Journal: Biologics : Targets & Therapy

Article Title: Globular domain of adiponectin: promising target molecule for detection of atherosclerotic lesions

doi: 10.2147/BTT.S22863

Figure Lengend Snippet: Binding efficiency of the full-length adiponectin subfractions to atherosclerotic plaques is much lower than that of globular adiponectin. Aortic sections of apoprotein E-deficient mice were incubated with unspecific rat antihuman IgG2a labeled with Atto655 as negative control (left) or full-length adiponectin subfraction-Atto655 30 μg/mL (middle) or globular adiponectin-Atto655 3 μg/mL (right). The inserts shown in the left and middle panels on the left side are positive controls, staining atherosclerotic plaques with an AlexaFluor488-prelabeled antibody against the macrophage marker CD68. Note: Scale bars: 50 μm.

Article Snippet: A rabbit antihuman collagen type I antibody (Rockland Immunochemicals, Philadelphia, PA) was used as a positive control, and nonspecific rat antihuman IgG2a as a negative control, similar in molecular weight (150 kDa) to the fAd-Sf hexamer (167 kDa).

Techniques: Binding Assay, Incubation, Labeling, Negative Control, Staining, Marker

Journal: Biologics : Targets & Therapy

Article Title: Globular domain of adiponectin: promising target molecule for detection of atherosclerotic lesions

doi: 10.2147/BTT.S22863

Figure Lengend Snippet: Globular adiponectin accumulates in lipid-stress injured human endothelial colony forming cells. Endothelial colony forming cells were cultured in the presence or absence (control) of acetylated low density lipoprotein and incubated with 10 μg/mL globular adiponectin-Atto655 or nonspecific IgG-Atto655 for 1 hour at 37°C. Imaging was performed by confocal laser scanning microscopy. Unstained (blank) samples are shown as inserts in the middle panels. Yellow arrows indicate globular adiponectin accumulations. Note: Scale bar: 25 μm. Abbreviations: acLDL, acetylated low-density lipoprotein; unspec., unspecific.

Article Snippet: A rabbit antihuman collagen type I antibody (Rockland Immunochemicals, Philadelphia, PA) was used as a positive control, and nonspecific rat antihuman IgG2a as a negative control, similar in molecular weight (150 kDa) to the fAd-Sf hexamer (167 kDa).

Techniques: Cell Culture, Control, Incubation, Imaging, Confocal Laser Scanning Microscopy

Journal: Journal of Functional Biomaterials

Article Title: Paeonia lactiflora Callus-Derived Polynucleotides Enhance Collagen Accumulation in Human Dermal Fibroblasts

doi: 10.3390/jfb17010056

Figure Lengend Snippet: Nuclear NF-κB and MMP expression were reduced by PL-PN treatment in human dermal fibroblast cells. Data represented mean ± SD of ≥3 biological replicates, and the arrow indicates the expected molecular weight. ( A ) HDF treated with PL-PN and PDRN for 72 h, NF-κB protein expression between cytosol and nucleus was examined using a cell fractionation assay. Each protein was normalized with Histone H3. Data represented three biological replications. ( B ) Immunostaining of nucleus (DAPI, blue) and NF-κB (green) in the presence of PL-PN and PDRN at 72 h. The scale bar was 20 μm. They indicated the mean ± SD of the NF-κB nucleus intensity determined. Data represented mean ± SD of ≥3 biological replicates. ( C ) mRNA expression of MMP1, MMP3, MMP9, and MMP13 in HDF cells. Each mRNA was normalized with GAPDH as a housekeeping gene. Data represented mean ± SD of ≥3 biological replicates. * p < 0.05, ** p < 0.01 vs. Ctrl.

Article Snippet: Normal human adult primary dermal fibroblasts cells (HDF) were obtained from American Type Culture Collection (ATCC; PCS-201-012, Manassas, VA, USA) and grown in DMEM (Welgene, Daegu, Republic of Korea) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Welgene) and 1% penicillin–streptomycin (Welgene).

