human nasal epithelial cells hnecs primary hnecs  (PromoCell)

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