Journal: NPJ Precision Oncology

Article Title: IKKα promotes lung adenocarcinoma growth through ERK signaling activation via DARPP-32-mediated inhibition of PP1 activity

doi: 10.1038/s41698-023-00370-3

Figure Lengend Snippet: a-c Human lung adenocarcinoma cell lines HCC827 ( a ), PC9 ( b ), and H1975 ( c ) stably overexpressing FLAG-tagged human DARPP-32 isoforms were lysed and subjected to immunoprecipitation using anti-IKKα antibodies. Immunoprecipitated lysates were separated in SDS-PAGE and immunoblotted with antibodies against IKKα, FLAG (that detects exogenously overexpressed DARPP-32), and α-tubulin (loading control).

Article Snippet: Phosphorylation of DARPP-32 by kinase-activated human IKKα protein was validated via immunoblotting using monoclonal primary antibodies against phosphorylated DARPP-32 (T34 and T75, CST).

Techniques: Stable Transfection, Immunoprecipitation, SDS Page

Journal: NPJ Precision Oncology

Article Title: IKKα promotes lung adenocarcinoma growth through ERK signaling activation via DARPP-32-mediated inhibition of PP1 activity

doi: 10.1038/s41698-023-00370-3

Figure Lengend Snippet: a Human A549 cell lines stably overexpressing FLAG-tagged human DARPP-32 isoforms (DARPP-32 and t-DARPP) were lysed and subjected to immunoprecipitation using anti-FLAG antibody–conjugated agarose beads. Immunoprecipitated lysates were used to perform nonradioactive in vitro kinase assays following incubation with commercially available active IKKα protein. At the end, the reaction mixtures were subjected to immunoblotting using antibodies against DARPP-32 phosphorylated on Thr-34 or Thr-75 and total DARPP-32 protein. b , c Human HCC827, PC9, and H1975 lung adenocarcinoma cell lines retrovirally transduced with either FLAG-tagged human DARPP-32 ( b ) or t-DARPP ( c ) cDNA plasmids were lysed, immunoprecipitated, incubated with active IKKα protein, and subjected to western blotting using anti-phospho (Thr-34 or Thr-75) DARPP-32 and anti-DARPP-32 antibodies. Data from one experimental replicate are shown. The experiments were repeated three times independently; each circle in a bar represents one experiment. Error bars indicate SEM. * P < 0.05; ns not significant.

Article Snippet: Phosphorylation of DARPP-32 by kinase-activated human IKKα protein was validated via immunoblotting using monoclonal primary antibodies against phosphorylated DARPP-32 (T34 and T75, CST).

Techniques: Stable Transfection, Immunoprecipitation, In Vitro, Incubation, Western Blot, Transduction

Journal: NPJ Precision Oncology

Article Title: IKKα promotes lung adenocarcinoma growth through ERK signaling activation via DARPP-32-mediated inhibition of PP1 activity

doi: 10.1038/s41698-023-00370-3

Figure Lengend Snippet: a , b Human lung cancer cells, HCC827 ( a ) and H1650 ( b ), transfected with GFP (control), constitutively active IKKα, or kinase-dead IKKα were lysed using 1× RIPA buffer supplemented with protease and phosphatase inhibitors. Equal amounts of proteins were separated with 4–20% SDS-PAGE and transferred to polyvinyl difluoride membranes. Antigen-coated membranes were incubated overnight with primary antibodies against IKKα, phosphorylated DARPP-32 (Thr-34), total DARPP-32, phosphorylated PP1α (Thr320), total PP1α, phosphorylated ERK (Thr202/Tyr204), total ERK, and α-tubulin (loading control). c , d Vehicle (DMSO)- or calyculin A-treated human HCC827 ( c ) and H1650 ( d ) cells were lysed with 1× RIPA buffer and subjected to immunoblotting using anti-phospho PP1α (Thr320), -total PP1α, -phospho ERK (Thr202/Tyr204), -total ERK, -DARPP-32, and -α-tubulin (loading control) antibodies. Chemiluminescence signals were detected after incubating membranes with HRP-tagged secondary antibodies. Representative images from one experiment are shown, but results were validated by performing three independent biological repeats. Bar graphs at the right show quantification of the results from the three western blotting experiments. Error bars indicate SEM. * P < 0.05; ns not significant.

Article Snippet: Phosphorylation of DARPP-32 by kinase-activated human IKKα protein was validated via immunoblotting using monoclonal primary antibodies against phosphorylated DARPP-32 (T34 and T75, CST).

Techniques: Transfection, SDS Page, Incubation, Western Blot

Journal: NPJ Precision Oncology

Article Title: IKKα promotes lung adenocarcinoma growth through ERK signaling activation via DARPP-32-mediated inhibition of PP1 activity

doi: 10.1038/s41698-023-00370-3

Figure Lengend Snippet: a – d Human NSCLC HCC827 ( a , c ) and H1650 ( b , d ) cells transduced with retrovirus designed to overexpress either wild-type ( a , b ) or mutant (T34A) DARPP-32 ( c , d ) were transfected with GFP (control), kinase-dead (KD), full-length (FL), and constitutively active (CA) IKKα cDNAs were lysed using 1× RIPA buffer supplemented with protease inhibitors only. Equal amounts of proteins (500 ng) were immunoprecipitated using anti-PP1α antibodies. Immunoprecipitated cell lysates were subjected to in vitro phosphatase assays following incubation with either PP1α substrate or histone H1 peptide (control). Released phosphates in each reaction tube were determined by using a phosphate detection reagent. In vitro phosphatase experiments were repeated three times independently. Bar graphs represent the mean ± SEM of the three repeats, with each circle in a bar representing an independent experiment. A value of P ≤ 0.05 was considered significant, ns not significant, one-way ANOVA followed by Dunnett’s test. e – h Immunoprecipitated HCC827 ( e , g ) and H1650 ( f , h ) cell lysates separated with 4–20% SDS-PAGE were subjected to western blotting using anti-PP1α antibodies. Input cell lysates were blotted with antibodies against IKKα, DARPP-32, and α-tubulin (loading control).

