Journal: Archives of Gynecology and Obstetrics

Article Title: Glycosylated fibronectin as a first trimester marker for gestational diabetes

doi: 10.1007/s00404-020-05670-8

Figure Lengend Snippet: a Glycosylated fibronectin concentrations in BMI subgroups of 20–25 and 30–35 kg/m 2 in the study and the control group. b Effect of smoking on concentrations of glycosylated fibronectin in the control and GDM groups. c Multiple of median (MoM) values for fibronectin and glycosylated fibronectin between control and GDM groups. d Multiple of median (MoM) values for adiponectin and glycosylated adiponectin between control and GDM groups

Article Snippet: Calibrators for both assays were made from recombinant human adiponectin (1065-AF, R&D Systems, Abingdon, UK).

Techniques: Control

Journal: Journal of Inflammation Research

Article Title: Unleashing AdipoRon’s Potential: A Fresh Approach to Tackle Pseudomonas aeruginosa Infections in Bronchiectasis via Sphingosine Metabolism Modulation

doi: 10.2147/JIR.S483689

Figure Lengend Snippet: The expression of adiponectin and AdipoR1 within bronchial epithelium determined in Pseudomonas aeruginosa infected mouse models. ( A ) Comparison of the expression of adiponectin between Pseudomonas aeruginosa infected mouse models and the uninfected mouse models by immunofluorescence (×200). ( B ) Comparison of the expression of AdipoR1 between Pseudomonas aeruginosa infected mouse models and the uninfected mouse models by immunofluorescence (×200). ( C ) Comparison of the expression of AdipoR1 between Pseudomonas aeruginosa infected mouse models and the uninfected mouse models by Western blot. ( D ) The expression levels of AdipoR1 in the bronchial epithelium of mice in different groups (DMSO control group, AdipoRon high-dose group, AdipoRon low-dose group and adiponectin Acrp30 recombinant protein group) were compared by immunofluorescence (×200). Data are expressed as the means ± standard deviation of three independent experiments. **indicates P <0.01.

Article Snippet: Treatment of the five groups: (i) Uninfected control: mice were anesthetized by intraperitoneal injection and instilled with 35 μL of PBS via the airway; (ii) Pseudomonas aeruginosa infection with DMSO control: 8 hours prior, mice were given an intravenous injection of DMSO solution as a control; (iii) Pseudomonas aeruginosa infection with low-dose AdipoRon: 8 hours prior, mice were treated with AdipoRon (5 mg/kg, intravenous injection); (iv) Pseudomonas aeruginosa infection with high-dose AdipoRon: 8 hours prior, mice were treated with AdipoRon (50 mg/kg, intravenous injection); and (v) Pseudomonas aeruginosa infection with adiponectin Acrp30 recombinant protein: 4 hours prior, mice were treated with adiponectin Acrp30 recombinant protein (HY-P7358, MedChemExpress, USA, 1 mg/kg, intravenous injection) as described.

Techniques: Expressing, Infection, Comparison, Immunofluorescence, Western Blot, Control, Recombinant, Standard Deviation

Journal: Journal of Inflammation Research

Article Title: Unleashing AdipoRon’s Potential: A Fresh Approach to Tackle Pseudomonas aeruginosa Infections in Bronchiectasis via Sphingosine Metabolism Modulation

doi: 10.2147/JIR.S483689

Figure Lengend Snippet: AdipoRon reduced the load of Pseudomonas aeruginosa in the airway of mice. ( A ) HE staining was performed on lung tissue specimens from mouse models with different treatment (control group without Pseudomonas aeruginosa infection, DMSO control group with Pseudomonas aeruginosa infection, AdipoRon low-dose (5 mg/kg) pretreatment group with Pseudomonas aeruginosa infection, AdipoRon high-dose (50 mg/kg) pretreatment group with Pseudomonas aeruginosa infection, and ACRP30 recombinant protein pretreatment group with Pseudomonas aeruginosa infection. ( B ) A semi-quantitative grading method of inflammation score was used to evaluate the severity of peribronchial inflammatory cell infiltration: 0, normal; 1, few cells; 2, a ring of inflammatory cells 1 cell layer deep; 3, a ring of inflammatory cells 2–4 cells deep; 4, a ring of inflammatory cells of > 4 cells deep. The inflammation scores of different groups were compared. ( C and D ) The CFU of Pseudomonas aeruginosa was counted in the lung tissue homogenates of mice in different treatment groups. ( E ) The bacterial load of Pseudomonas aeruginosa in the lung tissue of mice in different treatment groups was detected by immunofluorescence with the antibody against Pseudomonas aeruginosa (PA1-73116). Data are expressed as the means ± standard deviation of three independent experiments. **indicates P <0.01.

