Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Endoplasmic Reticulum Stress Induced by Surfactant Protein C BRICHOS Mutants Promotes Proinflammatory Signaling by Epithelial Cells

doi: 10.1165/rcmb.2009-0382oc

Figure Lengend Snippet: Figure 3. SP-CDexon4 and SP-CL188Q induce IL-8 cytokine release. A549 cells were transiently transfected with the indicated EGFP-tagged SP-C WT or mutant construct, using Lipofectamine 2000 reagent (Invitro- gen). Untreated (UT) cells were used as a negative control sample, and overnight treatment of 10 ng/ml TNFa was used for a positive control sample. Media were collected 48 hours after transfection, and IL-8 was measured by ELISA, as described in MATERIALS AND METHODS. Data are expressed as mean IL-8 concentration 6 SD of triplicate samples, and are representative of three separate experiments. The intra-assay coefficients of variation (%CV) for the three experiments were 2.7%, 3.0%, and 2.1%, whereas the interassay %CV was 12.2%. *P , 0.05 and 1P , 0.05 versus WT control.

Article Snippet: Media collected 12–48 hours after transfection were used for the determination of released IL-8, measured using the DuoSet Human IL-8 ELISA Assay according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN).

Techniques: Transfection, Mutagenesis, Construct, Negative Control, Positive Control, Enzyme-linked Immunosorbent Assay, Concentration Assay, Intra Assay, Control

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Endoplasmic Reticulum Stress Induced by Surfactant Protein C BRICHOS Mutants Promotes Proinflammatory Signaling by Epithelial Cells

doi: 10.1165/rcmb.2009-0382oc

Figure Lengend Snippet: Figure 7. 4–Phenylbutyric acid (4-PBA) blocks NFkB activation, but not the release of IL-8. (A) The A549–NFkB–luc cell line was transiently transfected with the indicated control or SP-C constructs. pRL-SV40 was transfected into all samples as an internal control to normalize transfection efficiencies. Eight hours after transfection, increasing concentrations of 4-PBA (PBA) were added to SP-CDexon4–transfected cells, and overnight treatment with 10 ng/ml TNF-a was used for a positive control sample. Cells were harvested and lysed 48 hours after transfection. Firefly and Renilla luciferase activities were measured using luciferin substrates, as in Figure 5. The ratio of mean Firefly to Renilla luciferase was determined, and expressed as fold change over WT control. Graph represents the mean 6 SD of triplicate experiments. (B) A549 cells were transiently transfected with the indicated control or EGFP–SP-C mutant constructs using Lipofect- amine 2000 reagent. Eight hours after transfection, increasing concentra- tions of 4-PBA, or a combination of 5 mM PBA and 10 mM SP600125, was added to SP-CDexon4 transfected cells, as indicated. Media were collected 48 hours after transfection, and IL-8 was measured by ELISA as described in MATERIALS AND METHODS. Graph represents the mean 6 SD of triplicate samples, and is representative of three separate experiments. The intra- assay %CVs for the three experiments were 1.4%, 3.4%, and 1.6%, whereas the interassay %CV was 2.3%. *P , 0.05 versus WT control. fP , 0.05 versus SP-CDexon4 sample. 1P , 0.0001 versus WT control.

Article Snippet: Media collected 12–48 hours after transfection were used for the determination of released IL-8, measured using the DuoSet Human IL-8 ELISA Assay according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN).

Techniques: Activation Assay, Transfection, Control, Construct, Positive Control, Luciferase, Mutagenesis, Enzyme-linked Immunosorbent Assay, Intra Assay

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Endoplasmic Reticulum Stress Induced by Surfactant Protein C BRICHOS Mutants Promotes Proinflammatory Signaling by Epithelial Cells

doi: 10.1165/rcmb.2009-0382oc

Figure Lengend Snippet: Figure 6. IL-8 release induced by SP-C BRICHOS mutants is blocked by the inhibition of JNK. A549 cells were transiently transfected with the indicated control or HA-tagged SP-C mutant construct, using Lipofect- amine 2000 reagent. Twenty-four hours after transfection, 10 mM of the JNK-specific inhibitor SP600125 was added to the indicated samples. Overnight treatment with 10 ng/ml TNFa was used for a positive control sample. Media were collected 48 hours after trans- fection (18 hours after TNF-a treatment), and IL-8 was measured by ELISA, as described in MATERIALS AND METHODS. Data are expressed as mean IL-8 concentration 6 SD of triplicate samples, and are represen- tative of three separate experiments. The intra-assay %CVs for the three experiments were 2.1%, 1.5%, and 1.6%, whereas the interassay %CV was 6.0%. *P , 0.05 versus WT control. fP , 0.05 versus SP-CDexon4

Article Snippet: Media collected 12–48 hours after transfection were used for the determination of released IL-8, measured using the DuoSet Human IL-8 ELISA Assay according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN).

Techniques: Inhibition, Transfection, Control, Mutagenesis, Construct, Positive Control, Enzyme-linked Immunosorbent Assay, Concentration Assay, Intra Assay