Journal: Cells

Article Title: Caspase-Dependent HMGB1 Release from Macrophages Participates in Peripheral Neuropathy Caused by Bortezomib, a Proteasome-Inhibiting Chemotherapeutic Agent, in Mice

doi: 10.3390/cells10102550

Figure Lengend Snippet: Involvement of HMGB1-targeted receptors in the CIPN caused by bortezomib. Bortezomib at 0.4 mg/kg or vehicle was administered i.p. on days 0, 2, 5, 7, 9, and 12. ( A , B ) Preventive ( A ) or therapeutic ( B ) effect of FPS-ZM1, TAK-242, and AMD3100, antagonists of RAGE, TLR4, and CXCR4, respectively. The mice received repeated i.p. administration of FPS-ZM1 at 1 mg/kg, TAK-242 at 3 mg/kg, AMD3100 at 8 mg/kg, or vehicle, 30 min before each dose of bortezomib ( A ), or single i.p. administration of each of them on day 14 ( B ). ( C , D ) Protein levels of RAGE, TLR4, and CXCR4 in the dorsal root ganglion ( C ) and sciatic nerves ( D ) on day 14 after the onset of bortezomib treatment. ( E , F ) Lack of preventive ( E ) and therapeutic ( F ) effects of TH1020, a TLR5 antagonist. The mice received repeated ( E ) or single ( F ) i.p. administration of TH1020 at 1 mg/kg or vehicle, in mice treated with bortezomib according to the above-mentioned schedules. Data show the mean with S.E.M for 5–6 ( A , B , F ), 5–7 ( C ), 6–7 ( D ), or 6 ( E ) mice. V, vehicle; BTZ, bortezomib; DRG, dorsal root ganglion; * p < 0.05, ** p < 0.01 vs. V ( C ) or V in V-treated mice ( A , B , E , F ). † p < 0.05, †† p < 0.01 vs. V in BTZ-treated mice.

Article Snippet: Primary antibodies were: an anti-HMGB1 rabbit polyclonal antibody (Abcam, Cambridge, UK) (1: 5000 dilution), anti-RAGE rabbit polyclonal antibody (Abcam) (1:1000 dilution), anti-TLR4 rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) (1:10000 dilution), anti-CXCR4 rabbit polyclonal antibody (Novus Biologicals, Littleton, CO, USA) (1:1000 dilution), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc.) (1:5000 dilution).

Techniques:

Journal: Cells

Article Title: Caspase-Dependent HMGB1 Release from Macrophages Participates in Peripheral Neuropathy Caused by Bortezomib, a Proteasome-Inhibiting Chemotherapeutic Agent, in Mice

doi: 10.3390/cells10102550

Figure Lengend Snippet: Scheme for bortezomib-induced caspase-dependent HMGB1 release from macrophages and CIPN development, in contrast to caspase-independent mechanisms for paclitaxel. ( A ) Inhibition of proteasome by bortezomib causes caspase-dependent apoptosis of macrophages followed by the release of HMGB1, which in turn causes neuronal sensitization via activation of RAGE and acceleration of CXCL12/CXCR4 signals, leading to CIPN. ( B ) Paclitaxel causes HMGB1 release from macrophages through activation of the ROS/p38MAPK/NF-κB pathway , independently of caspase (see D), and the extracellular HMGB1 develops CIPN in a manner dependent on RAGE and CXCR4 , as shown in the CIPN caused by bortezomib.

Article Snippet: Primary antibodies were: an anti-HMGB1 rabbit polyclonal antibody (Abcam, Cambridge, UK) (1: 5000 dilution), anti-RAGE rabbit polyclonal antibody (Abcam) (1:1000 dilution), anti-TLR4 rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) (1:10000 dilution), anti-CXCR4 rabbit polyclonal antibody (Novus Biologicals, Littleton, CO, USA) (1:1000 dilution), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc.) (1:5000 dilution).

Techniques: Inhibition, Activation Assay

Journal: Developmental Cell

Article Title: Lumen Expansion Facilitates Epiblast-Primitive Endoderm Fate Specification during Mouse Blastocyst Formation

doi: 10.1016/j.devcel.2019.10.011

Figure Lengend Snippet: EPI and PrE Expression Levels Are Reduced in ATP1-Inhibited Embryos (A) Immunofluorescence images of TE (Cdx2), EPI (Sox2), and PrE (Gata4) fate in pre-treatment control (E3.5 WT), Atp1 inhibited (E4.0 500 μM and E4.0 250 μM), and end-stage control (E4.0 DMSO) embryos. Lumen boundaries outlined by dashed white line and mean lumen volume in white text. Scale bars, 10 μm. (B) Boxplot of lumen volume for E3.5 WT (N = 21), E4.0 DMSO (N = 24), E4.0 250 μM Atp1 inhibited (N = 14) and E4.0 500 μM Atp1 inhibited (N = 31) embryos indicating that the impact on lumen volume is concentration dependent. (C) Boxplot of fluorescence levels of Cdx2 (gray), Sox2 (green), and Gata4 (magenta) in E4.0 500 μM Atp1 inhibited embryos compared to E4.0 DMSO controls. (D) Schematic 2D representation of 3D analysis method for spatial segregation of ICM lineages. P 1,2,3,4 are 3D points. L ↔ is a 3D line ( P 1 P 2 ↔ equivalent) that defines the embryonic-abembryonic axis. d ‾ is the 3D line segment ( P 3 P 4 ‾ equivalent) that measures the perpendicular distance from the center of a cell to L ↔ . See Image Analysis for formal definitions of all geometric entities. (E) Boxplot of spatial overlap between EPI and PrE lineages within E4.0 control (DMSO, N = 15), E4.0 Atp1 inhibited (500 μM, N =13) and simulated data of maximal overlap in E4.0 WT embryos (Simulation, N = 27). ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05. n.s., not significant. For boxplots: central mark indicates the median; lower edge, 25%; upper edge, 75%; lower whisker, Q1 − (1.5 × IQR), where IQR = Q3 − Q1; upper whisker, Q3 + (1.5 × IQR). See also and ; .

Article Snippet: The following primary antibodies were used in this study: rabbit anti-pERM (Cell Signaling, 3726), mouse anti-Cdx2 (BioGenex, MU392A-UC), goat anti-Sox2 (Santa Cruz Biotechnology, sc-17320), goat anti-Sox2 (R&D Systems, AF2018-SP), rabbit anti-Sox2 (Cell Signaling, 23064), rabbit anti Gata4 (Santa Cruz Biotechnology, sc-9053), goat anti-Gata4 (R&D Systems, AF2606-SP), rabbit anti-GFP (MBL, 598), mouse anti-Rab11 (BD Biosciences; 610656), rat anti-integrin-β1 (Merck Millipore, MAB1997), goat anti-biotinylated Gata4 (R&D Systems, BAF2606) mouse anti-Oct3/4 (Santa Cruz Biotechnology, sc-5279) and mouse anti-Hsp47 (Enzo Life Sciences, M16.10A1).

Techniques: Expressing, Immunofluorescence, Control, Concentration Assay, Fluorescence, Whisker Assay

Journal: Developmental Cell

Article Title: Lumen Expansion Facilitates Epiblast-Primitive Endoderm Fate Specification during Mouse Blastocyst Formation

doi: 10.1016/j.devcel.2019.10.011

Figure Lengend Snippet: PrE Specification and Spatial Segregation of ICM Lineages Is Impaired by Mechanical Inhibition of Lumen Expansion (A) Brightfield images of mechanical deflation. Magenta asterisk marks the needle tip. Dotted magenta line indicates lumen boundary. (B) Immunofluorescence images of EPI (Sox2) and PrE (Gata4) fate in pre-manipulation control (E3.5 WT), E4.0 post-manipulation control (E4.0 WT), and E4.0 mechanically inhibited (E4.0 Mechanical) embryos. Magenta arrowheads indicate the position of cells expressing high levels of Gata4 within the ICM. White dotted line indicates lumen boundaries. Average lumen volume in white text. Scale bars, 10 μm. (C) Boxplot of fluorescence levels of Sox2 (green) and Gata4 (magenta) in mechanically inhibited (Mech., N = 33) and post-manipulation control (WT, N = 28) E4.0 embryos. (D) Boxplot of spatial overlap between EPI and PrE lineages within post-manipulation control (WT, N = 27), mechanically inhibited (Mech., N = 33), E4.0 procedural control (Control, N = 11), and E4.0 simulation of complete overlap in WT conditions (Simulation, N = 27). ∗∗∗∗ p < 0.0001, ∗∗ p < 0.01. n.s., not significant. For boxplots: central mark indicates the median; lower edge, 25%; upper edge, 75%; lower whisker, Q1 − (1.5 × IQR), where IQR = Q3 − Q1; upper whisker, Q3 + (1.5 × IQR). See also Figure S6 and .

Article Snippet: The following primary antibodies were used in this study: rabbit anti-pERM (Cell Signaling, 3726), mouse anti-Cdx2 (BioGenex, MU392A-UC), goat anti-Sox2 (Santa Cruz Biotechnology, sc-17320), goat anti-Sox2 (R&D Systems, AF2018-SP), rabbit anti-Sox2 (Cell Signaling, 23064), rabbit anti Gata4 (Santa Cruz Biotechnology, sc-9053), goat anti-Gata4 (R&D Systems, AF2606-SP), rabbit anti-GFP (MBL, 598), mouse anti-Rab11 (BD Biosciences; 610656), rat anti-integrin-β1 (Merck Millipore, MAB1997), goat anti-biotinylated Gata4 (R&D Systems, BAF2606) mouse anti-Oct3/4 (Santa Cruz Biotechnology, sc-5279) and mouse anti-Hsp47 (Enzo Life Sciences, M16.10A1).

Techniques: Inhibition, Immunofluorescence, Control, Expressing, Fluorescence, Whisker Assay

Journal: Developmental Cell

Article Title: Lumen Expansion Facilitates Epiblast-Primitive Endoderm Fate Specification during Mouse Blastocyst Formation

doi: 10.1016/j.devcel.2019.10.011

Figure Lengend Snippet: Perturbation of FGF4 Signaling in the Lumen Impacts Molecular Specification of EPI and PrE Lineages (A) Immunofluorescence images of EPI (Sox2) and PrE (Gata4) fate in E3.5 post-FGF4 deposition (E3.5 FGF4 Inj.), E3.5 post-PD173074 deposition (E3.5 PD Inj.), and E3.5 post-PBS deposition (E3.5 PBS Inj.). White dotted line indicates lumen boundaries. Average lumen volume in white text. Scale bars, 10 μm. (B) Boxplot of fluorescence levels of Sox2 (green) and Gata4 (magenta) in E3.5 post-FGF4 deposition (FGF4 Inj., N = 24), E3.5 post-PD173074 deposition (PD Inj., N = 21), and E3.5 post-PBS deposition (PBS Inj., N = 13) embryos. (C) Boxplot of luminal volume in E3.5 post-FGF4 deposition (FGF4 Inj., N = 24), E3.5 post-PD173074 deposition (PD Inj., N = 21), and E3.5 post-PBS deposition (PBS Inj., N = 13). ∗∗∗∗ p < 0.0001, ∗∗ p < 0.01. n.s., not significant. For boxplots: central mark indicates the median; lower edge, 25%; upper edge, 75%; lower whisker, Q1 − (1.5 × IQR) where IQR = Q3 − Q1; upper whisker, Q3 + (1.5 × IQR). See also Figure S7 .

