Journal: Frontiers in Endocrinology

Article Title: Thrombospondin 1 (THBS1) Promotes Follicular Angiogenesis, Luteinization, and Ovulation in Primates

doi: 10.3389/fendo.2019.00727

Figure Lengend Snippet: Thrombospondin production by the ovulatory follicle. Granulosa cells were aspirated from monkey ovarian follicles after ovarian stimulation before (0), 12, 24, or 36 h after administration of an ovulatory dose of hCG; additional monkeys received hCG and the PTGS2 inhibitor celecoxib (36+C) 36 h before follicle aspiration. Granulosa cells were assessed by qPCR for mRNA levels of THBS1 (A) , THBS2 (B) , and THBS4 (C) . All THBS mRNA levels are expressed relative to BACT . Granulosa cell lysates were assessed for THBS1 (D,E) and THBS4 (D,F) by western blotting and are expressed relative to pan-actin (D) . For (A–C,E,F) , data are expressed as mean + SEM, n = 3–7 samples/treatment. Within each panel, groups with no common letters are different by ANOVA and Duncans post hoc test, p < 0.05.

Article Snippet: On the next day, intrafollicular injection of an antibody against THBS1 (R&D Systems, Minneapolis, MN; AF3074; n = 4) or control IgG antibody (Abbiotec, San Diego, CA; n = 4) was performed during aseptic surgery; an estimated 10 μg of antibody protein was delivered to each follicle at injection ( ).

Techniques: Western Blot

Journal: Frontiers in Endocrinology

Article Title: Thrombospondin 1 (THBS1) Promotes Follicular Angiogenesis, Luteinization, and Ovulation in Primates

doi: 10.3389/fendo.2019.00727

Figure Lengend Snippet: THBS1 and THBS4 immunodetection in monkey ovarian follicles. Immunocytochemical detection (brown) of THBS1 (A–G) and THBS4 (H–N) was localized to the granulosa cell layer of ovulatory follicles obtained before (0; A,H) and 12 (B,I) , 24 (C,J) , and 36 (D,K) hours (h) after hCG administration as well as 36 h after administration of hCG and celecoxib (36+C h; E,L ). Images shown are representative of n-3-6 ovaries/treatment. Granulosa cell immunodetection of THBS1 (F) and THBS4 (M) was reduced after preabsorption of the primary antibody with recombinant human THBS1 or THBS4 and similar to staining observed with no primary antibody (G,N) . Nuclei are counterstained blue. All panels are oriented as shown in (A) , with stroma (st) in lower left, granulosa cell (gc, arrow) layer central, and follicle antrum (an) in upper right. Arrowheads indicate stromal staining with antibody against THBS1 (A,E) , THBS4 (J) , and no primary antibody (G,N) . Images in (A–N) are at the same magnification; bar in (A) is 50 μm. Lower magnification images shows immunocytochemical detection of THBS1 (O) and no primary antibody (P) stained sections of an ovary obtained after ovarian stimulation and 36 h hCG. Staining is apparent in stromal vessels ( O , arrows). Non-specific staining is indicated near the granulosa cell basement membrane and in vessel lumens in (O,P) (arrowheads); bar in (A) is 100 μm for images in (O,P) .

Article Snippet: On the next day, intrafollicular injection of an antibody against THBS1 (R&D Systems, Minneapolis, MN; AF3074; n = 4) or control IgG antibody (Abbiotec, San Diego, CA; n = 4) was performed during aseptic surgery; an estimated 10 μg of antibody protein was delivered to each follicle at injection ( ).

Techniques: Immunodetection, Recombinant, Staining, Membrane

Journal: Frontiers in Endocrinology

Article Title: Thrombospondin 1 (THBS1) Promotes Follicular Angiogenesis, Luteinization, and Ovulation in Primates

