Journal: World Journal of Gastrointestinal Oncology

Article Title: MiRNA-145-5p inhibits gastric cancer progression via the serpin family E member 1- extracellular signal-regulated kinase-1/2 axis

doi: 10.4251/wjgo.v16.i5.2123

Figure Lengend Snippet: Primer sequences used in this study

Article Snippet: The proteins were then boiled at 100°C for 10 min with 100 μL of loading buffer (Beyotime Biotechnology, China) and separated by SDS-PAGE at 120 V. After electrophoresis, the proteins were transferred onto PVDF membranes and blocked using 5% BSA/TBST for 60 min. the primary rabbit polyclonal antibodies, SERPINE1 (Proteintech, United States), α-smooth muscle actin (α-SMA) (Proteintech, United States), β-catenin (Proteintech, United States), vimentin (HuaBio, China), ERK1/2 (HuaBio, China), phosphorylation extracellular signal-regulated kinase1/2 (p-ERK1/2) (HuaBio, China) and actin (ZSGB-BIO, China), and the mouse monoclonal antibody E-cad (HuaBio, China), were added to the membranes and incubated overnight at 4 °C.

Techniques:

Journal: World Journal of Gastrointestinal Oncology

Article Title: MiRNA-145-5p inhibits gastric cancer progression via the serpin family E member 1- extracellular signal-regulated kinase-1/2 axis

doi: 10.4251/wjgo.v16.i5.2123

Figure Lengend Snippet: Serpin family E member 1 is a target of microRNA-145-5p. A: miRNA-145-5p and serpin family E member 1 ( SERPINE1 ) 3'- UTR binding sites were predicted using TargetScan; B: WT-SERPINE1 and MUT-SERPINE1 sequences; C: Relative luciferase activity of 293T cells transfected with either WT-SERPINE1 or MUT-SERPINE1 . c P < 0.001.

Article Snippet: The proteins were then boiled at 100°C for 10 min with 100 μL of loading buffer (Beyotime Biotechnology, China) and separated by SDS-PAGE at 120 V. After electrophoresis, the proteins were transferred onto PVDF membranes and blocked using 5% BSA/TBST for 60 min. the primary rabbit polyclonal antibodies, SERPINE1 (Proteintech, United States), α-smooth muscle actin (α-SMA) (Proteintech, United States), β-catenin (Proteintech, United States), vimentin (HuaBio, China), ERK1/2 (HuaBio, China), phosphorylation extracellular signal-regulated kinase1/2 (p-ERK1/2) (HuaBio, China) and actin (ZSGB-BIO, China), and the mouse monoclonal antibody E-cad (HuaBio, China), were added to the membranes and incubated overnight at 4 °C.

Techniques: Binding Assay, Luciferase, Activity Assay, Transfection

Journal: World Journal of Gastrointestinal Oncology

Article Title: MiRNA-145-5p inhibits gastric cancer progression via the serpin family E member 1- extracellular signal-regulated kinase-1/2 axis

doi: 10.4251/wjgo.v16.i5.2123

Figure Lengend Snippet: Serpin family E member 1 is a potential target gene of microRNA-145-5p and is highly expressed in gastric cancer tissues and cells. A: A Venn diagram of overlapping mRNAs from the differentially expressed mRNAs in The Cancer Genome Atlas (TCGA)-STAD data and miRNA-145-5p ( miR-145-5p ) target genes predicted from the two bioinformatics databases; B: Box diagram showing the differential expression of serpin family E member 1 ( SERPINE1 ) in the normal and tumor data in TCGA-STAD; C: Overall survival curves of patients in the high (red) and low (blue) SERPINE1 expression groups; D: Differential expression of SERPINE1 mRNA (left panel) and protein (middle panel) in gastric cancer (GC) tissue and SERPINE1 mRNA (right panel) in the HGC27 and SGC7901 GC cell lines; scale bar = 20 μm; E: SERPINE1 mRNA and protein expression in the miR-145-5p mimic group in SGC7901 cells and the inhibitor group in HGC27 cells. b P < 0.01; c P < 0.001; d P < 0.0001.