Article Snippet: The following primary antibodies were used in this study: rabbit anti-pERM (Cell Signaling, 3726), mouse anti-Cdx2 (BioGenex, MU392A-UC), goat anti-Sox2 (Santa Cruz Biotechnology, sc-17320), goat anti-Sox2 (R&D Systems, AF2018-SP), rabbit anti-Sox2 (Cell Signaling, 23064), rabbit anti Gata4 (Santa Cruz Biotechnology, sc-9053), goat anti-Gata4 (R&D Systems, AF2606-SP), rabbit anti-GFP (MBL, 598), mouse anti-Rab11 (BD Biosciences; 610656), rat anti-integrin-β1 (Merck Millipore, MAB1997), goat anti-biotinylated Gata4 (R&D Systems, BAF2606) mouse anti-Oct3/4 (Santa Cruz Biotechnology, sc-5279) and mouse anti-Hsp47 (Enzo Life Sciences, M16.10A1).

Techniques: Immunofluorescence, Fluorescence, Whisker Assay

Journal: Developmental Cell

Article Title: Lumen Expansion Facilitates Epiblast-Primitive Endoderm Fate Specification during Mouse Blastocyst Formation

doi: 10.1016/j.devcel.2019.10.011

Figure Lengend Snippet: Luminal Deposition of FGF4 Partially Rescues EPI-PrE Specification in ATP1-Inhibited Embryos (A) Immunofluorescence images of EPI (Sox2) and PrE (Gata4) fate in E3.5 post-FGF4 deposition and Atp1 inhibition (E3.5 FGF4 250 μM), E3.5 post-PBS deposition and Atp1 inhibition (E3.5 PBS 250 μM), and E3.5 control embryos (E3.5 DMSO). White dotted line indicates lumen boundaries. Average lumen volume in white text. Magenta arrowhead indicates cell with high Gata4 expression relative to neighboring cells. Scale bars, 10 μm. (B) Boxplot of fluorescence levels of Sox2 (green) and Gata4 (magenta) in E3.5 post-FGF4 deposition and Atp1 inhibition (250 μM FGF4 Inj., N = 42 embryos), E3.5 post-PBS deposition and Atp1 inhibition (250 μM PBS Inj., N = 12), and E3.5 control embryos (DMSO, N = 12). (C) Boxplot of luminal volume in E3.5 post-FGF4 deposition and Atp1 inhibition (250 μM FGF4 Inj., N = 49 embryos), E3.5 post-PBS deposition and Atp1 inhibition (250μM PBS Inj., N = 9), and E3.5 control embryos (DMSO, N = 38). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001. n.s., not significant. For boxplots: central mark indicates the median; lower edge, 25%; upper edge, 75%; lower whisker, Q1 − (1.5 × IQR), where IQR = Q3 − Q1; upper whisker, Q3 + (1.5 × IQR).

Article Snippet: The following primary antibodies were used in this study: rabbit anti-pERM (Cell Signaling, 3726), mouse anti-Cdx2 (BioGenex, MU392A-UC), goat anti-Sox2 (Santa Cruz Biotechnology, sc-17320), goat anti-Sox2 (R&D Systems, AF2018-SP), rabbit anti-Sox2 (Cell Signaling, 23064), rabbit anti Gata4 (Santa Cruz Biotechnology, sc-9053), goat anti-Gata4 (R&D Systems, AF2606-SP), rabbit anti-GFP (MBL, 598), mouse anti-Rab11 (BD Biosciences; 610656), rat anti-integrin-β1 (Merck Millipore, MAB1997), goat anti-biotinylated Gata4 (R&D Systems, BAF2606) mouse anti-Oct3/4 (Santa Cruz Biotechnology, sc-5279) and mouse anti-Hsp47 (Enzo Life Sciences, M16.10A1).

Techniques: Immunofluorescence, Inhibition, Control, Expressing, Fluorescence, Whisker Assay

Journal: Developmental Cell

Article Title: Lumen Expansion Facilitates Epiblast-Primitive Endoderm Fate Specification during Mouse Blastocyst Formation

doi: 10.1016/j.devcel.2019.10.011

Figure Lengend Snippet:

Article Snippet: The following primary antibodies were used in this study: rabbit anti-pERM (Cell Signaling, 3726), mouse anti-Cdx2 (BioGenex, MU392A-UC), goat anti-Sox2 (Santa Cruz Biotechnology, sc-17320), goat anti-Sox2 (R&D Systems, AF2018-SP), rabbit anti-Sox2 (Cell Signaling, 23064), rabbit anti Gata4 (Santa Cruz Biotechnology, sc-9053), goat anti-Gata4 (R&D Systems, AF2606-SP), rabbit anti-GFP (MBL, 598), mouse anti-Rab11 (BD Biosciences; 610656), rat anti-integrin-β1 (Merck Millipore, MAB1997), goat anti-biotinylated Gata4 (R&D Systems, BAF2606) mouse anti-Oct3/4 (Santa Cruz Biotechnology, sc-5279) and mouse anti-Hsp47 (Enzo Life Sciences, M16.10A1).

Techniques: Recombinant, Software

Journal: Circulation

Article Title: Enhanced Inhibition of Neointimal Hyperplasia by Genetically Engineered Endothelial Progenitor Cells

doi: 10.1161/01.cir.0000121732.85572.6f

Figure Lengend Snippet: Figure 1. Characterization of rabbit EPCs and comparison with HUVECs. A, B, Cobblestone morphology of EPCs and HUVEC 10 days after plating (40); C, D, immunohistochemical detec- tion of CD31; immunofluorescence detection of (E, F), VE-cadherin; (G, H), eNOS; (I, J), vWF. Preparations were viewed at 400 unless otherwise indicated. Preparations in G, H, I, and J were counterstained with Hoechst 33342.

Article Snippet: The cells were incubated with 1:50 dilution of monoclonal anti human CD31 (PECAM-1, Dako, Carpinteria, Calif), or polyclonal goat anti-mouse VE-cadherin or anti-human eNOS (R&D Systems Inc, Minneapolis, Minn), or anti-human von Willebrand factor (vWF) (Dako).

Techniques: Comparison, Immunohistochemical staining

Journal: Circulation

Article Title: Enhanced Inhibition of Neointimal Hyperplasia by Genetically Engineered Endothelial Progenitor Cells

doi: 10.1161/01.cir.0000121732.85572.6f

Figure Lengend Snippet: Figure 2. Ex vivo retroviral transduction and transgene expression in EPCs. A, Confluent monolayer of EPCs trans- duced with MSCV retrovirus expressing GFP viewed under normal (white) light (100); B, view of same field under green fluorescent light; C, D, human eNOS transgene mRNA and protein expression in untransduced and trans- duced EPCs, respectively; E, F, HO-1 mRNA and protein expression in untransduced and transduced EPCs, respectively.

Article Snippet: The cells were incubated with 1:50 dilution of monoclonal anti human CD31 (PECAM-1, Dako, Carpinteria, Calif), or polyclonal goat anti-mouse VE-cadherin or anti-human eNOS (R&D Systems Inc, Minneapolis, Minn), or anti-human von Willebrand factor (vWF) (Dako).

Techniques: Ex Vivo, Retroviral, Transduction, Expressing

Journal: Circulation

Article Title: Enhanced Inhibition of Neointimal Hyperplasia by Genetically Engineered Endothelial Progenitor Cells

doi: 10.1161/01.cir.0000121732.85572.6f

Figure Lengend Snippet: Figure 3. In vivo transgene expression after transplantation of EPCs onto injured carotid arteries. A, Immunohistochemical detection of eNOS (arrows) in frozen sections from denuded vessel trans- planted with eNOS-EPCs (250). B, eNOS immunoreactivity in sections from control vessel transplanted with GFP- EPCs (250); C, immunohistochemical detection of human HO-1 (arrows) in fro- zen sections from vessel transplanted with HO-1-EPCs (250); D, control ves- sels transplanted with GFP-EPCs (250); E, detection of human eNOS transgene mRNA expression by RT-PCR; F, human eNOS protein expression in homoge- nates from eNOS-EPC–transplanted ves- sels; G, human HO-1 transgene mRNA expression; H, HO-1 protein expression in homogenates from HO-1-EPC–trans- planted vessels. GFP-EPC–transplanted vessels served as controls.

Article Snippet: The cells were incubated with 1:50 dilution of monoclonal anti human CD31 (PECAM-1, Dako, Carpinteria, Calif), or polyclonal goat anti-mouse VE-cadherin or anti-human eNOS (R&D Systems Inc, Minneapolis, Minn), or anti-human von Willebrand factor (vWF) (Dako).

Techniques: In Vivo, Expressing, Transplantation Assay, Immunohistochemical staining, Control, Reverse Transcription Polymerase Chain Reaction

Journal: Circulation

Article Title: Enhanced Inhibition of Neointimal Hyperplasia by Genetically Engineered Endothelial Progenitor Cells

doi: 10.1161/01.cir.0000121732.85572.6f

Figure Lengend Snippet: Figure 4. Reendothelialization of injured arteries after transplan- tation of EPCs expressing GFP, eNOS, or HO-1 genes. Low- power (100) magnification of vessel profiles stained with anti- CD31 antibody in A, unseeded; B, GFP-EPCs; C, eNOS-EPCs; and D, HO-1-EPCs. Red CD31-positive staining indicates pres- ence of an endothelial monolayer. E, Quantification of endotheli- alization by planar morphometry. Endothelialization was enhanced in EPC-transplanted vessels compared with unseeded saline-treated controls (*P0.05, n4/group).

Article Snippet: The cells were incubated with 1:50 dilution of monoclonal anti human CD31 (PECAM-1, Dako, Carpinteria, Calif), or polyclonal goat anti-mouse VE-cadherin or anti-human eNOS (R&D Systems Inc, Minneapolis, Minn), or anti-human von Willebrand factor (vWF) (Dako).

Techniques: Expressing, Staining, Saline

Journal: Circulation

Article Title: Enhanced Inhibition of Neointimal Hyperplasia by Genetically Engineered Endothelial Progenitor Cells

doi: 10.1161/01.cir.0000121732.85572.6f

Figure Lengend Snippet: Figure 5. Inhibition of neointimal proliferation by EPCs in injured carotid arteries. All sections were stained with Accustain elastic stain. Low (40, left) and high (200, right) magnification of vessel profiles. A, B, Saline-injured artery transplanted with eNOS-EPCs (n6); C, D, balloon-injured artery transplanted with EPC expressing GFP (n6); E, F, balloon-injured artery transplanted with EPC expressing eNOS (n6); G, H, balloon- injured artery transplanted with HO-1-EPCs (n6); I, neointima/ media ratios in unseeded and EPC-transplanted injured vessels. Arrows indicate neointima. *Saline vs GFP-EPCs, eNOS-EPCs, HO-1-EPCs; †eNOS-EPCs vs GFP-EPCs, P0.05.