doi: 10.3389/fendo.2019.00727

Figure Lengend Snippet: THBS1 is pro-angiogenic in vitro . Monkey ovarian microvascular endothelial cells (mOMECs) were treated with human THBS1 protein at concentrations of 0.001–1 nM or no THBS1 (0 nM) as indicated. (A–C) Migration was assessed 24 h after plating on a porous membrane and treatment with THBS1. mOMECs (arrow) which migrated through pores (arrowhead) were stained with hematoxylin and eosin, photographed, and counted. Representative membranes from 0 nM (B) and 1 nM (C) treatment groups are shown. (D–F) Proliferation was assessed by Ki67 immunodetection in mOMECs cultured for 24 h with THBS1. Ki67 positive (arrows) and negative (arrowhead) cells are indicated in representative images from mOMECs treated with 0 nM (E) and 1 nM (F) THBS1. Data are expressed as a percentage of Ki67 positive cells among all cells counted. (G–M) Sprout formation in response to THBS1 treatment was determined after 1 day (G,I) and 2 days (H,J,L,M) in vitro . mOMECs coating a polymer bead in vitro before THBS1 treatment (Day 0; K ) shows absence of sprouts. Arrows indicate representative sprouts on Day 2 of treatment with no THBS1 (L) and 0.1 nM THBS1 (M) . Images were quantified for both the number of sprouts (sprouts/bead; G,H ) and sprout length in μm (H,J) . For (A,D,G–J) , data are expressed as mean + SEM, n = 3–5 samples/treatment. Within each panel, groups with no common letters are different by ANOVA and Duncans post hoc test, p < 0.05.

Article Snippet: On the next day, intrafollicular injection of an antibody against THBS1 (R&D Systems, Minneapolis, MN; AF3074; n = 4) or control IgG antibody (Abbiotec, San Diego, CA; n = 4) was performed during aseptic surgery; an estimated 10 μg of antibody protein was delivered to each follicle at injection ( ).

Techniques: In Vitro, Migration, Membrane, Staining, Immunodetection, Cell Culture, Polymer

Journal: Frontiers in Endocrinology

Article Title: Thrombospondin 1 (THBS1) Promotes Follicular Angiogenesis, Luteinization, and Ovulation in Primates

doi: 10.3389/fendo.2019.00727

Figure Lengend Snippet: Confirmatory experiments for the intrafollicular antibody injections. Serum levels of estradiol (A) and progesterone (B) were not different between animals receiving intrafollicular injection of the THBS1 antibody and animals receiving control IgG. For each day, hormone levels were compared by unpaired t -test. Data are expressed as mean + SEM, n = 4/group. (C) THBS1 antibody reduces THBS1 (1 nM)-stimulated mOMEC migration in vitro . Within each panel, groups with no common letters are different by ANOVA with 1 repeated measure and Duncans post hoc test, p < 0.05. Data are expressed as mean + SEM, n = 3/group.

Article Snippet: On the next day, intrafollicular injection of an antibody against THBS1 (R&D Systems, Minneapolis, MN; AF3074; n = 4) or control IgG antibody (Abbiotec, San Diego, CA; n = 4) was performed during aseptic surgery; an estimated 10 μg of antibody protein was delivered to each follicle at injection ( ).

Techniques: Injection, Control, Migration, In Vitro

Journal: Frontiers in Endocrinology

Article Title: Thrombospondin 1 (THBS1) Promotes Follicular Angiogenesis, Luteinization, and Ovulation in Primates

doi: 10.3389/fendo.2019.00727

Figure Lengend Snippet: Follicle rupture and oocyte release are compromised after intrafollicular injection with an antibody against THBS1. Ovarian surface at the time of ovary removal after intrafollicular injection of control IgG (A) or THBS1 antibody (B–D) . Oocytes were located within a THBS1 antibody-injected follicle (E) and on the surface of a THBS1 antibody-injected follicle (F) . Rupture site after intrafollicular injection of control IgG (G) . Rupture sites in follicles injected with the THBS1 antibody were either small (H) or absent (I) . Tissues shown in (E–I) were stained with hematoxylin and eosin. Oocytes (E,F) shown at approximately maximal diameter. Images in (G–I) are at the same magnification; bar in (I) = 0.5 mm. (J) Rupture site area after intrafollicular injection with control IgG (IgG) or THBS1 antibody. Groups are different by two-tailed unpaired t -test as indicated by * p < 0.05. Data are expressed as mean + SEM, n = 4/group.

Article Snippet: On the next day, intrafollicular injection of an antibody against THBS1 (R&D Systems, Minneapolis, MN; AF3074; n = 4) or control IgG antibody (Abbiotec, San Diego, CA; n = 4) was performed during aseptic surgery; an estimated 10 μg of antibody protein was delivered to each follicle at injection ( ).

