Journal: Cell stem cell

Article Title: Stress-induced changes in bone marrow stromal cell populations revealed through single-cell protein expression mapping

doi: 10.1016/j.stem.2019.06.003

Figure Lengend Snippet: Multi-Dimensional Single-Cell Mass Cytometry analysis reveals distinguishable clusters of bone marrow stromal cells

Article Snippet: Chondrocyte, adipocyte and osteoblast differentiation CD105 − NGFR high stromal cells were cultured under hypoxic condition 2% O 2 in a 96 well plate format, in 100ml of α-MEM (GE Healthcare Life Sciences, SH30265.01) complemented with 10% FBS (Gibco by Life Technologies, A31605–01), 1% Penicillin/Streptomycin (Gibco by Life Technologies, 15140–122).

Techniques: Mass Cytometry, Purification, Affinity Purification, Functional Assay, Recombinant, Electron Microscopy, Modification, Conjugation Assay, Labeling, Isolation, Imaging, Expressing, Software

Journal: International Journal of Molecular Medicine

Article Title: Hypoxic mast cells accelerate the proliferation, collagen accumulation and phenotypic alteration of human lung fibroblasts

doi: 10.3892/ijmm.2019.4400

Figure Lengend Snippet: Effects of hypoxia on the proliferation of HMC-1 cells. (A) Following culture under hypoxic conditions, the expression of HIF-1α in HMC-1 cells was determined by western blotting and used to confirm that cells exist within a hypoxic environment. (B) The proliferation of HMC-1 cells under hypoxic and normoxic conditions was examined by the Cell Counting Kit-8 assay for the indicated time periods. There were 4 biological replicates/condition. Error bars represent the standard deviation. * P<0.05, ** P<0.01. HMC-1, human mast cells; HIF-1α, hypoxia inducible factor-1α; OD, optical density.

Article Snippet: Subsequently, HMC-1 cells were incubated with fresh IMDM (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS under hypoxic conditions, and HFL-1 cells were cultured in conditioned medium from hypoxic or normoxic HMC-1 cells for different times.

Techniques: Expressing, Western Blot, Cell Counting, Standard Deviation

Journal: International Journal of Molecular Medicine

Article Title: Hypoxic mast cells accelerate the proliferation, collagen accumulation and phenotypic alteration of human lung fibroblasts

doi: 10.3892/ijmm.2019.4400

Figure Lengend Snippet: Effects of hypoxia on the secretion of cytokines by HMC-1 cells. (A) HMC-1 cells were cultured under hypoxic conditions for the indicated time periods and then subjected to reverse transcription-quantitative polymerase chain reaction to detect the expression of cytokines IL-1β, IL-6, IL-15 and TNF-α. β-tubulin was used as an internal control. (B-E) Following culture under hypoxic conditions, the levels of the secreted cytokines IL-1β, IL-6, IL-15 and TNF-α in the conditioned medium from HMC-1 cells were determined by ELISA. There were 4-6 biological replicates/condition. Error bars depict the standard deviation. * P<0.05, ** P<0.01, NS, not significant. IL, interleukin; TNF, tumor necrosis factor; HMC-1, human mast cells.

Article Snippet: Subsequently, HMC-1 cells were incubated with fresh IMDM (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS under hypoxic conditions, and HFL-1 cells were cultured in conditioned medium from hypoxic or normoxic HMC-1 cells for different times.

Techniques: Cell Culture, Real-time Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: International Journal of Molecular Medicine

Article Title: Hypoxic mast cells accelerate the proliferation, collagen accumulation and phenotypic alteration of human lung fibroblasts

doi: 10.3892/ijmm.2019.4400

Figure Lengend Snippet: Differential expression of mRNA in HFL-1 cells cultured in hypoxic and normoxic conditioned medium from HMC-1 cells. (A) Box plot of RNA sequencing dataset after normalization. (B) Hierarchical clustering of differentially expressed mRNAs in HFL-1 hypoxic group and normoxic controls. (C) Scatter plots demonstrated differential mRNA expression between hypoxic and normoxic samples. The red circles indicate upregulated genes, the green circles represent downregulated genes, and black circles indicate unchanged genes. (D) Volcano plots were generated to visualize the mRNA expression profiles between hypoxic and normoxic samples. (E and F) GO enrichment and KEGG analysis of differentially expressed mRNAs in HFL-1 cells cultured in the hypoxic and normoxic conditioned medium from HMC-1 cells. HFL-1, human lung fibroblasts; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; H, hypoxia; N, normoxia; HMC-1, human mast cells; HFL-1, human fetal lung fibroblasts.