Article Snippet: Treatment of the five groups: (i) Uninfected control: mice were anesthetized by intraperitoneal injection and instilled with 35 μL of PBS via the airway; (ii) Pseudomonas aeruginosa infection with DMSO control: 8 hours prior, mice were given an intravenous injection of DMSO solution as a control; (iii) Pseudomonas aeruginosa infection with low-dose AdipoRon: 8 hours prior, mice were treated with AdipoRon (5 mg/kg, intravenous injection); (iv) Pseudomonas aeruginosa infection with high-dose AdipoRon: 8 hours prior, mice were treated with AdipoRon (50 mg/kg, intravenous injection); and (v) Pseudomonas aeruginosa infection with adiponectin Acrp30 recombinant protein: 4 hours prior, mice were treated with adiponectin Acrp30 recombinant protein (HY-P7358, MedChemExpress, USA, 1 mg/kg, intravenous injection) as described.

Techniques: Staining, Control, Infection, Recombinant, Immunofluorescence, Standard Deviation

Journal: Journal of Inflammation Research

Article Title: Unleashing AdipoRon’s Potential: A Fresh Approach to Tackle Pseudomonas aeruginosa Infections in Bronchiectasis via Sphingosine Metabolism Modulation

doi: 10.2147/JIR.S483689

Figure Lengend Snippet: AdipoRon increased sphingosine level in the lung of mouse infection model. ( A )The levels of sphingosine were compared between the Pseudomonas aeruginosa -infected mouse model group and the uninfected control group. ( B ) The metabolites in the lung tissue of mice in 3 groups were compared by PCA analysis: DMSO control group, adipoRon low-dose group and adipoRon high-dose group. ( C ) The levels of sphingosine in 3 groups were compared. ( D ) The metabolites in the lung tissue of mice were compared between adiponectin Acrp30 recombinant protein pretreatment group and the DMSO control group by PCA analysis. ( E ) The levels of sphingosine in 2 groups were compared. *** P <0.001.

Article Snippet: Treatment of the five groups: (i) Uninfected control: mice were anesthetized by intraperitoneal injection and instilled with 35 μL of PBS via the airway; (ii) Pseudomonas aeruginosa infection with DMSO control: 8 hours prior, mice were given an intravenous injection of DMSO solution as a control; (iii) Pseudomonas aeruginosa infection with low-dose AdipoRon: 8 hours prior, mice were treated with AdipoRon (5 mg/kg, intravenous injection); (iv) Pseudomonas aeruginosa infection with high-dose AdipoRon: 8 hours prior, mice were treated with AdipoRon (50 mg/kg, intravenous injection); and (v) Pseudomonas aeruginosa infection with adiponectin Acrp30 recombinant protein: 4 hours prior, mice were treated with adiponectin Acrp30 recombinant protein (HY-P7358, MedChemExpress, USA, 1 mg/kg, intravenous injection) as described.

Techniques: Infection, Control, Recombinant

Journal: Journal of Inflammation Research

Article Title: Unleashing AdipoRon’s Potential: A Fresh Approach to Tackle Pseudomonas aeruginosa Infections in Bronchiectasis via Sphingosine Metabolism Modulation

doi: 10.2147/JIR.S483689

Figure Lengend Snippet: AdipoRon activated P-AMPKα/PGC1α, inhibited TLR4/P-NF-κB p65, and reduced expression of bax in Pseudomonas aeruginosa -infected mouse model. ( A and B ) Western blot was used to quantitatively analyze the expression levels of several major indicators in the lung tissues of mice in three treatment groups (DMSO control group, AdipoRon low-dose group and AdipoRon high-dose group). Image J software was used to analyze the gray levels of different protein imprinting bands and to make statistical analysis. ( C and D ) Western blot was used to quantitatively analyze the expression levels of several major indexes in the lung tissues of mice in two groups: DMSO control group and Acrp30 recombinant protein pretreatment group. Data are expressed as the means ± standard deviation of three independent experiments. *indicates P <0.05, **indicates P <0.01.