Article Snippet: The cells were incubated with 1:50 dilution of monoclonal anti human CD31 (PECAM-1, Dako, Carpinteria, Calif), or polyclonal goat anti-mouse VE-cadherin or anti-human eNOS (R&D Systems Inc, Minneapolis, Minn), or anti-human von Willebrand factor (vWF) (Dako).

Techniques: Inhibition, Staining, Saline, Expressing

Journal: Cell Death Discovery

Article Title: Transcription factor 7-like 1 dysregulates keratinocyte differentiation through upregulating lipocalin 2

doi: 10.1038/cddiscovery.2016.28

Figure Lengend Snippet: The effect of Tcf7l1 depletion on LCN2 expression in HFKs. HFKs were stimulated with calcium chloride (1.2 mM, 7 days) after siRNA transfection. ( a and b ) Intracellular ( a ) and soluble ( b ) LCN2 levels were evaluated by western blot and ELISA, respectively. ( c ) By immunofluorescence, the expression of involucrin and loricrin were detected in HFKs that were transfected with Tcf7l1 or control siRNA. ( d and e ) The expression of involucrin and loricrin was also detected in HFKs by western blot. Data show mean±S.E.M. of triplicate cultures ( n =3). Statistical differences were tested with one-way ANOVA followed by a two-tailed t -test. Representative images are shown. Scale bar=10 μ m. * P <0.05; ** P <0.01.

Article Snippet: For confocal analysis, rabbit anti-involucrin (Alexa Fluor 555-conjugated; Bioss), anti-loricrin (Cy5-conjugated) (Biorbyt LLC, San Francisco, CA, USA) or anti-cytokeratin 1 (Alexa Fluor 488-conjugated) (Abcam, Cambridge, MA, USA) antibodies were used by following the standard method.

Techniques: Expressing, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Two Tailed Test

Journal: Cell Death Discovery

Article Title: Transcription factor 7-like 1 dysregulates keratinocyte differentiation through upregulating lipocalin 2

doi: 10.1038/cddiscovery.2016.28

Figure Lengend Snippet: The effect of Tcf7l1 overexpression on HFKs. After the transfection of Tcf7l1-inserted pcDNA vector, HFKs were stimulated with calcium chloride (1.2 mM, 7 days). ( a and b ) By qRT-PCR ( a ) and western blot ( b ), HFKs expressed more lipocalin 2 (LCN2) upon Tcf7l1 pcDNA transfection. ( c and d ) Both cell apoptosis ( c ) and proliferation ( d ) were determined in HFKs that were either transfected with Tcf7l1 pcDNA or not. Cell apoptosis and proliferation were also visualized by TUNEL and fluorescent Ki-67 staining, respectively. ( e ) qRT-PCR was performed for the mRNA levels of involucrin, loricrin and caspase-14 in HFKs. Data show mean±S.E.M. of triplicate cultures ( n =3). Statistical differences were tested with one-way ANOVA followed by a two-tailed t -test. Scale bar=10 μ m. In ( e ), * P <0.05, ** P <0.01, compared with nontreated groups accordingly; Δ P <0.05, compared with calcium alone-treated HFKs accordingly.

Article Snippet: For confocal analysis, rabbit anti-involucrin (Alexa Fluor 555-conjugated; Bioss), anti-loricrin (Cy5-conjugated) (Biorbyt LLC, San Francisco, CA, USA) or anti-cytokeratin 1 (Alexa Fluor 488-conjugated) (Abcam, Cambridge, MA, USA) antibodies were used by following the standard method.

Techniques: Over Expression, Transfection, Plasmid Preparation, Quantitative RT-PCR, Western Blot, TUNEL Assay, Staining, Two Tailed Test

Journal: Cell Death Discovery

Article Title: Transcription factor 7-like 1 dysregulates keratinocyte differentiation through upregulating lipocalin 2

doi: 10.1038/cddiscovery.2016.28

Figure Lengend Snippet: The effect of LCN2 inhibition on HFKs. ( a and b ) By western blot, involucrin and loricrin expression was determined in Tcf7l1-transfected HFKs that were then treated with LCN2 or control siRNA. ( c and d ) Similarly, involucrin and loricrin were assessed in E6/E7-transfected HFKs that were treated with neutralizing anti-LCN2 antibody or isotype control (2 μ g/ml, 2 days). ( e and f ) By immunofluorescence ( e ) and western blot ( f ), keratin 1 was detected in HFKs that were transfected with Tcf7l1 vector and then LCN2 or control siRNA. ( g ) HFKs were pre-transfected with Tcf7l1 vector, and then transfected with LCN2 siRNA. Cell apoptosis was quantitated by the CaspaTag caspase 3, 7 method. Data show mean±S.E.M. of triplicate cultures ( n =3). Statistical differences were tested with one-way ANOVA followed by a two-tailed t -test. Representative images are shown. Scale bar=10 μ m. * P <0.05; ** P <0.01.

Article Snippet: For confocal analysis, rabbit anti-involucrin (Alexa Fluor 555-conjugated; Bioss), anti-loricrin (Cy5-conjugated) (Biorbyt LLC, San Francisco, CA, USA) or anti-cytokeratin 1 (Alexa Fluor 488-conjugated) (Abcam, Cambridge, MA, USA) antibodies were used by following the standard method.

Techniques: Inhibition, Western Blot, Expressing, Transfection, Immunofluorescence, Plasmid Preparation, Two Tailed Test

Journal: Journal of Nuclear Medicine

Article Title: Clinical Translation of a 68 Ga-Labeled Integrin α v β 6 –Targeting Cyclic Radiotracer for PET Imaging of Pancreatic Cancer

doi: 10.2967/jnumed.119.237347

Figure Lengend Snippet: (A) Chemical structure of DOTA-cycratide. (B) Inhibition of 64Cu-cycratide binding to integrin αvβ6 on BxPC-3 cells by cycratide, DOTA-cycratide, and linear-pep. Data are shown as mean ± SD, n = 4. (C) Binding of 68Ga-cycratide to BxPC-3 with or without blocking of cold cycratide or linear-pep. %AD/106 cells = percentage of total added dose per million cells. Data are shown as mean ± SD, n = 4. (D) Metabolic stability of 68Ga-cycratide in blood and urine of BALB/c mice (data are representative of 3 independent experiments). **P < 0.01.

Article Snippet: After endogenous peroxidase activity had been abolished using 0.3% hydrogen peroxide and antigen had been retrieved by microwave, tumor tissues were incubated with rabbit antihuman integrin α v β 6 antibody (bs-5791r; Bioss) overnight at 4°C.

Techniques: Inhibition, Binding Assay, Blocking Assay

Journal: Journal of Nuclear Medicine

Article Title: Clinical Translation of a 68 Ga-Labeled Integrin α v β 6 –Targeting Cyclic Radiotracer for PET Imaging of Pancreatic Cancer

doi: 10.2967/jnumed.119.237347

Figure Lengend Snippet: (A) PET imaging of orthotopic pancreatic cancer lesions in nude mice at 0.5 h after injection of 68Ga-cycratide without or with blocking dose of cold cycratide. Tumors are indicated by arrows. (B) Hematoxylin and eosin (H&E) staining of tumor tissues harvested from orthotopic tumor model. (C) Left, immunofluorescence staining of integrin αvβ6 from tumor tissues harvested from orthotopic tumor model. Right, negative control with secondary antibody only.

Article Snippet: After endogenous peroxidase activity had been abolished using 0.3% hydrogen peroxide and antigen had been retrieved by microwave, tumor tissues were incubated with rabbit antihuman integrin α v β 6 antibody (bs-5791r; Bioss) overnight at 4°C.

Techniques: Imaging, Injection, Blocking Assay, Staining, Immunofluorescence, Negative Control

Journal: Journal of Nuclear Medicine

Article Title: Clinical Translation of a 68 Ga-Labeled Integrin α v β 6 –Targeting Cyclic Radiotracer for PET Imaging of Pancreatic Cancer

doi: 10.2967/jnumed.119.237347

Figure Lengend Snippet: (A) PET/CT images of female patient with suspected pancreatic cancer. Images were obtained at 1 h after intravenous administration of 68Ga-cycratide or 18F-FDG. Tumors are indicated by arrows. (B) Immunohistochemical (IHC) staining for integrin αvβ6 in tumor sample from same patient as in A. (C) Contrast-enhanced CT (CECT) image and PET/CT images of male patient with pancreatic cancer 7 mo after surgery at 1 h after administration of 68Ga-cycratide or 18F-FDG, as well as CECT image of same patient 3 mo later (10 mo after surgery). Occupancy lesions in CT are indicated by arrows.

Article Snippet: After endogenous peroxidase activity had been abolished using 0.3% hydrogen peroxide and antigen had been retrieved by microwave, tumor tissues were incubated with rabbit antihuman integrin α v β 6 antibody (bs-5791r; Bioss) overnight at 4°C.

Techniques: Positron Emission Tomography-Computed Tomography, Immunohistochemical staining, Immunohistochemistry

Journal: Cell reports

Article Title: Neurokinin-1 Receptor Signaling Is Required for Efficient Ca 2+ Flux in T-Cell-Receptor-Activated T Cells

doi: 10.1016/j.celrep.2020.02.054

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-mouse / rat / human Substance P (rabbit polyclonal), Cy3 , Bioss USA , Cat # bs-0064R-Cy3.

Techniques: Functional Assay, Recombinant, Staining, Avidin-Biotin Assay, Blocking Assay, Plasmid Preparation, Inhibition, Fluorescence, SYBR Green Assay, Activation Assay, Bicinchoninic Acid Protein Assay, In Situ, Enzyme-linked Immunosorbent Assay, Activity Assay, Labeling, Software

Journal: Cells

Article Title: The Inhibition on MDFIC and PI3K/AKT Pathway Caused by miR-146b-3p Triggers Suppression of Myoblast Proliferation and Differentiation and Promotion of Apoptosis

doi: 10.3390/cells8070656

Figure Lengend Snippet: Primers used for RT-PCR.

Article Snippet: The antibodies used for Western blots and their dilutions are as follows: Cleaved Caspase-8 (Asp391) (18C8) Rabbit mAb (Cell Signaling Technology, Boston, MA, USA; 1:1000), Anti-Caspase-9 antibody [E23] (Abcam, London, UK; 1:1000), mouse anti-MyHC antibody (Bioss, Beijing, China; 1:1000), rabbit anti-Lamin B antibody (Bioss, Beijing, China; 1:500), rabbit anti-AKT1 (Bioss, Beijing, China; 1:500), rabbit anti-phospho-AKT1 (Bioss, Beijing, China; 1:500), rabbit Anti-phospho-AKT antibody (Bioss, Beijing, China; 1:300), and mouse anti-GAPDH (Boster, Wuhan, China; 1:2000).

