primary human nasal epithelial cells (hnec)  (iCell Bioscience Inc)

Journal: International Journal of Molecular Sciences

Article Title: Ganglioside GD3 Regulates Inflammation and Epithelial-to-Mesenchymal Transition in Human Nasal Epithelial Cells

doi: 10.3390/ijms25074054

Figure Lengend Snippet: TNF-α treatment induced the inflammatory response, EMT process, and ganglioside GD3 expression. ( A ) Expression of inflammation-related genes, IL-6, IL-8, and TNF-α, in hNECs after treatment with TNF-α. ( B ) Comparison of EMT-related gene expression after treatment of hNECs with TNF-α. The mRNA expression levels of epithelial markers (E-cadherin) and mesenchymal markers (N-cadherin, SLUG, and MMP-9) were compared using qPCR. ( C ) Comparison of EMT-related protein expression after treatment of hNECs with TNF-α. Epithelial marker (E-cadherin) and mesenchymal marker (N-cadherin and SLUG) protein expression were compared via Western blotting. ( D ) Immunofluorescence staining of E-cadherin (Alexa 488, green), N-cadherin, and SLUG (Alexa 555, red) in TNF-α-treated hNECs. DAPI staining was used to evaluate the nuclei. Scale bar, 100 μm. ( E ) Comparison of GD3 synthase gene expression after treatment of hNECs with TNF-α. ST8SIA1 mRNA expression was evaluated using qPCR. mRNA and protein expression levels were normalized to that of the housekeeping gene β-actin. Expression levels are presented as the mean ± SD from three independent experiments and analyzed using Student’s t -test. ** p < 0.01 and *** p < 0.001 compared with non-treated hNECs. ( F ) High-performance thin-layer chromatography analysis of ganglioside expression in TNF-α-treated hNECs. M1, M2, and M3 are ganglioside standard markers. EMT, epithelial-to-mesenchymal transition; hNECs, human nasal epithelial cells; SLUG, zinc finger protein SNAI2; MMP-9, matrix metallopeptidase 9; ST8SIA1 , ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase.

Article Snippet: Primary human nasal epithelial cells (hNECs) from healthy donors were purchased from PromoCell (Heidelberg, Germany). hNECs were cultured in commercially available airway epithelial cell growth medium with supplements (PromoCell, Catalog No. C-21060, Heidelberg, Germany) in a 75 cm 2 T-flask in a humidified incubator at 37 °C in 5% CO 2 .

Techniques: Expressing, Comparison, Marker, Western Blot, Immunofluorescence, Staining, High Performance Thin Layer Chromatography

Journal: International Journal of Molecular Sciences

Article Title: Ganglioside GD3 Regulates Inflammation and Epithelial-to-Mesenchymal Transition in Human Nasal Epithelial Cells

doi: 10.3390/ijms25074054

Figure Lengend Snippet: Change in inflammatory response and EMT process upon ST8SIA1 knockdown in hNECs. ( A ) ST8SIA1 (GD3 synthase) gene expression after treatment of ST8SIA1 -knockdown hNECs with TNF-α. ( B ) High-performance thin-layer chromatography analysis of ganglioside expression in ST8SIA1 -knockdown hNECs with or without TNF-α treatment. M1 and M2 are ganglioside standard markers. ( C ) Inflammation-related gene expression after treatment of ST8SIA1 -knockdown hNECs with TNF-α was determined by qPCR. ( D ) NF-κB/P65 (cytoplasmic and nuclear) were analyzed by Western blotting. EMT-related ( E ) gene and ( F ) protein expression after treatment of ST8SIA1 -knockdown hNECs with TNF-α. mRNA and protein expression levels were normalized to that of the housekeeping gene β-actin. ( G ) Wound healing assay. ST8SIA1-knockdown hNECs were placed on 6-well plate and wounded by a blue pipette tip and then treated with 20 ng/mL of TNF-α for 24 h. Scale bar 200 μm Expression levels are presented as the mean ± SD from three independent experiments and analyzed using Student’s t -test. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with siNC or siST8SIA1 directly transfected hNECs. EMT, epithelial-to-mesenchymal transition; hNECs, human nasal epithelial cells; ST8SIA1, ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase; Cyt., cytoplasmic; Nuc., nuclear.

Article Snippet: Primary human nasal epithelial cells (hNECs) from healthy donors were purchased from PromoCell (Heidelberg, Germany). hNECs were cultured in commercially available airway epithelial cell growth medium with supplements (PromoCell, Catalog No. C-21060, Heidelberg, Germany) in a 75 cm 2 T-flask in a humidified incubator at 37 °C in 5% CO 2 .