Techniques: Expressing, Molecular Weight, Cell Fractionation, Immunostaining

Journal: Journal of Functional Biomaterials

Article Title: Paeonia lactiflora Callus-Derived Polynucleotides Enhance Collagen Accumulation in Human Dermal Fibroblasts

doi: 10.3390/jfb17010056

Figure Lengend Snippet: PL-PN was increased TGF-β/Smad pathway and increased collagen and elastin in human dermal fibroblast cells. ( A ) Protein expression levels of TGF-β (pro-form and mature form), p-SMAD2/3, and β-actin after 72 h of treatment. The bar graphs showed the ratio of TGF-β mature form to pro-form protein. Data represented mean ± SD of ≥4 biological replicates, and the arrow indicates the expected molecular weight. ( B ) mRNA expression of COL1A1, COL3A1, Elastin in HDF cells. Each mRNA was normalized with GAPDH as a housekeeping gene. ( C ) Sirius red stained collagen in HDF cells during 1 μg/mL PL-PN and 10 μg/mL S-PDRN treatment at 72 h. Objective: 20×; scale bar: 50 µm. ( D ) Elastin and collagen in HDF cells were stained during 1 μg/mL PL-PN and 10 μg/mL PDRN treatment at 72 h for elastin (black or blue black) and collagen (red). Objective: 20×; scale bar: 50 µm. Data represented mean ± SD of ≥ 3 biological replicates. * p < 0.05, ** p < 0.01 vs. Ctrl.

Article Snippet: Normal human adult primary dermal fibroblasts cells (HDF) were obtained from American Type Culture Collection (ATCC; PCS-201-012, Manassas, VA, USA) and grown in DMEM (Welgene, Daegu, Republic of Korea) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Welgene) and 1% penicillin–streptomycin (Welgene).

Techniques: Expressing, Molecular Weight, Staining

Journal: Nature Communications

Article Title: Nuclear PTEN functions as an essential regulator of SRF-dependent transcription to control smooth muscle differentiation

doi: 10.1038/ncomms10830

Figure Lengend Snippet: ( a , b ) PTEN (top) or SRF (bottom) proteins were immunoprecipitated (IP) from WCL of cultured human ( a ) or rat ( b ) aortic SMCs serum restricted for 48 h. About 10% of input WCL and co-immunoprecipitating SRF or PTEN were detected by immunoblotting (IB). A non-specific IgG was used for IPs as a negative control. ( c ) SMCs stably expressing control (Ctrl) or PTEN-specific shRNA were transiently transduced with empty vector adenovirus (EV) or adenoviruses encoding wild-type PTEN (WT) or phosphatase-inactive PTEN (MT) (multiplicity of infection=100). PTEN was immunoprecipitated (IP) and co-immunoprecipitating SRF was detected by immunoblotting (IB). ( d ) Cytoplasmic (cyto) and nuclear (nuc) SMC extracts were analysed by western blotting for SRF and PTEN levels. β-Tubulin and Lamin A/C were used as cytoplasmic and nuclear loading controls, respectively. ( e ) PTEN was immunoprecipitated from cytoplasmic and nuclear SMC extracts and co-immunoprecipitating SRF was detected by immunoblotting. A non-specific IgG was used for IPs as a negative control. ( f ) Recombinant His-tagged PTEN and GST-tagged SRF were purified, incubated together, and PTEN (top) or SRF (bottom) were immunoprecipitated. Co-immunoprecipitating SRF or PTEN was detected by immunoblotting. About 10% of protein mixture was immunoblotted to control for input. A non-specific IgG was used for IPs as a negative control. ( g ) Myocardin (left), Elk-1 (middle) and SRF or PTEN (right) proteins were immunoprecipitated from WCL of SMCs serum restricted for 48 h. About 10% of input WCL and co-immunoprecipitating SRF, PTEN or Elk-1 were detected by immunoblotting. Shown are representative blots from a minimum of three independent experiments. *, heavy chain IgG. Molecular weight markers were cropped out for final SRF blots; please see .

