Journal: Journal of Immunology Research

Article Title: TLR4-NLRP3-GSDMD-Mediated Pyroptosis Plays an Important Role in Aggravated Liver Injury of CD38 −/− Sepsis Mice

doi: 10.1155/2021/6687555

Figure Lengend Snippet: Escherichia coli can induce liver injury in mice. WT mice were injected with PBS or 3 × 10 8 cfu/ml E. coli intraperitoneally and sacrificed 3 hours later. Liver pathological injuries were observed with hematoxylin and eosin staining (a–d). The yellow arrows indicate edema, the blue arrows indicate inflammatory cell infiltration, the black arrows indicate punctate necrosis, and the green arrows indicate binucleate hepatocytes. Bacteria from the liver of PBS- or E. coli -stimulated WT mice were cultured in MH medium (e, f) overnight and identified by Gram staining (g). The serum AST (h) and ALT (i) concentrations were determined by using the detection kits. The mRNA of liver inflammatory cytokines TLR4 (j), NLRP3 (k), IL-1 β (l), and IL-18 (m) of PBS- or 3 × 10 8 cfu/ml E. coli -stimulated mice were measured by RT-qPCR. Data are presented as means ± standard deviation. Statistical significance was determined by the paired t -test ( n = 3, ∗ p < 0.05, ∗∗ p < 0.01).

Article Snippet: The primary antibodies anti-TLR4 rabbit mAb (1 : 500) (CST, USA), anti-TRIF rabbit mAb (1 : 1000) (Proteintech, USA), anti-MyD88 mouse mAb (1 : 2000) (Proteintech, USA), anti-NF- κ B p65 rabbit mAb (1 : 1000) (CST, USA), anti-phospho-NF- κ B p65 rabbit mAb (1 : 500) (CST, USA), anti-IL-6 mouse mAb (1 : 2000) (Proteintech, USA), anti-iNOS rabbit mAb (1 : 1000) (CST, USA), anti-BAX rabbit mAb (1 : 5000) (Proteintech, USA), anti-NLRP3 rabbit mAb (1 : 500) (Boster, China), anti-ASC rabbit mAb (1 : 500) (Affinity, China), anti-caspase-1 rabbit mAb (1 : 500) (Abcam, UK), anti-IL-1 β rabbit mAb (1 : 1000) (CST, USA), anti-IL-18 rabbit mAb (1 : 1000) (Affinity, China), anti-caspase-3 rabbit mAb (1 : 500) (CST, USA), anti-GSDMD rabbit mAb (1 : 500) (Affinity, China), anti-ERK1/2 rabbit mAb (1 : 1000) (CST, USA), anti-phospho-ERK1/2 rabbit mAb (1 : 2000) (CST, USA), and anti-GAPDH rabbit mAb (1 : 5000) (Proteintech, USA) were used.

Techniques: Injection, Staining, Bacteria, Cell Culture, Quantitative RT-PCR, Standard Deviation

Journal: Journal of Immunology Research

Article Title: TLR4-NLRP3-GSDMD-Mediated Pyroptosis Plays an Important Role in Aggravated Liver Injury of CD38 −/− Sepsis Mice

doi: 10.1155/2021/6687555

Figure Lengend Snippet: The expression levels of pyroptosis-related markers were detected by Western blot. The expressions of liver pyroptosis proteins in WT, CD38 −/− , and CD38 −/− TLR4 mut mice were detected at 3 hours after E. coli stimulation by Western blot. The expressions of NLRP3, ASC, procaspase-1, cleaved caspase-1, IL-1 β , IL-18, procaspase-3, and cleaved caspase-3 were measured (a). And relative levels of NLRP3 to GAPDH (b), ASC to GAPDH (c), cleaved to procaspase-1 (d), IL-1 β to GAPDH (e), IL-18 to GAPDH (f), and cleaved to procaspase-3 (g) were analyzed by ImageJ software. Data are presented as means ± standard deviation. Statistical significance was determined by one-way ANOVA ( n = 3, ∗ p < 0.05, ∗∗ p < 0.01).

