Journal: Frontiers in Oncology

Article Title: Knockdown of m6A Reader IGF2BP3 Inhibited Hypoxia-Induced Cell Migration and Angiogenesis by Regulating Hypoxia Inducible Factor-1α in Stomach Cancer

doi: 10.3389/fonc.2021.711207

Figure Lengend Snippet: IGF2BP3 knockdown hampered hypoxia-induced cell migration and angiogenesis in SC. (A) MKN-45 and HGC-27 cells were cultured in normoxic or hypoxic conditions for 24 h Next, the IGF2BP3 protein level was measured by western blot assay. (B) MKN-45 and HGC-27 cells were transfected with si-NC, si-IGF2BP3#1, si-IGF2BP3#2, or si-IGF2BP3#3. Next, the IGF2BP3 mRNA level was measured by RT-qPCR assay at 48 h after transfection. (C–F) MKN-45 and HGC-27 cells were transfected with si-NC or si-IGF2BP3#1 for 48 h and then maintained in hypoxic conditions for another 24 h Cells in the normoxia group were maintained in normoxic conditions for 72 h Cells in the hypoxia group were cultured in normoxia for 48 h and then exposed to hypoxia for an additional 24 h (C, D) Cell migratory potential was assessed by Transwell migration (C) and wound healing (D) assays. (E) VEGF level in cell culture supernatants was detected using a commercial kit. (F) The conditioned medium of MKN-45 and HGC-27 cells were collected after normoxia/hypoxia treatment or/and transfection. Next, HUVECs were cultured in a mixed medium of ECM and conditioned medium (volume ratio=1:1), followed by the measurement of tube formation ability at 12 h after incubation. * indicate that the difference is significant at 0.05 level. ** p < 0.01, *** p < 0.001, ## p < 0.01, ### p < 0.001 compared with the normoxia group.

Article Snippet: MKN-45 cells and human umbilical vein endothelial cells (HUVECs) were purchased from the China Center for Type Culture Collection (Wuhan, China).

Techniques: Knockdown, Migration, Cell Culture, Western Blot, Transfection, Quantitative RT-PCR, Incubation

Journal: Frontiers in Oncology

Article Title: Knockdown of m6A Reader IGF2BP3 Inhibited Hypoxia-Induced Cell Migration and Angiogenesis by Regulating Hypoxia Inducible Factor-1α in Stomach Cancer

doi: 10.3389/fonc.2021.711207

Figure Lengend Snippet: IGF2BP3 exerted its functions by up-regulating HIF1A. (A) MKN-45 and HGC-27 cells were transfected with pcDNA3.1 or pcDNA-HIF1A. Next, HIF1A mRNA level was measured by RT-qPCR assay at 48 h upon transfection. (B–E) MKN-45 and HGC-27 cells were transfected with si-NC+pcDNA3.1, si-IGF2BP3#1+pcDNA3.1, or si-IGF2BP3#1+pcDNA-HIF1A for 48 h and then maintained in hypoxic conditions for another 24 h, followed by the examination of cell migratory potential (B, C) and VEGF secretion level (D) . (E) MKN-45 and HGC-27 cells were transfected with si-NC+pcDNA3.1, si-IGF2BP3#1+pcDNA3.1, or si-IGF2BP3#1+pcDNA-HIF1A for 48 h and then maintained in hypoxic conditions for another 24 h, followed by the collection of conditioned medium. Next, HUVECs were cultured in a mixed medium of ECM and conditioned medium (volume ratio=1:1) and tube formation potential was examined at 12 h after incubation. ** p < 0.01, *** p < 0.001, ## p < 0.01, ### p < 0.001.

Article Snippet: MKN-45 cells and human umbilical vein endothelial cells (HUVECs) were purchased from the China Center for Type Culture Collection (Wuhan, China).