Article Snippet: Treatment of the five groups: (i) Uninfected control: mice were anesthetized by intraperitoneal injection and instilled with 35 μL of PBS via the airway; (ii) Pseudomonas aeruginosa infection with DMSO control: 8 hours prior, mice were given an intravenous injection of DMSO solution as a control; (iii) Pseudomonas aeruginosa infection with low-dose AdipoRon: 8 hours prior, mice were treated with AdipoRon (5 mg/kg, intravenous injection); (iv) Pseudomonas aeruginosa infection with high-dose AdipoRon: 8 hours prior, mice were treated with AdipoRon (50 mg/kg, intravenous injection); and (v) Pseudomonas aeruginosa infection with adiponectin Acrp30 recombinant protein: 4 hours prior, mice were treated with adiponectin Acrp30 recombinant protein (HY-P7358, MedChemExpress, USA, 1 mg/kg, intravenous injection) as described.

Techniques: Expressing, Infection, Western Blot, Control, Software, Recombinant, Standard Deviation

Journal: Annals of Dermatology

Article Title: Adiponectin Upregulates Filaggrin Expression via SIRT1-Mediated Signaling in Human Normal Keratinocytes

doi: 10.5021/ad.2017.29.4.407

Figure Lengend Snippet: Adiponectin stimulation increases filaggrin ( FLG ) expression in a time-dependent and dose-dependent manner without affecting the viability. Human normal epidermal keratinocytes (NHEKs) were treated with Acrp30 for 24 h at the indicated concentrations or with 10 µg/ml Acrp30 at the indicated time lines. (A) The MTT assay was performed to determine NHEK viability after Acrp30 treatment. (B, C) Cell lysates were generated with described lysis buffer and silent mating type information regulation 2 homolog (SIRT1), aryl hydrocarbon receptor nuclear translocator (ARNT), and FLG expression level was analyzed by immunoblotting. Representative results are shown from 3 independent experiments. NT: no treatment, O.D: optical density.

Article Snippet: Full-length recombinant human adiponectin was obtained from Biobud (Seoungnam, Korea).

Techniques: Expressing, MTT Assay, Generated, Lysis, Western Blot

Journal: Investigative Ophthalmology & Visual Science

Article Title: Lower Levels of Adiponectin and Its Receptor Adipor1 in the Uveal Melanomas With Monosomy-3

doi: 10.1167/iovs.61.5.12

Figure Lengend Snippet: Objective quantification of the immunohistochemical staining and the assessment of M3 status. ( a ) IHC on the paraffin sections of primary UM was performed using HRP-conjugated secondary antibodies and the HRP-green substrate, which yields a blue-green reaction product (left panel). The right panel demonstrates the negative control of the same tumor, which was incubated without the primary antibodies. Nuclei were counterstained with nuclear fast red. For each patient, light microscopy images of the entire tumor area were acquired under 200 × magnification. Image deconvolution was then performed using the Fiji software to separate the layers of nuclei, pigmentation, and IHC reaction with minimal overlap and background. The gray value of the IHC layer (as demonstrated for adiponectin) was inverted so that the regions exhibiting a stronger immunoreactivity would appear lighter and acquire higher pixel intensities compared to the background. The tumor area was then circumscribed on the original image (as demonstrated by the black lines ), and the integrated density (area × intensity) of the selected region was determined by redirecting the measurement to the inverted IHC image. This approach enabled the measurement of IHC intensity even in the samples that exhibited a very weak immunoreactivity, such as the negative control in the right panel. The mean IHC-intensities of the circumscribed areas in the positive and negative stainings were measured as 44.728 and 1.975 gray values, respectively, using this method. ( b ) Melan-A was selected as a suitable marker for the distinction of UM borders from the adjacent tissues by fluorescence microscopy. This marker was used in the subsequent Immuno-FISH assays to ensure that the nuclei that were quantified for chromosome 3 belonged to the UM cells. ( c ) Immuno-FISH for chromosome 3 was also performed on the retinal tissue of three enucleation samples as a positive control for diploid cells. Arrow indicates the autofluorescence in photoreceptor outer segments. GCL, ganglion cell layer; INL, inner nerve layer; ONL, outer nerve layer. Scale bars in ( b ) and ( c ) = 25 µm.

Article Snippet: Recombinant human adiponectin (Peprotech, Hamburg, Germany; 450-24) was reconstituted in sterile triple-distilled water.