Article Snippet: The primary antibodies anti-TLR4 rabbit mAb (1 : 500) (CST, USA), anti-TRIF rabbit mAb (1 : 1000) (Proteintech, USA), anti-MyD88 mouse mAb (1 : 2000) (Proteintech, USA), anti-NF- κ B p65 rabbit mAb (1 : 1000) (CST, USA), anti-phospho-NF- κ B p65 rabbit mAb (1 : 500) (CST, USA), anti-IL-6 mouse mAb (1 : 2000) (Proteintech, USA), anti-iNOS rabbit mAb (1 : 1000) (CST, USA), anti-BAX rabbit mAb (1 : 5000) (Proteintech, USA), anti-NLRP3 rabbit mAb (1 : 500) (Boster, China), anti-ASC rabbit mAb (1 : 500) (Affinity, China), anti-caspase-1 rabbit mAb (1 : 500) (Abcam, UK), anti-IL-1 β rabbit mAb (1 : 1000) (CST, USA), anti-IL-18 rabbit mAb (1 : 1000) (Affinity, China), anti-caspase-3 rabbit mAb (1 : 500) (CST, USA), anti-GSDMD rabbit mAb (1 : 500) (Affinity, China), anti-ERK1/2 rabbit mAb (1 : 1000) (CST, USA), anti-phospho-ERK1/2 rabbit mAb (1 : 2000) (CST, USA), and anti-GAPDH rabbit mAb (1 : 5000) (Proteintech, USA) were used.

Techniques: Expressing, Western Blot, Software, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Ticagrelor Exerts Immune-Modulatory Effect by Attenuating Neutrophil Extracellular Traps

doi: 10.3390/ijms21103625

Figure Lengend Snippet: Ticagrelor inhibits NET release induced by polyP in vitro. ( a ). Fluorescence microscopy for cit-H3/NE staining in control neutrophils treated with synthetic or platelet-derived (natural) polyP, with or without pre-treatment with Ticagrelor or Clopidogrel. One representative out of six independent experiments is shown. Original magnification: 400×, Scale bar: 5μm. Blue: DAPI, Green: NE, Red: cit-H3. ( b ). Percentage of NET-releasing neutrophils as assessed by immunofluorescence. ( c ). MPO-DNA complex levels in NET structures from these stimulations, as assessed by ELISA. Relative integrated optical density was calculated compared to control NETs value. Data from six independent experiments presented as mean ± SD. Statistical significance *, # p < 0.05. All conditions were compared to untreated/control condition and statistical significance is indicated by the symbol “*”. Any further statistical significance of other comparisons is indicated by the symbol “#”. ( d ). Annexin V/Propidium Iodide flow cytometry of control neutrophils in the presence or absence of Ticagrelor/Clopidogrel. One representative out of six independent experiments is shown. Polymorphonuclear neutrophils (PMNs).

Article Snippet: Samples were stained using a rabbit anti-citrullinated H3 (R2+R8+R17) (1/200 dilution, Abcam, ab5103, Cambridge, UK) and a mouse anti-neutrophil elastase (NE) mAb (1/200 dilution, Santa Cruz Biotechnology Inc, sc-55548, TX, USA), or a mouse anti-TF mAb (1/200 dilution; Sekisui Diagnostics, 4509, MS, USA) and a rabbit anti-neutrophil elastase (NE) mAb (1/200 dilution, Santa Cruz Biotechnology Inc, sc-25621, TX, USA), or a rabbit anti-Microtubule-Associated Protein 1 Light Chain Beta (LC3B) polyclonal Ab (1/200 dilution, Sigma-Aldrich, L7543, Munich, Germany) or a mouse anti- BECN1 mAb (1/200 dilution, Santa Cruz Biotechnology Inc, sc-48381).