Techniques: Knockdown, Expressing, High Performance Thin Layer Chromatography, Western Blot, Wound Healing Assay, Transferring, Transfection

Journal: International Journal of Molecular Sciences

Article Title: Ganglioside GD3 Regulates Inflammation and Epithelial-to-Mesenchymal Transition in Human Nasal Epithelial Cells

doi: 10.3390/ijms25074054

Figure Lengend Snippet: Effect of treatment with the ganglioside GD3 inhibitor NEU3 on TNF-α-treated hNECs. ( A ) ST8SIA1 gene expression in TNF-α-treated hNECs with or without NEU3 treatment. ( B ) High-performance thin-layer chromatography analysis of ganglioside expression in TNF-α-treated hNECs with or without NEU3 treatment. M1 and M2 are ganglioside standard markers. ( C ) mRNA expression levels of inflammation-related genes in TNF-α-treated hNECs with or without NEU3 treatment. ( D ) NF-κB/P65 (cytoplasmic and nuclear) were analyzed by Western blotting. ( E ) mRNA and ( F ) protein expression of epithelial-to-mesenchymal transition-related genes in TNF-α-treated hNECs with or without NEU3 treatment. mRNA and protein expression levels were normalized to that of the housekeeping gene β-actin. ( G ) Wound healing assay. hNECs were placed on 6-well plate and were treated with or without NEU3 (50 ng/mL) for 1 h and wounded by a blue pipette tip. And then treated with 20 ng/mL of TNF-α for 24 h. Scale bar 100 μm. Expression levels are presented as the mean ± SD from three independent experiments and analyzed using Student’s t -test. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with non-treated, TNF-α-treaed or NEU3-treaed hNECs. hNECs, human nasal epithelial cells; ST8SIA1 , ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase; NEU3, neuraminidase 3; Cyt., cytoplasmic; Nuc., nuclear.

Article Snippet: Primary human nasal epithelial cells (hNECs) from healthy donors were purchased from PromoCell (Heidelberg, Germany). hNECs were cultured in commercially available airway epithelial cell growth medium with supplements (PromoCell, Catalog No. C-21060, Heidelberg, Germany) in a 75 cm 2 T-flask in a humidified incubator at 37 °C in 5% CO 2 .

Techniques: Expressing, High Performance Thin Layer Chromatography, Western Blot, Wound Healing Assay, Transferring

Journal: Bioengineered

Article Title: Exosomal lncRNA Nuclear Paraspeckle Assembly Transcript 1 (NEAT1)contributes to the progression of allergic rhinitis via modulating microRNA-511/Nuclear Receptor Subfamily 4 Group A Member 2 (NR4A2) axis

doi: 10.1080/21655979.2021.1982313

Figure Lengend Snippet: NEAT1 expression is elevated in nasal mucosal tissues from AR patients and positively correlated to IL-13 stimulation. (a) RT-qPCR showed NEAT1 expression levels in mucosal tissues from 30 patients with perennial AR and 30 patients with nonallergic rhinitis (NAR) were measured. (b) RT-qPCR showed NEAT1 expression in the AR-EXO and control-EXO. (c) Correlation of lncRNA NEAT1 expression with TNSS score. (d) RT-qPCR showed NEAT1 expression in human HNECs treated with 50 ng/mL IL-13 for 12, 24, or 48 h. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: Primary human nasal epithelial cells (HNECs) were purchased from iCell Bioscience (Shanghai, China), and cultured in RPMI-1640 medium (Gibco, USA) supplemented with 10% FBS (Gibco, USA), 1% penicillin (Invitrogen, USA) and 1% streptomycin (Invitrogen, USA) maintained at 37°C and 5% CO 2 .

Techniques: Expressing, Quantitative RT-PCR, Control

Journal: Bioengineered

Article Title: Exosomal lncRNA Nuclear Paraspeckle Assembly Transcript 1 (NEAT1)contributes to the progression of allergic rhinitis via modulating microRNA-511/Nuclear Receptor Subfamily 4 Group A Member 2 (NR4A2) axis

doi: 10.1080/21655979.2021.1982313

Figure Lengend Snippet: NEAT1 knockdown regulates IL-13-triggered inflammatory cytokine, mucus production, and apoptosis in HNECs. (a). RT-qPCR showed the level of NEAT1 in human HNECstransfected with si-NEAT1 or si-NC. (b–g) RT-qPCR and ELISA assay showed the expression levels of GM-CSF, eotaxin-1, and MUC5AC in IL-13-stimulated HNECs transfected with si-NEAT1 or si-NC. (h and i) CCK-8 and TUNEL assays indicated the cell viability and apoptosis in IL-13-treated HNECs transfected with si-NEAT1 or si-NC. *P < 0.05, **P < 0.01

Article Snippet: Primary human nasal epithelial cells (HNECs) were purchased from iCell Bioscience (Shanghai, China), and cultured in RPMI-1640 medium (Gibco, USA) supplemented with 10% FBS (Gibco, USA), 1% penicillin (Invitrogen, USA) and 1% streptomycin (Invitrogen, USA) maintained at 37°C and 5% CO 2 .