Article Snippet: The proteins were then boiled at 100°C for 10 min with 100 μL of loading buffer (Beyotime Biotechnology, China) and separated by SDS-PAGE at 120 V. After electrophoresis, the proteins were transferred onto PVDF membranes and blocked using 5% BSA/TBST for 60 min. the primary rabbit polyclonal antibodies, SERPINE1 (Proteintech, United States), α-smooth muscle actin (α-SMA) (Proteintech, United States), β-catenin (Proteintech, United States), vimentin (HuaBio, China), ERK1/2 (HuaBio, China), phosphorylation extracellular signal-regulated kinase1/2 (p-ERK1/2) (HuaBio, China) and actin (ZSGB-BIO, China), and the mouse monoclonal antibody E-cad (HuaBio, China), were added to the membranes and incubated overnight at 4 °C.

Techniques: Quantitative Proteomics, Expressing

Journal: World Journal of Gastrointestinal Oncology

Article Title: MiRNA-145-5p inhibits gastric cancer progression via the serpin family E member 1- extracellular signal-regulated kinase-1/2 axis

doi: 10.4251/wjgo.v16.i5.2123

Figure Lengend Snippet: Effects of serpin family E member 1 overexpression on gastric cancer cell proliferation, invasion, migration, and apoptosis. A: Serpin family E member 1 ( SERPINE1 ) mRNA and protein expression in lentivirus transfected SGC7901 cells; B: SERPINE1 overexpression promotes SGC7901 cell proliferation; C: SERPINE1 overexpression promotes SGC7901 cell colony formation; D: SERPINE1 promotes SGC7901 cell invasion; scale bar = 100 μm; E: SERPINE1 promotes SGC7901 cell migration; scale bar = 100 μm; F: SERPINE1 overexpression reduces SGC7901 cell apoposis. a P < 0.05; b P < 0.01; c P < 0.001.

Article Snippet: The proteins were then boiled at 100°C for 10 min with 100 μL of loading buffer (Beyotime Biotechnology, China) and separated by SDS-PAGE at 120 V. After electrophoresis, the proteins were transferred onto PVDF membranes and blocked using 5% BSA/TBST for 60 min. the primary rabbit polyclonal antibodies, SERPINE1 (Proteintech, United States), α-smooth muscle actin (α-SMA) (Proteintech, United States), β-catenin (Proteintech, United States), vimentin (HuaBio, China), ERK1/2 (HuaBio, China), phosphorylation extracellular signal-regulated kinase1/2 (p-ERK1/2) (HuaBio, China) and actin (ZSGB-BIO, China), and the mouse monoclonal antibody E-cad (HuaBio, China), were added to the membranes and incubated overnight at 4 °C.

Techniques: Over Expression, Migration, Expressing, Transfection

Journal: World Journal of Gastrointestinal Oncology

Article Title: MiRNA-145-5p inhibits gastric cancer progression via the serpin family E member 1- extracellular signal-regulated kinase-1/2 axis

doi: 10.4251/wjgo.v16.i5.2123

Figure Lengend Snippet: Serpin family E member 1 reverses microRNA-145-5p-mediated gastric cancer cell proliferation, invasion, metastasis, epithelial-mesenchymal transition, and apoptosis. A: Serpin family E member 1 protein expression in co-transfected cells; B: Co-transfected cell proliferation; C: Co-transfected cell colony formation; D: Co-transfected cell migration; scale bar = 100 μm; E: Co-transfected cell invasion; scale bar = 100 μm; F: Epithelial-mesenchymal transition-related marker protein (E-cadherin, β-catenin, Vimentin, and α-smooth muscle actin) expression in co-transfected cells; G: Apoptosis of co-transfected cells. a P < 0.05; b P < 0.01; c P < 0.001.

Article Snippet: The proteins were then boiled at 100°C for 10 min with 100 μL of loading buffer (Beyotime Biotechnology, China) and separated by SDS-PAGE at 120 V. After electrophoresis, the proteins were transferred onto PVDF membranes and blocked using 5% BSA/TBST for 60 min. the primary rabbit polyclonal antibodies, SERPINE1 (Proteintech, United States), α-smooth muscle actin (α-SMA) (Proteintech, United States), β-catenin (Proteintech, United States), vimentin (HuaBio, China), ERK1/2 (HuaBio, China), phosphorylation extracellular signal-regulated kinase1/2 (p-ERK1/2) (HuaBio, China) and actin (ZSGB-BIO, China), and the mouse monoclonal antibody E-cad (HuaBio, China), were added to the membranes and incubated overnight at 4 °C.