Article Snippet: Phosphorylation of DARPP-32 by kinase-activated human IKKα protein was validated via immunoblotting using monoclonal primary antibodies against phosphorylated DARPP-32 (T34 and T75, CST).

Techniques: Transduction, Mutagenesis, Transfection, Immunoprecipitation, In Vitro, Incubation, SDS Page, Western Blot

Journal: NPJ Precision Oncology

Article Title: IKKα promotes lung adenocarcinoma growth through ERK signaling activation via DARPP-32-mediated inhibition of PP1 activity

doi: 10.1038/s41698-023-00370-3

Figure Lengend Snippet: a Representative images of HCC827, PC9, and H1650 cells transduced with lentivirus encoding either LacZ shRNA or IKKα shRNAs forming colonies on soft-agar cell culture dishes 1 to 2 weeks after plating. b Human NSCLC HCC827, PC9, and H1650 cells transduced with lentivirus designed to silence LacZ (control) or IKKα protein expression were subjected to soft-agar colony formation assays to determine anchorage-independent cell growth. ImageJ was used to count colonies on the cell culture dishes after 1 to 2 weeks of incubation, and the number of counted colonies was plotted. Each circle on a graph represents an independent experiment. Soft-agar colony formation experiments were repeated at least six times. Error bars indicate SEM ( n = 6). Scale bar 200 µm. A value of P ≤ 0.05 was considered significant, one-way ANOVA followed by Dunnett’s test. c , d Lysates from HCC827 ( c ) and PC9 ( d ) cells transduced with either LacZ shRNA or IKKα shRNA were subjected to immunoblotting with primary antibodies against IKKα, phosphorylated DARPP-32 (Thr-34), total DARPP-32, phosphorylated PP1α (Thr320), total PP1α, phosphorylated ERK (Thr202/Tyr204), total ERK, and α-tubulin (loading control). Bar graphs at the right show values obtained from the densitometric quantification of the results from three western blotting experiments. * P < 0.05; Two-tailed unpaired t -test.

Article Snippet: Phosphorylation of DARPP-32 by kinase-activated human IKKα protein was validated via immunoblotting using monoclonal primary antibodies against phosphorylated DARPP-32 (T34 and T75, CST).

Techniques: Transduction, shRNA, Cell Culture, Expressing, Incubation, Western Blot, Two Tailed Test

Journal: NPJ Precision Oncology

Article Title: IKKα promotes lung adenocarcinoma growth through ERK signaling activation via DARPP-32-mediated inhibition of PP1 activity

doi: 10.1038/s41698-023-00370-3

Figure Lengend Snippet: a Luciferase-labeled IKKα-depleted human HCC827 cells were orthotopically injected into the left thorax of SCID mice and imaged for luminescence on the indicated days. Total luminescence intensity (photon count) was calculated using molecular imaging software and plotted as a line graph. Error bars are shown as dotted lines indicating SEM. A value of P ≤ 0.05 was considered significant, two-way ANOVA followed by Sidak’s test. b Images of anesthetized mice were captured to detect luminescence signals on the indicated days. c Tumor tissue lysates obtained from either LacZ shRNA- or IKKα shRNA-transduced human HCC827 cells-derived xenografts were subjected to immunoblotting using primary antibodies against IKKα, phosphorylated PP1α (Thr320), total PP1α, phosphorylated ERK (Thr202/Tyr204), total ERK, and α-tubulin (loading control). Bar graphs show densitometric quantification values of the results from three western blotting experiments. * P < 0.05; Two-tailed unpaired t -test. d Overall morphological evaluation was performed on formalin‐fixed, paraffin‐embedded lung tissues ( n = 5 mice per group) obtained from human HCC827 cells-derived lung tumor xenograft mice model using hematoxylin and eosin (H&E) dye. Scale bar 50 µm.

Article Snippet: Phosphorylation of DARPP-32 by kinase-activated human IKKα protein was validated via immunoblotting using monoclonal primary antibodies against phosphorylated DARPP-32 (T34 and T75, CST).

Techniques: Luciferase, Labeling, Injection, Imaging, Software, shRNA, Derivative Assay, Western Blot, Two Tailed Test, Formalin-fixed Paraffin-Embedded

Journal: Frontiers in Immunology

Article Title: Human monocyte-derived suppressive cells (HuMoSC) for cell therapy in giant cell arteritis

doi: 10.3389/fimmu.2023.1137794

Figure Lengend Snippet: Effect of HuMoSC in the mTOR pathway. (A, B) Confocal microscopy analysis of temporal arteries affected by GCA cultivated during 5 days alone or in the presence of PDGF (20 ng/mL), HuMoSC (250.10 3 /mL) or supernatant of HuMoSC (25%). Pictures show staining of α-SMA (red), p-rpS6 (green) and nuclei (DAPI, blue). p-rpS6, whose expression is shown by the white arrows, is a metabolite of mTOR. Thus, the intensity of the green staining reflects the activation of the mTOR complex. Pictures show the entire artery (A) and magnifications (x 40) in the media (B) . (C) Mean ± SEM fluorescence intensity of p-rpS6 staining normalized to background noise was calculated in the neo-intima and the media of each temporal artery using ImageJ fiji software (untreated TAB+ [n=5], HuMoSC [n=3], HuMoSC supernatant [n=3], PDGF [n=4], PDGF + HuMoSC [n=2], PDGF + HuMoSC supernatant [n=2]).

Article Snippet: Then, 10 μm cryostat sections were fixed, blocked and incubated with primary antibodies against human phospho-S6 ribosomal protein (p-rpS6, Ser 235/236, Cell Signaling, at 1:200 dilution) and α-smooth muscle actin (polyclonal, Abcam, at 1:500 dilution), and then donkey secondary antibodies were conjugated with fluorochromes A488 (green) and A647 (red) (Abcam, at 1:300 dilution).

