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. Author manuscript; available in PMC: 2017 Jan 15.
Published in final edited form as: J Immunol. 2015 Dec 7;196(2):655–667. doi: 10.4049/jimmunol.1501708

FIGURE 1. A Diptericin-luciferase reporter construct enables a genome-wide screen for genes that mediate hypercapnic immune suppression.

FIGURE 1

(A) The Dipt-luc reporter containing 2.2 kb of the Dipt promoter region driving firefly luciferase (23) in S2* cells closely parallels expression of the endogenous Dipt locus in air and in neutral hypercapnia (13% CO2, pH 7.1: 5 h in 13% CO2, then 5 h PGN in 13% CO2). A reporter containing the promoter for the PolIII 128 subunit gene driving Renilla luciferase (25) is not responsive to CO2 and was used to calculate normalized activity of the Dipt-luc reporter. Dipt mRNA levels were assessed using qPCR normalized to RP49 mRNA levels.

(B, C) The Dipt-luc reporter closely parallels endogenous Dipt mRNA expression in PGN-stimulated S2* cells in (B) neutral mild hypercapnia (pH 7.1; 5 h in 5% CO2, then 5 h PGN in 5% CO2), in sustained neutral hypercapnia (13% CO2, pH 7.1: 19 h in 13% CO2, then 5 h PGN in 13% CO2) and (C) over an 8-h time course of PGN treatment (13% CO2, pH 7.1: 5 h pre-exposure in 13% CO2).

(D) dsRNAs targeting bsk (Drosophila JNK) or carbonic anhydrases, do not up-regulate Dipt-luc in elevated CO2 (results for additional candidate genes in pilot screening shown in Supplemental Table SI).

(E) Workflow for the genome-wide screen to identify genes that mediate hypercapnic immune suppression.

For all panels, **, p<0.01.