 experiments.

Article Snippet: The following antibodies were used in this study: BCL9L (sheep polyclonal, # AF4967, R&D Systems; HPA 049370, Atlas Antibodies), α-tubulin (mouse monoclonal, B-5-1-2, #T5168, Sigma Aldrich), β-catenin (rabbit polyclonal, H102, #sc-7199, Santa Cruz), E-cadherin (goat polyclonal, #AF648, R&D Systems), α-catenin (mouse monoclonal, 6D202, #C2069-44H, USBiological).

Techniques: Control, Stable Transfection, shRNA, Transfection, Knockdown, Plasmid Preparation, Western Blot, Quantitative RT-PCR, Expressing, Immunofluorescence, Staining, Clinical Proteomics, Membrane, Translocation Assay, Standard Deviation, Immunoprecipitation, Binding Assay

Journal: Oncotarget

Article Title: Role of BCL9L in transforming growth factor-β (TGF-β)-induced epithelial-to-mesenchymal-transition (EMT) and metastasis of pancreatic cancer

doi: 10.18632/oncotarget.12455

Figure Lengend Snippet: ( A ) Panc-1 cells stably transduced with control vector or BCL9L shRNA (shBCL9L) were treated with 5 ng/ml TGF-β or left untreated for 72 h and subsequently visualized using a phase-contrast microscope. Control cells responded to TGF-β treatment by adopting a mesenchymal, spindle-like phenotype whereas BCL9L-knockdown cells largely retained the cobblestone-like epithelial morphology. ( B ) Western Blot analysis of E-cadherin and α-tubulin (loading control) protein levels in Panc-1 cells treated with 5 ng/ml TGF-β for 72 h. shRNA- and siRNA-mediated knockdown of BCL9L (shBCL9L, siRNA1, siRNA2, siRNA pool) induced an upregulation of E-cadherin expression in comparison to controls (Vector ctrl, siCTR). Shown are representative images of 2 experiments. mRNA expression levels of epithelial (CDH1) and mesenchymal (SNAI2, VIM) genes were quantified in BCL9L-knockdown and control Panc-1 cells treated or not with TGF-β for 24 h ( C ), 48 h ( D ), 72 h ( E ) and 96 h ( F ), respectively. Data shown represent mean and standard deviation of 3 independent experiments. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 (Student's t-test ).

Article Snippet: The following antibodies were used in this study: BCL9L (sheep polyclonal, # AF4967, R&D Systems; HPA 049370, Atlas Antibodies), α-tubulin (mouse monoclonal, B-5-1-2, #T5168, Sigma Aldrich), β-catenin (rabbit polyclonal, H102, #sc-7199, Santa Cruz), E-cadherin (goat polyclonal, #AF648, R&D Systems), α-catenin (mouse monoclonal, 6D202, #C2069-44H, USBiological).

Techniques: Stable Transfection, Transduction, Control, Plasmid Preparation, shRNA, Microscopy, Knockdown, Western Blot, Expressing, Comparison, Standard Deviation

Journal: Viruses

Article Title: Processing Hundreds of SARS-CoV-2 Samples with an In-House PCR-Based Method without Robotics

doi: 10.3390/v13091712

Figure Lengend Snippet: CFX plate loading scheme. Shown is the pipetting strategy for the transfer of extracted RNA from the 96-well elution plate to the 384-well qPCR plate using a 12-channel pipette in order to run a full plate RT-qPCR experiment that includes 88 samples measured with three primer/probe sets like N1 (blue), N2 (orange) and RP (purple). Four dilutions (10 5 , 10 4 , 10 3 , and 10 2 copies) of the RNA control as well as two no-template control reactions (NTC) for each primer/probe set are included.

Article Snippet: The RT-qPCR reactions were set up in an optical 384-well plate (Bio-Rad, Hercules, CA, USA, #HSP3865) on ice and sealed with adhesive optical clear qPCR plate seals (Bio-Rad, #MSB1001), briefly mixed and centrifuged for 2 min at 2000× g . The RT-qPCR run was performed using a CFX384 Real-Time System type C1000 thermocycler (Bio-Rad) with the following thermal conditions: 25 °C for 2 min, 50 °C for 20 min, 95 °C for 3 min and 45 cycles of 95 °C for 15 s and 58 °C for 10 s including a plate read to detect for the FAM/Cy5/HEX signal, depending on the probes used.

Techniques: Transferring, Quantitative RT-PCR, Control

Journal: PLoS ONE

Article Title: Abrogation of Plasminogen Activator Inhibitor-1-Vitronectin Interaction Ameliorates Acute Kidney Injury in Murine Endotoxemia

doi: 10.1371/journal.pone.0120728

Figure Lengend Snippet: (A) Immunolocalization of neutrophils (indicated by arrows) in mice kidneys using anti-NIMP-R14 antibody (Abcam). Magnification: 40x (scale bar, 100 μm). (B) Neutrophil infiltration was assessed by measuring renal MPO activity as previously described [ , ]. Data are expressed as mean ± SEM (N = 3 mice per group), * P <0.05. Real time RT-PCR (C) and Western blot (D) analysis of ICAM-1 expression in renal tissues after LPS challenge. Western blot analysis was performed using anti-ICAM-1 and anti-β-actin antibody as described. Densitometric analysis of relative ICAM-1 expression after normalization with β-actin is shown in the lower panel. Data are expressed as the mean ± SEM (N = 5–6 mice per group), * P <0.05.

Article Snippet: The primary antibodies used were: monoclonal mouse-anti-β-actin (GenScript, Piscataway, NJ), polyclonal goat-anti-mouse ICAM-1 (R&D Systems, Minneapolis, MN), polyclonal rabbit-anti-rat ERK1/2 and polyclonal rabbit-anti-human phospho-ERK1/2 (Thr202/Tyr204) (Cell Signaling), polyclonal sheep-anti-mouse PC (Haematologic Technologies, Essex Junction, VT), and polyclonal rabbit-anti-mouse PAI-1 (ABCAM).

Techniques: Activity Assay, Quantitative RT-PCR, Western Blot, Expressing