Techniques: Confocal Microscopy, Staining, Expressing, Activation Assay, Fluorescence, Software

Journal: Patterns

Article Title: Restaining-based annotation for cancer histology segmentation to overcome annotation-related limitations among pathologists

doi: 10.1016/j.patter.2023.100688

Figure Lengend Snippet: Antibodies used in this study

Article Snippet: The following primary antibodies were used, as summarized in : monoclonal mouse immunoglobulin G (IgG) anti-pan-cytokeratin, clone AE1/AE3 (without dilution; IS05330-2J; DAKO, Carpinteria, CA, USA); monoclonal mouse IgG anti-human αSMA, clone 1A4 (1:200 dilution; M085129-2; DAKO); monoclonal mouse IgG anti-human CD45RB, leukocyte common antigen, clones 2B11 + PD7/26 (1:200 dilution; IR75161-2J; DAKO); monoclonal mouse IgG anti-human N-terminal ERG, clone 9FY (without dilution; PM421AA; Biocare Medical, Concord, CA, USA); monoclonal mouse IgG anti-glycophorin A, clone JC159 (1:200 dilution; MA5-12484; Thermo Fisher Scientific, Waltham, MA, USA); polyclonal rabbit anti-human MNDA (1:1,000 dilution; HPA034532-100UL; Sigma-Aldrich); monoclonal rabbit IgG anti-human MIST1/bHLHa15 protein, clone D7N4B (1:100 dilution; #14896; Cell Signaling Technology, Beverly, MA, USA); polyclonal mouse anti-human CD3 (1:200 dilution; IS50330-2J; DAKO); and monoclonal mouse IgG anti-human CD20cy, clone L26 (1:200 dilution; IS60430-2J; DAKO).

Techniques:

Journal: Patterns

Article Title: Restaining-based annotation for cancer histology segmentation to overcome annotation-related limitations among pathologists

doi: 10.1016/j.patter.2023.100688

Figure Lengend Snippet: Dataset summary

Article Snippet: The following primary antibodies were used, as summarized in : monoclonal mouse immunoglobulin G (IgG) anti-pan-cytokeratin, clone AE1/AE3 (without dilution; IS05330-2J; DAKO, Carpinteria, CA, USA); monoclonal mouse IgG anti-human αSMA, clone 1A4 (1:200 dilution; M085129-2; DAKO); monoclonal mouse IgG anti-human CD45RB, leukocyte common antigen, clones 2B11 + PD7/26 (1:200 dilution; IR75161-2J; DAKO); monoclonal mouse IgG anti-human N-terminal ERG, clone 9FY (without dilution; PM421AA; Biocare Medical, Concord, CA, USA); monoclonal mouse IgG anti-glycophorin A, clone JC159 (1:200 dilution; MA5-12484; Thermo Fisher Scientific, Waltham, MA, USA); polyclonal rabbit anti-human MNDA (1:1,000 dilution; HPA034532-100UL; Sigma-Aldrich); monoclonal rabbit IgG anti-human MIST1/bHLHa15 protein, clone D7N4B (1:100 dilution; #14896; Cell Signaling Technology, Beverly, MA, USA); polyclonal mouse anti-human CD3 (1:200 dilution; IS50330-2J; DAKO); and monoclonal mouse IgG anti-human CD20cy, clone L26 (1:200 dilution; IS60430-2J; DAKO).

Techniques:

Journal: Patterns

Article Title: Restaining-based annotation for cancer histology segmentation to overcome annotation-related limitations among pathologists

doi: 10.1016/j.patter.2023.100688

Figure Lengend Snippet: Selection of the antibodies and target tissues in SegPath (A) Gene expression specificities of selected antibodies. Gene expression data were retrieved from single-cell transcriptome profiles in the Human Protein Atlas. , Target cell type is indicated by a red asterisk on the bar. ACTA2 expression in Sertoli cells, indicated by a green octothorpe, was high in this dataset, but a pathologist could not confirm the positive staining of anti-α-smooth muscle actin (SMA) antibody; therefore, testicular tissues were included in the dataset. ERG expression in microglial cells, indicated by a blue octothorpe, was higher in this dataset. This is highly likely to be an erroneous annotation of the single-cell transcriptome profile, as confirmed by a pathologist; therefore, brain tissues were included in the dataset. (B) H&E-stained image and IF staining of anti-MPO antibody, which targets neutrophils. Antigens spread around the target cells, as indicated by arrowheads, prevent accurate mask generation. (C) IF staining of anti-MIST1 antibody, which targets plasma cells. It unexpectedly stained the nuclei of some glandular epithelia, including the salivary gland and gastric epithelium. These tissues were excluded from SegPath.

Article Snippet: The following primary antibodies were used, as summarized in : monoclonal mouse immunoglobulin G (IgG) anti-pan-cytokeratin, clone AE1/AE3 (without dilution; IS05330-2J; DAKO, Carpinteria, CA, USA); monoclonal mouse IgG anti-human αSMA, clone 1A4 (1:200 dilution; M085129-2; DAKO); monoclonal mouse IgG anti-human CD45RB, leukocyte common antigen, clones 2B11 + PD7/26 (1:200 dilution; IR75161-2J; DAKO); monoclonal mouse IgG anti-human N-terminal ERG, clone 9FY (without dilution; PM421AA; Biocare Medical, Concord, CA, USA); monoclonal mouse IgG anti-glycophorin A, clone JC159 (1:200 dilution; MA5-12484; Thermo Fisher Scientific, Waltham, MA, USA); polyclonal rabbit anti-human MNDA (1:1,000 dilution; HPA034532-100UL; Sigma-Aldrich); monoclonal rabbit IgG anti-human MIST1/bHLHa15 protein, clone D7N4B (1:100 dilution; #14896; Cell Signaling Technology, Beverly, MA, USA); polyclonal mouse anti-human CD3 (1:200 dilution; IS50330-2J; DAKO); and monoclonal mouse IgG anti-human CD20cy, clone L26 (1:200 dilution; IS60430-2J; DAKO).