Techniques: In Vitro, Fluorescence, Microscopy, Staining, Control, Derivative Assay, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Ticagrelor Exerts Immune-Modulatory Effect by Attenuating Neutrophil Extracellular Traps

doi: 10.3390/ijms21103625

Figure Lengend Snippet: Neutrophils from individuals receiving Ticagrelor were more resistant to NETotic stimulation from polyP. ( a – c ). Fluorescence microscopy for cit-H3/NE staining in neutrophils isolated from a patient with a previous acute coronary syndrome and stent placement that receives Ticagrelor or Clopidogrel as a main antiplatelet treatment and neutrophils from a healthy individual, with or without synthetic polyP. One representative out of five independent experiments is shown. Original magnification: 600×, Scale bar: 5 μm. Blue: DAPI, Green: NE, Red: cit-H3. ( d ). Percentage of NET-releasing neutrophils as assessed by immunofluorescence. ( e ). MPO-DNA complex levels in NET structures from these stimulations, as assessed by ELISA. Data from five independent experiments presented as mean ± SD. Statistical significance * p < 0.05. All conditions were compared to untreated/control condition and statistical significance is indicated by the symbol “*”. Polymorphonuclear neutrophils (PMNs).

Article Snippet: Samples were stained using a rabbit anti-citrullinated H3 (R2+R8+R17) (1/200 dilution, Abcam, ab5103, Cambridge, UK) and a mouse anti-neutrophil elastase (NE) mAb (1/200 dilution, Santa Cruz Biotechnology Inc, sc-55548, TX, USA), or a mouse anti-TF mAb (1/200 dilution; Sekisui Diagnostics, 4509, MS, USA) and a rabbit anti-neutrophil elastase (NE) mAb (1/200 dilution, Santa Cruz Biotechnology Inc, sc-25621, TX, USA), or a rabbit anti-Microtubule-Associated Protein 1 Light Chain Beta (LC3B) polyclonal Ab (1/200 dilution, Sigma-Aldrich, L7543, Munich, Germany) or a mouse anti- BECN1 mAb (1/200 dilution, Santa Cruz Biotechnology Inc, sc-48381).

Techniques: Fluorescence, Microscopy, Staining, Isolation, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Ticagrelor Exerts Immune-Modulatory Effect by Attenuating Neutrophil Extracellular Traps

doi: 10.3390/ijms21103625

Figure Lengend Snippet: Ticagrelor does not signal through the P2Y12 or the autophagy pathway in neutrophil. ( a ). mRNA fold expression for P2Y12 receptor in control neutrophils treated with synthetic polyP and IRA plasma. Data from three independent experiments presented as mean ± SD. There is no statistical significance. ( b ). Western blot of mTOR p2448 in control neutrophils treated with synthetic polyP, with or without pre-treatment with Ticagrelor. One representative out of four independent experiments is shown. Relative integrated optical density was calculated compared to untreated PMNs value. Data from four independent experiments presented as mean ± SD. Statistical significance * p < 0.05. All conditions were compared to untreated/control conditions and statistical significance is indicated by the symbol “*”. ( c , d ). Confocal microscopy for DAPI/LC3B or DAPI/BECLIN 1 staining in control neutrophils treated with synthetic polyP, with or without pre-treatment with Ticagrelor. One representative out of six independent experiments is shown. Original magnification: 1000×, Scale bar: 5 μm. Blue: DAPI. Red: LC3B. Green: BECLIN 1. Polymorphonuclear neutrophils (PMNs).

Article Snippet: Samples were stained using a rabbit anti-citrullinated H3 (R2+R8+R17) (1/200 dilution, Abcam, ab5103, Cambridge, UK) and a mouse anti-neutrophil elastase (NE) mAb (1/200 dilution, Santa Cruz Biotechnology Inc, sc-55548, TX, USA), or a mouse anti-TF mAb (1/200 dilution; Sekisui Diagnostics, 4509, MS, USA) and a rabbit anti-neutrophil elastase (NE) mAb (1/200 dilution, Santa Cruz Biotechnology Inc, sc-25621, TX, USA), or a rabbit anti-Microtubule-Associated Protein 1 Light Chain Beta (LC3B) polyclonal Ab (1/200 dilution, Sigma-Aldrich, L7543, Munich, Germany) or a mouse anti- BECN1 mAb (1/200 dilution, Santa Cruz Biotechnology Inc, sc-48381).