Techniques: Immunohistochemical staining, Staining, Negative Control, Incubation, Light Microscopy, Software, Marker, Fluorescence, Microscopy, Positive Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Lower Levels of Adiponectin and Its Receptor Adipor1 in the Uveal Melanomas With Monosomy-3

doi: 10.1167/iovs.61.5.12

Figure Lengend Snippet: Adiponectin levels in the primary UM samples with regard to the M3 status. ( a ) IHC for adiponectin demonstrated a strong and relatively homogenous distribution of this protein in both the margins and the center of a primary UM, which was classified as having „low M3“ (obtained from Patient 3, male, 67 years). In contrast, the “high M3” tumor (derived from patient 14, female, 48 years) exhibited significantly less adiponectin in the periphery, which decreased further toward the center. The adiponectin-positive cells in the center of this latter tumor, which constituted less than 10% of the cell population, were mainly detected in the vicinity of blood vessels (indicated by the black arrows ). Patient 3 received stereotactic radiotherapy 17 months before undergoing endoresection, whereas patient 14 underwent enucleation without irradiation. Patient 14 developed liver metastases within 2 years, whereas no metastases or extraocular growth were detected in Patient 3 during the follow-up time of 9 years. ( b ) Immuno-FISH assay demonstrating the copy number of chromosome 3 in the Melan-A positive cells of these tumors. Note the lower number of chromosome 3 signals despite the higher nuclear density in the “high M3” tumor. Examples of cells with M3 are indicated by the white arrows in both tumors. ( c ) Quantification of adiponectin in the entire tumor area with respect to the M3 status. Tumors with a higher percentage of M3 positive cells (n = 16) had significantly lower levels of adiponectin compared to the low M3 tumors (n = 22) as determined by logistic regression analysis. ( d ) Patients with a higher percentage of M3 in their CMC (n = 15) also had significantly less adiponectin in their primary tumor compared with the patients with lower levels of M3-positive CMC (n = 16) as determined by logistic regression. ( e ) Examples of some of the CMC detected in patient 14 at the time of diagnosis with primary UM. Arrows indicate the immunobeads. No CMC were detected in patient 3. Scale bars in ( b ) and ( e ) = 10 µm. MCSP, Melanoma-associated chondroitin sulfate proteoglycan.

Article Snippet: Recombinant human adiponectin (Peprotech, Hamburg, Germany; 450-24) was reconstituted in sterile triple-distilled water.

Techniques: Derivative Assay, Irradiation, Biomarker Discovery

Journal: Investigative Ophthalmology & Visual Science

Article Title: Lower Levels of Adiponectin and Its Receptor Adipor1 in the Uveal Melanomas With Monosomy-3

doi: 10.1167/iovs.61.5.12

Figure Lengend Snippet: Expression of Adipor1 in primary UM samples with regard to the M3 status. ( a ) The irradiated, low M3 tumor of patient 3 exhibited a strong and mainly homogenous expression of Adipor1 in both the periphery and center. In contrast, Adipor1 was detected at significantly weaker levels in the periphery and was almost absent in the center of the nonirradiated, high M3 tumor of patient 14. Arrow indicates stronger Adipor1 expression in the proximity of a blood vessel in the latter tumor. Quantification of Adipor1 intensity in the entire tumor area demonstrated significantly lower levels of this protein in the patients who had a high extent of M3 in their primary tumor ( b ), a higher number of CMC ( c ), and a higher percentage of CMC with M3 ( d ). Adipor1 expression correlated positively with the adiponectin levels in the corresponding primary UM ( e ). The P values in ( b – e ) were determined by logistic regression analysis.

Article Snippet: Recombinant human adiponectin (Peprotech, Hamburg, Germany; 450-24) was reconstituted in sterile triple-distilled water.

Techniques: Expressing, Irradiation

Journal: Investigative Ophthalmology & Visual Science

Article Title: Lower Levels of Adiponectin and Its Receptor Adipor1 in the Uveal Melanomas With Monosomy-3

doi: 10.1167/iovs.61.5.12

Figure Lengend Snippet: Validation of the differential protein levels of adiponectin and Adipor1 with regard to the M3 status by immunoblotting. Freshly frozen tissue was available from two samples that were classified as having low versus high M3 (patients 5 and 6; female; age: 53 and 69 years, respectively). The remaining halves of the tissues were fixed in formalin and processed for immunostaining and immuno-FISH. Both patients underwent irradiation before the acquisition of the tumor samples. ( A ) IHC for adiponectin and Adipor1 demonstrated a reduction in the levels of both proteins in the high M3 sample. Arrows indicate several cells with M3 detected by immuno-FISH. Immunoblotting of the lysates of these tumors also revealed the stronger presence of ( B ) adiponectin and ( C ) Adipor1 in the low M3 sample. Expression of Melan-A was analyzed as a positive control for the abundance of melanoma cells in the tissue lysates. Patient 6 with the high M3 sample developed metastases within 5 years, whereas no metastases were detected in patient 5 with the low M3-tumor during the follow-up time of 8 years. Scale bar = 25 µm.

