Journal: PLoS ONE

Article Title: Activation of Hypoxia Inducible Factor 1 Is a General Phenomenon in Infections with Human Pathogens

doi: 10.1371/journal.pone.0011576

Figure Lengend Snippet: (A) In uninfected skin, rare and faint nuclear HIF-1α staining occurred in the epidermis whereas the dermis was negative for HIF-1α (A1). (B to L) In all cutaneous infections, a strong nuclear HIF-1α signal was detected in keratinocytes, predominantly of the spinal cell layer (examplified in L). Moreover, endothelial cells of dermal capillaries frequently stained positive (arrows in B1, C1, D1, E1, G, I1). Arrowheads: (B2) border zone of the positive abscess; (C2): positive neutrophils; (F): positive dermal lympho-histiocytic infiltrates; (F1): large positive nuclei of a multi-nuclear giant cell; (F2): skin lesion surrounded by a HIF-1 positive inflammatory infiltrate; (H): positive sub-corneal neutrophils (higher power in H1); (I): positive intra-epidermal infiltrate; (K): intradermal nodules containing leishmaniae. (A) Control: uninfected sample; (B–E): bioptic samples of patients suffering from infections with humanpathogenic bacteria [(B) S. aureus , (C) S. agalactiae and S. aureus , (D) A. baumanii and E. coli and (E) B. burgdorferi ], (F, G) with humanpathogenic Herpes-viruses [(F) Varicella zoster virus (VZV) and (G) Human Herpes Virus-8 (HHV 8)], (H–J) with humanpathogenic fungi [(H+I) T. rubrum and (J) C. albicans ] or (K, L) with the protozoic pathogen L. donovani . Magnifications: all 250×, except for A1, B1, B2, C1, D1, E1, F2, I1 (all 400×), and C2, F1, H1, L (all 1,000×).

Article Snippet: Peritoneal HIF-1α activation was analyzed by immunoblotting of the shock-frozen peritoneal specimens using the Nuclear Extract Kit (Active Motif; Rixensart, Belgium), a mouse monoclonal anti-HIF-1α (H1α67) antibody (Novus Biologicals, Littleton, USA) and horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG antibodies (Dako).

Techniques: Staining, Control, Bacteria, Virus

Journal: PLoS ONE

Article Title: Activation of Hypoxia Inducible Factor 1 Is a General Phenomenon in Infections with Human Pathogens

doi: 10.1371/journal.pone.0011576

Figure Lengend Snippet: Four to six hours upon infection, HIF-1 activation was determined by Western blotting (loading control: actin). pO 2 levels were quantified in the medium of control and infected cells (A: S. aureus ATCC 33592, MOI 20, infection time: six hours; B: S. epidermidis ATCC 12228, MOI 20, infection time: six hours; C: E. coli ATCC 25992, MOI 10, infection time: four hours). Negative control: uninfected cells, positive control: hypoxia or DFO (200 µmol/L).

Article Snippet: Peritoneal HIF-1α activation was analyzed by immunoblotting of the shock-frozen peritoneal specimens using the Nuclear Extract Kit (Active Motif; Rixensart, Belgium), a mouse monoclonal anti-HIF-1α (H1α67) antibody (Novus Biologicals, Littleton, USA) and horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG antibodies (Dako).

Techniques: Infection, Activation Assay, Western Blot, Control, Negative Control, Positive Control

Journal: PLoS ONE

Article Title: Activation of Hypoxia Inducible Factor 1 Is a General Phenomenon in Infections with Human Pathogens

doi: 10.1371/journal.pone.0011576

Figure Lengend Snippet: Four to six hours upon infection, HIF-1 activation was determined by Western blotting (loading control: actin). pO 2 levels were quantified in the medium of control and infected cells (D: P. aeruginosa ATCC 27853, MOI 10, infection time: four hours; E: S. agalactiae SK 43, MOI 200, infection time: five hours; F: C. albicans ATCC 90028, MOI 20, infection time: six hours). Negative control: uninfected cells, positive control: hypoxia or DFO (200 µmol/L).