Techniques: Transfection, Quantitative RT-PCR, Cell Culture, Incubation

Journal: PPAR Research

Article Title: Stimulation of Alpha 1 -Adrenergic Receptor Ameliorates Cellular Functions of Multiorgans beyond Vasomotion through PPAR δ

doi: 10.1155/2020/3785137

Figure Lengend Snippet: The effects of α 1 -AR stimulation on the expression of mitochondrial energetic molecules, oxidative phosphorylation, and biological functions in skeletal and cardiac muscle cells and liver cells. (a, b) The expression of p-AMPK and PPAR δ in C2C12, HL1, and HepG2 cells was stimulated with 1–30 μ M midodrine for the indicated times. (c) Cytosolic calcium mobilization after midodrine treatment in C2C12 and HL1 cells. Each cell type was pretreated with the calcium reactive dye Fluo-3 AM for 45 min and then stimulated with 30 μ M midodrine for the indicated times. Green fluorescence emitted by Fluo-3 AM was detected using confocal microscopy. (d) The phosphorylation of AMPK α at Thr172 and expression of PPAR δ in C2C12 and HL1 cells after pretreatment with the calcium/calmodulin-dependent protein kinase kinase antagonist STO-609 for 25 min and treatment with midodrine. (e) Fluorescence after using the CytoPainter mitochondrial staining kit in midodrine-treated and control C2C12 cells. Original magnification was 200x. (f) The measured activity of succinate dehydrogenase (SDH) in C2C12 cells. (G) Oxygen consumption rate (OCR) in C2C12 cells treated with midodrine (30 μ M), as measured by a Seahorse XFp analyzer. (h) ATP content in C2C12 cells treated with midodrine (30 μ M) cultured with low-glucose (5.56 mM) medium. (i) Glucose transporter (GLUT) 4 protein expression in C2C12 cells treated with high glucose (HG) and midodrine (HG+Mido), HG and insulin (HG+Insulin), and the control treatment (Ctrl). (j) The uptake of 2-deoxyglucose in C2C12 skeletal muscle cells treated with midodrine. (k) OCR (measured by the Seahorse XFp analyzer) in H9C2 cells treated with midodrine (30 μ M) and cultured with low-glucose (5.56 mM) medium. (l) ATP content in H9C2 cells treated with midodrine (30 μ M). Data are expressed as the mean ± standard deviation of triplicate experiments. AMPK: adenosine monophosphate-activated protein kinase; p-AMPK: phosphorylated AMPK; PPAR δ : peroxisome proliferator-activated receptor delta; PGC-1 α : peroxisome proliferator-activated receptor gamma coactivator 1-alpha; mGLUT4: GLUT4 expression of the cell membrane; tGLUT4: total cellular expression of GLUT4; Ctrl: an untreated control group; Mido: midodrine-treated group.

Article Snippet: L6 rat skeletal muscle, C2C12 mouse skeletal muscle, HL1 and H9C2 cardiac muscle, HUVEC human umbilical vein endothelial cell line, RAW 264.7 macrophages, and 3T3-L1 mouse preadipocyte cells were purchased from a Korean cell line bank (Seoul, Korea).

Techniques: Expressing, Phospho-proteomics, Fluorescence, Confocal Microscopy, Staining, Control, Activity Assay, Cell Culture, Standard Deviation, Membrane

Journal: PPAR Research

Article Title: Stimulation of Alpha 1 -Adrenergic Receptor Ameliorates Cellular Functions of Multiorgans beyond Vasomotion through PPAR δ