Article Snippet: Recombinant human adiponectin (Peprotech, Hamburg, Germany; 450-24) was reconstituted in sterile triple-distilled water.

Techniques: Biomarker Discovery, Western Blot, Immunostaining, Irradiation, Expressing, Positive Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Lower Levels of Adiponectin and Its Receptor Adipor1 in the Uveal Melanomas With Monosomy-3

doi: 10.1167/iovs.61.5.12

Figure Lengend Snippet: Expression of adiponectin and Adipor1 with regard to the BAP1 levels in the primary tumor of our UM patients. The immunoreactivity for BAP1 in the ( A ) cytoplasm and ( B ) nucleus was graded at a scale of 0–3 (0: positive staining in less than 10% of cells; 1: positive staining in 11%–33% of cells; 2: positive staining in 34%–66% of cells; 3: positive staining in more than 67% of cells). Images were acquired under 200x magnification. The immunoreactivity for ( C ) adiponectin and ( D ) Adipor1 was significantly reduced in the tumors with lower cytoplasmic levels of BAP1 (n = 14 and n = 23 samples with low versus high BAP1, respectively; Mann-Whitney U test).

Article Snippet: Recombinant human adiponectin (Peprotech, Hamburg, Germany; 450-24) was reconstituted in sterile triple-distilled water.

Techniques: Expressing, Staining, MANN-WHITNEY

Journal: Investigative Ophthalmology & Visual Science

Article Title: Lower Levels of Adiponectin and Its Receptor Adipor1 in the Uveal Melanomas With Monosomy-3

doi: 10.1167/iovs.61.5.12

Figure Lengend Snippet: Association of the Metastases/Extraocular Growth With Clinical Parameters and Adiponectin/Adipor1 Protein Levels (Follow-Up Time: 2–9 Years)

Article Snippet: Recombinant human adiponectin (Peprotech, Hamburg, Germany; 450-24) was reconstituted in sterile triple-distilled water.

Techniques: Biomarker Discovery, Irradiation

Journal: Investigative Ophthalmology & Visual Science

Article Title: Lower Levels of Adiponectin and Its Receptor Adipor1 in the Uveal Melanomas With Monosomy-3

doi: 10.1167/iovs.61.5.12

Figure Lengend Snippet: Expression of the major genes involved in adiponectin-mediated signaling in the UM cohort of the TCGA study (n = 80 patients). Each column represents a tumor sample, which was clustered according to the copy number of chromosome 3p and 3q ( red : normal, blue : loss) in the uppermost two rows. Tumors with the loss of both 3p and 3q were considered as having M3, whereas these data were incomplete for n = 3 samples. The copy number of chromosomes 1q, 9q, and 12, which harbor several of the genes of interest, as well as the survival status are also demonstrated in the upper group of rows, whereas the middle rows indicate the presence of genetic alterations. The lower rows construct the expression heatmap, which was generated from the mRNA z-scores, with blue and red representing mRNA levels that were up to three standard deviations lower or higher than the mean, respectively, whereas black indicates an expression at the mean. The loci of the analyzed genes are indicated in parentheses next to the gene symbol in the heatmap. Expression of BAP1 was included as a reference.

Article Snippet: Recombinant human adiponectin (Peprotech, Hamburg, Germany; 450-24) was reconstituted in sterile triple-distilled water.