Techniques:

Journal: Cells

Article Title: The Inhibition on MDFIC and PI3K/AKT Pathway Caused by miR-146b-3p Triggers Suppression of Myoblast Proliferation and Differentiation and Promotion of Apoptosis

doi: 10.3390/cells8070656

Figure Lengend Snippet: Oligonucleotide sequences used in this study.

Article Snippet: The antibodies used for Western blots and their dilutions are as follows: Cleaved Caspase-8 (Asp391) (18C8) Rabbit mAb (Cell Signaling Technology, Boston, MA, USA; 1:1000), Anti-Caspase-9 antibody [E23] (Abcam, London, UK; 1:1000), mouse anti-MyHC antibody (Bioss, Beijing, China; 1:1000), rabbit anti-Lamin B antibody (Bioss, Beijing, China; 1:500), rabbit anti-AKT1 (Bioss, Beijing, China; 1:500), rabbit anti-phospho-AKT1 (Bioss, Beijing, China; 1:500), rabbit Anti-phospho-AKT antibody (Bioss, Beijing, China; 1:300), and mouse anti-GAPDH (Boster, Wuhan, China; 1:2000).

Techniques:

Journal: Cells

Article Title: The Inhibition on MDFIC and PI3K/AKT Pathway Caused by miR-146b-3p Triggers Suppression of Myoblast Proliferation and Differentiation and Promotion of Apoptosis

doi: 10.3390/cells8070656

Figure Lengend Snippet: Primers used for plasmids construction.

Article Snippet: The antibodies used for Western blots and their dilutions are as follows: Cleaved Caspase-8 (Asp391) (18C8) Rabbit mAb (Cell Signaling Technology, Boston, MA, USA; 1:1000), Anti-Caspase-9 antibody [E23] (Abcam, London, UK; 1:1000), mouse anti-MyHC antibody (Bioss, Beijing, China; 1:1000), rabbit anti-Lamin B antibody (Bioss, Beijing, China; 1:500), rabbit anti-AKT1 (Bioss, Beijing, China; 1:500), rabbit anti-phospho-AKT1 (Bioss, Beijing, China; 1:500), rabbit Anti-phospho-AKT antibody (Bioss, Beijing, China; 1:300), and mouse anti-GAPDH (Boster, Wuhan, China; 1:2000).

Techniques:

Journal: Cells

Article Title: The Inhibition on MDFIC and PI3K/AKT Pathway Caused by miR-146b-3p Triggers Suppression of Myoblast Proliferation and Differentiation and Promotion of Apoptosis

doi: 10.3390/cells8070656

Figure Lengend Snippet: miR-146b-3p targets AKT1 and MDFIC and downregulates PI3K/AKT pathway activity. ( a ) Relative mRNA expression of AKT1 and MDFIC in QM-7 cells after overexpression or inhibition of miR-146b-3p. ( b ) The phosphorylation levels of AKT and AKT1 and the protein expression of AKT1 after transfecting miR-146b-3p mimic or inhibitor in CPMs. ( c ) Gray value analysis of protein bands in (b). ( d ) Dual-luciferase report assay performed after co-transfecting the wild type or mutant 3′UTR of AKT1 with miR-146b-3p mimic or mimic NC in DF-1 cells. ( e ) Dual-luciferase report assay performed after co-transfecting the wild type or mutant 3′UTR of MDFIC with miR-146b-3p mimic or mimic NC in DF-1 cells. ( f ) Relative mRNA expression of the cell differentiation-related genes after co-transfection. ( g ) Relative mRNA expression of the cell apoptosis-related genes after co-transfection. ( h ) Cell cycle analysis of QM-7 cells after co-transfection. Results of all groups are shown as mean ± S.E.M. of three independent assessment methods. Statistical significance of the mean difference was assessed using unpaired two-sample t -tests. * p < 0.05; ** p < 0.01. NC, negative control.

Article Snippet: The antibodies used for Western blots and their dilutions are as follows: Cleaved Caspase-8 (Asp391) (18C8) Rabbit mAb (Cell Signaling Technology, Boston, MA, USA; 1:1000), Anti-Caspase-9 antibody [E23] (Abcam, London, UK; 1:1000), mouse anti-MyHC antibody (Bioss, Beijing, China; 1:1000), rabbit anti-Lamin B antibody (Bioss, Beijing, China; 1:500), rabbit anti-AKT1 (Bioss, Beijing, China; 1:500), rabbit anti-phospho-AKT1 (Bioss, Beijing, China; 1:500), rabbit Anti-phospho-AKT antibody (Bioss, Beijing, China; 1:300), and mouse anti-GAPDH (Boster, Wuhan, China; 1:2000).

Techniques: Activity Assay, Expressing, Over Expression, Inhibition, Luciferase, Mutagenesis, Cell Differentiation, Cotransfection, Cell Cycle Assay, Negative Control

Journal: Cells

Article Title: The Inhibition on MDFIC and PI3K/AKT Pathway Caused by miR-146b-3p Triggers Suppression of Myoblast Proliferation and Differentiation and Promotion of Apoptosis

doi: 10.3390/cells8070656

Figure Lengend Snippet: AKT1 and MDFIC promote myoblast proliferation. ( a ) Cell cycle analysis of CPMs after overexpression or inhibition of AKT1 . ( b ) Cell cycle analysis of CPMs after overexpression or inhibition of MDFIC . ( c ) Relative mRNA expression of the cell cycle-related genes after overexpression or inhibition of AKT1 in CPMs. ( d ) Relative mRNA expression of the cell cycle-related genes after overexpression or inhibition of MDFIC in CPMs. ( e ) EdU staining of CPMs after overexpression or inhibition of AKT1 . ( f ) The fold change of proliferation rates of CPMs with pcDNA3.1- AKT1 or si- AKT1 . ( g ) EdU staining of CPMs after overexpression or inhibition of MDFIC . ( h ) The fold change of proliferation rates of CPMs with pcDNA3.1- MDFIC or si- MDFIC . Results of all groups are shown as mean ± S.E.M. of three independent assessment methods. Statistical significance of the mean difference was assessed using unpaired two-sample t -tests. * p < 0.05; ** p < 0.01. NC, negative control.

Article Snippet: The antibodies used for Western blots and their dilutions are as follows: Cleaved Caspase-8 (Asp391) (18C8) Rabbit mAb (Cell Signaling Technology, Boston, MA, USA; 1:1000), Anti-Caspase-9 antibody [E23] (Abcam, London, UK; 1:1000), mouse anti-MyHC antibody (Bioss, Beijing, China; 1:1000), rabbit anti-Lamin B antibody (Bioss, Beijing, China; 1:500), rabbit anti-AKT1 (Bioss, Beijing, China; 1:500), rabbit anti-phospho-AKT1 (Bioss, Beijing, China; 1:500), rabbit Anti-phospho-AKT antibody (Bioss, Beijing, China; 1:300), and mouse anti-GAPDH (Boster, Wuhan, China; 1:2000).

Techniques: Cell Cycle Assay, Over Expression, Inhibition, Expressing, Staining, Negative Control

Journal: Cells

Article Title: The Inhibition on MDFIC and PI3K/AKT Pathway Caused by miR-146b-3p Triggers Suppression of Myoblast Proliferation and Differentiation and Promotion of Apoptosis

doi: 10.3390/cells8070656

Figure Lengend Snippet: Both AKT1 and MDFIC promote myoblast differentiation, while suppress myoblast apoptosis. ( a ) Relative expression of AKT1 in Xinghua chicken leg muscle from E11 to E18. ( b ) Relative expression of MDFIC in Xinghua chicken leg muscle from E11 to E18. ( c ) Relative mRNA expression of AKT1 during CPMs induced differentiation. ( d ) Relative mRNA expression of MDFIC during CPMs-induced differentiation. ( e , f ) Relative mRNA expression of the cell differentiation-related genes after overexpression or inhibition of AKT1 or MDFIC in CPMs. ( g , i ) Immunofluorescence of MyHC after overexpression or knockdown of AKT1 and MDFIC in CPMs. ( h , j ) Comparison of the area of myotubes described in ( g , i ). Results of all groups are shown as mean ± S.E.M. of three independent assessment methods. Statistical significance of the mean difference was assessed using unpaired two-sample t -tests. * p < 0.05; ** p < 0.01. NC, negative control.

Article Snippet: The antibodies used for Western blots and their dilutions are as follows: Cleaved Caspase-8 (Asp391) (18C8) Rabbit mAb (Cell Signaling Technology, Boston, MA, USA; 1:1000), Anti-Caspase-9 antibody [E23] (Abcam, London, UK; 1:1000), mouse anti-MyHC antibody (Bioss, Beijing, China; 1:1000), rabbit anti-Lamin B antibody (Bioss, Beijing, China; 1:500), rabbit anti-AKT1 (Bioss, Beijing, China; 1:500), rabbit anti-phospho-AKT1 (Bioss, Beijing, China; 1:500), rabbit Anti-phospho-AKT antibody (Bioss, Beijing, China; 1:300), and mouse anti-GAPDH (Boster, Wuhan, China; 1:2000).

Techniques: Expressing, Cell Differentiation, Over Expression, Inhibition, Immunofluorescence, Negative Control

Journal: Cells

Article Title: The Inhibition on MDFIC and PI3K/AKT Pathway Caused by miR-146b-3p Triggers Suppression of Myoblast Proliferation and Differentiation and Promotion of Apoptosis

doi: 10.3390/cells8070656

Figure Lengend Snippet: AKT1 shows an inhibitory effect on myoblast apoptosis and so does MDFIC . ( a , b ) Flow cytometry analysis of Annexin V-FITC and PI dual staining detecting the apoptosis of CPMs after transfection of pcDNA3.1- AKT1 or si- AKT1 . ( c ) Relative mRNA expression of the cell apoptosis-related genes after transfection of pcDNA3.1- AKT1 or si- AKT1 in CPMs. ( d ) Relative mRNA expression of the cell apoptosis-related genes after transfection of pcDNA3.1- MDFIC or si- MDFIC in CPMs. ( e , f ) The protein levels of MyHC, cleaved-caspase 8, and cleaved-caspase 9 after the overexpression or silence of AKT1 and MDFIC in CPMs. ( g , h ) Gray value analysis of protein bands in ( e , f ). Results of all groups are shown as mean ± S.E.M. of three independent assessment methods. Statistical significance of the mean difference was assessed using unpaired two-sample t -tests. * p < 0.05; ** p < 0.01. NC, negative control.

Article Snippet: The antibodies used for Western blots and their dilutions are as follows: Cleaved Caspase-8 (Asp391) (18C8) Rabbit mAb (Cell Signaling Technology, Boston, MA, USA; 1:1000), Anti-Caspase-9 antibody [E23] (Abcam, London, UK; 1:1000), mouse anti-MyHC antibody (Bioss, Beijing, China; 1:1000), rabbit anti-Lamin B antibody (Bioss, Beijing, China; 1:500), rabbit anti-AKT1 (Bioss, Beijing, China; 1:500), rabbit anti-phospho-AKT1 (Bioss, Beijing, China; 1:500), rabbit Anti-phospho-AKT antibody (Bioss, Beijing, China; 1:300), and mouse anti-GAPDH (Boster, Wuhan, China; 1:2000).

