Fig 1.
Schematic of experimental workflow evaluating mRNA and protein expression of lung epithelial cells following LPS exposure.
a) Timeline of experiments. Two cell stocks at the same passage number were thawed and cultured. After one passage, stocks were split into six flasks each (12 total lineages). At five timepoints over 11 passages, samples were collected from each lineage. b) At each of the five time points, each lineage was seeded in two wells of a 24-well plate. One well was treated with 10 μg/mL LPS diluted in media, the other received fresh media without LPS (untreated), and all were incubated for 24 hours at 37°C with 5% CO2 prior to harvesting of mRNA and supernatant. mRNA levels were evaluated using reverse-transcription quantitative polymerase chain reaction (RT-qPCR) and protein levels were measured in supernatants using immunoassays. Figure created with BioRender.com.
Fig 2.
mRNA and protein expression profiles in response to LPS exposure.
Each column represents an individual biological replicate for (a) mRNA and (b) protein analysis. mRNA values are displayed as ΔCT values following normalization to the housekeeping gene B2M CT value (see S1 Methods in S1 File). Protein values are displayed as log2 pg/mL following normalization to negative control (see S1 Methods in S1 File). Student’s paired t-tests were performed comparing LPS-treated to untreated samples of the same plate and lineage (***p<0.001; **p<0.01; NS, not significant). IL, interleukin; CCL, chemokine ligand; CXCL, chemokine (C-X-C motif) ligand; TNFα, tumor necrosis factor alpha; IFNγ, interferon gamma; LTβ, lymphotoxin beta; CSF, colony stimulating factor.
Fig 3.
LPS exposure results in discrepancies in mRNA and protein expression profiles of key cytokines and chemokines.
(a) Fold-changes in mRNA (x-axis) versus protein (y-axis) expression comparing LPS-treated to untreated controls. Dashed line indicates equivalent RNA and protein fold-changes. (b) p-values from pairwise Student’s t-test for RNA (x-axis) versus protein (y-axis) fold-changes. Dashed lines indicate p = 0.05. Cytokines and chemokines without significant changes in expression of mRNA and/or protein were not displayed. Significance was calculated using paired Student’s t-test comparing LPS-treated cells to untreated controls from the same lineage and plate. Individual plots comparing fold-changes in cytokines and chemokines significantly upregulated in both mRNA and protein expression for (c) CCL2, (d) CSF3, (e) CXCL5, (f) CXCL8, and (g) IL6. Values represent median signal and bars represent 95% confidence interval. Significance was calculated using pairwise-Student’s t-test matching mRNA and protein fold changes from the same lineage and plate. CXCL5 values represent values obtained via ELISA data (see S1 Methods and S4 Fig). No significant difference was found between mRNA and protein levels. IL, interleukin; CCL, chemokine ligand; CXCL, chemokine (C-X-C motif) ligand; TNF, tumor necrosis factor alpha; CX3CL, (C-X3-C) chemokine ligand; CSF, colony stimulating factor.