Article Snippet: Human primary aortic SMCs (ATCC PCS-100-012) were maintained in vascular cell basal medium (ATCC PCS-100-030; ATCC PCS-100-042) according to ATCC specifications.

Techniques: Immunoprecipitation, Cell Culture, Western Blot, Negative Control, Stable Transfection, Expressing, Control, shRNA, Transduction, Plasmid Preparation, Infection, Recombinant, Purification, Incubation, Molecular Weight

Journal: Cancers

Article Title: Impact of Hyaluronic Acid on the Cutaneous T-Cell Lymphoma Microenvironment: A Novel Anti-Tumor Mechanism of Bexarotene

doi: 10.3390/cancers17020324

Figure Lengend Snippet: HA overexpression in CTCL. ( A ) Serum HA levels of CTCL patients were compared with those of HCs (n = 16 each). ( B ) Immunofluorescent staining of HABP on MF lesion and HC skin sections (n = 5 for HC, patch, and plaque; n = 4 for tumor). Magnified images indicated by squares are shown on the right. Bar = 300 µm. ( C ) mRNA expression of NHDFs cocultured with HuT78 cells (n = 6 each). ( D – G ) Immunohistochemistry analysis of HAS2, HYAL1, HYAL2, and CEMIP2 expression in MF lesion and HC skin sections (n = 5 for HC, patch, and plaque; n = 4 for tumor). Fibroblasts are indicated by arrowheads. Bar = 50 µm. Representative images are shown. HC, healthy control; CTCL, cutaneous T-cell lymphoma; HABP, hyaluronic acid binding protein; NT, no treatment; AU, arbitrary unit.

Article Snippet: To evaluate the effect of HA, semi-confluent normal human dermal fibroblasts (NHDFs) (American Type Culture Collection) were serum-starved overnight in a serum-free medium and stimulated with 1 mg/mL of LMWHA or HMWHA (250 kDa and 2000 kDa each; both from Echelon Biosciences) for 24 h. NHDFs and HuT78 cells were serum-starved overnight and then cultured with 100 ng/mL of bexarotene for the next 24 h to assess the effect of bexarotene stimulation.

Techniques: Over Expression, Staining, Expressing, Immunohistochemistry, Control, Binding Assay

Journal: Cancers

Article Title: Impact of Hyaluronic Acid on the Cutaneous T-Cell Lymphoma Microenvironment: A Novel Anti-Tumor Mechanism of Bexarotene

doi: 10.3390/cancers17020324

Figure Lengend Snippet: Response of NHDFs to LMWHA. ( A ) mRNA expression of HA-producing and depolymerizing molecules in NHDFs stimulated with LMWHA or HMWHA (n = 6 each). HAS3 expression is shown at the protein level (n = 4 each). ( B ) NHDFs were similarly treated with HA, and the mRNA expression of extracellular matrix-related molecules was measured (n = 6 each). The result of immunoblotting of CTGF is shown. AU, arbitrary unit; NT, no treatment; LMW, low-molecular-weight; HMW, high-molecular-weight. Original western blots are presented in .

Article Snippet: To evaluate the effect of HA, semi-confluent normal human dermal fibroblasts (NHDFs) (American Type Culture Collection) were serum-starved overnight in a serum-free medium and stimulated with 1 mg/mL of LMWHA or HMWHA (250 kDa and 2000 kDa each; both from Echelon Biosciences) for 24 h. NHDFs and HuT78 cells were serum-starved overnight and then cultured with 100 ng/mL of bexarotene for the next 24 h to assess the effect of bexarotene stimulation.