Techniques: Expressing, Construct, Generated

Journal: Investigative Ophthalmology & Visual Science

Article Title: Lower Levels of Adiponectin and Its Receptor Adipor1 in the Uveal Melanomas With Monosomy-3

doi: 10.1167/iovs.61.5.12

Figure Lengend Snippet: Expression of adiponectin and its receptors in the cultured human choroidal melanocytes (hCM) and UM cells. ( a ) Culture purity was evaluated by the levels of melanocyte- and melanoma-specific markers. The hCM1 cells were derived from the choroid of a patient without an ocular tumor, whereas the hCM2 cells were obtained from the tumor-free choroidal region of a patient with posterior UM. The UM1 and UM2 cells were isolated from the primary tumors of patients 19 and 14, respectively, in our study. The donor of UM1 cells (male, 82 years) had posterior UM which was classified as a high M3 tumor. UM2 cells were derived from the high M3 tumor of the patient (female, 48 years), who was referred to in the and , as well. ( b ) Expression of adiponectin, as well as its receptors Adipor1 and Adipor2 in the cells grown in normal medium with 10% FBS. Expression of the receptors on the cell surface was analyzed by immunocytochemistry on non-permeabilized cells. Adipor1 could be detected on all the cell types whereas the expression of Adipor2 was considerably stronger in the UM cultures. Scale bar = 50 µm. ( c ) Immunoblotting under non-reducing conditions demonstrated the expression of adiponectin in the whole lysates of Mel-270 and OMM-2.5 cells that were either grown in normal medium with 10% FBS or serum-deprived for 2 days. The total protein loading in the lanes was detected by stain-free gel imaging. ( d ) Immunocytochemical analysis of adiponectin levels in the Mel-270 and OMM-2.5 cells that were serum-deprived for 2 days. The negative controls were incubated without the primary antibody. Scale bar = 25 µm.

Article Snippet: Recombinant human adiponectin (Peprotech, Hamburg, Germany; 450-24) was reconstituted in sterile triple-distilled water.

Techniques: Expressing, Cell Culture, Derivative Assay, Isolation, Immunocytochemistry, Western Blot, Staining, Imaging, Incubation

Journal: Investigative Ophthalmology & Visual Science

Article Title: Lower Levels of Adiponectin and Its Receptor Adipor1 in the Uveal Melanomas With Monosomy-3

doi: 10.1167/iovs.61.5.12

Figure Lengend Snippet: Basal adiponectin levels in the cultured UM cells with M3 versus disomy-3. ( a ) Immuno-FISH was performed for the codetection of adiponectin and chromosome 3 on the cytospins of intact UM1 and UM2 cells (both at passage 2). Images presented were acquired from the UM1 cells. Notice the larger nucleolar area (which appear darker in the nuclear stainings and were marked by the white lines ) in the cells with M3. ( b ) The mean gray value of the adiponectin staining was quantified by circumscribing the cytoplasmic region (excluding the nuclei) in a total of 112–180 nonoverlapping UM2 and UM1 cells, respectively, with clear signals for chromosome 3. The number of M3-positive cells that could be quantified was 43–69 for the UM2 and UM1 cells, respectively. The intensity of the cells with disomy-3 was taken as 100%. Data represent the mean ± standard deviation of the measurements for the UM1 and UM2 cells. P value was determined by two-sided t -test assuming equal variance.

Article Snippet: Recombinant human adiponectin (Peprotech, Hamburg, Germany; 450-24) was reconstituted in sterile triple-distilled water.

Techniques: Cell Culture, Staining, Standard Deviation

Journal: Investigative Ophthalmology & Visual Science

Article Title: Lower Levels of Adiponectin and Its Receptor Adipor1 in the Uveal Melanomas With Monosomy-3