Techniques: Expressing, Control, Clinical Proteomics, Western Blot, Confocal Microscopy, Staining

Journal: International Journal of Molecular Sciences

Article Title: Ticagrelor Exerts Immune-Modulatory Effect by Attenuating Neutrophil Extracellular Traps

doi: 10.3390/ijms21103625

Figure Lengend Snippet: Τicagrelor is not able to inhibit intracellular expression of tissue factor (TF). ( a ). Fluorescence microscopy for TF/NE staining in control neutrophils treated with IRA plasma, with or without pre-treatment with Ticagrelor or Clopidogrel. One representative out of six independent experiments is shown. Original magnification: 400×, Scale bar: 5 μm. Blue: DAPI, Green: TF, Red: NE. ( b ). Representative fluorescence-activated cell-sorting analysis and ( c ) relative MFIs of TF in control neutrophils treated with IRA plasma, with or without pre-treatment with Ticagrelor or Clopidogrel. Data from six independent experiments presented as mean ± SD. Statistical significance * p < 0.05. n.s.: non-significant. All conditions were compared to the untreated/control condition and statistical significance is indicated by the symbol “*”. Any further non-statistical significance of other comparisons is indicated by the symbol “n.s.”. MFI—mean fluorescence intensity. ( a , b ) One representative out of six independent experiments is shown. Polymorphonuclear neutrophils (PMNs).

Article Snippet: Samples were stained using a rabbit anti-citrullinated H3 (R2+R8+R17) (1/200 dilution, Abcam, ab5103, Cambridge, UK) and a mouse anti-neutrophil elastase (NE) mAb (1/200 dilution, Santa Cruz Biotechnology Inc, sc-55548, TX, USA), or a mouse anti-TF mAb (1/200 dilution; Sekisui Diagnostics, 4509, MS, USA) and a rabbit anti-neutrophil elastase (NE) mAb (1/200 dilution, Santa Cruz Biotechnology Inc, sc-25621, TX, USA), or a rabbit anti-Microtubule-Associated Protein 1 Light Chain Beta (LC3B) polyclonal Ab (1/200 dilution, Sigma-Aldrich, L7543, Munich, Germany) or a mouse anti- BECN1 mAb (1/200 dilution, Santa Cruz Biotechnology Inc, sc-48381).

Techniques: Expressing, Fluorescence, Microscopy, Staining, Control, Clinical Proteomics, FACS

Journal: International Journal of Molecular Sciences

Article Title: Ticagrelor Exerts Immune-Modulatory Effect by Attenuating Neutrophil Extracellular Traps

doi: 10.3390/ijms21103625

Figure Lengend Snippet: Coronary stents induce NET formation. ( a ). Fluorescence microscopy for cit-H3/NE staining in control neutrophils treated with stent and with or without pre-treatment with Ticagrelor or Clopidogrel. One representative out of six independent experiments is shown. Original magnification: 400×, Scale bar: 5 μm. Blue: DAPI, Green: NE, Red: cit-H3. ( b ). MPO-DNA complex levels in NET structures from control neutrophils treated with stent, with or without pre-treatment with Ticagrelor or Clopidogrel, as assessed by ELISA. Relative integrated optical density was calculated when compared to control NETs value. Data from six independent experiments presented as mean ± SD. Statistical significance *, # p < 0.05. All conditions were compared to untreated/control conditions and statistical significance is indicated by the symbol “*”. Any further statistical significance of other comparisons is indicated by the symbol “#”. ( c ). Fluorescence microscopy for TF/NE staining in control neutrophils treated with stent and IRA plasma. One representative out of six independent experiments is shown. Original magnification: 400×, Scale bar: 5 μm. Blue: DAPI, Green: TF, Red: NE. Polymorphonuclear neutrophils (PMNs).