Techniques: Expressing, Western Blot, Molecular Weight, High Molecular Weight

Journal: Cancers

Article Title: Impact of Hyaluronic Acid on the Cutaneous T-Cell Lymphoma Microenvironment: A Novel Anti-Tumor Mechanism of Bexarotene

doi: 10.3390/cancers17020324

Figure Lengend Snippet: Bexarotene inhibits HA expression in vivo and in vitro. ( A ) Amount of HA extracted from mouse tumor tissue treated with or without bexarotene (n = 8 each). ( B ) Immunohistochemistry analysis of HABP expression in murine tumor skin sections treated with bexarotene (n = 8 each). Bar = 300 μm. ( C ) Volcano plot of RNA sequencing. Gene expression in bexarotene-treated tumor tissue specimens was compared with that of non-treated specimens (n = 4 each). Up-regulated genes are shown in blue, and down-regulated genes are shown in red. Above the horizontal solid line indicates p < 0.05. The fold-change cut-off was set at 2.0. ( D ) Heatmap of related gene expression based on log 2 TPM. ( E ) HA concentration in cell culture supernatant and qRT-PCR and immunoblotting results in HuT78 cells treated with bexarotene or vehicle (n = 6 each). ( F ) HA concentration in cell culture supernatant and qRT-PCR and immunoblotting results of NHDFs treated with bexarotene or no treatment (n = 6 each). NT, no treatment; Bex, bexarotene; TPM, transcripts per kilobase million; AU, arbitrary unit. Original western blots are presented in .

Article Snippet: To evaluate the effect of HA, semi-confluent normal human dermal fibroblasts (NHDFs) (American Type Culture Collection) were serum-starved overnight in a serum-free medium and stimulated with 1 mg/mL of LMWHA or HMWHA (250 kDa and 2000 kDa each; both from Echelon Biosciences) for 24 h. NHDFs and HuT78 cells were serum-starved overnight and then cultured with 100 ng/mL of bexarotene for the next 24 h to assess the effect of bexarotene stimulation.

Techniques: Expressing, In Vivo, In Vitro, Immunohistochemistry, RNA Sequencing, Gene Expression, Concentration Assay, Cell Culture, Quantitative RT-PCR, Western Blot

Journal:

Article Title: Immunolocalization of the Ca 2+ -Activated K + Channel Slo1 in Axons and Nerve Terminals of Mammalian Brain and Cultured Neurons

doi: 10.1002/cne.20931

Figure Lengend Snippet: A, immunoblot analysis of brain membrane fractions from adult rats, and wild-type and Slo1-deficient mice. Proteins were fractionated on 7.5% SDS-PAGE and transferred to nitrocellulose membranes and probed with mouse monoclonal (L6/60, 10 µg/ml) or rabbit polyclonal anti-Slo1 (Alomone, 1:500; Chemicon, 1:200), or mouse monoclonal anti-Kv2.1 (K89/41, TC supe 1:2) antibodies as noted. Numbers to left denote mobility of prestained molecular weight standards in kD.

Article Snippet: Proteins were fractionated on 7.5% SDS-PAGE and transferred to nitrocellulose membranes and probed with mouse monoclonal (L6/60, 10 µg/ml) or rabbit polyclonal anti-Slo1 (Alomone, 1:500; Chemicon, 1:200), or mouse monoclonal anti-Kv2.1 (K89/41, TC supe 1:2) antibodies as noted.

Techniques: Western Blot, SDS Page, Molecular Weight

Journal:

Article Title: Immunolocalization of the Ca 2+ -Activated K + Channel Slo1 in Axons and Nerve Terminals of Mammalian Brain and Cultured Neurons