Techniques: Injection, Control, Staining, Two Tailed Test

Journal: Frontiers in Endocrinology

Article Title: Thrombospondin 1 (THBS1) Promotes Follicular Angiogenesis, Luteinization, and Ovulation in Primates

doi: 10.3389/fendo.2019.00727

Figure Lengend Snippet: Oocyte retention and follicle rupture.

Article Snippet: On the next day, intrafollicular injection of an antibody against THBS1 (R&D Systems, Minneapolis, MN; AF3074; n = 4) or control IgG antibody (Abbiotec, San Diego, CA; n = 4) was performed during aseptic surgery; an estimated 10 μg of antibody protein was delivered to each follicle at injection ( ).

Techniques:

Journal: Frontiers in Endocrinology

Article Title: Thrombospondin 1 (THBS1) Promotes Follicular Angiogenesis, Luteinization, and Ovulation in Primates

doi: 10.3389/fendo.2019.00727

Figure Lengend Snippet: Angiogenesis and luteinization are compromised after intrafollicular injection with an antibody against THBS1. Histological sections of ovaries shown in were immunostained for VWF (brown) to assess angiogenesis; hematoxylin counterstain. (A,B) Endothelial cell invasion into the granulosa cell layer of a control IgG-injected follicle (A) and THBS1 antibody-injected follicle (B) shows VWF+ cells within the granulosa cell (gc) layer. Arrows indicate VWF+ cells which have invaded furthest from the stroma into the granulosa cell layer. Single arrowheads indicate stromal vessels. Double arrowheads indicate capillary luminal spaces with red blood cells in the control-IgG injected follicle only. (C) shows representative measurements of granulosa cell layer thickness (brown lines) and endothelial cell invasion (green lines). (A–C) are oriented as shown in (A) , with stroma (st) at bottom, granulosa cell (gc) layer central, and follicle antrum (an) at top. Images in (A–C) are at the same magnification; bar in (B) is 100 μm. Granulosa cell layer thickness (D) , endothelial cell invasion (E) , and the percent of the granulosa cell layer penetrated by endothelial cells (F) after intrafollicular injection with control IgG (IgG) or THBS1 antibody. Groups are different by two-tailed unpaired t -test as indicated by * p < 0.05. Data are expressed as mean + SEM, n = 4/group. (H,J) 3D modeling of endothelial cells (white on black background) is shown alongside (G,I) representative VWF immunostained ovarian sections after intrafollicular injection with control IgG (G,H) or THBS1 antibody (I,J) . Green arrows indicate stromal vessels, green arrowheads indicate capillary-like structures that connect to a stromal vessel, and yellow arrowheads indicate endothelial cells that lack connect to a stromal vessel. (G–J) are oriented with antrum at the top, granulosa cells central, and stroma at the bottom of each image/model; (G,I) at the same magnification.

Article Snippet: On the next day, intrafollicular injection of an antibody against THBS1 (R&D Systems, Minneapolis, MN; AF3074; n = 4) or control IgG antibody (Abbiotec, San Diego, CA; n = 4) was performed during aseptic surgery; an estimated 10 μg of antibody protein was delivered to each follicle at injection ( ).

Techniques: Injection, Control, Two Tailed Test

Journal: PLoS ONE

Article Title: The clinicopathological significance of Thrombospondin-4 expression in the tumor microenvironment of gastric cancer

doi: 10.1371/journal.pone.0224727

Figure Lengend Snippet: THBS4 was stained at the cytoplasm and cell membrane of stroma cells. The stromal cells with THBS4 expression were much to the cells showing αSMA or Podoplanin expression. cytokeratin was stained in cancer cells.

Article Snippet: After blocking endogenous peroxidase activity, the samples were incubated with anti-human THBS4 (R&D Systems, Minneapolis, MN: MAB2390; 1:200), anti-Pan-Keratin (Proteintech, Rosemont, IL: 26411-1-AP; 1:3000), anti-Podoplanin (Santa Cruz Biotechnology, Dallas, TX: sc-376695; 1:300) or anti-human αSMA (Leica Biosystems Newcastle, Newcastle, UK: NCL-L-SMA; 1:200).