doi: 10.1155/2020/3785137

Figure Lengend Snippet: The effect of midodrine on the endothelial expression of p-AMPK and p-eNOS in HUVECs; OCR analyses in H9C2 cells; intracellular fat and the expression of PPAR δ , p-AMPK, and PGC-1 α in differentiated 3T3-L1 cells; and the effects of midodrine on mRNA levels of PPAR δ , AMPK α 1 , and mannose receptor and protein levels of mannose receptor and hexokinase II in RAW 264.7 macrophage cells treated with different concentrations of midodrine. (a) The expression of phosphorylated AMPK (p-AMPK) and phosphorylated endothelial nitric oxide synthase (p-eNOS) proteins in human umbilical vein endothelial cells (HUVECs) treated with cholesterol and palmitate, and the effects from the addition of GSK0660, a PPAR δ antagonist. Ctrl: the control group; CP: the cholesterol- and palmitate-treated group; CPM: the cholesterol-, palmitate-, and midodrine-treated group. (b) The maximal oxygen consumption rate (OCR) analysis as estimated using a Seahorse XFp analyzer and ATP content measured by ELISA in H9C2 cells. (c) The effect of compound C (1 μ M) on p-AMPK expression and PPAR δ expression. (d) The effect of midodrine on intracellular lipid deposits (Oil Red O staining result) and the protein levels of PPAR δ , AMPK, and PGC-1 α in differentiated 3T3-L1 cells treated with midodrine and GSK0660. (e) The effects of midodrine on mRNA levels of PPAR δ , AMPK α 1 , and mannose receptor and protein levels of mannose receptor and hexokinase II in RAW 264.7 macrophage cells treated with different concentrations of midodrine. Ctrl: untreated control group; Mido: midodrine-treated group; Mido+GSK0660: midodrine- and GSK0660-treated group.

Article Snippet: L6 rat skeletal muscle, C2C12 mouse skeletal muscle, HL1 and H9C2 cardiac muscle, HUVEC human umbilical vein endothelial cell line, RAW 264.7 macrophages, and 3T3-L1 mouse preadipocyte cells were purchased from a Korean cell line bank (Seoul, Korea).

Techniques: Expressing, Control, Enzyme-linked Immunosorbent Assay, Staining

Journal: Frontiers in Cardiovascular Medicine

Article Title: Therapeutic Effect of Pericytes for Diabetic Wound Healing

doi: 10.3389/fcvm.2022.868600

Figure Lengend Snippet: The expression of pericyte markers by flow cytometry was reduced in the diabetic condition. (A) Analysis of pericyte surface markers in the non-diabetic and diabetic conditions using flow cytometry [CD146-fluorescein isothiocyanate (FITC), Nestin-phycoerythrin (PE), and NG2-PE]. The results provided here are representative of those obtained in three independent studies. In the diabetic condition, Nestin and NG2-PE were seen to decrease by more than half. (B) Overlay of diabetic condition (green line) and non-diabetic (red line) condition flow cytometry. The diabetic condition showed the three pericyte markers to be decreased.

Article Snippet: Each well contained 300 μl of the human umbilical vein endothelial cells (HUVECs) and pericytes (10:1 ratio) cell suspension in the VascuNet Basal Assay Medium (ESI BIO, USA) ( ).

Techniques: Expressing, Flow Cytometry

Journal: Frontiers in Cardiovascular Medicine

Article Title: Therapeutic Effect of Pericytes for Diabetic Wound Healing

doi: 10.3389/fcvm.2022.868600

Figure Lengend Snippet: The group with pericytes showed improved tube formation and cell migration ability in the high glucose condition. (A) The in-vitro vascular, tube formation assay showed effects of exposure on FITC-tagged human umbilical vein endothelial cell (HUVEC) tube formation with and without PE-tagged human pericyte cells. The optimal observation time for tube formation was 8 h after seeding. In high glucose conditions, HUVECs with pericytes (10:1 ratio) showed active tube formation even compared to the without pericyte group. (B) The number of pieces in the assay was measured. (** P < 0.01 and *** P < 0.001 according to the Student's t -test). HUVEC tube formation capacity showed no significant difference in low glucose conditions. However, in high glucose conditions, the group with pericytes had a statistically significant increase in tube formation. (C) Photomicrographs were taken at 0, 12, and 24 h following initiation of the wound. HUVEC migration, often termed as wound closure ability, was more remarkable in the group with pericytes in both the high and low glucose conditions. (D) The quantitative approach applied to the wound closure assay, showing a histogram calculated based on the group breath ratio of the control group and the group with pericytes.