Techniques: Knockdown, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing, Transfection, CCK-8 Assay, TUNEL Assay

Journal: Bioengineered

Article Title: Exosomal lncRNA Nuclear Paraspeckle Assembly Transcript 1 (NEAT1)contributes to the progression of allergic rhinitis via modulating microRNA-511/Nuclear Receptor Subfamily 4 Group A Member 2 (NR4A2) axis

doi: 10.1080/21655979.2021.1982313

Figure Lengend Snippet: Extracellular NEAT1 was transferred via incorporation in exosomes in HNECs. (a) The expression of NEAT1 was detected by RT-qPCR after cells were treated with RNase A or RNase A + 0.1% Triton X100 for 30 min. (b) The exosomes images secreted by IL-13-treated HNECs were showed by TEM scanning. (c) Size distribution of exosomes ranged from 30 to 120 nm. (d) The levels of exosomal marker proteins CD9 and CD63 were measured by Western blot in HNECs. (e) RT-qPCR analysis showed the expression of NEAT1 in exosomes extracted from IL-13-treated HNECs cells. **P < 0.01

Article Snippet: Primary human nasal epithelial cells (HNECs) were purchased from iCell Bioscience (Shanghai, China), and cultured in RPMI-1640 medium (Gibco, USA) supplemented with 10% FBS (Gibco, USA), 1% penicillin (Invitrogen, USA) and 1% streptomycin (Invitrogen, USA) maintained at 37°C and 5% CO 2 .

Techniques: Expressing, Quantitative RT-PCR, Marker, Western Blot

Journal: Bioengineered

Article Title: Exosomal lncRNA Nuclear Paraspeckle Assembly Transcript 1 (NEAT1)contributes to the progression of allergic rhinitis via modulating microRNA-511/Nuclear Receptor Subfamily 4 Group A Member 2 (NR4A2) axis

doi: 10.1080/21655979.2021.1982313

Figure Lengend Snippet: NEAT1 silence attenuates the exosome-induced inflammatory response and apoptosis of HNECs. (a) RT-qPCR showed NEAT1 level in HNECs treated with PBS, exosome, exosome+si-NC, and exosome+si-NEAT1. (b–g) RT-qPCR and ELISA assay showed the mRNA expression levels of GM-CSF, eotaxin-1, and MUC5AC in HNECs treated with exosome, exosome+si-NC, and exosome+si-NEAT1. (h and i) CCK-8 and TUNEL assays indicated cell viability and apoptosis in different groups. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: Primary human nasal epithelial cells (HNECs) were purchased from iCell Bioscience (Shanghai, China), and cultured in RPMI-1640 medium (Gibco, USA) supplemented with 10% FBS (Gibco, USA), 1% penicillin (Invitrogen, USA) and 1% streptomycin (Invitrogen, USA) maintained at 37°C and 5% CO 2 .

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing, CCK-8 Assay, TUNEL Assay

Journal: Bioengineered

Article Title: Exosomal lncRNA Nuclear Paraspeckle Assembly Transcript 1 (NEAT1)contributes to the progression of allergic rhinitis via modulating microRNA-511/Nuclear Receptor Subfamily 4 Group A Member 2 (NR4A2) axis

doi: 10.1080/21655979.2021.1982313

Figure Lengend Snippet: NEAT1 acts as a molecule sponge for miR-511. (a) The putative binding sites between NEAT1 and miR-511 were predicted by starBase website. (b) The luciferase activity of NEAT1-WT and NEAT1-Mut were measured in HNECs transfected with NC mimics, miR-511 mimics, NC inhibitor, or miR-511 inhibitor. (c) Correlation analysis between miR-511 and NEAT1 in nasal mucosal tissues from AR patients. (d) The expression of miR-511 was detected by RT-qPCR. (e) RT-qPCR showed miR-511 expression in HNECs transfected with si-NEAT1 or si-NC. **P < 0.01

Article Snippet: Primary human nasal epithelial cells (HNECs) were purchased from iCell Bioscience (Shanghai, China), and cultured in RPMI-1640 medium (Gibco, USA) supplemented with 10% FBS (Gibco, USA), 1% penicillin (Invitrogen, USA) and 1% streptomycin (Invitrogen, USA) maintained at 37°C and 5% CO 2 .

