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recombinant human adiponectin 450-24  (PeproTech)


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    PeproTech recombinant human adiponectin 450-24
    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 <t>adiponectin)</t> 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.
    Recombinant Human Adiponectin 450 24, supplied by PeproTech, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/recombinant+human+adiponectin+450-24/human+recombinant+adiponectin/pmc07405622-229-0-3
    Average 90 stars, based on 1 article reviews
    recombinant human adiponectin 450-24 - by Bioz Stars, 2026-10
    90/100 stars

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    1) Product Images from "Lower Levels of Adiponectin and Its Receptor Adipor1 in the Uveal Melanomas With Monosomy-3"

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

    Journal: Investigative Ophthalmology & Visual Science

    doi: 10.1167/iovs.61.5.12

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

    Techniques Used: Derivative Assay, Irradiation, Biomarker Discovery

    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.
    Figure Legend 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.

    Techniques Used: Expressing, Irradiation

    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.
    Figure Legend 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.

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

    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).
    Figure Legend 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).

    Techniques Used: Expressing, Staining, MANN-WHITNEY

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

    Techniques Used: Biomarker Discovery, Irradiation

    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.
    Figure Legend 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.

    Techniques Used: Expressing, Construct, Generated

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

    Techniques Used: Cell Culture, Staining, Standard Deviation

    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.
    Figure Legend 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.

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

    Related Articles

    Recombinant:

    Article Title: Lower Levels of Adiponectin and Its Receptor Adipor1 in the Uveal Melanomas With Monosomy-3
    Article Snippet: .. Recombinant human adiponectin (Peprotech, Hamburg, Germany; 450-24) was reconstituted in sterile triple-distilled water. ..

    Sterility:

    Article Title: Lower Levels of Adiponectin and Its Receptor Adipor1 in the Uveal Melanomas With Monosomy-3
    Article Snippet: .. Recombinant human adiponectin (Peprotech, Hamburg, Germany; 450-24) was reconstituted in sterile triple-distilled water. ..



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    recombinant human adiponectin 450-24  (PeproTech)
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    PeproTech recombinant human adiponectin 450-24
    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 <t>adiponectin)</t> 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.
    Recombinant Human Adiponectin 450 24, supplied by PeproTech, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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.

    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

    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.

    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

    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.

    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

    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.

    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

    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).

    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

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

    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

    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.

    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

    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.

    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

    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.

    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

    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.

    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