Article Snippet: Each well contained 300 μl of the human umbilical vein endothelial cells (HUVECs) and pericytes (10:1 ratio) cell suspension in the VascuNet Basal Assay Medium (ESI BIO, USA) ( ).

Techniques: Migration, In Vitro, Tube Formation Assay, Wound Closure Assay, Control

Journal: Frontiers in Cardiovascular Medicine

Article Title: Therapeutic Effect of Pericytes for Diabetic Wound Healing

doi: 10.3389/fcvm.2022.868600

Figure Lengend Snippet: The diabetes mellitus (DM)-with-pericyte group exhibited substantial wound healing in a mouse wound model. (A) Illustration of injection of collagen type 1 scaffold + 5 × 10 5 pericytes in the mouse wound. (B) Streptozotocin (STZ)-induced diabetic mice on blood glucose confirmed successful DM induction (**** P < 0.0001). DM-induced mice ( n = 10), control ( n = 10). (C) Incisions with a diameter of 4 mm on either side of the midline were produced. To avoid wound contraction, the wounds were circumscribed with donut-shaped silicone splints. As observed on day 7, wounds of the DM-with-pericyte group healed, whereas those of the DM-without-pericyte and control groups did not heal. (D) The Y-axis of the graph represents the wound size, when measured on days 0, 2, 7, and 14. It was verified that all the groups, except the DM-without-pericyte group, had wound closure by the time of sacrifice on day 14 and approximately 3.6 mm of the wound of the DM-without-pericyte group remained. The pericyte and DM-with-pericyte groups showed the same wound size on days 0, 2, 7, and 14. (E) Quantification of wound size at sacrifice time points (** P < 0.01). In comparison to the DM-without-pericyte group, the DM-with-pericyte group exhibited substantial wound healing.

Article Snippet: Each well contained 300 μl of the human umbilical vein endothelial cells (HUVECs) and pericytes (10:1 ratio) cell suspension in the VascuNet Basal Assay Medium (ESI BIO, USA) ( ).

Techniques: Injection, Control, Produced, Comparison

Journal: Frontiers in Cardiovascular Medicine

Article Title: Therapeutic Effect of Pericytes for Diabetic Wound Healing

doi: 10.3389/fcvm.2022.868600

Figure Lengend Snippet: The DM-with-pericyte group showed successful repair of the collagen layer and higher blood vessel formation capacity compared to the DM-without-pericyte group. (A) H&E staining. (B) Masson's trichrome staining for wound analysis at 14 days. The black square indicates the regenerated dermal collagen layer. Scale bars = 200 μm. (C) CD31 and Ki67 immunofluorescence of wounds at 14 days. In diabetes wounds, new vessels were significantly less visible than in normal mice. However, when diabetic mice were injected with pericytes, new vessels were observed more often. (D) α-smooth muscle actin (α-SMA), collagen 1 (Col 1), and Nestin immunohistochemistry of wounds at 14 day. Scale bar = 50 μm.

Article Snippet: Each well contained 300 μl of the human umbilical vein endothelial cells (HUVECs) and pericytes (10:1 ratio) cell suspension in the VascuNet Basal Assay Medium (ESI BIO, USA) ( ).

Techniques: Staining, Immunofluorescence, Injection, Immunohistochemistry

Journal: eLife

Article Title: GIPC proteins negatively modulate Plexind1 signaling during vascular development

doi: 10.7554/eLife.30454

Figure Lengend Snippet:

Article Snippet: Cell line ( Homo sapiens ) , HUVEC (Normal Primary Human Umbilical Vein Endothelial Cells) , Lifeline Cell Technology , Cat. #FC-0003 , https://www.lifelinecelltech.com/shop/cells/human-endothelial-cells/umbilical-vein-endothelial-cells/huvec-fc-0003/.

Techniques: Transgenic Assay, Plasmid Preparation, Mutagenesis, Derivative Assay, Selection, Stable Transfection, Knock-Out, Recombinant, Control, shRNA, Sequencing, Construct, Synthesized, Positive Control, Sterility, Concentration Assay