doi: 10.1167/iovs.61.5.12

Figure Lengend Snippet: Adiponectin induces a more quiescent phenotype in the UM cells by suppressing proliferation and energy production. ( a ) Immunostaining for the proliferation marker Ki-67 on the Mel-270 and OMM-2.5 cells after a 1-day incubation in normal medium with 10% FBS, serum-free medium (no FBS), or serum-free medium with 30 µg/ml Adiponectin (no FBS + A). Scale bar = 50 µm. ( b ) Quantification of the Ki-67 immunostaining was performed on a minimum of 205 nuclei per group. Cells with a mean Ki-67 intensity that was above the cutoff value of negative cells were defined as being positive. Data represent the mean ± SEM of n = 3 independent experiments. * P < 0.05 compared to the corresponding no FBS group as determined by the two-sided t -test. ( c ) YAP-immunostaining demonstrating a gradual decline in the levels of this protein in response to serum deprivation ± adiponectin. The intensity of the nuclear staining also underwent a progressive decrease, suggesting the impairment of DNA duplication. Scale bar = 25 µm. ( d ) Immunoblotting for Ki-67 and YAP in the whole cell lysates after a 1-day exposure to the indicated treatments. Images are representative of n = 3 experiments. ( e ) Silver staining of nucleolar organizer regions (AgNOR, indicated by the arrow ), demonstrating the reduction in the number and area of the nucleolar structures in response to serum deprivation + Adiponectin. Scale bar = 25 µm. ( f ) Quantification of the AgNOR area. Data represent the mean ± SEM of n = 3 experiments with a minimum of n = 202 quantified nuclei per group. * P < 0.05 compared to the corresponding no FBS group as determined by the two-sided t -test. ( g ) Cell-cycle analysis after a 1-day exposure to the indicated treatments. Adiponectin could mainly impair the progression of UM cells into the G2/M phase and induce a slight but significant accumulation of the cells in the Sub G1 phase. The Mel-270 and OMM-2.5 cells also differed in their cell-cycle rates, with the former cell type apparently requiring more time for the G2/M phase, as demonstrated by the higher percentage of untreated Mel-270 cells in the G2/M phase and the lower cell density of this group compared with the untreated OMM-2.5 cells in panel ( a ). Data represent the mean ± SEM of n = 3 independent experiments. * P < 0.05 compared to the no FBS group of the corresponding cell as determined by the two-sided t -test. ( h ) ATP levels in the serum deprived cells treated with or without adiponectin for 8 to 10 hours as measured by a luminescence assay. The values of untreated cells were taken as 100%. Data represent the mean ± SEM of n = 3 independent experiments with triplicate wells for each group. * P < 0.05 compared to the no FBS group of the corresponding cell as determined by the two-sided t -test.

Article Snippet: Recombinant human adiponectin (Peprotech, Hamburg, Germany; 450-24) was reconstituted in sterile triple-distilled water.

Techniques: Immunostaining, Marker, Incubation, Staining, Western Blot, Silver Staining, Cell Cycle Assay, Luminescence Assay

Journal: PLoS ONE

Article Title: A Saccharomyces cerevisiae Assay System to Investigate Ligand/AdipoR1 Interactions That Lead to Cellular Signaling

doi: 10.1371/journal.pone.0065454

Figure Lengend Snippet: (A) The components of the AdipoR1 signaling pathway of mammalian cells are shown in black font and their S. cerevisiae homologs, if known are shown in red font. In mammalian cells, adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain and leucine zipper motif 1 (APPL1) interacts directly with AdipoR1. Interaction of adiponectin (ADPN) with AdipoR1 stimulates AdipoR1-APPL1 interaction. This results in release of Ca 2+ from the ER to the cytosol and also increases export of LKB1 kinase from the nucleus. Influx of extracellular Ca 2+ to the cytosol is also stimulated by adiponectin, although the mechanism by which this occurs remains to be clarified. Increase in cytosolic Ca 2+ concentration activates Ca 2+ /calmodulin-dependent protein kinase kinase (CaMKK) which in turn activates AMP activated protein kinase (AMPK) by phosphorylating its α subunit. However, phosphorylation of AMPK α subunit by the cytosol-localized kinase LKB1 is the major pathway for activation of AMPK. Adiponectin-AdipoR1 interaction also increases cellular ceramidase activity which in turn leads to phosphorylation of AMPKα subunit. The details of this pathway are not clear yet. Activation of AMPK is required for many of the anti-diabetic and anti-atherosclerotic effects of adiponectin. In vascular endothelial cells, interaction of AdipoR1 with adiponectin activates protein kinase A (PKA) which has the effect of lowering accumulation of reactive oxygen species (ROS) and thereby reducing inflammation. (B) Subunit structure of S. cerevisiae AMPK (ScAMPK). Like mammalian AMPK, ScAMPK is a trimer composed of an α, β, and γ subunit. The genes encoding the β subunit isoforms as well as the sole α and γ subunits are indicated. (C) Components of the IZH2 -mediated signaling pathways in S. cerevisiae are shown in red font. Interaction of IZH2 (homolog of AdipoRs) with osmotin (OSM) activates PKA via a RAS2-cAMP pathway. Overexpression of IZH2 , overexpression of AdipoR1 , treatment of S. cerevisiae cells expressing AdipoR1 with adiponectin and treatment of S. cerevisiae cells expressing various levels of IZH2 with thaumatin (THN, a homolog of osmotin) has been shown to activate PKA by increasing cellular ceramidase activity. Activation of PKA leads to decreased transcription from a stress responsive promoter element ( STRE ) and increased cellular ROS content. Activated PKA promotes export of ScAMPK from the nucleus which leads to decreased transcription from the ferroxidase (FET3 ) promoter. Activated PKA also represses FET3 transcription via the stress-responsive transcription factors MSN2/4. Mutational analyses show that genes encoding the APPL1-lke protein Sip3, the LKB1-like protein Sak1, the ScAMPK β subunit Sip1 and the ScAMPK γ subunit Snf4 are components of the pathway leading from IZH2 (or AdipoR1 ) to FET3 repression in S. cerevisiae .

