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