Techniques: Staining, Membrane, Expressing

Journal: PLoS ONE

Article Title: The clinicopathological significance of Thrombospondin-4 expression in the tumor microenvironment of gastric cancer

doi: 10.1371/journal.pone.0224727

Figure Lengend Snippet: Correlation between the expression of THBS4 in tumor stromal cells and clinicopathologic features in 584 patients with gastric carcinoma.

Article Snippet: After blocking endogenous peroxidase activity, the samples were incubated with anti-human THBS4 (R&D Systems, Minneapolis, MN: MAB2390; 1:200), anti-Pan-Keratin (Proteintech, Rosemont, IL: 26411-1-AP; 1:3000), anti-Podoplanin (Santa Cruz Biotechnology, Dallas, TX: sc-376695; 1:300) or anti-human αSMA (Leica Biosystems Newcastle, Newcastle, UK: NCL-L-SMA; 1:200).

Techniques: Expressing

Journal: PLoS ONE

Article Title: The clinicopathological significance of Thrombospondin-4 expression in the tumor microenvironment of gastric cancer

doi: 10.1371/journal.pone.0224727

Figure Lengend Snippet: (A) The Kaplan-Meier survival curve indicates that the OS of all patients with high THBS4 expression in stromal cells was significantly worse than that of the patients with low THBS4 expression (p<0.001). (B–E) The Kaplan-Meier survival curve for each stage. The 5-year OS rates of the Stage I patients with high THBS4 expression were poorer than those of the Stage I patients with low THBS4 expression (p<0.001), and the same was true of the Stage III patients (p = 0.001).

Article Snippet: After blocking endogenous peroxidase activity, the samples were incubated with anti-human THBS4 (R&D Systems, Minneapolis, MN: MAB2390; 1:200), anti-Pan-Keratin (Proteintech, Rosemont, IL: 26411-1-AP; 1:3000), anti-Podoplanin (Santa Cruz Biotechnology, Dallas, TX: sc-376695; 1:300) or anti-human αSMA (Leica Biosystems Newcastle, Newcastle, UK: NCL-L-SMA; 1:200).

Techniques: Expressing

Journal: PLoS ONE

Article Title: The clinicopathological significance of Thrombospondin-4 expression in the tumor microenvironment of gastric cancer

doi: 10.1371/journal.pone.0224727

Figure Lengend Snippet: Univariate and multivariate Cox multiple regression analysis with respect to overall survival after surgery in 584 patients with gastric carcinoma.

Article Snippet: After blocking endogenous peroxidase activity, the samples were incubated with anti-human THBS4 (R&D Systems, Minneapolis, MN: MAB2390; 1:200), anti-Pan-Keratin (Proteintech, Rosemont, IL: 26411-1-AP; 1:3000), anti-Podoplanin (Santa Cruz Biotechnology, Dallas, TX: sc-376695; 1:300) or anti-human αSMA (Leica Biosystems Newcastle, Newcastle, UK: NCL-L-SMA; 1:200).

Techniques:

Journal: PLoS ONE

Article Title: The clinicopathological significance of Thrombospondin-4 expression in the tumor microenvironment of gastric cancer

doi: 10.1371/journal.pone.0224727

Figure Lengend Snippet: (A), Representative picture of Podoplanin staining. Podoplanin was mainly stained at the cytoplasm fibroblasts. The expression level of Podoplanin was higher in CAFs, in compared with that in NFs. (B), THBS4 expression. CAFs expressed THBS4, but NFs and cancer cells did not.

Article Snippet: After blocking endogenous peroxidase activity, the samples were incubated with anti-human THBS4 (R&D Systems, Minneapolis, MN: MAB2390; 1:200), anti-Pan-Keratin (Proteintech, Rosemont, IL: 26411-1-AP; 1:3000), anti-Podoplanin (Santa Cruz Biotechnology, Dallas, TX: sc-376695; 1:300) or anti-human αSMA (Leica Biosystems Newcastle, Newcastle, UK: NCL-L-SMA; 1:200).