Article Snippet: Subsequently, HMC-1 cells were incubated with fresh IMDM (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS under hypoxic conditions, and HFL-1 cells were cultured in conditioned medium from hypoxic or normoxic HMC-1 cells for different times.

Techniques: Expressing, Cell Culture, RNA Sequencing Assay, Generated

Journal: International Journal of Molecular Medicine

Article Title: Hypoxic mast cells accelerate the proliferation, collagen accumulation and phenotypic alteration of human lung fibroblasts

doi: 10.3892/ijmm.2019.4400

Figure Lengend Snippet: Top 20 upregulated and downregulated mRNAs in human fetal lung fibroblasts (HFL-1) cultured in hypoxic conditioned medium from human mast cells (HMC-1) and normoxic controls.

Article Snippet: Subsequently, HMC-1 cells were incubated with fresh IMDM (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS under hypoxic conditions, and HFL-1 cells were cultured in conditioned medium from hypoxic or normoxic HMC-1 cells for different times.

Techniques: Cell Culture

Journal: International Journal of Molecular Medicine

Article Title: Hypoxic mast cells accelerate the proliferation, collagen accumulation and phenotypic alteration of human lung fibroblasts

doi: 10.3892/ijmm.2019.4400

Figure Lengend Snippet: Effects of conditioned medium from HMC-1 cells on the proliferation and migration of HFL-1 cells. (A) After treatment with normoxic or hypoxic conditioned medium from HMC-1 cells, the proliferation of HFL-1 cells was assessed by the Cell Counting Kit-8 assay. (B) Migration of HFL-1 cells after culture in normoxic or hypoxic conditioned medium from HMC-1 cells was determined by scratch assay. Representative images before and after inflicting the wound are shown and quantified. There were 6 biological replicates/condition. Error bars depict the standard deviation. * P<0.05, ** P<0.01, NS, non-significant. Bar, 1 mm. N-CM, normoxic conditioned medium; H-CM, hypoxic conditioned medium; HMC-1, human mast cells; HFL-1, human fetal lung fibroblasts.

Article Snippet: Subsequently, HMC-1 cells were incubated with fresh IMDM (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS under hypoxic conditions, and HFL-1 cells were cultured in conditioned medium from hypoxic or normoxic HMC-1 cells for different times.

Techniques: Migration, Cell Counting, Wound Healing Assay, Standard Deviation

Journal: International Journal of Molecular Medicine

Article Title: Hypoxic mast cells accelerate the proliferation, collagen accumulation and phenotypic alteration of human lung fibroblasts

doi: 10.3892/ijmm.2019.4400

Figure Lengend Snippet: Effects of conditioned medium from HMC-1 cells on the production of collagens and MMPs by HFL-1 cells. (A) Following culture in normoxic or hypoxic conditioned medium from HMC-1 cells, the mRNA expression of collagen type I and III, MMP-9, MMP-13 and TIMP-1 in HFL-1 cells were assessed by reverse transcription-quantitative polymerase chain reaction analysis, normalized to the expression of β-tubulin, and expressed as fold change relative to the expression of the control group. (B) Western blot analysis of collagen type I and III, MMP-9, MMP-13 and TIMP-1 protein expression in HFL-1 cells cultured in conditioned medium from HMC-1 cells. There were 4 biological replicates/condition. Error bars depict the standard deviation. * P<0.05, ** P<0.01, NS, not significant. COL I, collagen type I; COL III, collagen type III; MMP-9, matrix metalloproteinase-9; TIMP-1, tissue inhibitor of metalloproteinase-1; N-CM, normoxic conditioned medium; H-CM, hypoxic conditioned medium; HMC-1, human mast cells; HFL-1, human fetal lung fibroblasts.