Techniques: Selection, Expressing, Staining

Journal: Mediators of Inflammation

Article Title: Allicin Alleviates Inflammation of Trinitrobenzenesulfonic Acid-Induced Rats and Suppresses P38 and JNK Pathways in Caco-2 Cells

doi: 10.1155/2015/434692

Figure Lengend Snippet: Levels of cytokines in the serum. Serum TNF- α and IL-1 β levels of TNBS-induced rats were dramatically decreased by drug treatment, and mesalazine + allicin group was the lowest (a and b); sulfasalazine and mesalazine + allicin rose serum IL-4 level; however, these drugs did not alter serum IL-10 level (c and d). Data were presented as mean ± SD. * P < 0.05 versus TNBS group.

Article Snippet: After 24 h serum-free starvation, cells were treated with 0.1, 1, or 10 ng/mL of human recombinant IL-1 β (Cell Signaling Technology, MA, USA) for 12 h, 24 h, or 48 h. Untreated Caco-2 cells served as control.

Techniques:

Journal: Mediators of Inflammation

Article Title: Allicin Alleviates Inflammation of Trinitrobenzenesulfonic Acid-Induced Rats and Suppresses P38 and JNK Pathways in Caco-2 Cells

doi: 10.1155/2015/434692

Figure Lengend Snippet: Expression of colon IL-1 β mRNA level in rats. (a) Total RNA was isolated from colon tissue. Colon IL-1 β mRNA levels were presented on an agarose gel. (b) The gel was carried out by densitometric and statistical analysis. Results were reported as means ± SD from 3 independent experiments. * P < 0.01 versus TNBS group.

Article Snippet: After 24 h serum-free starvation, cells were treated with 0.1, 1, or 10 ng/mL of human recombinant IL-1 β (Cell Signaling Technology, MA, USA) for 12 h, 24 h, or 48 h. Untreated Caco-2 cells served as control.

Techniques: Expressing, Isolation, Agarose Gel Electrophoresis

Journal: Mediators of Inflammation

Article Title: Allicin Alleviates Inflammation of Trinitrobenzenesulfonic Acid-Induced Rats and Suppresses P38 and JNK Pathways in Caco-2 Cells

doi: 10.1155/2015/434692

Figure Lengend Snippet: The influence of IL-1 β and allicin on IL-8 in the supernatant of Caco-2 cells. (a) 1 ng/mL IL-1 β significantly increased concentrations of IL-8 in the medium of Caco-2 cells at 12 and 24 h; (b) IL-8 level was not changed by allicin on IL-1 β -induced Caco-2 cells.

Article Snippet: After 24 h serum-free starvation, cells were treated with 0.1, 1, or 10 ng/mL of human recombinant IL-1 β (Cell Signaling Technology, MA, USA) for 12 h, 24 h, or 48 h. Untreated Caco-2 cells served as control.

Techniques:

Journal: Mediators of Inflammation

Article Title: Allicin Alleviates Inflammation of Trinitrobenzenesulfonic Acid-Induced Rats and Suppresses P38 and JNK Pathways in Caco-2 Cells

doi: 10.1155/2015/434692

Figure Lengend Snippet: NF- κ B p65 was increased in IL-1 β -induced Caco-2 cells (a and b): IL-1 β induced the expression of NF- κ B p65 in nucleus of Caco-2 cells at 12 and 24 h. * P < 0.05 versus 1 ng/mL IL-1 β group. Allicin influenced inflammation caused by IL-1 β in Caco-2 cells (c and d): 25 ng/mL allicin decreased the expression of NF- κ B p65 in nucleus of IL-1 β -induced Caco-2 cells at 12 and 24 h. * P < 0.05 1 ng/mL IL-1 β group versus 1 ng/mL IL-1 β + 25 μ g/mL allicin group. # P < 0.05 1 ng/mL 1 ng/mL IL-1 β + 10 μ g/mL allicin group versus 1 ng/mL IL-1 β + 25 μ g/mL allicin group. Data were presented as mean ± SD and repeated 3 times.

Article Snippet: After 24 h serum-free starvation, cells were treated with 0.1, 1, or 10 ng/mL of human recombinant IL-1 β (Cell Signaling Technology, MA, USA) for 12 h, 24 h, or 48 h. Untreated Caco-2 cells served as control.

Techniques: Expressing

Journal: Mediators of Inflammation

Article Title: Allicin Alleviates Inflammation of Trinitrobenzenesulfonic Acid-Induced Rats and Suppresses P38 and JNK Pathways in Caco-2 Cells

doi: 10.1155/2015/434692

Figure Lengend Snippet: The change of P38, ERK, and JNK of allicin on IL-1 β -induced Caco-2 cells. Results were repeated 3 times and presented by SDS-PAGE (a) and densitometric analysis (b). 25 μ g/mL allicin attenuated IL-1 β -induced phosphorylations P38 and JNK pathways. Both 1 ng/mL IL-1 β and 25 μ g/mL activated ERK pathway. * P < 0.05 1 ng/mL IL-1 β + 25 μ g/mL allicin versus 1 ng/mL (IL-1 β p-P38 and p-JNK). # P < 0.05 versus 25 μ g/mL allicin (p-ERK).

Article Snippet: After 24 h serum-free starvation, cells were treated with 0.1, 1, or 10 ng/mL of human recombinant IL-1 β (Cell Signaling Technology, MA, USA) for 12 h, 24 h, or 48 h. Untreated Caco-2 cells served as control.

Techniques: SDS Page

Journal: Journal of Neuroinflammation

Article Title: Plasminogen activator inhibitor type 1 regulates microglial motility and phagocytic activity

doi: 10.1186/1742-2094-9-149

Figure Lengend Snippet: Expression of plasminogen activator inhibitor type 1 (PAI-1) mRNA and protein in mixed glial cultures. (A) Cell lysates or conditioned medium were prepared from either untreated or lipopolysaccharide (LPS)/interferon (IFN)-γ-treated mixed glial cultures (24 hours treatment). Western blotting analysis was conducted to evaluate the expression level of the 47 kDa PAI-1 protein. Ponceau S staining images are shown for comparison. (B) The mixed glial cells, microglia, and astrocytes were stimulated with a combination of LPS (100 ng/ml) and IFN-γ (50 U/ml) for 24 hours, and conditioned medium were collected. PA1-1 secretion was measured by ELISA. Results are given as mean ± SD ( n = 3). * P < 0.05, ** P < 0.01. (C, D) Real-time PCR was performed to detect PAI-1 mRNA expression in glial cells treated for 6 hours with LPS (100 ng/ml) and IFN-γ (50 U/ml) either alone or in combination as indicated. GAPDH was used as an internal control. Results are given as mean ± SD ( n = 3). * P < 0.05, ** P < 0.01, NS = not significant; compared with the untreated control cells.