Techniques: Flow Cytometry, Staining, Transfection, Expressing, Over Expression, Negative Control

Journal: Cells

Article Title: The Inhibition on MDFIC and PI3K/AKT Pathway Caused by miR-146b-3p Triggers Suppression of Myoblast Proliferation and Differentiation and Promotion of Apoptosis

doi: 10.3390/cells8070656

Figure Lengend Snippet: Model of miR-146b-3p mediated regulatory mechanism in myoblast proliferation, differentiation, and apoptosis. In simple terms, miR-146b-3p downregulates the expression of AKT1 and MDFIC by targeting the 3′UTR of their mRNA. Both the phosphorylation of AKT1 and AKT can trigger the activation of PI3K/AKT pathway, thereby promoting the expression of cell cycle-related genes and myoblast differentiation-related genes and suppress cell apoptosis-related genes, which in turn facilitate cell proliferation and differentiation and inhibit apoptosis. In addition, the expression of MDFIC can also lead to the same effect.

Article Snippet: The antibodies used for Western blots and their dilutions are as follows: Cleaved Caspase-8 (Asp391) (18C8) Rabbit mAb (Cell Signaling Technology, Boston, MA, USA; 1:1000), Anti-Caspase-9 antibody [E23] (Abcam, London, UK; 1:1000), mouse anti-MyHC antibody (Bioss, Beijing, China; 1:1000), rabbit anti-Lamin B antibody (Bioss, Beijing, China; 1:500), rabbit anti-AKT1 (Bioss, Beijing, China; 1:500), rabbit anti-phospho-AKT1 (Bioss, Beijing, China; 1:500), rabbit Anti-phospho-AKT antibody (Bioss, Beijing, China; 1:300), and mouse anti-GAPDH (Boster, Wuhan, China; 1:2000).

Techniques: Expressing, Activation Assay

Journal: The Journal of General Physiology

Article Title: The zinc-binding motif of TRPM7 acts as an oxidative stress sensor to regulate its channel activity

doi: 10.1085/jgp.202012708

Figure Lengend Snippet: H 2 O 2 inhibited mouse and human TRPM7 current in a [Mg 2+ ] i -dependent manner without affecting expression on the plasma membrane . (A) Time course of whole-cell currents in the presence of various [Mg 2+ ] i in full-length mTRPM7-overexpressing HEK293 cells. Each symbol represents the mean ± SEM of recordings at [Mg 2+ ] i , which are shown on the right. The currents at 2 min (control; black vertical dashed line) and 6 min (H 2 O 2 ; red vertical dashed line) relative to the mean current at 2 min in the absence of Mg 2+ are plotted in . Each symbol represents the mean ± SEM (vertical bar) of 14, 11, 9, 6, 8, 10, 18, 4, and 6 observations in the absence or presence of 2.8 µM, 7.4 µM, 20.9 µM 0.2 mM, 0.5 mM, 1.0 mM, 2.0 mM, and 5.0 mM [Mg 2+ ] i , respectively. (B) Localization of TRPM7 in full-length mTRPM7-wt–overexpressing HEK293 cells. Control conditions (left), 5-min treatment with H 2 O 2 (500 µM; middle), or NMM (100 µM; right). Confocal images show the localization of TRPM7 (green) and nuclei (blue). Scale bar, 20 µm. (C) The effect of oxidative stress induced by H 2 O 2 on the hTRPM7 current. Representative traces of whole-cell currents showing the time course of the current inhibition by H 2 O 2 (500 µM) in the absence (open circles) or presence (closed circles) of 0.2 mM [Mg 2+ ] i in hTRPM7-expressing HEK293 cells. (D) H 2 O 2 (500 µM) inhibited the hTRPM7 current in the presence of 0.2 mM [Mg 2+ ] i ( n = 6), but not in the absence of intracellular Mg 2+ ( n = 5). Each bar represents the mean ± SEM (vertical bar). *, P = 0.00040 versus control. (E) NMM (100 µM) inhibited the hTRPM7 current in the presence of 0.2 mM [Mg 2+ ] i ( n = 5), but not in the absence of intracellular Mg 2+ ( n = 4). Each bar represents the mean ± SEM (vertical bar). *, P = 0.031 versus control.

Article Snippet: To detect full-length TRPM7, its mutants, and M7cd, rabbit anti-TRPM7 antibody (ACC-047, epitope 1146–1165 of hTRPM7; Alomone Labs) was used.

Techniques: Expressing, Clinical Proteomics, Membrane, Control, Inhibition

Journal: The Journal of General Physiology

Article Title: The zinc-binding motif of TRPM7 acts as an oxidative stress sensor to regulate its channel activity

doi: 10.1085/jgp.202012708

Figure Lengend Snippet: Screening of cysteines in mTRPM7 that were responsible for the oxidative stress–induced inhibition. (A) Schematic diagram of 34 cysteines that are conserved in mTRPM7 and hTRPM7. S1107 and D1510 are also depicted (red dots). (B) Expression of TRPM7-wt and its mutants. Immunoblotting of TRPM7 and calnexin (loading control) was performed with HEK293 whole-cell lysates. Numbers on the left represent molecular mass of standards (in kD). (C) The effect of H 2 O 2 (500 µM) on mutant TRPM7 current at 0.2 mM [Mg 2+ ] i . Each bar represents the mean ± SEM (vertical bar). *, P < 0.05 by unpaired t test versus Dox[−] cells. The number of observations ( n ) and exact P values for each mutant are shown in .

Article Snippet: To detect full-length TRPM7, its mutants, and M7cd, rabbit anti-TRPM7 antibody (ACC-047, epitope 1146–1165 of hTRPM7; Alomone Labs) was used.

Techniques: Inhibition, Expressing, Western Blot, Control, Mutagenesis

Journal: The Journal of General Physiology

Article Title: The zinc-binding motif of TRPM7 acts as an oxidative stress sensor to regulate its channel activity

doi: 10.1085/jgp.202012708

Figure Lengend Snippet: Localization of TRPM7 or its mutants in HEK293 cells. Representative confocal images showing the localization of TRPM7 (green) in doxycycline-untreated (Dox[−]) and mTRPM7-wt (WT)–, -C721A–, -C738A–, -C721S–, -C738S–, -C1809S–, and -C1813S–expressing HEK293 cells. Phase-contrast images are also shown. Scale bar, 20 µm.

Article Snippet: To detect full-length TRPM7, its mutants, and M7cd, rabbit anti-TRPM7 antibody (ACC-047, epitope 1146–1165 of hTRPM7; Alomone Labs) was used.

Techniques: Expressing

Journal: The Journal of General Physiology

Article Title: The zinc-binding motif of TRPM7 acts as an oxidative stress sensor to regulate its channel activity

doi: 10.1085/jgp.202012708

Figure Lengend Snippet: Functional expression of the M7cd in HEK293 cells. (A) Time course of M7cd currents in the absence (open circles; n = 15) or presence of 2.8 µM (blue squares; n = 9), 20.9 µM (green triangles; n = 14), and 0.2 mM [Mg 2+ ] i (black circles; n = 16) in HEK293 cells that were treated with doxycycline. Each symbol represents the mean ± SEM (vertical bar). (B) Representative I-V relationship of M7cd current recorded in the presence of various [Mg 2+ ] i at 2 min after break-in. (C) [Mg 2+ ] i -dependent inhibition of M7cd current at 2 min. The current was inhibited by intracellular free Mg 2+ with an IC 50 of 3.0 µM. Each symbol represents the mean ± SEM (vertical bar) of 15, 36, 19, 27, 14, and 20 recordings in the absence or presence of 0.8 µM, 2.8 µM, 7.4 µM, 20.9 µM, and 0.2 mM [Mg 2+ ] i , respectively. The dashed line is the [Mg 2+ ] i -dependent curve of the full-length TRPM7-wt current (from ). (D) M7cd-S1107E current density was similar in the absence (open inversed triangles; n = 10) and presence of 0.2 mM [Mg 2+ ] i (closed inversed triangles; n = 7). Each symbol represents the mean ± SEM (vertical bar). (E) The effect of H 2 O 2 (500 µM) on M7cd current in the absence of intracellular Mg 2+ (open circles). Each symbol represents the mean ± SEM (vertical bar) of 19 recordings. (F) The time course of the M7cd current in the presence of 0.8 µM [Mg 2+ ] i with (red circles; n = 17) or without (black circles; n = 35) H 2 O 2 application at 2 min. Each symbol represents the mean ± SEM (vertical bar). The current decreased over time even in the control conditions (black circles), and repeated-measures ANOVA revealed that H 2 O 2 has no significant effect on the current decrease (degrees of freedom were corrected using the Greenhouse-Geisser estimate of epsilon; P = 0.247).

Article Snippet: To detect full-length TRPM7, its mutants, and M7cd, rabbit anti-TRPM7 antibody (ACC-047, epitope 1146–1165 of hTRPM7; Alomone Labs) was used.

Techniques: Functional Assay, Expressing, Inhibition, Control

Journal: The Journal of General Physiology

Article Title: The zinc-binding motif of TRPM7 acts as an oxidative stress sensor to regulate its channel activity

doi: 10.1085/jgp.202012708

Figure Lengend Snippet: Functional reconstitution of the TRPM7 current by coexpression of M7kd in M7cd-expressing cells. (A) Transient expression of M7kd-wt in M7cd-expressing cells resulted in an increase in the M7cd current density that was inhibited by H 2 O 2 (500 µM) in the presence of 0.2 mM [Mg 2+ ] i (closed circles; n = 8), but not in the absence of intracellular Mg 2+ (open circles; n = 9). Each symbol represents the mean ± SEM (vertical bar). (B and C) Representative I-V relationships of the M7cd current that was recorded in M7cd-expressing cells transfected with M7kd-wt in the presence (B) or absence of 0.2 mM [Mg 2+ ] i (C), before (black line) or 4 min after (red line) the application of H 2 O 2 (500 µM). (D) [Mg 2+ ] i -dependent inhibition of M7cd with coexpression of M7kd-wt in the control (black squares) and H 2 O 2 -treated (red squares) conditions. The data fit well with a biphasic concentration–response curve, assuming the fraction of high-affinity inhibition of 0.314 estimated for full-length TRPM7-wt (in ). Under control conditions, the current was inhibited by intracellular free Mg 2+ with an IC 50(1) of 7.6 µM and an IC 50(2) of 986 µM. After H 2 O 2 treatment, the current was inhibited by intracellular free Mg 2+ with an IC 50 of 3.0 µM. Each symbol represents the mean ± SEM (vertical bar) of 28, 9, 13, 16, 19, 12, 5, and 6 observations in the absence or presence of 2.8 µM, 7.4 µM, 20.9 µM, 0.2 mM, 1 mM, 2 mM, and 5 mM [Mg 2+ ] i , respectively. (E) The time course of the M7cd current when M7kd-wt was transfected by either lipofection (closed circles; n = 20) or baculovirus infection (open squares; n = 8) in the presence of 0.2 mM [Mg 2+ ] i . Each symbol represents the mean ± SEM (vertical bar). (F) The effect of H 2 O 2 (500 µM) on the M7cd current in the presence of 0.2 mM [Mg 2+ ] i in cells that were transfected with M7kd-wt by baculovirus infection. Each symbol represents the mean ± SEM (vertical bar) of eight recordings. (G) The effect of NMM (100 µM) on M7cd current in the presence of 0.2 mM [Mg 2+ ] i in cells that were transfected with M7kd-wt by baculovirus infection. Each symbol represents the mean ± SEM (vertical bar) of six recordings. (H) The effect of coexpression with kinase-inactive mutant M7kd-1645R on M7cd current. Each symbol represents the mean ± SEM (vertical bar) of four recordings. (I) The effect of M6kd coexpression on M7cd current. Each symbol represents the mean ± SEM (vertical bar) of eight recordings.