Article Snippet: Subsequently, HMC-1 cells were incubated with fresh IMDM (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS under hypoxic conditions, and HFL-1 cells were cultured in conditioned medium from hypoxic or normoxic HMC-1 cells for different times.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Cell Culture, Standard Deviation

Journal: International Journal of Molecular Medicine

Article Title: Hypoxic mast cells accelerate the proliferation, collagen accumulation and phenotypic alteration of human lung fibroblasts

doi: 10.3892/ijmm.2019.4400

Figure Lengend Snippet: Effects of conditioned medium from HMC-1 cells on the transition of HFL-1 cells from fibroblasts to myofibroblasts. (A) After treatment with nor-moxic or hypoxic conditioned medium from HMC-1 cells, the expression of α-SMA mRNA was determined by reverse transcription-quantitative polymerase chain reaction analysis. β-tubulin was used as an internal control. (B) Western blotting was performed to assess the level of α-SMA protein in HFL-1 cells cultured in normoxic or hypoxic conditioned medium from HMC-1 cells. (C) After treatment with conditioned medium from HMC-1 cells, HFL-1 cells were stained for immunofluorescence for α-SMA. Nuclei were counterstained with DAPI. There were 4-6 biological replicates/condition. Error bars represent the standard deviation. * P<0.05, ** P<0.01. Bar, 50 µ m. α-SMA, α-smooth muscle actin; DAPI, 4′,6-diamidino-2-phenylindole; HMC-1, human mast cells; HFL-1, human fetal lung fibroblasts.

Article Snippet: Subsequently, HMC-1 cells were incubated with fresh IMDM (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS under hypoxic conditions, and HFL-1 cells were cultured in conditioned medium from hypoxic or normoxic HMC-1 cells for different times.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Cell Culture, Staining, Immunofluorescence, Standard Deviation

Journal: PLoS ONE

Article Title: Up-Regulation of ENO1 by HIF-1α in Retinal Pigment Epithelial Cells after Hypoxic Challenge Is Not Involved in the Regulation of VEGF Secretion

doi: 10.1371/journal.pone.0147961

Figure Lengend Snippet: Serum-starved ARPE-19 cells were challenged with anoxia or CoCl 2 -induced hypoxia. LDH activities in the culture medium were detected after 24 hr of treatment. No significant differences in the LDH activities were observed in cells under normoxic, anoxic and CoCl 2 -induced hypoxic conditions. This implied that no remarkable cell death (LDH release less than 5%) resulted from the anoxic or hypoxic stress in the cultured ARPE-19 cells. (n = 4).

Article Snippet: For hypoxic treatment, the culture medium was supplemented with 300μM cobalt (II) chloride CoCl 2 (Sigma, St. Louis, USA), a commonly used hypoxia-mimetic chemical.

Techniques: Cell Culture

Journal: PLoS ONE

Article Title: Up-Regulation of ENO1 by HIF-1α in Retinal Pigment Epithelial Cells after Hypoxic Challenge Is Not Involved in the Regulation of VEGF Secretion

doi: 10.1371/journal.pone.0147961

Figure Lengend Snippet: ARPE-19 cells were cultured with deoxygenated DMEM medium or supplemented with 300μM CoCl 2 for 24 hr. (A. B) Expression of HIF-1α (116kDa) and (C, D) ENO1 (46kDa) were assayed by Western blotting. β-actin (42kDa) and p84 (84kDa) were used, respectively as the internal control for equal loading of proteins. In ARPE-19 cells, HIF-1α protein level was barely detectable under normoxia. (A) Upon 24 hr of anoxia, HIF-1α expression rose significantly (n = 3, *p < 0.05 versus normoxia). (B) CoCl 2 -induced hypoxia also successfully increased HIF-1α protein levels in ARPE-19 cells (n = 6, ****p<0.001 versus normoxia). In contrast to the normoxic control, the protein level of ENO1 increased significantly under both treatment conditions. (C) It approximately doubled after 24 hr of anoxia (n = 3, **p<0.01 versus normoxia) while it was tripled in the CoCl 2 -treated cells. (n = 6, ***p<0.001 versus normoxia) (D).

Article Snippet: For hypoxic treatment, the culture medium was supplemented with 300μM cobalt (II) chloride CoCl 2 (Sigma, St. Louis, USA), a commonly used hypoxia-mimetic chemical.