Article Snippet: Samples were stained using a rabbit anti-citrullinated H3 (R2+R8+R17) (1/200 dilution, Abcam, ab5103, Cambridge, UK) and a mouse anti-neutrophil elastase (NE) mAb (1/200 dilution, Santa Cruz Biotechnology Inc, sc-55548, TX, USA), or a mouse anti-TF mAb (1/200 dilution; Sekisui Diagnostics, 4509, MS, USA) and a rabbit anti-neutrophil elastase (NE) mAb (1/200 dilution, Santa Cruz Biotechnology Inc, sc-25621, TX, USA), or a rabbit anti-Microtubule-Associated Protein 1 Light Chain Beta (LC3B) polyclonal Ab (1/200 dilution, Sigma-Aldrich, L7543, Munich, Germany) or a mouse anti- BECN1 mAb (1/200 dilution, Santa Cruz Biotechnology Inc, sc-48381).

Techniques: Fluorescence, Microscopy, Staining, Control, Enzyme-linked Immunosorbent Assay, Clinical Proteomics

Journal: Materials Today Bio

Article Title: Dual-functional biomimetic periosteum incorporating engineered small extracellular vesicles for treating critical bone defect with soft tissue fenestration via TGF-beta1/SMAD pathway

doi: 10.1016/j.mtbio.2025.101892

Figure Lengend Snippet: Construction and characterization of engineered sEVs. A Schematic preparation of porous titanium-preconditioned MSCs-derived sEVs. B Light microscopy images of representative images of MSCs cultured on the cell culture plate. C Scanning electron microscopy (SEM) images of the porous titanium surface. D SEM images of adhesion elongation of preconditioned MSCs on the titanium surface. E Transmission electron microscopy (TEM) images of Con-sEVs and Ti-sEVs. F Particle size distributions of Con-sEVs and Ti-sEVs in PBS. G Representative western blots of sEVs markers (CD63 and TSG101) and a negative marker (CALNEXIN). H Representative western blots of proteins related to sEVs secretion (SMPD3 and SYTL4), soft tissue and bone repair (COL1A1, MMP1, VEGFA and TGF-β1). I Relative protein expression evaluation related to sEVs-loaded proteins for Con-sEVs and Ti-sEVs (reference protein: TSG101, a sEVs specific marker). J Relative protein expression evaluation for Con-hMSCs and Ti-hMSCs (reference protein: CALNEXIN). Data were presented as the mean ± standard deviation (n = 3). Statistical analyses were performed using Student's t -test. ns, no significance, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Primary antibodies, including anti-COL1A1 rabbit mAb (1:1,000; Zenbio, China), anti-MMP1 rabbit pAb (1:1,000; Affinity, USA), anti-VEGFA rabbit pAb (1:1,000; Affinity, USA), anti-TGF-β1 rabbit mAb (1:1,000; Zenbio, China), anti-SMPD3 rabbit pAb (1:1,000; ABclonal, China), anti-SYTL4 rabbit pAb (1:1,000; Immunoway, USA), anti-CALNEXIN rabbit pAb (1:1,000), and anti-TSG101 rabbit pAb (1:1,000), were applied.

Techniques: Derivative Assay, Light Microscopy, Cell Culture, Electron Microscopy, Transmission Assay, Western Blot, Marker, Expressing, Standard Deviation

Journal: Materials Today Bio

Article Title: Dual-functional biomimetic periosteum incorporating engineered small extracellular vesicles for treating critical bone defect with soft tissue fenestration via TGF-beta1/SMAD pathway

