Supplementary Figure 3: Physiological and immunological consequences of rLC loss of function.
From: The regulatory isoform rPGRP-LC induces immune resolution via endosomal degradation of receptors
(a) eGFP mRNA expression (representing LC transcripts from P[acman] constructs only) shows that deletion of exon 5 from the engineered P[acman]-GFP -LC locus does not affect PGRP-LC expression in cis. Tested on unchallenged flies (initial insertion lines, carrying a wild-type PGRP-LC locus on chromosome 3, plus one genomic copy of P[acman]-GFP-LC at the attP landing site 51C on chromosome 2).
(b) Quantification of adults hatching per female per 24h egg lay shows that reproductive output is not significantly affected in rLC loss-of-function flies (resc(LCΔex5)) compared to WT or resc(LCwt) flies. Each dot represents counts from one fly vial.
(c) Quantification of Diptericin (Dpt) mRNA expression (relative to ribosomal (RpL32) mRNA) in flies infected with Erwinia carotovora carotovora strain 15 (Ecc15) shows that rLCx is necessary and sufficient for resolution of IMD activation, and that rLCx-mediated regulation is dose-dependent. Efficiency of regulation correlated with genomic copy number of rLC (compare efficiency of rescue constructs in the ΔLCE12 background, which has zero genomic rLC copies, versus rescue in the ΔLCird7(1) background, which has two genomic rLC copies). See also Fig. 2d, e for a visual explanation of genotypes.
(d) Quantification of Dpt mRNA expression in dissected tissues of unchallenged flies shows that loss of rLC has no impact on PGRP-LE-dependent basal IMD activation by commensals in the midgut of conventionally reared flies, but leads to increased immune activation in whole guts where PGRP-LC contributes to sensing (see also Fig. 1d for receptor expression levels). Loss of rLC did not cause any significant changes in baseline IMD activation in the fat body of conventionally reared but uninfected flies.
(e) Quantification of Dpt mRNA expression over time in in flies with (resc(LCwt)) or without (resc(LCΔex5)) rLC or controls (WT = w1118 and ΔLCE12) infected with heat-killed E.coli (see also Fig. 3b).
(f) Survival of rLC overexpressing flies (c564-Gal4 > UAS-PGRP-LCx) compared to controls (c564-Gal4) infected with Ecc15.
NS P > 0.05, * P < 0.05, **P < 0.01, *** P < 0.001 (Student’s t test (a), one-way ANOVA, Tukey’s post hoc test (b, c), two-way ANOVA, Bonferroni post hoc test (d, e); in f, P = 0.0002 (log-rank test). Data are pooled from three (b, c, d, e) or four (a, f) independent experiments and represent mean ± s.e.m (a, b, c, d, e).