doi: 10.1002/cne.20931

Figure Lengend Snippet: A: Immunoblot analysis of the effects of alkaline phosphatase (AP) digestion on adult rat brain Slo1. Adult rat brain membranes treated without (−) or with 0.1 U/ml AP (+) for 3 h at 37°C were separated on 7.5% SDS-PAGE and transferred to nitrocellulose membranes, then probed with anti-Kv2.1 (K89/41 mouse mAb, TC supe 1:2), anti-Kv1.4 (K13/31 mouse mAb, TC supe 1:2), anti-GluR1 (rabbit polyclonal antibody, Upstate 1:1000), or anti-Slo1 (L6/60, 10 µg/ml) antibodies. Slo1 exhibited AP- shifts from Mr ≈ 135 to ≈ 128 kD (bands 1 and 2 indicated on the right). Brain Kv2.1 also shifted in Mr upon AP treatment, whereas Kv1.4 and GluR1 did not. B: Immunoblot analysis of developmental rat brain membrane samples. L6/60 (10 µg/ml) immunostaining at postnatal day 2 (P2) revealed at least three distinct bands as indicated in the left margin (1, 135 kD; 2, 131 kD; 3, 124 kD), which changed in their relative proportions during postnatal development. A=adult, C=adult sample incubated 3 h at 37°C without AP, AP=adult sample incubated 3 h at 37°C with AP. Numbers to left denote mobility of prestained molecular weight standards in kD.

Article Snippet: Proteins were fractionated on 7.5% SDS-PAGE and transferred to nitrocellulose membranes and probed with mouse monoclonal (L6/60, 10 µg/ml) or rabbit polyclonal anti-Slo1 (Alomone, 1:500; Chemicon, 1:200), or mouse monoclonal anti-Kv2.1 (K89/41, TC supe 1:2) antibodies as noted.

Techniques: Western Blot, SDS Page, Immunostaining, Incubation, Molecular Weight

Journal:

Article Title: Immunolocalization of the Ca 2+ -Activated K + Channel Slo1 in Axons and Nerve Terminals of Mammalian Brain and Cultured Neurons

doi: 10.1002/cne.20931

Figure Lengend Snippet: A–C: L6/60 staining in adult rat hippocampus A: overview of L6/60 (0.6 µg/ml) immunoperoxidase staining in hippocampus. B, C: higher magnification images of the staining shown in A. B: stratum lucidum of the CA3 region of the hippocampus. C: magnified view of mossy fiber axons. Arrows in A–C highlight anatomical landmarks that specify the regions magnified. D–E: double label immunofluorescence labeling of brain sections from wild-type (D, WT) and Slo1-deficient (E, KO) mice. Brain sections from these mice were stained with L6/60 (24 µg/ml) mAb in red, and anti-Kv2.1 (KC rabbit polyclonal, 1:100) antibody in green. Images were taken from the hilus of the dentate gyrus and CA3 stratum lucidum. Scale bars, A:, 500 µm; B, C: 100 µm; D: 50 µm.

Article Snippet: Proteins were fractionated on 7.5% SDS-PAGE and transferred to nitrocellulose membranes and probed with mouse monoclonal (L6/60, 10 µg/ml) or rabbit polyclonal anti-Slo1 (Alomone, 1:500; Chemicon, 1:200), or mouse monoclonal anti-Kv2.1 (K89/41, TC supe 1:2) antibodies as noted.

Techniques: Staining, Immunoperoxidase Staining, Immunofluorescence, Labeling

Journal:

Article Title: Immunolocalization of the Ca 2+ -Activated K + Channel Slo1 in Axons and Nerve Terminals of Mammalian Brain and Cultured Neurons

doi: 10.1002/cne.20931

Figure Lengend Snippet: Magnified view of stratum lucidum of CA3 region of hippocampus. A–C: somata and apical dendrites of CA3 pyramidal neurons were negatively stained with L6/60 (A, 24 µg/ml) as well as with anti-Kv1.4 antibody (B, K13/31 mouse mAb, TC supe 1:2), which overlapped in mossy fibers (C). D–F: in contrast, somatodendritic Kv2.1 (D, KC rabbit polyclonal antibody, 1:100) staining interdigitated with L6/60 staining (E), indicating the presence of Slo1 on synaptic terminals on the Kv2.1-positive apical dendrites of CA3 pyramidal neurons (F, overlay). Scale bars, 10 µm.