doi: 10.1016/j.mtbio.2025.101892

Figure Lengend Snippet: Effect of Ti-sEVs on the uptake efficiency and scarless soft tissue repair ability of human skin fibroblasts (HSF) in vitro. A Fluorescence images of PKH26-labeled sEVs internalization by human skin fibroblasts at 0, 6, 12 and 24 h (red: sEVs; blue: living cell nuclei stained by Hoechst). B Brightfield images of wound healing assay using human skin fibroblasts treated with sEVs at 0, 6, 12, 24 and 36 h. C Gene expression evaluation of human skin fibroblasts related to scarless soft tissue repair (COL1A1, TRAM2, VEGFA and MMP1; reference gene: GAPDH) treated with sEVs after 7 days by qRT-PCR (n = 3). D Representative western blot bands of proteins related to scarless soft tissue repair (COL1A1, TRAM2, TGF-β1, VEGFA and MMP1; reference protein: GAPDH) for human skin fibroblasts treated with sEVs after 7 days. E Relative protein expression evaluation for the western blot bands (n = 3). F Representative western blot bands of proteins related to scarless soft tissue repair related pathways (p-AKT/AKT, p-ERK/ERK and p-SMAD/SMAD; reference protein: β-ACTIN) for human skin fibroblasts treated with sEVs after 7 days. G Relative protein expression evaluation for the western blot bands (n = 3). Statistical analyses were performed using one-way ANOVA with the Bonferroni correction. Different letters, a, b, c above the bar indicate significant difference compared to the Blank, Con-sEVs and Ti-sEVs groups respectively, p < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Primary antibodies, including anti-COL1A1 rabbit mAb (1:1,000; Zenbio, China), anti-MMP1 rabbit pAb (1:1,000; Affinity, USA), anti-VEGFA rabbit pAb (1:1,000; Affinity, USA), anti-TGF-β1 rabbit mAb (1:1,000; Zenbio, China), anti-SMPD3 rabbit pAb (1:1,000; ABclonal, China), anti-SYTL4 rabbit pAb (1:1,000; Immunoway, USA), anti-CALNEXIN rabbit pAb (1:1,000), and anti-TSG101 rabbit pAb (1:1,000), were applied.

Techniques: In Vitro, Fluorescence, Labeling, Staining, Wound Healing Assay, Gene Expression, Quantitative RT-PCR, Western Blot, Expressing

Journal: Materials Today Bio

Article Title: Dual-functional biomimetic periosteum incorporating engineered small extracellular vesicles for treating critical bone defect with soft tissue fenestration via TGF-beta1/SMAD pathway

doi: 10.1016/j.mtbio.2025.101892

Figure Lengend Snippet: Effect of Ti-sEVs on the osteogenic capability of human BMSCs (hBMSCs) in vitro. A Fluorescence images of PKH26-labeled sEVs internalization by human BMSCs at 0, 6, 12 and 24 h (red: sEVs; blue: living cell nuclei stained by Hoechst). B Brightfield images of wound healing assay using human BMSCs treated with sEVs at 0, 6, 12, 24 and 36 h. C Gene expression evaluation of human BMSCs related to bone repair (COL1A1, VEGFA, RUNX2, ALP, OPN, OCN and RANKL; reference gene: GAPDH) treated with sEVs after 7 days by qRT-PCR (n = 3). Statistical analyses were performed using one-way ANOVA with the Bonferroni correction. Different letters, a, b, c above the bar indicate significant difference compared to the Blank, Con-sEVs and Ti-sEVs groups respectively, p < 0.05. D Representative western blot bands of proteins related to bone repair (COL1A1, TGF-β1, VEGFA, RUNX2, OPN, ALP, OCN and RANKL; reference protein: GAPDH) and sEVs secretion (SMPD3 and SYTL4; reference protein: GAPDH) for human BMSCs treated with sEVs after 7 days. E Representative western blot bands of proteins related to bone repair related pathways (p-AKT/AKT, p-ERK/ERK and p-SMAD/SMAD; reference protein: β-ACTIN) for BMSCs treated with sEVs after 7 days. F Relative protein expression evaluation for the western blot bands (n = 3). Statistical analyses were performed using one-way ANOVA with the Bonferroni correction. Different letters, a, b, c above the bar indicate significant difference compared to the Blank, Con-sEVs and Ti-sEVs groups respectively, p < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Primary antibodies, including anti-COL1A1 rabbit mAb (1:1,000; Zenbio, China), anti-MMP1 rabbit pAb (1:1,000; Affinity, USA), anti-VEGFA rabbit pAb (1:1,000; Affinity, USA), anti-TGF-β1 rabbit mAb (1:1,000; Zenbio, China), anti-SMPD3 rabbit pAb (1:1,000; ABclonal, China), anti-SYTL4 rabbit pAb (1:1,000; Immunoway, USA), anti-CALNEXIN rabbit pAb (1:1,000), and anti-TSG101 rabbit pAb (1:1,000), were applied.