Article Snippet: To detect full-length TRPM7, its mutants, and M7cd, rabbit anti-TRPM7 antibody (ACC-047, epitope 1146–1165 of hTRPM7; Alomone Labs) was used.

Techniques: Functional Assay, Expressing, Transfection, Inhibition, Control, Concentration Assay, Infection, Mutagenesis

Journal: The Journal of General Physiology

Article Title: The zinc-binding motif of TRPM7 acts as an oxidative stress sensor to regulate its channel activity

doi: 10.1085/jgp.202012708

Figure Lengend Snippet: Schematic illustration of TRPM7 regulation by oxidative stress. Left: Under basal conditions, the current inhibition by Mg 2+ binding to the high-affinity site (indicated by an orange arrow) is attenuated possibly via the interdomain interaction between the channel domain and the kinase domain. The structural integrity of the kinase domain is guaranteed by the zinc-binding motif that coordinates Zn 2+ (indicated by brown) via C1809, C1813, H1750, and H1807. For simplicity, the low-affinity Mg 2+ -binding site that is assumed to exist in the kinase domain is not shown here. Right: Oxidative stress causes the oxidation of the cysteines (C1809 and C1813) in the zinc-binding motif, which may disrupt the interaction between the channel domain and the kinase domain and enhance the inhibition of TRPM7 by intracellular Mg 2+ .

Article Snippet: To detect full-length TRPM7, its mutants, and M7cd, rabbit anti-TRPM7 antibody (ACC-047, epitope 1146–1165 of hTRPM7; Alomone Labs) was used.

Techniques: Inhibition, Binding Assay

Journal: Journal of Neuroinflammation

Article Title: Inhibition of HMGB1 reduces rat spinal cord astrocytic swelling and AQP4 expression after oxygen-glucose deprivation and reoxygenation via TLR4 and NF-κB signaling in an IL-6-dependent manner

doi: 10.1186/s12974-017-1008-1

Figure Lengend Snippet: Spinal cord astrocyte identification and high mobility group box-1 (HMGB1) knockdown. a Spinal cord astrocytes were identified using immunofluorescence. The percentage of cells stained with the astrocytic marker S100β, which were identified as astrocytes, was more than 95% of the total cells (three replicates). b HMGB1 knockdown efficiency in the plasma membrane and cytoplasm of spinal cord astrocytes was evaluated using Western blot for HMGB1 protein levels. Results were obtained after 72 h of specific HMGB1 shRNA treatment. HMGB1 protein levels were decreased to approximately 30% of normal levels with shRNA multiplicity of infection 60 as compared to normal astrocytes. * P < 0.05 vs. normal group (three replicates)

Article Snippet: Primary antibodies include rabbit polyclonal anti-HMGB1 antibody for rat, mouse, and human (Abcam, Cat# ab18256, RRID:AB_444360, Cambridge, UK); rabbit polyclonal anti-AQP4 antibody for rat, mouse, human, and pig (Abcam, Cat# ab46182, RRID: AB_955676); mouse monoclonal anti-TLR4 antibody for rat, mouse, human, pig, baboon, bovine, and Chinese hamster (Novus, Cat# 76B357.1, RRID: AB_839000, Littleton, CO, USA); rabbit polyclonal anti-TLR4 antibody for rat, mouse, human, and rabbit (Boster, Cat# BA1717, RRID:AB_2716293); rabbit polyclonal anti-myeloid differentiation primary response gene 88 (MyD88) antibody for rat and human (Abcam, Cat# ab131071, RRID: AB_11156885); mouse monoclonal anti-IκBα antibody for rat, mouse, human, monkey, bovine, pig, and guinea pig (Cell Signaling Technology, Cat# 4814, RRID: AB_390781, Boston, MA, USA); mouse monoclonal anti-p-IκBα antibody for rat, mouse, human, and monkey (Cell Signaling Technology, Cat# 9246, RRID:AB_2267145); rabbit monoclonal anti-NF-κB antibody for rat, mouse, human, monkey, and bovine (Cell Signaling Technology, Cat# 4764, RRID:AB_823578); mouse monoclonal anti-GAPDH antibody for rat, mouse, and human (Beyotime, Cat# AF0006, RRID: AB_2715590, Shanghai, China); mouse monoclonal anti-Histone H3 antibody for rat, mouse, and human (Beyotime, Cat# AF0009, RRID: AB_2715593); and mouse monoclonal anti-S100β antibody for rat, mouse, human, rabbit, and pig (Boster, Cat# BM0120, RRID:AB_2716291).

Techniques: Immunofluorescence, Staining, Marker, Western Blot, shRNA, Infection

Journal: Journal of Neuroinflammation

Article Title: Inhibition of HMGB1 reduces rat spinal cord astrocytic swelling and AQP4 expression after oxygen-glucose deprivation and reoxygenation via TLR4 and NF-κB signaling in an IL-6-dependent manner

doi: 10.1186/s12974-017-1008-1

Figure Lengend Snippet: Effects of oxygen-glucose deprivation/reoxygenation (OGD/R) on cellular swelling, high mobility group box-1 (HMGB1), and aquaporin-4 (AQP4) expression in cultured spinal cord astrocytes as well as levels of HMGB1 and interleukin-6 (IL-6) released into the surrounding medium. a Astrocyte volume measurement was performed using a Live Cell Imaging System. Cellular volume was calculated by the average value of four measured diameters of the largest compiled Z-slice image. Cellular volumes of spinal cord astrocytes were significantly increased at 2, 6, 12, 24, and 48 h during reoxygenation after OGD when compared with normal astrocytes. # P < 0.05 vs. normal group; * P < 0.05 vs. OGD6h/R24h group (three replicates). b Membrane and cytoplasmic HMGB1 expression was significantly increased in spinal cord astrocytes at different time points after OGD/R. # P < 0.05 vs. normal group; * P < 0.05 vs. OGD6h/R24h group (three replicates). c Membrane and cytoplasmic AQP4 expression was significantly increased in spinal cord astrocytes at different time points after OGD/R. # P < 0.05 vs. normal group; * P < 0.05 vs. OGD6h/R24h group (three replicates). d HMGB1 levels in the surrounding medium of spinal cord astrocytes were significantly increased at 6, 12, and 24 h during reoxygenation after OGD/R. # P < 0.05 vs. normal group; * P < 0.05 vs. OGD6h/R24h group (three replicates). e IL-6 levels in the surrounding medium of spinal cord astrocytes were significantly increased at 6, 12, and 24 h during reoxygenation after OGD/R. # P < 0.05 vs. normal group; * P < 0.05 vs. OGD6h/R24h group (three replicates)

Article Snippet: Primary antibodies include rabbit polyclonal anti-HMGB1 antibody for rat, mouse, and human (Abcam, Cat# ab18256, RRID:AB_444360, Cambridge, UK); rabbit polyclonal anti-AQP4 antibody for rat, mouse, human, and pig (Abcam, Cat# ab46182, RRID: AB_955676); mouse monoclonal anti-TLR4 antibody for rat, mouse, human, pig, baboon, bovine, and Chinese hamster (Novus, Cat# 76B357.1, RRID: AB_839000, Littleton, CO, USA); rabbit polyclonal anti-TLR4 antibody for rat, mouse, human, and rabbit (Boster, Cat# BA1717, RRID:AB_2716293); rabbit polyclonal anti-myeloid differentiation primary response gene 88 (MyD88) antibody for rat and human (Abcam, Cat# ab131071, RRID: AB_11156885); mouse monoclonal anti-IκBα antibody for rat, mouse, human, monkey, bovine, pig, and guinea pig (Cell Signaling Technology, Cat# 4814, RRID: AB_390781, Boston, MA, USA); mouse monoclonal anti-p-IκBα antibody for rat, mouse, human, and monkey (Cell Signaling Technology, Cat# 9246, RRID:AB_2267145); rabbit monoclonal anti-NF-κB antibody for rat, mouse, human, monkey, and bovine (Cell Signaling Technology, Cat# 4764, RRID:AB_823578); mouse monoclonal anti-GAPDH antibody for rat, mouse, and human (Beyotime, Cat# AF0006, RRID: AB_2715590, Shanghai, China); mouse monoclonal anti-Histone H3 antibody for rat, mouse, and human (Beyotime, Cat# AF0009, RRID: AB_2715593); and mouse monoclonal anti-S100β antibody for rat, mouse, human, rabbit, and pig (Boster, Cat# BM0120, RRID:AB_2716291).