Article Snippet: Peritoneal HIF-1α activation was analyzed by immunoblotting of the shock-frozen peritoneal specimens using the Nuclear Extract Kit (Active Motif; Rixensart, Belgium), a mouse monoclonal anti-HIF-1α (H1α67) antibody (Novus Biologicals, Littleton, USA) and horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG antibodies (Dako).

Techniques: Infection, Activation Assay, Western Blot, Control, Negative Control, Positive Control

Journal: PLoS ONE

Article Title: Activation of Hypoxia Inducible Factor 1 Is a General Phenomenon in Infections with Human Pathogens

doi: 10.1371/journal.pone.0011576

Figure Lengend Snippet: Six hours upon infection, HIF-1 activation was analyzed in (A) Western blots (loading control: actin) and by determining (B) the induction of the HIF-1-dependent luciferase activity. HeLa-229 cells were transfected with a 4xHRE-luc reporter construct 24 hours before infection (triplicate means given; for details see ). Induction was determined by chemiluminescence. Bacterial induced (C) VEGF and (D) HIF-1α transcription. Total mRNA was prepared six hours upon infection, transcribed into cDNA and quantified by real-time Light-Cycler-PCR (ratio: VEGF/actin or HIF-1α/actin transcripts; triplicate means given). Negative control: uninfected cells; S. aureus wt (8325-4); S. aureus Δ hemB ( S. aureus ermB Ω hemB 8325-4, SCV); S. aureus Δ hemB , hemB -complemented ( S. aureus pCX19Ω hemB 8325-4, complemented SCV mutant) ; positive control: DFO (200 µmol/L). Multiplicity of infection (MOI): 20. * significant difference to control cells (P<0.05).

Article Snippet: Peritoneal HIF-1α activation was analyzed by immunoblotting of the shock-frozen peritoneal specimens using the Nuclear Extract Kit (Active Motif; Rixensart, Belgium), a mouse monoclonal anti-HIF-1α (H1α67) antibody (Novus Biologicals, Littleton, USA) and horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG antibodies (Dako).

Techniques: Infection, Activation Assay, Western Blot, Control, Luciferase, Activity Assay, Transfection, Construct, Negative Control, Mutagenesis, Positive Control

Journal: PLoS ONE

Article Title: Activation of Hypoxia Inducible Factor 1 Is a General Phenomenon in Infections with Human Pathogens

doi: 10.1371/journal.pone.0011576

Figure Lengend Snippet: The HIF-1α protein was analyzed in Western blots six hours upon infection (loading control: actin) of HeLa 229 cells with the following bacterial strains: (A) S. aureus 8325-4 (wt), S. aureus ermB Ω hemB 8325-4 (SCV), S. aureus SA113 (wt), S. aureus SA113 Δ ypf::ermB (reduced in lipoteichoic acid synthesis), S. aureus SA113 Δ tagO (defective in producing wall teichoic acid); (B) S. aureus ATCC 25923 (wt), S. epidermidis ATCC 12228 (wt), S. aureus Newman (wt), S. aureus Newman mAH12 (defective in producing the extracellular adherence protein EAP). Negative control: uninfected cells, positive control: DFO (200 µM). Multiplicity of infection (MOI): 20.

Article Snippet: Peritoneal HIF-1α activation was analyzed by immunoblotting of the shock-frozen peritoneal specimens using the Nuclear Extract Kit (Active Motif; Rixensart, Belgium), a mouse monoclonal anti-HIF-1α (H1α67) antibody (Novus Biologicals, Littleton, USA) and horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG antibodies (Dako).

Techniques: Western Blot, Infection, Control, Negative Control, Positive Control

Journal: PLoS ONE

Article Title: Activation of Hypoxia Inducible Factor 1 Is a General Phenomenon in Infections with Human Pathogens

doi: 10.1371/journal.pone.0011576

Figure Lengend Snippet: The HIF-1α protein was analyzed in Western blots six hours upon infection (loading control: actin). The following patient isolates were used: S. aureus patient isolate 1 (wt; S. aureus A22223 I), S. aureus patient isolate 2 (clinical SCV; S. aureus A22223 II), S. aureus patient isolate 3 (wt; S. aureus OM 1a), S. aureus patient isolate 4 (clinical SCV; S. aureus OM 1b). Negative control: uninfected cells, positive control: hypoxia. Multiplicity of infection (MOI): 20.