Article Snippet: Unless specified otherwise, the pure recombinant Escherichia coli expressed N-terminal (His) 6 -tagged full length human adiponectin used in these studies was the gift of Dr. A. Sharkhuu (KAUST, Saudi Arabia) or Dr.

Techniques: Binding Assay, Concentration Assay, Phospho-proteomics, Activation Assay, Activity Assay, Protein-Protein interactions, Over Expression, Expressing

Journal: PLoS ONE

Article Title: A Saccharomyces cerevisiae Assay System to Investigate Ligand/AdipoR1 Interactions That Lead to Cellular Signaling

doi: 10.1371/journal.pone.0065454

Figure Lengend Snippet: Cells of strain BY4741carrying pESC-URA-CLuc-AdipoR1-APPL1-NLuc were grown for 16 h at 30°C in selective minimal medium at the indicated galactose concentrations, treated for 4 h at 30°C with the indicated test compounds and then assayed for Luc activity. (A) Imaging of Luc activity. (B) Quantitative measurement of Luc activity as a function of osmotin concentration. A representative image of relative Luc activity at the different osmotin concentrations is shown for each galactose concentration. Data represent the means ± SD from three experiments with triplicate samples. For each galactose concentration, significant differences by a Student’s t-test between osmotin treated samples and untreated control are indicated by asterisks. Symbols: PBS, 1/8 X PBS; f-ADPN, bacterially expressed full length adiponectin; BSA, bovine serum albumin, OSM, osmotin; g-ADPN, bacterially expressed globular adiponectin; **, p <0.01; ***, p <0.001.

Article Snippet: Unless specified otherwise, the pure recombinant Escherichia coli expressed N-terminal (His) 6 -tagged full length human adiponectin used in these studies was the gift of Dr. A. Sharkhuu (KAUST, Saudi Arabia) or Dr.

Techniques: Activity Assay, Imaging, Concentration Assay, Control

Journal: PLoS ONE

Article Title: A Saccharomyces cerevisiae Assay System to Investigate Ligand/AdipoR1 Interactions That Lead to Cellular Signaling

doi: 10.1371/journal.pone.0065454

Figure Lengend Snippet: (A) Cell lysates (100 µg protein) of strain BWG1-7a and an isogenic Δsnf1::Kan_MX line were fractionated by 10% SDS-PAGE. Shown are blots probed with anti-phospho-AMPK(Thr-172) antibody. The expected size of Snf1p is 72 kDa. (B, C) Cells (about 10 8 /mL) of strain BWG1-7a Δizh2::Kan_MX transformed with p426GPD (Vec), p426GPD- AdipoR1 (pAdipoR1) or p426GPD- AdipoR2 (pAdipoR2) were treated with indicated osmotin and adiponectin concentrations for 30 min at 30°C in YPD. Aliquots were withdrawn for viable counts determination before the cell lysates were prepared for analysis by10% SDS-PAGE. Shown in the top panels are blots probed first with phospho-AMPK(Thr-172) antibody, then stripped and probed with actin antibody (100 µg total protein per lane). Shown in the middle panels are relative band intensities in the depicted gels. ‘Relative band intensity’ was defined as the ratio of the intensity of phospho-Snf1p signal to actin signal for each lane when the value of this ratio for the corresponding untreated Vec sample was arbitrarily assigned the value 1.0. Shown in the bottom panels are viable counts in each sample at the end of the treatments. The experiments were performed twice with comparable results and the results of one experiment are shown.

Article Snippet: Unless specified otherwise, the pure recombinant Escherichia coli expressed N-terminal (His) 6 -tagged full length human adiponectin used in these studies was the gift of Dr. A. Sharkhuu (KAUST, Saudi Arabia) or Dr.

Techniques: SDS Page, Transformation Assay