Article Snippet: Cells were treated with the recombinant human PAI-1 protein (100 ng/ml), mouse PAI-1 protein (0.1 to 1 μg/ml), BSA (2.2 or 22.0 nmol/l), monoclonal anti-mouse TLR2 antibody, polyclonal anti-mouse integrin β3 (ITGB3) antibody (Cell Signaling Technology), and vitronectin (1 μg/ml) for 1 hour in serum-free DMEM.

Techniques: Expressing, Western Blot, Staining, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

Journal: Journal of Neuroinflammation

Article Title: Plasminogen activator inhibitor type 1 regulates microglial motility and phagocytic activity

doi: 10.1186/1742-2094-9-149

Figure Lengend Snippet: Plasminogen activator inhibitor type 1 (PAI-1) promoted the migration of microglia in a concentration-dependent manner. (A) BV-2 microglial cells were seeded at a density of 8.0 × 10 4 cells/well in 96-well plates. When the BV-2 microglial cells had reached 80 to 90% confluence, a single scratch wound was made by using a 200 μl pipette tip, and the cell debris was removed by washing with PBS. Cells were treated with mouse PAI-1 protein (0 to 1000 ng/ml). At 0 and 24 hours, phase-contrast pictures of the wounds at three different locations were taken, and then the fold increase of migration distance was measured in three independent experiments. Results are given as mean ± SD ( n = 3). * P < 0.05, NS = not significant, compared with the untreated control (lower panel). Representative images are shown (upper panel; original magnification,× 150). (B) The Boyden chamber assay was also performed to evaluate cell migration. BV-2 microglial cells (5 × 10 4 cells/upper well) placed in the Boyden chambers were exposed to mouse PAI-1 protein (0 to 1000 ng/ml), and then incubated at 37°C for 6 hours. Microglial cells that migrated through a membrane were stained and counted. Results are given as mean ± SD ( n = 3). * P < 0.01; compared with the untreated control (lower panel). Representative images are also shown (upper panel; original magnification, × 100). (C) BV-2 microglial cells were treated with BSA (0 to 22 nmol/l) or PAI-1 (2.2 nmol/l; 100 ng/ml), followed by the wound-healing assay as described above. Results are given as mean ± SD ( n = 3). * P < 0.05, NS = not significant, compared with the untreated control. (D) BV-2 microglial cells were seeded at the density of 5.0 × 10 3 cells/well in 96-well plate. Cells were treated with a mouse PAI-1 protein (100 ng/ml) and incubated at 37°C for 12–72 hours to evaluate cell proliferation. Proliferation curves are based on the 2,5-diphenyltetrazolium bromide (MTT) assay. Results represent the mean ± SD (n = 3). Proliferation of PAI-1-treated cells (open circle) was compared with the untreated control (filled square). (E) The Boyden chamber assay was performed to evaluate primary microglial cell migration. Primary microglia cultures (5 × 10 4 cells/upper well) placed in the Boyden chambers were exposed to mouse PAI-1 protein (100 ng/ml), and then incubated at 37°C for 24 hours. Microglial cells that migrated through a membrane were stained and counted. Results are mean ± SD ( n = 3). * P < 0.05; compared with the untreated control (lower panel). Representative images are also shown (upper panel). (F) C6 glioma cells were seeded at the density of 8.0 × 10 4 cells/well in 96-well plate. Cells were treated with BSA (100 ng/ml) or PAI-1 (100 ng/ml), followed by the wound-healing assay as described above. Results are the mean ± SD ( n = 3). * P < 0.05; compared with the untreated control.

Article Snippet: Cells were treated with the recombinant human PAI-1 protein (100 ng/ml), mouse PAI-1 protein (0.1 to 1 μg/ml), BSA (2.2 or 22.0 nmol/l), monoclonal anti-mouse TLR2 antibody, polyclonal anti-mouse integrin β3 (ITGB3) antibody (Cell Signaling Technology), and vitronectin (1 μg/ml) for 1 hour in serum-free DMEM.

Techniques: Migration, Concentration Assay, Transferring, Boyden Chamber Assay, Incubation, Staining, Wound Healing Assay, MTT Assay

Journal: Journal of Neuroinflammation

Article Title: Plasminogen activator inhibitor type 1 regulates microglial motility and phagocytic activity

doi: 10.1186/1742-2094-9-149

Figure Lengend Snippet: No significant effects of plasminogen activator inhibitor type 1 (PAI-1) on microglial nitric oxide (NO) production or neurotoxicity after lipopolysaccharide (LPS) or interferon (IFN)-γ stimulation. (A) BV-2 microglial cells and (B) primary microglia cultures were treated with the indicated concentration of mouse PAI-1 protein, LPS (100 ng/ml), and IFN-γ (50 U/ml) for 24 hours. NO production was measured by the Griess reaction. Results are given as mean ± SD ( n = 3). * P < 0.01, NS = not significant. ( C) Primary microglia cultures (4.0 × 10 4 cells/well) were treated for 12 hours with PAI-1 (100 ng/ml), BSA (100 ng/ml), or LPS (100 ng/ml) as indicated. Afterwards, primary microglial cells were cocultured with (upper panel) 5-chloromethyl-fluoresceindiacetate (CMFDA)-labeled primary neuron cultures for 24 hours (co-culture scheme). (Lower panel) CMFDA-positive neurons in the five randomly chosen microscopic fields per well were counted under an inverted microscope. Results are given as mean ± SD ( n = 3). * P < 0.05, NS = not significant.