Article Snippet: Peritoneal HIF-1α activation was analyzed by immunoblotting of the shock-frozen peritoneal specimens using the Nuclear Extract Kit (Active Motif; Rixensart, Belgium), a mouse monoclonal anti-HIF-1α (H1α67) antibody (Novus Biologicals, Littleton, USA) and horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG antibodies (Dako).

Techniques: Western Blot, Infection, Control, Negative Control, Positive Control

Journal: PLoS ONE

Article Title: Activation of Hypoxia Inducible Factor 1 Is a General Phenomenon in Infections with Human Pathogens

doi: 10.1371/journal.pone.0011576

Figure Lengend Snippet: (A) Control and infected HeLa-229 cells were incubated with the hypoxic marker pimonidazole hydrochloride which was visualized by using specific Cy3 labelled antibodies (for details see ). Scale bare: 40 µm. (B) pO 2 levels were quantified in the medium of control and infected cells (triplicate means given). (C) Hela-229 cells were incubated in gas permeable (gas-perm) and conventional (conv) culture dishes and HIF 1α protein was analyzed in Western blots (loading control: actin). (D) pO 2 levels in the medium of control or S. aureus infected HeLa-229 cells (means of doubles). Negative control: uninfected cells; S. aureus wt (8325-4); S. aureus Δ hemB ( S. aureus ermB Ω hemB 8325-4, SCV); S. aureus Δ hemB , hemB -complemented ( S. aureus pCX19Ω hemB 8325-4, complemented SCV mutant). Multiplicity of infection (MOI): 20. * significant difference to control cells (P<0.05).

Article Snippet: Peritoneal HIF-1α activation was analyzed by immunoblotting of the shock-frozen peritoneal specimens using the Nuclear Extract Kit (Active Motif; Rixensart, Belgium), a mouse monoclonal anti-HIF-1α (H1α67) antibody (Novus Biologicals, Littleton, USA) and horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG antibodies (Dako).

Techniques: Control, Infection, Incubation, Marker, Western Blot, Negative Control, Mutagenesis

Journal: PLoS ONE

Article Title: Activation of Hypoxia Inducible Factor 1 Is a General Phenomenon in Infections with Human Pathogens

doi: 10.1371/journal.pone.0011576

Figure Lengend Snippet: (A) HIF 1α immunohistochemistry and (B) hematoxilin-eosin staining of renal mouse abscesses induced by intravenous infection with S. aureus seven days upon infection (following sections). HIF 1α positive nuclei are most abundant within the abscesses borders, some interstitial staining also occurs within the renal parenchyma. In addition, renal tubules in the vicinity of the abscess stain also positively (halfed arrow in B). A: arcuate artery, G: glomerulus, V: arcuate vein; arrowheads: margin of abscesses. Magnifications: (A) 250×, (B) 100×.

Article Snippet: Peritoneal HIF-1α activation was analyzed by immunoblotting of the shock-frozen peritoneal specimens using the Nuclear Extract Kit (Active Motif; Rixensart, Belgium), a mouse monoclonal anti-HIF-1α (H1α67) antibody (Novus Biologicals, Littleton, USA) and horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG antibodies (Dako).

Techniques: Immunohistochemistry, Staining, Infection

Journal: PLoS ONE

Article Title: Activation of Hypoxia Inducible Factor 1 Is a General Phenomenon in Infections with Human Pathogens