Techniques: Binding Assay, Luciferase, Activity Assay, Transfection, Expressing, Quantitative RT-PCR

Journal: Bioengineered

Article Title: Exosomal lncRNA Nuclear Paraspeckle Assembly Transcript 1 (NEAT1)contributes to the progression of allergic rhinitis via modulating microRNA-511/Nuclear Receptor Subfamily 4 Group A Member 2 (NR4A2) axis

doi: 10.1080/21655979.2021.1982313

Figure Lengend Snippet: miR-511 is a downstream regulator of NEAT1 in AR. (a) RT-qPCR showed miR-511 expression in HNECs transfected with si-NC, si-NEAT1, and si-NEAT1+miR-511 inhibitor. (b–g) RT-qPCR and ELISA assay showed the expression levels of GM-CSF, eotaxin-1, and MUC5AC in IL-13-treated HNECs transfected with si-NC, si-NEAT1, and si-NEAT1+ miR-511 inhibitor. (h and i) CCK-8 and TUNEL assays indicated the cell viability and apoptosis in IL-13-treated HNECs transfected with si-NC, si-NEAT1, and si-NEAT1+ miR-511 inhibitor. *P < 0.05, **P < 0.01

Article Snippet: Primary human nasal epithelial cells (HNECs) were purchased from iCell Bioscience (Shanghai, China), and cultured in RPMI-1640 medium (Gibco, USA) supplemented with 10% FBS (Gibco, USA), 1% penicillin (Invitrogen, USA) and 1% streptomycin (Invitrogen, USA) maintained at 37°C and 5% CO 2 .

Techniques: Quantitative RT-PCR, Expressing, Transfection, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, TUNEL Assay

Journal: Bioengineered

Article Title: Exosomal lncRNA Nuclear Paraspeckle Assembly Transcript 1 (NEAT1)contributes to the progression of allergic rhinitis via modulating microRNA-511/Nuclear Receptor Subfamily 4 Group A Member 2 (NR4A2) axis

doi: 10.1080/21655979.2021.1982313

Figure Lengend Snippet: NR4A2 is a target of miR-511. (a) The putative binding sites between NR4A2 and miR-511 were predicted by TargetScan. (b) The luciferase activity of NR4A2-WT and NR4A2-Mut were measured in HNECs cells transfected with NC mimics or miR-511 mimics. (c) RT-qPCR showed NR4A2 expression in HNECs transfected with NC mimics, miR-511 mimics, NC inhibitor, or miR-511 inhibitor. (d) Correlation analysis between miR-511 and NR4A2 in nasal mucosal tissues from AR patients. **P < 0.01

Article Snippet: Primary human nasal epithelial cells (HNECs) were purchased from iCell Bioscience (Shanghai, China), and cultured in RPMI-1640 medium (Gibco, USA) supplemented with 10% FBS (Gibco, USA), 1% penicillin (Invitrogen, USA) and 1% streptomycin (Invitrogen, USA) maintained at 37°C and 5% CO 2 .

Techniques: Binding Assay, Luciferase, Activity Assay, Transfection, Quantitative RT-PCR, Expressing

Journal: Bioengineered

Article Title: Exosomal lncRNA Nuclear Paraspeckle Assembly Transcript 1 (NEAT1)contributes to the progression of allergic rhinitis via modulating microRNA-511/Nuclear Receptor Subfamily 4 Group A Member 2 (NR4A2) axis

doi: 10.1080/21655979.2021.1982313

Figure Lengend Snippet: NEAT1 regulates IL-13-induced dysfunction of HNECs via miR-511/NR4A2 axis. (a) NR4A2 expression was detected by RT-qPCR in HNECs transfected with NC inhibitor, miR-511 inhibitor, miR-511 inhibitor+si-NR4A2. (b–g) The levels of GM-CSF, eotaxin-1, and MUC5AC were determined by RT-qPCR and ELISA in IL-13-treated HNECs transfected with NC inhibitor, miR-511 inhibitor, miR-511 inhibitor + si-NR4A2. (h and i) TUNEL and CCK-8 assays indicated the cell apoptosis and viability in IL-13-treated HNECs transfected with NC inhibitor, miR-511 inhibitor, miR-511 inhibitor + si-NR4A2. (j) NR4A2 expression was measured by RT-qPCR in HNECs transfected with pcDNA3.1, pcDNA3.1-NEAT1, pcDNA3.1-NEAT1+ miR-511 mimics. (k) Correlation analysis between NEAT1 and NR4A2 in nasal mucosal tissues from AR patients. *P < 0.05, **P < 0.01

Article Snippet: Primary human nasal epithelial cells (HNECs) were purchased from iCell Bioscience (Shanghai, China), and cultured in RPMI-1640 medium (Gibco, USA) supplemented with 10% FBS (Gibco, USA), 1% penicillin (Invitrogen, USA) and 1% streptomycin (Invitrogen, USA) maintained at 37°C and 5% CO 2 .

Techniques: Expressing, Quantitative RT-PCR, Transfection, Enzyme-linked Immunosorbent Assay, TUNEL Assay, CCK-8 Assay