Article Snippet: Cells were treated with the recombinant human PAI-1 protein (100 ng/ml), mouse PAI-1 protein (0.1 to 1 μg/ml), BSA (2.2 or 22.0 nmol/l), monoclonal anti-mouse TLR2 antibody, polyclonal anti-mouse integrin β3 (ITGB3) antibody (Cell Signaling Technology), and vitronectin (1 μg/ml) for 1 hour in serum-free DMEM.

Techniques: Concentration Assay, Labeling, Co-Culture Assay, Inverted Microscopy

Journal: Journal of Neuroinflammation

Article Title: Plasminogen activator inhibitor type 1 regulates microglial motility and phagocytic activity

doi: 10.1186/1742-2094-9-149

Figure Lengend Snippet: Plasminogen activator inhibitor type 1 (PAI-1) promoted microglial migration through low-density lipoprotein receptor-related protein (LRP)1. (A, B) BV-2 microglial cells were transiently transfected with control small interfering (si)RNA or LRP1-specific siRNA. After 48 hours, a scratch wound was made. Cells were treated with or without mouse PAI-1 protein (100 ng/ml), followed by (A) the wound-healing assay and (B) the Boyden chamber assay, as described in Figure . Results are given as mean ± SD ( n = 3). * P < 0.05, ** P < 0.01, NS = not significant (lower panel). (Upper panels) Representative images of each assay. (C, D) Knockdown of LRP1 gene expression by siRNA was confirmed by using (C) reverse transcriptase PCR, (D) dot blotting (upper panel), and western blotting (lower panel). β-actin and the α-tubulin were used as internal controls. (E) BV-2 microglial cells were treated with mouse PAI-1 protein (100 ng/ml) and RAP protein (5 μg/ml) as indicated. The fold increase in migration distance was measured using the wound-healing assay. Results are given as mean ± SD ( n = 3). * P < 0.05, NS = not significant, compared with the untreated control (lower panel). (Upper panel) Representative images also shown.

Article Snippet: Cells were treated with the recombinant human PAI-1 protein (100 ng/ml), mouse PAI-1 protein (0.1 to 1 μg/ml), BSA (2.2 or 22.0 nmol/l), monoclonal anti-mouse TLR2 antibody, polyclonal anti-mouse integrin β3 (ITGB3) antibody (Cell Signaling Technology), and vitronectin (1 μg/ml) for 1 hour in serum-free DMEM.

Techniques: Migration, Transfection, Wound Healing Assay, Boyden Chamber Assay, Expressing, Western Blot

Journal: Journal of Neuroinflammation

Article Title: Plasminogen activator inhibitor type 1 regulates microglial motility and phagocytic activity

doi: 10.1186/1742-2094-9-149

Figure Lengend Snippet: Janus kinase (JAK)/signal transducer and activator of transcription (STAT)-1 was involved in the plasminogen activator inhibitor type 1 (PAI-1)-enhanced microglial motility. (A) BV-2 microglial cells were treated with mouse PAI-1 protein (100 ng/ml) or interferon (IFN)-γ (50 U/ml), and cell lysates were collected at 30 minutes after the treatment. The levels of phosphorylated STAT1 (pSTAT1 at Tyr701) or total STAT1 protein were then evaluated by western blotting analysis. Ponceau S staining was performed to confirm the equal loading of the samples. (B) BV-2 microglial cells were transfected with control small interfering (si)RNA or low-density lipoprotein receptor-related protein (LRP)1 siRNA. The cells were harvested 48 hours after transfection and used for the experiments. Cells were treated with mouse PAI-1 protein (100 ng/ml) for 30 minutes. Phosphorylated STAT1 or total STAT1 was measured by western blotting analysis. α-tubulin detection was also performed to confirm the equal loading of the samples. Values indicate the results of densitometric quantification normalized to α-tubulin. (C) BV-2 microglial cells were transfected with control siRNA or LRP1 siRNA. The cells were harvested at 48 hours after transfection and then treated with mouse IFN-γ (50 U/ml) for 30 minutes. Phosphorylated STAT1 was detected by western blotting analysis. α-tubulin detection was also performed to confirm the equal loading of the samples. (D) BV-2 microglial cells were pretreated with AG490 (JAK-specific inhibitor; 20 μmol/l) for 30 minutes before the treatment with mouse PAI-1 protein (100 ng/ml), and then cell migration was evaluated by the wound-healing assay. Results are mean ± SD ( n = 3). * P < 0.05, NS = not significant, compared with the untreated control (lower panel). Representative images are shown (upper panel; original magnification × 150).

Article Snippet: Cells were treated with the recombinant human PAI-1 protein (100 ng/ml), mouse PAI-1 protein (0.1 to 1 μg/ml), BSA (2.2 or 22.0 nmol/l), monoclonal anti-mouse TLR2 antibody, polyclonal anti-mouse integrin β3 (ITGB3) antibody (Cell Signaling Technology), and vitronectin (1 μg/ml) for 1 hour in serum-free DMEM.

Techniques: Western Blot, Staining, Transfection, Migration, Wound Healing Assay

Journal: Journal of Neuroinflammation

Article Title: Plasminogen activator inhibitor type 1 regulates microglial motility and phagocytic activity

doi: 10.1186/1742-2094-9-149

Figure Lengend Snippet: Plasminogen activator inhibitor type 1 (PAI-1) promoted microglial migration in vivo . (A, B) Microglial cells around the protein injection sites were stained with anti-Iba-1 antibody at 48 hours after intrastriatal injection of vehicle (dialyzed elution buffer that was used for PAI-1 protein purification), denatured human PAI-1 wild-type, PAI-1 wild-type, or PAI-1 R346A mutant protein (1 μl; 1.5 μg/μl). Boxes indicate the 300 × 300 μm squares placed for cell counting. Immunohistochemistry results showed that Iba-1-positive cells were recruited into the injection site after injection of wild-type or R346 mutant PAI-1 protein. (B) Results are given as mean ± SD from three animals and six independent sections per animal. * P < 0.05, ** P < 0.01, NS = not significant. (A) Representative images (scale bar, 300 μm). Asterisks indicate the injection sites (inj.). Guide cannula was stereotaxically located in the intrastriatal region (dotted line).