Techniques: Expressing, Cell Culture, Live Cell Imaging

Journal: Journal of Neuroinflammation

Article Title: Inhibition of HMGB1 reduces rat spinal cord astrocytic swelling and AQP4 expression after oxygen-glucose deprivation and reoxygenation via TLR4 and NF-κB signaling in an IL-6-dependent manner

doi: 10.1186/s12974-017-1008-1

Figure Lengend Snippet: Effects of inhibiting high mobility group box-1 (HMGB1) on cellular swelling in cultured spinal cord astrocytes after oxygen-glucose deprivation/reoxygenation (OGD/R). a Astrocyte volume analysis was performed using a Live Cell Imaging System, and cellular volume was calculated by the average value of four measured diameters. Inhibiting HMGB1 using either HMGB1 shRNA or ethyl pyruvate (EP) significantly blocked increases in cellular volume of spinal cord astrocytes at 6, 12, and 24 h during reoxygenation when compared with astrocytes of the OGD/R group. * P < 0.05 vs. OGD/R group (three replicates). b — d Effects of inhibiting HMGB1 on spinal cord astrocytic morphology and ultrastructure were evaluated using transmission electron microscopy at 6, 12, and 24 h during reoxygenation after OGD. After OGD/R, spinal cord astrocytes showed swelling at 6, 12, and 24 h during reoxygenation. The mitochondrial (M) swelling, endoplasmic reticulum (ER) swelling and fragmentation, and an increase in the number of lysosomes (L) were concurrent with this observation. However, astrocytic swelling, mitochondrial (M) swelling, endoplasmic reticulum (ER) swelling and fragmentation, and the increase in lysosome (L) number after OGD/R were reduced by HMGB1 inhibition using either HMGB1 shRNA or EP (× 50,000, bar equal to 1 μm, three replicates)

Article Snippet: Primary antibodies include rabbit polyclonal anti-HMGB1 antibody for rat, mouse, and human (Abcam, Cat# ab18256, RRID:AB_444360, Cambridge, UK); rabbit polyclonal anti-AQP4 antibody for rat, mouse, human, and pig (Abcam, Cat# ab46182, RRID: AB_955676); mouse monoclonal anti-TLR4 antibody for rat, mouse, human, pig, baboon, bovine, and Chinese hamster (Novus, Cat# 76B357.1, RRID: AB_839000, Littleton, CO, USA); rabbit polyclonal anti-TLR4 antibody for rat, mouse, human, and rabbit (Boster, Cat# BA1717, RRID:AB_2716293); rabbit polyclonal anti-myeloid differentiation primary response gene 88 (MyD88) antibody for rat and human (Abcam, Cat# ab131071, RRID: AB_11156885); mouse monoclonal anti-IκBα antibody for rat, mouse, human, monkey, bovine, pig, and guinea pig (Cell Signaling Technology, Cat# 4814, RRID: AB_390781, Boston, MA, USA); mouse monoclonal anti-p-IκBα antibody for rat, mouse, human, and monkey (Cell Signaling Technology, Cat# 9246, RRID:AB_2267145); rabbit monoclonal anti-NF-κB antibody for rat, mouse, human, monkey, and bovine (Cell Signaling Technology, Cat# 4764, RRID:AB_823578); mouse monoclonal anti-GAPDH antibody for rat, mouse, and human (Beyotime, Cat# AF0006, RRID: AB_2715590, Shanghai, China); mouse monoclonal anti-Histone H3 antibody for rat, mouse, and human (Beyotime, Cat# AF0009, RRID: AB_2715593); and mouse monoclonal anti-S100β antibody for rat, mouse, human, rabbit, and pig (Boster, Cat# BM0120, RRID:AB_2716291).

Techniques: Cell Culture, Live Cell Imaging, shRNA, Transmission Assay, Electron Microscopy, Inhibition

Journal: Journal of Neuroinflammation

Article Title: Inhibition of HMGB1 reduces rat spinal cord astrocytic swelling and AQP4 expression after oxygen-glucose deprivation and reoxygenation via TLR4 and NF-κB signaling in an IL-6-dependent manner

doi: 10.1186/s12974-017-1008-1

Figure Lengend Snippet: Effects of inhibiting high mobility group box-1 (HMGB1) on HMGB1, aquaporin-4 (AQP4), and toll-like receptor-4 (TLR4) expression in cultured spinal cord astrocytes after oxygen-glucose deprivation/reoxygenation (OGD/R) as well as levels of HMGB1 and interleukin-6 (IL-6) release into the surrounding medium. a Inhibiting HMGB1 using either HMGB1 shRNA or ethyl pyruvate (EP) significantly suppressed the increased levels of HMGB1 in both the plasma membrane and cytoplasm of spinal cord astrocytes at 6, 12, and 24 h during reoxygenation after OGD. * P < 0.05 vs. OGD/R group (three replicates). b Inhibiting HMGB1 significantly suppressed the increased levels of AQP4 in both the plasma membrane and cytoplasm of spinal cord astrocytes at 6, 12, and 24 h during reoxygenation after OGD. * P < 0.05 vs. OGD/R group (three replicates). c Inhibiting HMGB1 significantly suppressed increased levels of TLR4 in both the plasma membrane and cytoplasm of spinal cord astrocytes at 6, 12, and 24 h during reoxygenation after OGD. * P < 0.05 vs. OGD/R group (three replicates). d HMGB1, AQP4, and TLR4 immunofluorescence on spinal cord astrocytes at 24 h into the reoxygenation process after OGD showed significantly increased membrane and cytoplasmic levels of HMGB1, AQP4, and TLR4 in the OGD/R group when compared with those in the normal group. These were markedly suppressed in both the OGD/R + HMGB1 shRNA and OGD/R + EP groups (× 200, bar equal to 100 μm). * P < 0.05 vs. OGD/R group (three replicates). e , f Inhibiting HMGB1 mitigated increases in levels of HMGB1 and IL-6 in the surrounding medium when compared with levels in the OGD/R group at 6, 12, and 24 h during reoxygenation after OGD. * P < 0.05 vs. OGD/R group (three replicates)

Article Snippet: Primary antibodies include rabbit polyclonal anti-HMGB1 antibody for rat, mouse, and human (Abcam, Cat# ab18256, RRID:AB_444360, Cambridge, UK); rabbit polyclonal anti-AQP4 antibody for rat, mouse, human, and pig (Abcam, Cat# ab46182, RRID: AB_955676); mouse monoclonal anti-TLR4 antibody for rat, mouse, human, pig, baboon, bovine, and Chinese hamster (Novus, Cat# 76B357.1, RRID: AB_839000, Littleton, CO, USA); rabbit polyclonal anti-TLR4 antibody for rat, mouse, human, and rabbit (Boster, Cat# BA1717, RRID:AB_2716293); rabbit polyclonal anti-myeloid differentiation primary response gene 88 (MyD88) antibody for rat and human (Abcam, Cat# ab131071, RRID: AB_11156885); mouse monoclonal anti-IκBα antibody for rat, mouse, human, monkey, bovine, pig, and guinea pig (Cell Signaling Technology, Cat# 4814, RRID: AB_390781, Boston, MA, USA); mouse monoclonal anti-p-IκBα antibody for rat, mouse, human, and monkey (Cell Signaling Technology, Cat# 9246, RRID:AB_2267145); rabbit monoclonal anti-NF-κB antibody for rat, mouse, human, monkey, and bovine (Cell Signaling Technology, Cat# 4764, RRID:AB_823578); mouse monoclonal anti-GAPDH antibody for rat, mouse, and human (Beyotime, Cat# AF0006, RRID: AB_2715590, Shanghai, China); mouse monoclonal anti-Histone H3 antibody for rat, mouse, and human (Beyotime, Cat# AF0009, RRID: AB_2715593); and mouse monoclonal anti-S100β antibody for rat, mouse, human, rabbit, and pig (Boster, Cat# BM0120, RRID:AB_2716291).

Techniques: Expressing, Cell Culture, shRNA, Immunofluorescence

Journal: Journal of Neuroinflammation

Article Title: Inhibition of HMGB1 reduces rat spinal cord astrocytic swelling and AQP4 expression after oxygen-glucose deprivation and reoxygenation via TLR4 and NF-κB signaling in an IL-6-dependent manner

doi: 10.1186/s12974-017-1008-1

Figure Lengend Snippet: Effects of either inhibiting high mobility group box-1 (HMGB1) or toll-like receptor-4 (TLR4) on oxygen-glucose deprivation/reoxygenation (OGD/R)-induced astrocytic swelling, TLR4, myeloid differentiation primary response gene 88 (MyD88), aquaporin-4 (AQP4) upregulation, and nuclear factor-kappa B (NF-κB) activation as well as levels of interleukin-6 (IL-6) released into the surrounding medium. a Inhibiting HMGB1 (using either HMGB1 shRNA or ethyl pyruvate (EP)) or TLR4 (using CLI-095 or C34) significantly reduced the increase in cellular volume of spinal cord astrocytes at 24 h during the reoxygenation process after OGD when compared with those in the OGD/R group. * P < 0.05 vs. OGD/R group (three replicates). b Inhibiting HMGB1 or TLR4 significantly suppressed the increased levels of TLR4, MyD88, and AQP4 in both the plasma membrane and cytoplasm of spinal cord astrocytes at 24 h during the reoxygenation process after OGD. * P < 0.05 vs. OGD/R group (three replicates). c Inhibiting HMGB1 or TLR4 significantly suppressed the increased nuclear levels of NF-κB and the upregulation of cytoplasmic p-IκBα in spinal cord astrocytes after 24 h of the reoxygenation process after OGD. * P < 0.05 vs. OGD/R group (three replicates). d Immunofluorescence results showed that either inhibiting HMGB1 or TLR4 decreased membrane and cytoplasmic TLR4 and AQP4 upregulation and attenuated the increases of nuclear NF-κB when compared with the OGD/R group at 24 h during reoxygenation (× 200, bar equal to 100 μm). * P < 0.05 vs. OGD/R group (three replicates). e Inhibiting HMGB1 or TLR4 reduced increased levels of IL-6 in the surrounding medium when compared with those of the OGD/R group after 24 h of the reoxygenation process after OGD. * P < 0.05 vs. OGD/R group (three replicates)

Article Snippet: Primary antibodies include rabbit polyclonal anti-HMGB1 antibody for rat, mouse, and human (Abcam, Cat# ab18256, RRID:AB_444360, Cambridge, UK); rabbit polyclonal anti-AQP4 antibody for rat, mouse, human, and pig (Abcam, Cat# ab46182, RRID: AB_955676); mouse monoclonal anti-TLR4 antibody for rat, mouse, human, pig, baboon, bovine, and Chinese hamster (Novus, Cat# 76B357.1, RRID: AB_839000, Littleton, CO, USA); rabbit polyclonal anti-TLR4 antibody for rat, mouse, human, and rabbit (Boster, Cat# BA1717, RRID:AB_2716293); rabbit polyclonal anti-myeloid differentiation primary response gene 88 (MyD88) antibody for rat and human (Abcam, Cat# ab131071, RRID: AB_11156885); mouse monoclonal anti-IκBα antibody for rat, mouse, human, monkey, bovine, pig, and guinea pig (Cell Signaling Technology, Cat# 4814, RRID: AB_390781, Boston, MA, USA); mouse monoclonal anti-p-IκBα antibody for rat, mouse, human, and monkey (Cell Signaling Technology, Cat# 9246, RRID:AB_2267145); rabbit monoclonal anti-NF-κB antibody for rat, mouse, human, monkey, and bovine (Cell Signaling Technology, Cat# 4764, RRID:AB_823578); mouse monoclonal anti-GAPDH antibody for rat, mouse, and human (Beyotime, Cat# AF0006, RRID: AB_2715590, Shanghai, China); mouse monoclonal anti-Histone H3 antibody for rat, mouse, and human (Beyotime, Cat# AF0009, RRID: AB_2715593); and mouse monoclonal anti-S100β antibody for rat, mouse, human, rabbit, and pig (Boster, Cat# BM0120, RRID:AB_2716291).