Techniques: Staining, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Elevated Levels of Active GSK3β in the Blood of Patients with Myotonic Dystrophy Type 1 Correlate with Muscle Weakness

doi: 10.3390/ijms262110760

Figure Lengend Snippet: Levels of active GSK3β are increased in PBMCs from patients with adult-onset DM1 relative to unaffected control patients. ( A ) The scheme shows the preparation of PBMC proteins for Western blot analyses. The whole blood samples were separated by centrifugation in the gradient of Biocoll. The buffy coats were collected, and PBMCs were spun down. The cell pellets were resuspended in SDS-loading buffer, boiled for 30 min, and analyzed by Western blot assay as described in the Methods section. ( B ) Western blot analyses of proteins from PBMCs from control and adult-onset DM1 patients. Control samples 1–3 are familial controls not affected by DM1 (a 56-year-old female, a 49-year-old male and 55-year-old male, respectively). Control samples 4–7 are not familial participants (a 59-year-old female, a 35-year-old female, a 66-year-old male and 28-year-old female). The labels for DM1 samples include the length of CTG repeats. The DM1–68, DM1–74, DM1–100, DM1–131, DM1–163, DM1–>200 and DM1–~500 are samples from participants with adult-onset DM1 (females of 48, 43, 59, 31, 40, 29 and 40 years, respectively). Coomassie blue staining of the proteins extracted from PBMCs is shown on the top. Active GSK3β, total GSK3β and THBS1 were measured. Ph to tot: numbers below the total GSK3β image represent the ratios of the active GSK3β signals to total GSK3β in the same DM1 blood samples. ( C ) Bar graphs of the active GSK3β signals shown in ( B ) relative to Coomassie signals. Standard deviations (SDs) calculated from three repeats are shown. ( D ) Weighted regression results of active GSK3β levels on CTG repeat number, using data from 1B. For each DM1 subject, the dependent variable is the mean active GSK3β, and the weight is the inverse of the variance. Predicted GSK3β is shown with shaded 95% confidence bands. Standard deviation bars are calculated from three replicates. Colors denote the patients. ( E ) Western blot analysis of additional unrelated control blood samples vs. DM1 samples with antibodies to active GSK3β. C8, C10, C11, C13 and C14 are females of 41, 28, 38 and 52 years of age, respectively. C9 and C12 are males of 69 and 62 years. DM1 samples from patients with 163 and >200 CTG repeats analyzed in ( B ) are used as positive controls. Coomassie staining of the proteins extracted from PBMCs is shown on the bottom. ( F ) Using the same approach applied to GSK3β, a weighted regression was performed with THBS1 levels from 1B as the dependent variable and CTG repeat number as a predictor. For control samples, the maximum limit of 50 CTG repeats was applied because the exact number of repeats in these unaffected controls is not known. Bars indicate THBS1 standard deviations from three repeats. Predicted THBS1 is shown with shaded 95% confidence bands.

Article Snippet: To examine GSK3β, THBS1, TGFβ and hnRNP-A3 in the human PBMCs; 10 μg of total proteins from PBMCs were separated by the 4–20% polyacrylamide gel electrophoresis; proteins were transferred onto a nitro cellulose membrane and probed with primary antibodies to phospho-GSK3β-Y216 (EpigenTek, New York, NY, USA), total GSK3β and β-actin (Santa Cruz Biotechnology Inc., Dallas, TX, USA), THBS1 (Proteintech Group, Inc., Rosemont, IL, USA), TGFβ (MilliporeSigma, Burlington, VT, USA) and hnRNP-A3 (ThermoFisher, Dallas, TX, USA) according to the manufactures’ suggestions overnight.

Techniques: Control, Western Blot, Centrifugation, Staining, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Elevated Levels of Active GSK3β in the Blood of Patients with Myotonic Dystrophy Type 1 Correlate with Muscle Weakness

