Journal: Open Access Rheumatology : Research and Reviews

Article Title: A suppressive effect of prostaglandin E 2 on the expression of SERPINE1 /plasminogen activator inhibitor-1 in human articular chondrocytes: An in vitro pilot study

doi:

Figure Lengend Snippet: Suppressive effect of PGE 2 on PAI-1 expression. A ) Western blot. Left: Chondrocytes were stimulated with PGE 2 (0.1, 1, or 10 nM) for 24 h, and the expression of PAI-1 was analyzed. NS: not stimulated. Representative results are shown. Bottom: Calculation of the relative intensity of the bands among four OA samples tested. B ) ELISA. Levels of PAI-1 in culture supernatants of chondrocytes after PGE 2 stimulation (0.1–10 nM) were measured. Notes: OA chondrocytes, N = 3, duplicate. Abbreviations: ELISA, enzyme-linked immunosorbent assay; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PAI-1, plasminogen activator inhibitor; PGE 2 , prostaglandin E 2 .

Article Snippet: The level of PAI-1 in the culture supernatant was measured by using an ELISA kit (AssayMax Human Plasminogen activator inhibitor-1 ELISA Kit™, AssayPro LLC, St. Charles, MO, USA).

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Open Access Rheumatology : Research and Reviews

Article Title: A suppressive effect of prostaglandin E 2 on the expression of SERPINE1 /plasminogen activator inhibitor-1 in human articular chondrocytes: An in vitro pilot study

doi:

Figure Lengend Snippet: The PAI-1 suppression by PGE 2 is delivered through EP4 receptor. The summary of PAI-1 ELISA with receptor antagonists are shown. Chondrocytes were stimulated with PGE 2 (10 nM) with or without AH6809 (10 ng/ml) or GW627368X (5 μM), and the levels of PAI-1 in culture supernatants were measured. Notes: OA chondrocytes, N = 6. Abbreviations: ELISA, enzyme-linked immunosorbent assay; PAI-1, plasminogen activator inhibitor; PGE 2 , prostaglandin E 2 .

Article Snippet: The level of PAI-1 in the culture supernatant was measured by using an ELISA kit (AssayMax Human Plasminogen activator inhibitor-1 ELISA Kit™, AssayPro LLC, St. Charles, MO, USA).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Open Access Rheumatology : Research and Reviews

Article Title: A suppressive effect of prostaglandin E 2 on the expression of SERPINE1 /plasminogen activator inhibitor-1 in human articular chondrocytes: An in vitro pilot study

doi:

Figure Lengend Snippet: Inhibition of p38 and ERK MAPK did not abrogate the effect of PGE 2 on PAI-1 downregulation. The summary of PAI-1 ELISA with receptor antagonists are shown. Chondrocytes were stimulated with PGE 2 (10 nM) with or without SB203580 or PD98059, and the levels of PAI-1 in culture supernatants were measured. Notes: OA chondrocytes, N = 3. *p <0.05; **p <0.02. Abbreviations: MAPK, mitogen-activated protein kinases, PAI-1, plasminogen activator inhibitor; PGE 2 , prostaglandin E 2 .

Article Snippet: The level of PAI-1 in the culture supernatant was measured by using an ELISA kit (AssayMax Human Plasminogen activator inhibitor-1 ELISA Kit™, AssayPro LLC, St. Charles, MO, USA).

Techniques: Inhibition, Enzyme-linked Immunosorbent Assay

Journal: Cancers

Article Title: Novel Function of Cancer Stem Cell Marker ALDH1A3 in Glioblastoma: Pro-Angiogenesis through Paracrine PAI-1 and IL-8.