Article Snippet: Cells were treated with the recombinant human PAI-1 protein (100 ng/ml), mouse PAI-1 protein (0.1 to 1 μg/ml), BSA (2.2 or 22.0 nmol/l), monoclonal anti-mouse TLR2 antibody, polyclonal anti-mouse integrin β3 (ITGB3) antibody (Cell Signaling Technology), and vitronectin (1 μg/ml) for 1 hour in serum-free DMEM.

Techniques: Migration, In Vivo, Injection, Staining, Protein Purification, Mutagenesis, Cell Counting, Immunohistochemistry

Journal: Journal of Neuroinflammation

Article Title: Plasminogen activator inhibitor type 1 regulates microglial motility and phagocytic activity

doi: 10.1186/1742-2094-9-149

Figure Lengend Snippet: Plasminogen activator inhibitor type 1 (PAI-1)-induced microglial migration was independent of fibrinolysis or vitronectin binding. BV-2 microglial cells were treated with 100 ng/ml of mouse PAI-1, human wild-type PAI-1, or two variants (Q123K, R346A) of PAI-1 proteins, followed by a wound-healing assay as described in Figure . The wound recovery areas were visualized under an inverted microscope (upper panel), and the fold increase in migration distance was measured. Results are given as mean ± SD ( n = 3). * P < 0.05, ** P < 0.01; compared with the untreated control (lower panel).

Article Snippet: Cells were treated with the recombinant human PAI-1 protein (100 ng/ml), mouse PAI-1 protein (0.1 to 1 μg/ml), BSA (2.2 or 22.0 nmol/l), monoclonal anti-mouse TLR2 antibody, polyclonal anti-mouse integrin β3 (ITGB3) antibody (Cell Signaling Technology), and vitronectin (1 μg/ml) for 1 hour in serum-free DMEM.

Techniques: Migration, Binding Assay, Wound Healing Assay, Inverted Microscopy

Journal: Journal of Neuroinflammation

Article Title: Plasminogen activator inhibitor type 1 regulates microglial motility and phagocytic activity

doi: 10.1186/1742-2094-9-149

Figure Lengend Snippet: Plasminogen activator inhibitor type 1 (PAI-1) enhanced 5-chloromethyl-fluoresceindiacetate (CMFDA)-labeled microglial migration in vivo. The effect of PAI-1 on microglial migration was examined using a stab-injury/cell-injection model. Primary microglia were treated with 1 μg/ml of PAI-1 protein (denatured wild-type protein (control), wild-type protein, R346A mutant protein) for 12 hours. Microglial cells were then labeled with CMFDA and injected into the mouse brain. (A) After 72 hours, three areas (cell-injection site (area 1), an intermediate location between the injection site and the stab-injury site (area 2), and the stab-injury site (area 3) were chosen for the analysis of CMFDA-labeled microglial cell migration (left panel). (A) A schematic diagram of the stab-injury/cell-injection model is also shown (right panel). The stab injury (3 mm long, 2 mm deep) was created 2 mm posterior to the bregma and 4 mm right lateral to the midline. (B) Representative images of CMFDA (green) and Iba-1 (red) staining. Scale bar, 20 μm. (C) Iba-1 microglial staining in the three areas of brain. Scale bar, 200 μm.

Article Snippet: Cells were treated with the recombinant human PAI-1 protein (100 ng/ml), mouse PAI-1 protein (0.1 to 1 μg/ml), BSA (2.2 or 22.0 nmol/l), monoclonal anti-mouse TLR2 antibody, polyclonal anti-mouse integrin β3 (ITGB3) antibody (Cell Signaling Technology), and vitronectin (1 μg/ml) for 1 hour in serum-free DMEM.

Techniques: Labeling, Migration, In Vivo, Injection, Mutagenesis, Staining

Journal: Journal of Neuroinflammation

Article Title: Plasminogen activator inhibitor type 1 regulates microglial motility and phagocytic activity

doi: 10.1186/1742-2094-9-149

Figure Lengend Snippet: Astrocyte-derived plasminogen activator inhibitor type 1 (PAI-1) promoted the migration of microglia. Primary astrocytes were left untreated or treated with lipopolysaccharide (LPS; 100 ng/ml) and interferon (IFN)-γ (50 U/ml) for 12 hours. Cells were then washed twice with PBS, and cultured in fresh DMEM for an additional 24 hours. The astrocyte-conditioned medium (ACM) was then collected. BV-2 microglial cells were treated for 24 hours with ACM in the presence or absence of PAI-1 neutralizing antibody (PAI-1 Ab; 2 μg/ml), or normal rabbit serum (2 μg/ml) as control. Microglial migration was assessed by wound-healing assay as described in Figure . At 0 and 24 hours, phase-contrast images of the wounds at three different locations were taken (upper panel, original magnification, × 150), and then fold increase in migration distance from three independent experiments was measured (lower panel). Results are given as mean ± SD ( n = 3). * P < 0.05, ** P < 0.01, NS = not significant.

Article Snippet: Cells were treated with the recombinant human PAI-1 protein (100 ng/ml), mouse PAI-1 protein (0.1 to 1 μg/ml), BSA (2.2 or 22.0 nmol/l), monoclonal anti-mouse TLR2 antibody, polyclonal anti-mouse integrin β3 (ITGB3) antibody (Cell Signaling Technology), and vitronectin (1 μg/ml) for 1 hour in serum-free DMEM.