Article Snippet: Proteins were fractionated on 7.5% SDS-PAGE and transferred to nitrocellulose membranes and probed with mouse monoclonal (L6/60, 10 µg/ml) or rabbit polyclonal anti-Slo1 (Alomone, 1:500; Chemicon, 1:200), or mouse monoclonal anti-Kv2.1 (K89/41, TC supe 1:2) antibodies as noted.

Techniques: Staining

Journal:

Article Title: Immunolocalization of the Ca 2+ -Activated K + Channel Slo1 in Axons and Nerve Terminals of Mammalian Brain and Cultured Neurons

doi: 10.1002/cne.20931

Figure Lengend Snippet: A: low magnification view of L6/60 (0.6 µg/ml) immunoperoxidase staining near the primary fissure. Note moderate to high levels of staining in the molecular layer relative to the granule cell layer, and intense staining at/near the Purkinje cell layer. B: higher magnification view of the area boxed in A, showing intense L6/60 staining in the Purkinje cell layer. The arrowheads point to a Purkinje cell soma, while the arrow points to a basket cell pinceau terminal onto a Purkinje cell initial segment. C–D: Double label immunofluorescence staining of brain sections from wild-type (C: WT) and Slo1-deficient (D: KO) mice. Brain sections from these mice were stained with L6/60 mAb (24 µg/ml) in red, and anti-Kv2.1 (KC rabbit polyclonal, 1:100) antibody in green. Images were taken from the Purkinje cell layer and reveal that staining in Purkinje cell somata, basket cell terminals, and the molecular layer is eliminated in the Slo1 knockout. Scale bars, A:, 100 µm; B, C: 500 µm; D: 10 µm.

Article Snippet: Proteins were fractionated on 7.5% SDS-PAGE and transferred to nitrocellulose membranes and probed with mouse monoclonal (L6/60, 10 µg/ml) or rabbit polyclonal anti-Slo1 (Alomone, 1:500; Chemicon, 1:200), or mouse monoclonal anti-Kv2.1 (K89/41, TC supe 1:2) antibodies as noted.

Techniques: Immunoperoxidase Staining, Staining, Immunofluorescence, Knock-Out

Journal:

Article Title: Immunolocalization of the Ca 2+ -Activated K + Channel Slo1 in Axons and Nerve Terminals of Mammalian Brain and Cultured Neurons

doi: 10.1002/cne.20931

Figure Lengend Snippet: Confocal images of double label immunofluorescence staining. A–C: Slo1 and Kv1.2 localization in basket cell terminals. L6/60 (A: 24 µg/ml) staining overlaps with that for Kv1.2 (B: K14/16 mouse mAb, 16 µg/ml) in basket cell terminals (C: overlay); D–F: L6/60 (D: 24 µg/ml) staining encircles axon initial segments of Purkinje cells, stained with anti-NF-155/186 antibody (E: L11A/41 mouse mAb, TC supe 1:2; F: overlay). G–H: dendrites of multiple Purkinje cells, filled with anti-calbindin (H: Sigma mouse monoclonal, 16 µg/ml) staining, were associated with membrane-associated L6/60 staining (G: 24 µg/ml; I: overlay). J–L: localization of Slo1 in Purkinje cell somata. Slo1 staining (J: L6/60; 24 µg/ml) on the somata of Purkinje cells partially overlapped with Kv2.1 (K: KC rabbit polyclonal antibody, 1:100) surface clusters (L: overlay). Scale bars, 10 µm.

Article Snippet: Proteins were fractionated on 7.5% SDS-PAGE and transferred to nitrocellulose membranes and probed with mouse monoclonal (L6/60, 10 µg/ml) or rabbit polyclonal anti-Slo1 (Alomone, 1:500; Chemicon, 1:200), or mouse monoclonal anti-Kv2.1 (K89/41, TC supe 1:2) antibodies as noted.

Techniques: Immunofluorescence, Staining