Techniques: Cell Culture, Expressing, Western Blot

Journal: PLoS ONE

Article Title: Up-Regulation of ENO1 by HIF-1α in Retinal Pigment Epithelial Cells after Hypoxic Challenge Is Not Involved in the Regulation of VEGF Secretion

doi: 10.1371/journal.pone.0147961

Figure Lengend Snippet: (A, B) Representative photomicrographs of ARPE-19 cells at 48 hr of transfection with FAM-labeled siGLO RISC-free siRNA. RPE cells were transfected with scramble control siRNA (siGLO) for 24 hr and subsequently cultured with 300μM CoCl 2 for another 24 hr. Cell images were captured at 48 hr under bright field (A) and fluorescence field with a FITC filter (B) at 200X magnification. Cells displayed green fluorescence, indicating that they were transfected with FAM-labeled RISC-independent siRNA (siGLO), which was used as both the scramble control and the transfection indicator. Under our experimental condition, over 60% of cells showed observable fluorescence suggesting successful transfection after 48 hr. (C) LDH analysis of transfected ARPE-19 cells under 24 hr of CoCl 2 -induced hypoxia. ARPE-19 cells were transfected with scramble control siRNA (siGLO), HIF-1α siRNA (siHIF), ENO1 siRNA (siENO), and co-transfected with both HIF-1α siRNA and ENO1 siRNA (siHIF + siENO) for 24 hr, followed by another 24 hr of CoCl 2 -induced hypoxia before LDH assay. Compared with the control group, lack of changes in the LDH activities were observed in the cells transfected with different targeting siRNA under CoCl 2 challenge. All cell survival rates were over 85%. (n = 4).

Article Snippet: For hypoxic treatment, the culture medium was supplemented with 300μM cobalt (II) chloride CoCl 2 (Sigma, St. Louis, USA), a commonly used hypoxia-mimetic chemical.

Techniques: Transfection, Labeling, Cell Culture, Fluorescence, Lactate Dehydrogenase Assay

Journal: PLoS ONE

Article Title: Up-Regulation of ENO1 by HIF-1α in Retinal Pigment Epithelial Cells after Hypoxic Challenge Is Not Involved in the Regulation of VEGF Secretion

doi: 10.1371/journal.pone.0147961

Figure Lengend Snippet: ARPE cells were transfected with scramble control (siGLO) or HIF-1α siRNA (siHIF) for 24 hr, and cultured with 300μM CoCl 2 for another 24 hr. Expressions of HIF-1α and ENO1 were assayed by Western blotting. β-actin and p84 were used as the internal controls, respectively. (A) Upon transfection with siHIF, the HIF-1α level under hypoxia condition was clearly reduced when compared with the control cells transfected with siGLO (n = 6, ****p< 0.0001). (B) The expression of ENO1 in the hypoxic ARPE-19 cells displayed a decrease by about 30% upon HIF-1α diminished (n = 6, ****p< 0.0001).

Article Snippet: For hypoxic treatment, the culture medium was supplemented with 300μM cobalt (II) chloride CoCl 2 (Sigma, St. Louis, USA), a commonly used hypoxia-mimetic chemical.

Techniques: Transfection, Cell Culture, Western Blot, Expressing

Journal: PLoS ONE

Article Title: Up-Regulation of ENO1 by HIF-1α in Retinal Pigment Epithelial Cells after Hypoxic Challenge Is Not Involved in the Regulation of VEGF Secretion

doi: 10.1371/journal.pone.0147961

Figure Lengend Snippet: (A) ARPE-19 cells were transfected with siENO for 48 hr. In the meantime, cells were exposed to CoCl 2 -induced hypoxia for the last 24 hr. The protein level of ENO1 managed to be reduced by about 40% in the hypoxic ARPE-19 cells. (B) ARPE cells were (co-)transfected with scramble control siRNA (siGLO), HIF-1α siRNA (siHIF) and/or ENO1 siRNA (siENO) for 24 hr, respectively and cultured with 300μM CoCl 2 for another 24 hr. ENO1 protein was successfully suppressed by transfecting with siENO (~40%), the expression of which was further knocked down by co-transfecting with both siHIF and siENO (~60%) (n = 4, ****p<0.0001 versus siGLO respectively; # p<0.0001). (C) Under hypoxia, HIF-1α protein was significantly decreased (~60%) in ARPE-19 cells with the transfection of siHIF. Co-transfection with siHIF and siENO did not further down-regulate the protein level of HIF-1α, which was nearly the same as that in the siHIF-transfected cells (n = 4, ****p<0.0001 versus siGLO respectively). (c, d).

Article Snippet: For hypoxic treatment, the culture medium was supplemented with 300μM cobalt (II) chloride CoCl 2 (Sigma, St. Louis, USA), a commonly used hypoxia-mimetic chemical.

Techniques: Transfection, Cell Culture, Expressing, Cotransfection