Techniques: In Vitro, Fluorescence, Labeling, Staining, Wound Healing Assay, Gene Expression, Quantitative RT-PCR, Western Blot, Expressing

Journal: PloS one

Article Title: Expression of MUC17 is regulated by HIF1α-mediated hypoxic responses and requires a methylation-free hypoxia responsible element in pancreatic cancer.

doi: 10.1371/journal.pone.0044108

Figure Lengend Snippet: Figure 1. MUC17 expression is enhanced by hypoxia. (A) AsPC1 cells were cultured under hypoxic conditions (1% O2) for the indicated times. MUC17 mRNA expression was examined by RT-PCR at each time point. (B) AsPC1 cells were cultured under normoxic or hypoxic conditions for the indicated times. Cell lysates were probed with anti-MUC17, HIF1a, and a-tubulin antibodies by Western blot analysis. The intensities of the bands were quantitated by densitometric scanning, and the ratio of MUC17 to a-tubulin expression is shown under each band as the relative intensity compared with that obtained in normoxic AsPC1 cells. (C) MUC17 promoter activity was measured by a Dual-Luciferase Reporter Assay. After transfection of the MUC17 reporter plasmid, AsPC1 cells were incubated under normoxic or hypoxic conditions for 24 h. Cell lysates were assayed using a luciferase assay kit in a Tristar multimode microplate reader LB941 (Berthold Technologies). Transformation efficiency was normalized on the basis of Renilla luciferase activity. The promoter activity under normoxic conditions was given a value of 1. P values were determined using the Student’s t-test. * P,0.05. doi:10.1371/journal.pone.0044108.g001

Article Snippet: The primary antibodies were as follows: anti-human MUC17 (rabbit pAb, 1:1000, generated by one of the authors, Dr. Surinder K. Batra, University of Nebraska Medical Center, Omaha, NE, USA); anti-human HIF1a (mouse mAb, 1:1000, H1alpha67, Novus Biologicals), and anti-human a-tubulin (mouse mAb, DM1A, Sigma).

Techniques: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Western Blot, Activity Assay, Luciferase, Reporter Assay, Transfection, Plasmid Preparation, Incubation, Transformation Assay

Journal: PloS one

Article Title: Expression of MUC17 is regulated by HIF1α-mediated hypoxic responses and requires a methylation-free hypoxia responsible element in pancreatic cancer.

doi: 10.1371/journal.pone.0044108

Figure Lengend Snippet: Figure 2. Hypoxic induction of MUC17 is dependent on HIF1a in AsPC1 cells. (A) After the transfection of HIF1A siRNAs, AsPC1 cells were cultured under normoxia (N) or hypoxia (H) for 24 h. The level of mRNA was measured by RT-PCR. (B) Cell lysates from AsPC1 cells treated with HIF1A siRNAs were immunoblotted with the indicated antibodies. a-tubulin served as a loading control. (C) The densities of the acquired bands from Western blotting analysis were quantified and expressed as relative fold increases compared with that obtained from mock cells under hypoxic culture conditions. ns, not significant. * P,0.05, ** P,0.005. doi:10.1371/journal.pone.0044108.g002

Article Snippet: The primary antibodies were as follows: anti-human MUC17 (rabbit pAb, 1:1000, generated by one of the authors, Dr. Surinder K. Batra, University of Nebraska Medical Center, Omaha, NE, USA); anti-human HIF1a (mouse mAb, 1:1000, H1alpha67, Novus Biologicals), and anti-human a-tubulin (mouse mAb, DM1A, Sigma).