doi: 10.1371/journal.pone.0011576

Figure Lengend Snippet: (A) Survival of NMRI mice after intraperitoneal infection with S. aureus (wt; S. aureus 8325-4; n = 8) or S. aureus Δ hemB (SCV, S. aureus erm Ω hemB 8325-4; n = 8). Note the higher susceptibility of mice infected with S. aureus 8325-4 compared to the mice infected with S. aureus Δ hemB . *significant difference: P<0.001. (B) Inhibition of DFO induced HIF-1 activation in HeLa-229 cells by 17-DMAG. Cells were incubated with 17 DMAG (10 µmol/L) for 16 hours following induction of HIF 1 activation by the iron chelator DFO (200 µmol/L) for six hours. HIF 1α protein was analyzed in Western blots (loading control: actin). Negative control: uninfected cells. (C) Survival rate of NMRI mice after intraperitoneal infection with S. aureus ( S. aureus 8325-4). One group of mice was treated 24 h and 16 h before infection with the HIF-1 inhibiting compound 17-DMAG (25 mg/g body weight). Note the higher survival rate of 17-DMAG-treated mice (n = 45) compared with control mice (n = 45). *significant difference: P = 0.005 (Kaplan-Meier analysis).

Article Snippet: Peritoneal HIF-1α activation was analyzed by immunoblotting of the shock-frozen peritoneal specimens using the Nuclear Extract Kit (Active Motif; Rixensart, Belgium), a mouse monoclonal anti-HIF-1α (H1α67) antibody (Novus Biologicals, Littleton, USA) and horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG antibodies (Dako).

Techniques: Infection, Inhibition, Activation Assay, Incubation, Western Blot, Control, Negative Control

Journal: EBioMedicine

Article Title: Bone marrow mesenchymal stem cells combined with ultra-purified alginate gel as a regenerative therapeutic strategy after discectomy for degenerated intervertebral discs

doi: 10.1016/j.ebiom.2020.102698

Figure Lengend Snippet: Cell sorting data for separation of caboxyfluorescein diactetate succinimidyl ester (CFDA-SE)-labeled bone-derived mesenchymal stem cells (BMSCs) and unlabeled nucleus pulposus cells (NPCs) following 7-day co-culture using UPAL gel. (a): Two-dimensional (2D) dot plot for co-cultured cells. The P1 gate was placed around single live cells. (b): 2D dot plot showing unlabeled NPCs in the P2 gate and CFDA-SE-labeled BMSCs in the P3 gate that were sorted. FSC-A: Forward scatter-area; FSC-W: Forward scatter-width; SSC-A: Side scatter-area; CFDA-SE-A: CFDA-SE-area. Gene expression for each cell was normalized to the housekeeping gene GAPDH and plotted on a log scale (y-axis). Data were averaged from four different rabbit NPC lines. (c): HIF-1α, (d): GLUT-1, (e): Brachyury, (f): CDMP-1, (g): TGF-β, (h): IGF-1, (i): type II collagen, and (j): aggrecan. Data are represented as the means ± SE. p -values were determined by one-way ANOVA with a post hoc Tukey–Kramer test.

Article Snippet: For HIF-1α, GLUT-1, and Brachyury staining, DyLight 550-conjugated rabbit polyclonal antibodies against HIF-1α (Novus Biologicals, Centennial, CO, USA; NB100-479R, RRID: AB_1642267), PE-conjugated rabbit polyclonal antibody against GLUT-1 (LS Bio, Seattle, WA, USA; LS-A109342-100), and unconjugated rabbit polyclonal antibodies against Brachyury (LS Bio; LS-C31179-100, RRID: AB_911118) were applied.

Techniques: FACS, Labeling, Derivative Assay, Co-Culture Assay, Cell Culture, Gene Expression

Journal: EBioMedicine

Article Title: Bone marrow mesenchymal stem cells combined with ultra-purified alginate gel as a regenerative therapeutic strategy after discectomy for degenerated intervertebral discs

doi: 10.1016/j.ebiom.2020.102698

Figure Lengend Snippet: Nucleus pulposus (NP) marker-positive cells in rabbit NPs. (a-c): Horizontal sections of rabbit intervertebral discs (IVDs) stained for HIF-1α, GLUT-1, and Brachyury at days 1, 7, and 28. Images are representative of four replicates (Intact control, Discectomy, and BMSCs + Gel, n = 4; days 1, 7, and 28). Scale bar = 50 μm. (d–f): Percentages of NP marker-positive cells relative to total cells. (g–i): Percentages of NP marker-positive cells relative to CFDA-SE-positive cells (representative of implanted BMSCs). Data are the means ± SE. p -values were determined using the paired t -test.