doi: 10.3390/cancers15174422

Figure Lengend Snippet: Figure 1. Upregulation of ALDH1A3 in oxGBM cells is associated with the increased expression and release of pro-angiogenic factors PAI-1 and IL-8. GBM cell lines were transduced with ALDH1A3 for overexpression (oxGBM) or with empty vector (evGBM). (A) Confirmation of up-regulation of ALDH1A3 mRNA level in oxGBM cells by RT2-PCR. (B) Confirmation of up-regulation of ALDH1A3 protein expression by Western blot. wt, wild-type cells. The uncropped blots are shown in Figure S7; (C) Angiogenesis array. The blots showed duplicated dots for 55 angiogenesis-related proteins in the media of oxU373 or evU373. 10 of 55 proteins were upregulated more than 2-fold in ox group compared to ev group (indicated by rectangle). They are: (1) Ang-1, (2) artemin, (3) TF, (4) ET-1, (5) GM-CSF, (6) IL-8, (7) PDGF-AA, (8) PAI-1, (9) PEDF, and (10) uPA. (D) Semi-quantification of the dots representing PAI-1 and IL-8. (E) Immunofluorescence staining of GBM cells. U373 (left panel) and LN229 (right panel). Co-localization of ALDH1A3 with PAI-1 and IL-8 was observed in oxGBM cells, whereas no immunoreactivity of PAI-1 and IL-8 was detected in evGBM cells. (F) mRNA expression of PAI-1 and IL-8 in transduced U373 cells and the effect of inhibitors. Tiplaxtinin (Tip, 30 µM) and reparixin (Rep, 1 µM) are the specific inhibitors of PAI-1 and IL-8 receptors CXCR1/2, respectively. (G) Detection of PAI-1 and potential signaling proteins by Western blot. IOD: optical density. The uncropped blots are shown in Figure S8. **, p < 0.01; ***, p < 0.001, compared with ev. ##, p < 0.01; ###, p < 0.001, compared with ox.

Article Snippet: To determine the effects of GBM-derived PAI-1 and IL-8 on EC behavior, the following inhibitors were used: Tiplaxtinin (cat# HY-15253; MedChemExpress, Monmouth Junction, NJ, USA), a small-molecule inhibitor of PAI-1, and reparixin (cat# HY-15251; Med-ChemExpress), an allosteric inhibitor of the IL-8 receptors CXCR1 and CXCR2.

Techniques: Expressing, Transduction, Over Expression, Plasmid Preparation, Western Blot, Staining

Journal: Cancers

Article Title: Novel Function of Cancer Stem Cell Marker ALDH1A3 in Glioblastoma: Pro-Angiogenesis through Paracrine PAI-1 and IL-8.

doi: 10.3390/cancers15174422

Figure Lengend Snippet: Figure 2. Overexpression of ALDH1A3 in GBM cells activated endothelial angiogenesis in indirect co-culture with endothelial cells (ECs), which was reversed by treatment with respective inhibitors of PAI-1 or IL-8 receptors CXCR1/2. Indirect co-culture was performed by culture of HBMECs in a conditioned medium (CM) containing the media derived from evGBMs or oxGBMs and ECGM in a ratio of 1:1. PAI-1 inhibitor tiplaxtinin (Tip, 30 µM) and CXCR1/2 inhibitor reparixin (Rep, 1 µM) or vehicle DMSO (0.1%) was added to CM, followed by EC behavior study. All data were reproduced in three independent experiments. (A) Proliferation assay in HBMEC and HUVEC. Indirect co-culture of HBMECs and HUVECs with CM derived from oxU373 and oxLN229 stimulated EC proliferation, which was completely reversed by the treatment of tiplaxtinin, not by reparixin. (B) Scratch assay in HBMEC. Left panel: images were acquired 24 h after scratching. Scale bar: 200 µm. Right panel: quantitative analysis. Culture of HBMECs with CM derived from oxU373 or oxLN229 (oxCM) significantly promoted HBMEC migration, which was reversed by the treatment of tiplaxtinin and reparixin, respectively. (C) Transwell invasion assay in HBMEC. Left panel: Representative images of invaded cells were acquired after 24 h of incubation. Scale bar: 100 µm. Right panel: quantitative analysis. Culture of HBMECs with oxCM accelerated HBMEC invasion. This effect was significantly inhibited by the treatment of reparixin but not by tiplaxtinin. (D) Tube formation assay in HBMEC. Left panel: representative images of tube formation. Scale bar: 200 µm. Right panel: quantitative analysis of branching points per field. Tube formation in HBMECs was stimulated by incubation with oxCM, which was completely diminished by both inhibitors. (E) Sprouting assay in HBMEC. Left panel: representative images of sprouting in HBMECs after 24 h of co-culture. Scale bar: 100 µm. A pronounced increase in sprouting was observed in HBMECs cultured in oxCM. Tiplaxtinin and reparixin suppressed the sprouting effect resulting from oxCM. *, p < 0.05; **, p < 0.01 and ***, p < 0.001, compared with evCM. #, p < 0.05; ##, p < 0.01 and ###, p < 0.001, compared with oxCM.