Techniques: Expressing, Transfection, Migration, Marker

Journal: World Journal of Gastrointestinal Oncology

Article Title: MiRNA-145-5p inhibits gastric cancer progression via the serpin family E member 1- extracellular signal-regulated kinase-1/2 axis

doi: 10.4251/wjgo.v16.i5.2123

Figure Lengend Snippet: Effect of serpin family E member 1 overexpression on gastric cancer cell proliferation and the signal-regulated kinase-1/2 pathway in vitro and in vivo . A: Expression of signal-regulated kinase-1/2 (ERK1/2) and p-ERK1/2 in SGC7901 co-transfected cells; B: Image of xenograft tumors at day 18 post-inoculation; C: Xenograft tumor growth, volume, and mass at day-18 post-inoculation. n = 5 mice per group; D: Ki67 expression in xenograft tumor tissue; Scale bar = 20 μm; E: E-cadherin, vimentin, serpin family E member 1, ERK1/2, and p-ERK1/2 expression in xenograft tumor tissues. a P < 0.05; b P < 0.01; c P < 0.001. SERPINE1 : Serpin family E member 1; ERK1/2: Signal-regulated kinase-1/2.

Article Snippet: The proteins were then boiled at 100°C for 10 min with 100 μL of loading buffer (Beyotime Biotechnology, China) and separated by SDS-PAGE at 120 V. After electrophoresis, the proteins were transferred onto PVDF membranes and blocked using 5% BSA/TBST for 60 min. the primary rabbit polyclonal antibodies, SERPINE1 (Proteintech, United States), α-smooth muscle actin (α-SMA) (Proteintech, United States), β-catenin (Proteintech, United States), vimentin (HuaBio, China), ERK1/2 (HuaBio, China), phosphorylation extracellular signal-regulated kinase1/2 (p-ERK1/2) (HuaBio, China) and actin (ZSGB-BIO, China), and the mouse monoclonal antibody E-cad (HuaBio, China), were added to the membranes and incubated overnight at 4 °C.

Techniques: Over Expression, In Vitro, In Vivo, Expressing, Transfection

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: Cells

Article Title: Silencing of Ago-2 Interacting Protein SERBP1 Relieves KCC2 Repression by miR-92 in Neurons

doi: 10.3390/cells11061052

Figure Lengend Snippet: SERBP1 or Ago2 silencing increases KCC2 expression. ( A ) Forty-eight hours after transfection with either control siRNA, Ago2 siRNA or SERBP1 siRNA (20 nM), SH-SY5Y cells were transfected with PSSG control plasmid or the Serpine1, or APP or KCC2 3′UTR firefly reporter plasmid. Firefly/renilla luminescence ratios were determined 24 h later. The data are presented as the fold increase of normalized luminescence ratios relative to cells transfected with control siRNA. Data represent the mean of three independent experiments +/− S.E.; * p < 0.05; ** p < 0.01. ( B ) Forty-eight hours after transfection with siAgo2 or siSERBP1 or a control siRNA (200 nM), hippocampal neurons were transfected with control plasmid or KCC2 3′UTR luciferase reporter. Firefly/renilla luminescence ratio was analyzed as described in ( A ). Data represent the mean of three independent experiments ± S.E. * p < 0.05. Individual data points for Ago2 (circles) and Serbp1 (triangles) siRNA transfected cells are indicated in ( A ) and ( B ) panels. ( C ) Representative Western blotting showing KCC2 upregulation in hippocampal neurons after SERBP1 silencing. Nitrocellulose membranes were hybridized with KCC2 or SERBP1 antibodies, as indicated. The GAPDH signal was used to normalize different samples. Means +/− S.E. of KCC2 and SERBP1 protein levels obtained from three independent experiments are presented relative to control cells. * p < 0.05; ** p < 0.01 ( t -test).

Article Snippet: Serpine1 3′UTR: The last 1332 nts of the Serpine1 3′UTR within the full-length clone SC119781 (Origene, Rockville, MD, USA) (SacII/NotI) and the first 509 nts of the Serpine1 3′UTR within the SG-Luc- Serpine1 plasmid (XbaI/SacII) (Amplicon start: chr7: 100362990; Amplicon end: chr7: 100363963 purchased from Switch Gear Genomic (Carlsbad, CA, USA) were cloned into the PSSG plasmid.

Techniques: Expressing, Transfection, Plasmid Preparation, Luciferase, Western Blot