Techniques: Transfection, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Control, Western Blot

Journal: PloS one

Article Title: Expression of MUC17 is regulated by HIF1α-mediated hypoxic responses and requires a methylation-free hypoxia responsible element in pancreatic cancer.

doi: 10.1371/journal.pone.0044108

Figure Lengend Snippet: Figure 3. Hypoxia enhances the recruitment of HIF1a to HRE and activates MUC17 transcription. (A) Binding of HIF1a to chromatin was confirmed by a ChIP assay. AsPC1 cells were cultured under normoxia or hypoxia for 24 h. PCR was performed with specific primers covering HRE. (B) The densities of the acquired bands in panel (A) were quantified using Image J (NIH) and normalized to Input included in each experiment. (C) To evaluate the transactivation activity of HIF1a through HRE, a dual luciferase assay was conducted. AsPC1 cells were transfected with wild-type or HRE mutant MUC17 promoter constructs under hypoxic conditions for 24 h. P values were determined using Student’s t-test. ns, not significant. * P,0.01, ** P,0.001. doi:10.1371/journal.pone.0044108.g003

Article Snippet: The primary antibodies were as follows: anti-human MUC17 (rabbit pAb, 1:1000, generated by one of the authors, Dr. Surinder K. Batra, University of Nebraska Medical Center, Omaha, NE, USA); anti-human HIF1a (mouse mAb, 1:1000, H1alpha67, Novus Biologicals), and anti-human a-tubulin (mouse mAb, DM1A, Sigma).

Techniques: Binding Assay, Cell Culture, Activity Assay, Luciferase, Transfection, Mutagenesis, Construct

Journal: PloS one

Article Title: Expression of MUC17 is regulated by HIF1α-mediated hypoxic responses and requires a methylation-free hypoxia responsible element in pancreatic cancer.

doi: 10.1371/journal.pone.0044108

Figure Lengend Snippet: Figure 5. Methylation status of the HRE site determines the sensitivity of MUC17 to HIF1a-induced transactivation. (A) Demethylation of CpG sites in the MUC17 promoter in BxPC3 and PANC1 cells after 1 mM 5-azadC treatment. MUC17-negative/low cell lines were treated with or without 5-azadC for 7 days. The methylation status of the MUC17 promoter harboring HRE was examined by MSP. The PCR products labeled M (methylated) were amplified by methylation-specific primers, and those labeled U (unmethylated) were amplified by primers specific for unmethylated DNA. (B) Restoration of MUC17 expression in pancreatic cancer cell lines by 5-azadC treatment. Cells were treated with or without 5-azadC for 7 days. During the last 24 h, each group was cultured under normoxic (N) or hypoxic (H) conditions. MUC17 expression was examined by Western blotting. doi:10.1371/journal.pone.0044108.g005

Article Snippet: The primary antibodies were as follows: anti-human MUC17 (rabbit pAb, 1:1000, generated by one of the authors, Dr. Surinder K. Batra, University of Nebraska Medical Center, Omaha, NE, USA); anti-human HIF1a (mouse mAb, 1:1000, H1alpha67, Novus Biologicals), and anti-human a-tubulin (mouse mAb, DM1A, Sigma).

Techniques: Methylation, Labeling, Amplification, Expressing, Cell Culture, Western Blot

Journal:

Article Title: The phagosomal transporter A couples threonine acquisition to differentiation and replication of Legionella pneumophila in macrophages

doi: 10.1073/pnas.0502767102

Figure Lengend Snippet: PhtA couples nutrient acquisition to microbial differentiation at two stages of the L. pneumophila life cycle. PhtA threonine transport triggers intracellular transmissive L. pneumophila to differentiate to the replicative form (Fig. 5). Threonine acquisition by means of PhtA also is essential for L. pneumophila to replicate in macrophages (Figs. ​(Figs.11 and ​and4).4). When threonine acquisition by replicating bacteria is inefficient, L. pneumophila prematurely express transmission traits (Fig. 6), perhaps due to an early accumulation of the alarmone ppGpp.

Article Snippet: Anti-lysosome-associated membrane protein 1 (LAMP-1) (1D4B) rat antibody (1:1,000) was acquired from Santa Cruz Biotechnology, anti-calnexin rabbit polyclonal antibody (1:200) was purchased from Stressgen Biotechnologies (Victoria, Canada), anti- L. pneumophila rabbit polyclonal antibody (1:2,000) was a generous gift from R. Isberg (Tufts University School of Medicine, Boston), and anti- L. pneumophila mouse monoclonal hybridoma cell line CRL-1765 (1:5) was acquired from American Type Culture Collection (ATCC).

Techniques: Bacteria, Transmission Assay