Article Snippet: To determine the effects of GBM-derived PAI-1 and IL-8 on EC behavior, the following inhibitors were used: Tiplaxtinin (cat# HY-15253; MedChemExpress, Monmouth Junction, NJ, USA), a small-molecule inhibitor of PAI-1, and reparixin (cat# HY-15251; Med-ChemExpress), an allosteric inhibitor of the IL-8 receptors CXCR1 and CXCR2.

Techniques: Over Expression, Co-Culture Assay, Derivative Assay, Proliferation Assay, Wound Healing Assay, Migration, Transwell Invasion Assay, Incubation, Tube Formation Assay, Cell Culture

Journal: Cancers

Article Title: Novel Function of Cancer Stem Cell Marker ALDH1A3 in Glioblastoma: Pro-Angiogenesis through Paracrine PAI-1 and IL-8.

doi: 10.3390/cancers15174422

Figure Lengend Snippet: Figure 3. Overexpression of ALDH1A3 in GBM cells stimulated EC proliferation, migration, and sprouting in direct co-culture with endothelial cells (ECs), which was inhibited by treatment with the specific inhibitors of PAI-1 or IL-8 receptors CXCR1/2. Direct co-culture was performed by co- culture of CellTrace™CFSE pre-labeled HBMECs with transduced U373 and LN229 in a ratio of 1:1 in ECGM. To inhibit PAI-1, tiplaxtinin (Tip, 30 µM) was pretreated with GBM cells 6 h in advance. For IL-8 receptor inhibition, reparixin (Rep, 1 µM) was pretreated with ECs 30 min in advance and added to ECGM in the co-culture, as well. All data were reproduced in three independent experiments. (A) Representative images of directly co-cultured CellTrace™CFSE-labeled HBMEC (green) with transduced U373 and LN229. Scale bar: 100 µm. (B) HBMEC proliferation. Direct co-culture of oxGBMs with HBMECs promoted the proliferation of HBMEC, which was completely reversed by the treatment of tiplaxtinin and reparixin. The fluorescence intensity of CFSE-labeled HBMECs, reflecting cell proliferation, was detected 48 h after direct co-culture at 485 nm of excitation wavelength and 535 nm of emission wavelength. (C) HBMEC migration. The images were acquired after 12 h of direct

Article Snippet: To determine the effects of GBM-derived PAI-1 and IL-8 on EC behavior, the following inhibitors were used: Tiplaxtinin (cat# HY-15253; MedChemExpress, Monmouth Junction, NJ, USA), a small-molecule inhibitor of PAI-1, and reparixin (cat# HY-15251; Med-ChemExpress), an allosteric inhibitor of the IL-8 receptors CXCR1 and CXCR2.

Techniques: Over Expression, Migration, Co-Culture Assay, Labeling, Inhibition, Cell Culture

Journal: Cancers

Article Title: Novel Function of Cancer Stem Cell Marker ALDH1A3 in Glioblastoma: Pro-Angiogenesis through Paracrine PAI-1 and IL-8.

doi: 10.3390/cancers15174422

Figure Lengend Snippet: Figure 5. Immunohistochemistry and immunofluorescence staining on GBM sections. (A,B) Im- munohistochemistry staining of ALDH1A3. The immunoreactivity of ALDH1A3 was detected in vessels (arrows in (A)) and peripheral cells of glomeruloid (arrowheads in (B)) as well as in tumor cells (asterisks in (A)). Scale bar: 50 µm in (A) and 20 µm in (B). (C–E) Double-immunofluorescence staining of ALDH1A3 (red) with PAI-1 (green) (C,D) or with IL-8 (green) (E,F). The co-localization of ALDH1A3 with PAI-1 or with IL-8 immunoreactivity was detected in microvessels (arrows), glomeruloid (arrowheads), and tumor cells (asterisks) in GBM sections.