Article Snippet: For HIF-1α, GLUT-1, and Brachyury staining, DyLight 550-conjugated rabbit polyclonal antibodies against HIF-1α (Novus Biologicals, Centennial, CO, USA; NB100-479R, RRID: AB_1642267), PE-conjugated rabbit polyclonal antibody against GLUT-1 (LS Bio, Seattle, WA, USA; LS-A109342-100), and unconjugated rabbit polyclonal antibodies against Brachyury (LS Bio; LS-C31179-100, RRID: AB_911118) were applied.

Techniques: Marker, Staining, Control

Journal: Scientific Reports

Article Title: Thyroid Transcription Factor 1 Reprograms Angiogenic Activities of Secretome

doi: 10.1038/srep19857

Figure Lengend Snippet: Western blots of NCI-H1299 cells subject to hypoxia or DFO show an increase of HIF1α ( A ). VEGF was quantified in the CM of NCI-H1299 (TTF-1 − ) and NCI-H2009 (TTF-1 + ) under normoxia, hypoxia, or DFO treatment ( B ). The CM of the A549 cells retrovirally transfected with EV , HDD , or TTF-1 was analyzed by VEGF ELISA assays after three types of treatments, normoxia, hypoxia, or DFO ( C ). The exosomes and EDMs of A549-based transfectant cells were analyzed for CD9 protein expression by immunoblotting ( D ) and VEGF ELISA assays ( E ).

Article Snippet: Membranes were blocked in 5% bovine serum albumin (Cell Signaling, #9998S) Tris-buffered saline and incubated overnight with primary antibodies: TTF-1 1:500 (sc-H190, Santa Cruz Biotechnologies #SC-13040), HIF1α 1:500 (clone 16H4L13, Fisher Scientific #70050), VEGF 1:500 (sc-152, Santa Cruz Biotechnology), CD9 1:1000 (D801A, Cell Signaling Technologies, #13174) or β-actin 1:20,000 (Cell Signaling, mouse #3700P, rabbit #4970P).

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

Journal: Genes & Development

Article Title: Discoidin domain receptor 1 (DDR1) ablation promotes tissue fibrosis and hypoxia to induce aggressive basal-like breast cancers