Techniques: Activation Assay, shRNA, Immunofluorescence

Journal: Journal of Neuroinflammation

Article Title: Inhibition of HMGB1 reduces rat spinal cord astrocytic swelling and AQP4 expression after oxygen-glucose deprivation and reoxygenation via TLR4 and NF-κB signaling in an IL-6-dependent manner

doi: 10.1186/s12974-017-1008-1

Figure Lengend Snippet: Effects of nuclear factor-kappa B (NF-κB) inhibition on oxygen-glucose deprivation/reoxygenation (OGD/R)-induced astrocytic swelling, NF-κB activation, and aquaporin-4 (AQP4) upregulation, as well as levels of interleukin-6 (IL-6) released into the surrounding medium. a NF-κB inhibition (using BAY 11-7082) significantly suppressed the increased nuclear levels of NF-κB and the upregulation of cytoplasmic p-IκBα in spinal cord astrocytes after 24 h of the reoxygenation phase after OGD. * P < 0.05 vs. OGD/R group (three replicates). b NF-κB and AQP4 immunofluorescence in spinal cord astrocytes after 24 h of the reoxygenation process after OGD showed significantly increased nuclear levels of NF-κB and membrane and cytoplasmic levels of AQP4 in the OGD/R group. Levels were markedly attenuated in the OGD/R + HMGB1 shRNA, OGD/R + BAY 11-7082, and OGD/R + EP groups (× 200, bar equal to 100 μm). * P < 0.05 vs. OGD/R group (three replicates). c NF-κB inhibition significantly reduced the increase in cellular volume of spinal cord astrocytes at 24 h during the reoxygenation process after OGD when compared with those of the OGD/R group. * P < 0.05 vs. OGD/R group (three replicates). d NF-κB inhibition significantly suppressed increased AQP4 levels in both the plasma membrane and cytoplasm of spinal cord astrocytes after 24 h of the reoxygenation process after OGD. * P < 0.05 vs. OGD/R group (three replicates). e NF-κB inhibition reduced increased levels of IL-6 in the surrounding medium when compared with those of the OGD/R group after 24 h of the reoxygenation process after OGD. * P < 0.05 vs. OGD/R group (three replicates)

Article Snippet: Primary antibodies include rabbit polyclonal anti-HMGB1 antibody for rat, mouse, and human (Abcam, Cat# ab18256, RRID:AB_444360, Cambridge, UK); rabbit polyclonal anti-AQP4 antibody for rat, mouse, human, and pig (Abcam, Cat# ab46182, RRID: AB_955676); mouse monoclonal anti-TLR4 antibody for rat, mouse, human, pig, baboon, bovine, and Chinese hamster (Novus, Cat# 76B357.1, RRID: AB_839000, Littleton, CO, USA); rabbit polyclonal anti-TLR4 antibody for rat, mouse, human, and rabbit (Boster, Cat# BA1717, RRID:AB_2716293); rabbit polyclonal anti-myeloid differentiation primary response gene 88 (MyD88) antibody for rat and human (Abcam, Cat# ab131071, RRID: AB_11156885); mouse monoclonal anti-IκBα antibody for rat, mouse, human, monkey, bovine, pig, and guinea pig (Cell Signaling Technology, Cat# 4814, RRID: AB_390781, Boston, MA, USA); mouse monoclonal anti-p-IκBα antibody for rat, mouse, human, and monkey (Cell Signaling Technology, Cat# 9246, RRID:AB_2267145); rabbit monoclonal anti-NF-κB antibody for rat, mouse, human, monkey, and bovine (Cell Signaling Technology, Cat# 4764, RRID:AB_823578); mouse monoclonal anti-GAPDH antibody for rat, mouse, and human (Beyotime, Cat# AF0006, RRID: AB_2715590, Shanghai, China); mouse monoclonal anti-Histone H3 antibody for rat, mouse, and human (Beyotime, Cat# AF0009, RRID: AB_2715593); and mouse monoclonal anti-S100β antibody for rat, mouse, human, rabbit, and pig (Boster, Cat# BM0120, RRID:AB_2716291).

Techniques: Inhibition, Activation Assay, Immunofluorescence, shRNA

Journal: Journal of Neuroinflammation

Article Title: Inhibition of HMGB1 reduces rat spinal cord astrocytic swelling and AQP4 expression after oxygen-glucose deprivation and reoxygenation via TLR4 and NF-κB signaling in an IL-6-dependent manner

doi: 10.1186/s12974-017-1008-1

Figure Lengend Snippet: Effects of recombinant HMGB1 (rHMGB1) on aquaporin-4 (AQP4) expression in cultured spinal cord astrocytes. Incubation of cultured spinal cord astrocytes with rHMGB1 (0, 0.1, 1, 10, and 20 ng/ml) for 24 h did not induce dose-dependent increases in the membrane and cytoplasmic AQP4 expression (three replicates)

Article Snippet: Primary antibodies include rabbit polyclonal anti-HMGB1 antibody for rat, mouse, and human (Abcam, Cat# ab18256, RRID:AB_444360, Cambridge, UK); rabbit polyclonal anti-AQP4 antibody for rat, mouse, human, and pig (Abcam, Cat# ab46182, RRID: AB_955676); mouse monoclonal anti-TLR4 antibody for rat, mouse, human, pig, baboon, bovine, and Chinese hamster (Novus, Cat# 76B357.1, RRID: AB_839000, Littleton, CO, USA); rabbit polyclonal anti-TLR4 antibody for rat, mouse, human, and rabbit (Boster, Cat# BA1717, RRID:AB_2716293); rabbit polyclonal anti-myeloid differentiation primary response gene 88 (MyD88) antibody for rat and human (Abcam, Cat# ab131071, RRID: AB_11156885); mouse monoclonal anti-IκBα antibody for rat, mouse, human, monkey, bovine, pig, and guinea pig (Cell Signaling Technology, Cat# 4814, RRID: AB_390781, Boston, MA, USA); mouse monoclonal anti-p-IκBα antibody for rat, mouse, human, and monkey (Cell Signaling Technology, Cat# 9246, RRID:AB_2267145); rabbit monoclonal anti-NF-κB antibody for rat, mouse, human, monkey, and bovine (Cell Signaling Technology, Cat# 4764, RRID:AB_823578); mouse monoclonal anti-GAPDH antibody for rat, mouse, and human (Beyotime, Cat# AF0006, RRID: AB_2715590, Shanghai, China); mouse monoclonal anti-Histone H3 antibody for rat, mouse, and human (Beyotime, Cat# AF0009, RRID: AB_2715593); and mouse monoclonal anti-S100β antibody for rat, mouse, human, rabbit, and pig (Boster, Cat# BM0120, RRID:AB_2716291).

Techniques: Recombinant, Expressing, Cell Culture, Incubation

Journal: Journal of Neuroinflammation

Article Title: Inhibition of HMGB1 reduces rat spinal cord astrocytic swelling and AQP4 expression after oxygen-glucose deprivation and reoxygenation via TLR4 and NF-κB signaling in an IL-6-dependent manner

doi: 10.1186/s12974-017-1008-1

Figure Lengend Snippet: Effects of interleukin-6 (IL-6) on aquaporin-4 (AQP4) expression in cultured spinal cord astrocytes. a Spinal cord astrocytes were exposed to exogenous IL-6 at 0, 0.1, 1, or 10 ng/ml. After 24 h exposure, the membrane and cytoplasmic AQP4 expression in spinal cord astrocytes were markedly increased in the IL-6 0.1 ng/ml group, IL-6 1 ng/ml group, and IL-6 10 ng/ml group. * P < 0.05 vs. 0 ng/ml group (three replicates). b IL-6 levels increased in the surrounding medium of the OGD/R group after 24 h of the reoxygenation process after OGD. In comparison, this increase was significantly reduced in the OGD/R + HMGB1 shRNA group. * P < 0.05 vs. OGD/R group (three replicates). c The effects of astrocyte conditioned medium (ACM) on AQP4 expression in cultured spinal cord astrocytes. Twenty-four hours exposure of spinal cord astrocytes to the ACM obtained from the OGD/R group significantly increased the membrane and cytoplasmic AQP4 expression when compared with astrocytes incubated with the ACM obtained from the OGD/R + HMGB1 shRNA group. * P < 0.05 vs. astrocytes + OGD6h/R24h ACM group (three replicates). d Western blot analysis showed that the neutralizing anti-rat-IL-6 antibody could significantly reverse the upregulation effect of exogenous IL-6 or OGD/R ACM containing increased IL-6 on AQP4 expression in cultured spinal cord astrocytes. # P < 0.05 vs. astrocytes + IL-6 group; * P < 0.05 vs. astrocytes + OGD6h/R24h ACM group (three replicates)

Article Snippet: Primary antibodies include rabbit polyclonal anti-HMGB1 antibody for rat, mouse, and human (Abcam, Cat# ab18256, RRID:AB_444360, Cambridge, UK); rabbit polyclonal anti-AQP4 antibody for rat, mouse, human, and pig (Abcam, Cat# ab46182, RRID: AB_955676); mouse monoclonal anti-TLR4 antibody for rat, mouse, human, pig, baboon, bovine, and Chinese hamster (Novus, Cat# 76B357.1, RRID: AB_839000, Littleton, CO, USA); rabbit polyclonal anti-TLR4 antibody for rat, mouse, human, and rabbit (Boster, Cat# BA1717, RRID:AB_2716293); rabbit polyclonal anti-myeloid differentiation primary response gene 88 (MyD88) antibody for rat and human (Abcam, Cat# ab131071, RRID: AB_11156885); mouse monoclonal anti-IκBα antibody for rat, mouse, human, monkey, bovine, pig, and guinea pig (Cell Signaling Technology, Cat# 4814, RRID: AB_390781, Boston, MA, USA); mouse monoclonal anti-p-IκBα antibody for rat, mouse, human, and monkey (Cell Signaling Technology, Cat# 9246, RRID:AB_2267145); rabbit monoclonal anti-NF-κB antibody for rat, mouse, human, monkey, and bovine (Cell Signaling Technology, Cat# 4764, RRID:AB_823578); mouse monoclonal anti-GAPDH antibody for rat, mouse, and human (Beyotime, Cat# AF0006, RRID: AB_2715590, Shanghai, China); mouse monoclonal anti-Histone H3 antibody for rat, mouse, and human (Beyotime, Cat# AF0009, RRID: AB_2715593); and mouse monoclonal anti-S100β antibody for rat, mouse, human, rabbit, and pig (Boster, Cat# BM0120, RRID:AB_2716291).

Techniques: Expressing, Cell Culture, shRNA, Incubation, Western Blot

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:

Article Title: Bicyclams, Selective Antagonists of the Human Chemokine Receptor CXCR4, Potently Inhibit Feline Immunodeficiency Virus Replication

doi:

Figure Lengend Snippet: Inhibition of binding of the anti-CXCR4 MAb to feline thymocytes in the presence (C) or absence (B) of AMD3100 at 1 μg/ml. In panel A, results obtained with an isotype control MAb are shown. The percentages of fluorescence-positive cells and the mean fluorescence intensity values are indicated in each histogram.

Article Snippet: AMD3100 had no inhibitory effect (at 100 μg/ml) on the binding of feline anti-CD4 MAb (MCA 1347; Serotec, Oxford, United Kingdom) and anti-CD8 MAb (MCA 1350; Serotec) in feline thymocytes. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG. 4 caption a7 Inhibition of binding of the anti-CXCR4 MAb to feline thymocytes in the presence (C) or absence (B) of AMD3100 at 1 μg/ml.

Techniques: Inhibition, Binding Assay, Fluorescence