ORMDL3 regulates poly I:C induced inflammatory responses in airway epithelial cells

Figures

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  Fig.1. TLR levels in ORMDL3 knockdown and over-expressing A549 cells. A549 cells were seeded at 40,000 cell/per well in 24 well culture plate. a Western Blotting images of ORMDL3 expression in knockdown and over-expressing A549 cells. Full-length blots were presented in Additional file 1: Western Bollting Images of ORMDL3. b 25 nM siRNA control and ORMDL3 siRNA were added into the cell culture medium. The cells were cultured for 48 h. c 250 ng/ml pcDNA3.1-ORMDL3 plasmid and control plasmid were added into the cell culture medium. Transcript levels of ORMDL3, TLR3 were quantified by qPCR and with GAPDH as internal control. ΔΔCT were compared with siRNA control samples. Student’s t test was calculated. ***Indicates P < 0.001. Each group contained triplicated experiments

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  Fig.2. Inflammatory response induced by poly I:C in A549 cells. A549 cells were seeded with full medium for 24 h, then changed to serum-free medium for 1 day. Poly I:C was added to the wells at different concentrations. Supernatants were collected for measurements for IL-6 and IL8 levels after 10 h. a The IL-6 levels of poly I:C titration, b the IL-8 levels of poly I:C titration. c The IL-6 levels in ORMDL3 knockdown cells after poly I:C stimulation for 10 h. d The IL-8 levels after ORMDL3 knockdown cells after poly I:C stimulation for 10 h. e The IL-6 levels in pcDNA3.1-ORMDL3 over-expressing cells after poly I:C stimulation for 10 h. f The IL-8 levels after pcDNA3.1-ORMDL3 over-expressing cell after poly I:C stimulation for 10 h.

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  Fig.3. Time course studies in epithelial cells stimulated with poly I:C. A549 cells and NHBE cells were used for time course studies. Cells were transfected with 25 nM ORMDL3 siRNA or 250 ng/ml pcDNA3.1-ORMDL3 for 2 days, then changed to serum free medium. Cells were stimulated with 10 μg/ml poly I:C for 6, 10, 16 and 24 h. The supernatants were collected. a The IL-6 levels in ORMDL3 knockdown A549 cell samples. b The IL-8 levels in ORMDL3 knockdown A549 cell samples. c The IL-6 levels in ORMDL3 over-expressing A549 cell samples. d The IL-8 levels in ORMDL3 over-expressing A549 cell samples. e The IL-6 levels in ORMDL3 knockdown NHBE cell samples. f The IL-8 levels in ORMDL3 knockdown NHBE cell samples. g The IL-6 levels in ORMDL3 over-expressing NHBE cell samples. h The IL-8 levels in ORMDL3 over-expressing A549 cell samples. Samples were analyzed by one way ANOVA. ***Indicates P < 0.001; **indicates P < 0.01; *indicates P < 0.05, each group contained triplicated experiments

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  Fig.4. Co-stimulation with poly I:C and IL-17A in A549 cells. A549 cells were used for co-stimulations with 10 µg/ml poly I:C and 100 µg/ml IL-17A for 10 h in ORMDL3 knockdown and ORMDL3 over-expression A549 cells for 10 h. a The IL-6 levels in ORMDL3 knockdown A549 cell samples. b The IL-8 levels in ORMDL3 knockdown A549 cells. c IL-6 levels in ORMDL3 over-expressing A549 cells. d IL-8 levels in ORMDL3 over-expressing A549 cell samples. Samples were analyzed by one way ANOVA. ***Indicates P < 0.001; ** indicates P < 0.01; *indicates P < 0.05, each group contained triplicated experiments. PIC: poly I:C, Co-stim: co-stimulation of poly I:C and IL-17A, No-stim: no-stimulation

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  Fig. 5. Gene transcript levels in ORMDL3 knockdown and over-expression before and after poly I:C stimulation. ORMDL3 knockdown or over-expression A549 cells and NHBE cells were stimulated with 10 μg/ml poly I:C for 24 h. RNAs were extracted and qPCRs were performed. a Gene transcript levels at ORMDL3 knockdown cells and control cells. b Gene transcript levels at ORMDL3 over-expressing cells and control cells. c Transcript levels at ORMDL3 knockdown cells and control cells after 24 h stimulation with poly I:C. d Gene transcript levels in ORMDL3 over-expressing cells and control cells after 24 h stimulations with poly I:C. ***indicates P < 0.001; **indicates P < 0.01; *indicates P < 0.05, each group contained triplicated experiments

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  Fig.6. IL-6 and IL-8 levels in ORDML3 knockdown A549 and BEAS-2B epithelial cells after HRV-16 infection. ORMDL3 knockdown A549 and BEAS-2B cells were infected with HRV-16 (5 MOI) and incubated for 48 h at 33 °C, 5% CO2. The cell-free supernatant was harvested and both IL-6 and IL-8 were measured. IL-6 and IL-8 levels in non-infection cells were utilized as comparison in each group. a IL-6 induction levels in ORMDL3 A549 cells after HRV-16 infection. b IL-8 induction levels in ORMDL3 A549 cells after HRV-16 infection. c IL-6 induction levels in ORMDL3 BEAS-2B cells after HRV-16 infection. d IL-8 induction levels in ORMDL3 BEAS-2B cells after HRV-16 infection. *Indicates P < 0.05, each group contained triplicated experiments excepting experiment A had six experiments