doi: 10.1101/gad.301366.117

Figure Lengend Snippet: DDR1 −/− mammary tumors grow faster and are more basal and necrotic. ( A ) Primary tumor burden of PyMT/DDR1 −/− mice compared with PyMT/DDR1 +/+ and PyMT/DDR1 +/− mice. PyMT/DDR1 −/− mammary tumors grow faster than control tumors. Data are shown as mean ± SD. n = 4–7. (*) P < 0.02, one-way ANOVA and unpaired Student's t -test. ( B ) DDR1 +/+ and DDR1 +/− mammary tumors showed large epithelial clusters, and DDR1 −/− tumors showed multiple small clusters and central necrosis (orange dots) by H&E staining. Representative photographs are shown (13 wk). n = 3. ( C ) The lengths of epithelial tumor clusters were measured. DDR1 −/− primary tumors show small clusters by H&E staining. Data are shown as mean ± SD. n = 3. (*) P < 0.05; (**) P < 0.02, one-way ANOVA and unpaired Student's t -test. ( D ) Percentage of necrotic clusters out of small clusters (200–300 µm in length) was measured. Necrotic small clusters increased in DDR1 −/− tumors. Data are shown as mean ± SD. n = 3. (*) P < 0.02, one-way ANOVA and unpaired Student's t -test. ( E , F ) Immunofluorescence of DDR1 +/+ and DDR1 −/− mammary tumor tissues using anti-keratin 8 (anit-K8) (green) and anti-phospho-histone H3 (phH3) (red) antibodies. (Blue) DAPI-stained nuclei. ( E ) Representative photographs are shown. PhH3 + cells were present mainly at the edge of the epithelial clusters. ( F ) The percentage of phH3 + cells per epithelial cell (K8 + and phH3 + ) shows that DDR1 −/− mammary tumor cells are more proliferative. Data are shown as mean ± SD. n = 3. (*) P < 0.02, unpaired Student's t -test. ( G ) Immunofluorescence of DDR1 +/+ and DDR1 −/− mammary tumor tissues using anti-vimentin antibody (red) and DAPI-stained nuclei (blue). Representative photographs are shown. ( H ) Expression levels of vimentin were quantified by using ImageJ software. Vimentin levels increased in DDR1 −/− epithelial clusters. Data are shown as mean ± SD. n = 4. (*) P < 0.05, unpaired Student's t -test. ( I , J ) Immunofluorescence of DDR1 +/+ , DDR1 +/− , and DDR1 −/− mammary tumor tissues using anti-E-cadherin (green) and anti-keratin 14 (anti-K14) (red) antibodies and DAPI-stained nuclei (blue). ( I ) K14 + basal cells were mainly at the edges of the epithelial clusters. Representative photographs are shown. ( J ) The ratio of K14 + basal cells per epithelial (E-cadherin + and K14 + ) cell increased ([*] P < 0.02; [**] P < 0.05, one-way ANOVA and unpaired Student's t -test) ( left panel) and expression of E-cadherin decreased ([**] P < 0.05, one-way ANOVA and unpaired Student's t -test) ( right panel) in DDR1 −/− epithelial clusters. Data are shown as mean ± SD. n = 3. ( K , L ) Immunofluorescence of the primary tumors was performed by using anti-K8 (green) and anti-DDR2 (red) antibodies. (Blue) DAPI-stained nuclei. ( K ) Representative photos of tumor epithelial clusters are shown. ( L ) The ratio of DDR2 + cells per epithelial cell increased significantly in DDR1 −/− epithelial clusters. Data are shown as mean ± SD. n = 3. (*) P < 0.01, one-way ANOVA and unpaired Student's t -test. ( M , N ) Immunofluorescence of DDR1 +/+ , DDR1 +/− , and DDR1 −/− mammary tumor tissues was conducted by using anti-K8 (green) and anti-K14 (red) antibodies. (Blue) DAPI-stained nuclei. ( M ) Representative photos of tumor necrotic areas are shown. ( N ) K14 + basal cells, including K8 + K14 + basal-like cells (yellow), significantly increased near necrotic fields of DDR1 −/− mammary tumor tissues. Data are shown as mean ± SD. n = 3. (*) P < 0.05, one-way ANOVA and unpaired Student's t -test. ( O ) Immunofluorescence of DDR1 −/− mammary tumor tissues using anti-E-cadherin (green) and anti-hypoxia-inducible factor α (anti-HIF1α) (red) antibodies and DAPI-stained nuclei (blue). ( Right panel) The white dots represent a border between an epithelial and a necrotic field. HIF1α is expressed and localized near necrosis.

Article Snippet: The following antibodies were used for immunofluorescence at the indicated concentrations: DDR1 (1:50; Santa Cruz Biotechnology, sc-532), E-cadherin (1:100; BD Biosciences, 610181), K8 (1:50; Developmental Studies Hybridoma Bank, TROMA-I), K14 (1:5000 [Convance, PRB-155P] and 1:100 [Santa Cruz Biotechnology, sc-17104]), α-SMA Cy3 conjugate (1:250; Sigma, C6198), vimentin (1:200; Sigma, V5255), DDR2 (1:50; LifeSpan BioSciences, LS-C164363), phH3 (1:100; Cell Signaling, 9701), HIF1α (1:50; Novus Biologicals, NB100-479), pMLC2 (1:100; Cell Signaling Technology, 3671), goat anti-mouse IgM μ chain Cy3 conjugate (1:200; Jackson ImmunoResearch, 115-166-075), and Alexa 488 anti-rat, Alexa 488 anti-rabbit, Alexa 488 anti-mouse, Alexa 568 anti-rabbit, and Alexa 647 anti-goat secondary antibodies (1:500; Molecular Probes, A11006, A24922, A24920, A21069, and A21447).

Techniques: Control, Staining, Immunofluorescence, Expressing, Software