Techniques: Derivative Assay, Migration, Cell Culture, Wound Healing Assay

Journal: Journal of Neuroinflammation

Article Title: Plasminogen activator inhibitor type 1 regulates microglial motility and phagocytic activity

doi: 10.1186/1742-2094-9-149

Figure Lengend Snippet: Plasminogen activator inhibitor type 1 (PAI-1) inhibited microglial phagocytosis of zymosan particles. (A) BV-2 microglial cells were treated with PAI-1 (100 or 1000 ng/ml) for 1 hour and then incubated with zymosan particles conjugated with Alexa Fluor 594 (red) for 3 hours. Cells were washed five times with ice-cold PBS to remove bound particles. (A) Images at five different locations were taken and then the percentage of phagocytic cells was calculated based on the number of microglial cells that phagocytosed the zymosan particles (lower panel). (Upper panel) Representative images are shown. Results are given as mean ± SD from three independent experiments. * P < 0.05, ** P < 0.01, different from untreated control. Scale bar = 20 μm. (B) BV-2 microglial cells were treated with BSA (2.2 or 22 nmol/l) or PAI-1 (2.2 nmol/l; 100 ng/ml) for 1 hour and then incubated with zymosan particles for 3 hours, followed by the phagocytosis assay as described above. Results are given as mean ± SD from three independent experiments. * P < 0.05, NS = not significant, compared with the untreated control. Scale bar = 20 μm. (C) Primary microglia were treated with BSA (100 ng/ml), PAI-1 (100 ng/ml), or RAP protein (5 μg/ml) for 1 hour and then incubated with zymosan particles for 90 minutes. Microglial phagocytosis was assessed as described above. Results are given as mean ± SD ( n = 3). * P < 0.05, NS = not significant.

Article Snippet: Cells were treated with the recombinant human PAI-1 protein (100 ng/ml), mouse PAI-1 protein (0.1 to 1 μg/ml), BSA (2.2 or 22.0 nmol/l), monoclonal anti-mouse TLR2 antibody, polyclonal anti-mouse integrin β3 (ITGB3) antibody (Cell Signaling Technology), and vitronectin (1 μg/ml) for 1 hour in serum-free DMEM.

Techniques: Incubation, Phagocytosis Assay

Journal: Journal of Neuroinflammation

Article Title: Plasminogen activator inhibitor type 1 regulates microglial motility and phagocytic activity

doi: 10.1186/1742-2094-9-149

Figure Lengend Snippet: Plasminogen activator inhibitor type 1 (PAI-1) downregulated Toll-like receptor (TLR)2/6 expression and its signaling. (A) BV-2 microglial cells were treated with PAI-1 (100 ng/ml) for 5 hours. TLR2/TLR6 and dectin-1 gene expression was detected by reverse transcriptase PCR. β-actin was used as an internal control. (B) Alternatively, BV-2 microglial cells were treated with PAI-1 (100 ng/ml) for 24 hours. The levels of TLR2, TLR6, and TLR9 protein were then evaluated by western blotting analysis. α-tubulin was used as an internal control. Values indicate the results of densitometric analysis normalized to either β-actin or α-tubulin. (C) Primary microglia cultures were treated with mouse PAI-1 protein (100 ng/ml), lipopolysaccharide (LPS; 100 ng/ml), interferon (IFN)-γ; 50 U/ml), and lipoteichoic acid (LTA; 1 μg/ml) as indicated for 24 hours. (Upper panel) NO production was measured by a Griess reaction. (Lower panel) Cell viability was measured by 2,5-diphenyltetrazolium bromide (MTT) reduction assays, and the results expressed as the percentage of surviving cells over the control cells . Results are given as mean ± SD ( n = 3). * P < 0.01, NS = not significant.

Article Snippet: Cells were treated with the recombinant human PAI-1 protein (100 ng/ml), mouse PAI-1 protein (0.1 to 1 μg/ml), BSA (2.2 or 22.0 nmol/l), monoclonal anti-mouse TLR2 antibody, polyclonal anti-mouse integrin β3 (ITGB3) antibody (Cell Signaling Technology), and vitronectin (1 μg/ml) for 1 hour in serum-free DMEM.

Techniques: Expressing, Western Blot

Journal: Journal of Neuroinflammation

Article Title: Plasminogen activator inhibitor type 1 regulates microglial motility and phagocytic activity

doi: 10.1186/1742-2094-9-149

Figure Lengend Snippet: Plasminogen activator inhibitor type 1 (PAI-1) inhibited microglial phagocytosis in a vitronectin-dependent manner. (A) BV-2 microglial cells were treated with 100 ng/ml of human wild-type PAI-1, the Q123K variant, or the R346A variant for 1 hour, and then incubated for 3 hours with zymosan particles. (A) Microglial phagocytosis of fluorescent zymosan particles was assessed (lower panel) as described above. (Upper panel) Representative images are shown. Results are given as mean ± SD ( n = 3). * P < 0.05, NS = not significantly different from untreated control. Scale bar = 20 μm. (B) BV-2 microglial cells were treated with human wild-type PAI-1 or the Q123K mutant (100 ng/ml) in the presence or absence of vitronectin (1 μg/ml) for 1 hour, and then incubated with zymosan particles for 3 hours, followed by phagocytosis assay. Results are given as mean ± SD ( n = 3). * P < 0.05, NS = not significant. (C) BV-2 microglial cells were incubated with zymosan particles in the presence or absence of Toll-like receptor (TLR)2 antibody (TLR2 Ab; 2 μg/ml), integrin (ITG)B3 antibody (ITGB3 Ab; 2 μg/ml), normal rabbit serum (2 μg/ml; negative control), or PAI-1 (100 ng/m; positive control). Microglial phagocytosis assay was performed. Results are given as mean ± SD ( n = 3). * P < 0.05, ** P < 0.01, compared with normal serum.

Article Snippet: Cells were treated with the recombinant human PAI-1 protein (100 ng/ml), mouse PAI-1 protein (0.1 to 1 μg/ml), BSA (2.2 or 22.0 nmol/l), monoclonal anti-mouse TLR2 antibody, polyclonal anti-mouse integrin β3 (ITGB3) antibody (Cell Signaling Technology), and vitronectin (1 μg/ml) for 1 hour in serum-free DMEM.

Techniques: Variant Assay, Incubation, Mutagenesis, Phagocytosis Assay, Negative Control, Positive Control