doi: 10.3390/ijms262110760

Figure Lengend Snippet: Correlation of the average levels of active GSK3β in blood with the muscle performance of DM1 patients with different severity of muscle disease. ( A ) Comparison of the average GSK3β levels in PBMCs from adult-onset DM1 patients with 68–100 CTG repeats without myotonia (n = 3) and in patients with 131–500 CTG repeats with myotonia and muscle weakness (n = 4). Western blot data and signal intensity analyses (using Image J2) are shown in B,C. A group of DM1 patients with mild muscle disease includes DM1 patients: DM1–68, DM1–74 and DM–100. A group of DM1 patients with moderate muscle weakness includes DM1–131, DM1–163, DM1 > 200 and DM1 ~ 500. The values of grip strength ( B , C ) and ankle strength ( D , E ) were compared in adult-onset DM1 patients with mild (n = 3) and moderate (n = 4) skeletal muscle weakness. The skeletal muscle outcomes for each patient with DM1 are shown in A,B. ( F ) Comparison of the blood levels of THBS1 in adult-onset DM1 patients with mild and moderate skeletal muscle disease. Western blot analyses of THBS1 in DM1 blood samples are shown in B. Standard deviations and the number of DM1 patients is shown. * and ** are p values < 0.05 and <0.01 for the DM1 patients with mild vs. moderate muscle weakness.

Article Snippet: To examine GSK3β, THBS1, TGFβ and hnRNP-A3 in the human PBMCs; 10 μg of total proteins from PBMCs were separated by the 4–20% polyacrylamide gel electrophoresis; proteins were transferred onto a nitro cellulose membrane and probed with primary antibodies to phospho-GSK3β-Y216 (EpigenTek, New York, NY, USA), total GSK3β and β-actin (Santa Cruz Biotechnology Inc., Dallas, TX, USA), THBS1 (Proteintech Group, Inc., Rosemont, IL, USA), TGFβ (MilliporeSigma, Burlington, VT, USA) and hnRNP-A3 (ThermoFisher, Dallas, TX, USA) according to the manufactures’ suggestions overnight.

Techniques: Comparison, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Elevated Levels of Active GSK3β in the Blood of Patients with Myotonic Dystrophy Type 1 Correlate with Muscle Weakness

doi: 10.3390/ijms262110760

Figure Lengend Snippet: The levels of active GSK3β are increased in PBMCs of patients with juvenile and congenital DM1 correlating with muscle weakness. ( A ) Western blot analyses of the active GSK3β and THBS1 in PBMCs from the familial control patients (C1 and C2 are a 56-year-old female and a 49-year-old male, respectively), patients with JDM1 (17- and 19-year-old females) and CDM1 (CDM1-1–4 are an 18-year-old female, a 16-year-old male, a 5-year-old female and a 5-month-old female, respectively). Coomassie staining of the PBMC proteins is shown below the Western blot images. ( B ) The average active GSK3β levels in the PBMCs from control, JDM1 and CDM1 patients were adjusted to the Coomassie-stained proteins shown in the box in ( A ). * and *** are p values < 0.05 and <0.001 for the average GSK3β signal in JDM1 compared to control and for the average GSK3β in CDM1 patients vs. control. ( C ) Comparison of the mean length of CTG repeat expansions in JDM1 and CDM1 patients. ** is p value < 0.01 for JDM1 vs. CDM1. ( D ) Western blot analysis of active GSK3β in pediatric and adolescent control blood samples and CDM1 samples. Coomassie staining is shown on the bottom. The average values of ankle dorsiflexion ( E , F ), grip strength ( G , H ) and manual strength ( I , J ) in patients with JDM1 and CDM1 are shown. The number of patients per group is also shown. * indicates a p value < 0.05 for JDM1 vs. CDM1. While GSK3β levels were evaluated in blood samples from four patients with CDM1, the muscle performance was analyzed in two patients with CDM1 aged 16 and 18. No muscle performance data were collected in two CDM1 patients due to early age (0.5 and 5 years).

Article Snippet: To examine GSK3β, THBS1, TGFβ and hnRNP-A3 in the human PBMCs; 10 μg of total proteins from PBMCs were separated by the 4–20% polyacrylamide gel electrophoresis; proteins were transferred onto a nitro cellulose membrane and probed with primary antibodies to phospho-GSK3β-Y216 (EpigenTek, New York, NY, USA), total GSK3β and β-actin (Santa Cruz Biotechnology Inc., Dallas, TX, USA), THBS1 (Proteintech Group, Inc., Rosemont, IL, USA), TGFβ (MilliporeSigma, Burlington, VT, USA) and hnRNP-A3 (ThermoFisher, Dallas, TX, USA) according to the manufactures’ suggestions overnight.

Techniques: Western Blot, Control, Staining, Comparison