Article Snippet: To determine the effects of GBM-derived PAI-1 and IL-8 on EC behavior, the following inhibitors were used: Tiplaxtinin (cat# HY-15253; MedChemExpress, Monmouth Junction, NJ, USA), a small-molecule inhibitor of PAI-1, and reparixin (cat# HY-15251; Med-ChemExpress), an allosteric inhibitor of the IL-8 receptors CXCR1 and CXCR2.

Techniques: Immunohistochemistry, Staining

Journal: Cancers

Article Title: Novel Function of Cancer Stem Cell Marker ALDH1A3 in Glioblastoma: Pro-Angiogenesis through Paracrine PAI-1 and IL-8.

doi: 10.3390/cancers15174422

Figure Lengend Snippet: Figure 6. Schematic illustration of the pro-angiogenic function of ALDH1A3 via paracrine PAI-1 and

Article Snippet: To determine the effects of GBM-derived PAI-1 and IL-8 on EC behavior, the following inhibitors were used: Tiplaxtinin (cat# HY-15253; MedChemExpress, Monmouth Junction, NJ, USA), a small-molecule inhibitor of PAI-1, and reparixin (cat# HY-15251; Med-ChemExpress), an allosteric inhibitor of the IL-8 receptors CXCR1 and CXCR2.

Techniques:

Journal: JCI insight

Article Title: Transcriptional heterogeneity of fibroblasts is a hallmark of the aging heart.

doi: 10.1172/jci.insight.131092

Figure Lengend Snippet: Figure 6. Age-dependent changes of fibroblast function on endothelial cells. (A and B) Experimental outline and tube formation assay of human umbilical vein endothelial cells (HUVECs) that were cultured in conditioned medium received from young (12 weeks) and aged (20 months) cardiac mouse fibroblasts. Accumulated tube length was measured in 5 randomly chosen microscopic fields with a computer-assisted microscope using Axiovision 4.5 (Zeiss) (scale bar: 100 μm; n = 4). (C) Capillary density of random healthy areas of young (12 weeks; n = 6) and old (n = 8) heart sections versus capillary density of fibrotic (n = 8) areas of aged hearts (20 months). Shown is the quantification of the Isolectin B4–positive area versus the total area (%). (D) Expression of Serpine1, Serpine2, and Efemp1 in fibroblasts displayed in log scale in trajectory plots. (E) Secretion of SerpinE1 in isolated young and aged cardiac fibroblasts cultured for 24 hours in serum-free medium. Data were derived using a mouse angiogenesis array (R&D Systems) of culture supernatants of isolated cardiac fibroblasts (n = 4). (F) Tube formation assay of HUVECs that were cultured in conditioned medium received from young (12 weeks) and aged (20 months) cardiac mouse fibroblasts. The aged phenotype was rescued by supplementing the aged fibroblast medium with 4 μg of an anti-Serpin antibody. Accumulated tube length was mea- sured in 5 randomly chosen microscopic fields with a computer-assisted microscope using Axiovision 4.5 (Zeiss) (scale bar: 200 μm; n = 4). (G) RNA expression

Article Snippet: For the recombinant serpin study, HUVECs were cultured in medium supplemented with 10 ng/mL serpin E1 (CSB-EP021081MO, Cusabio) or serpin E2 (2175-PI-010, R&D Systems) for 24 hours.

Techniques: Tube Formation Assay, Cell Culture, Microscopy, Expressing, Isolation, Derivative Assay, RNA Expression