(A,B) Quantification of relative dJNK phosphorylation in S2 cells incubated with 15,683 distinct dsRNAs prior to PGN exposure for 15min (A) or 60min (B). The relative P-JNK:f-actin z-score was determined for each dsRNA. The red dashed lines represent z-score values of + or − 2.58. Key was identified as the strongest suppressor of dJNK phosphorylation at 15min and 60min PGN exposure (A,B), whereas Tak1 was identified as the strongest enhancer of dJNK phoshorylation at 15min PGN exposure (B). (C) dsRNAs that modified dJNK phosphorylation in response to PGN were grouped according to biological functions. The biological functions for enhancers of dJNK phosphorylation with a z-score below −2.58 at 15min PGN exposure are presented (left panel). Additionally, the biological functions for suppressors of dJNK phosphorylation with a z-score above 2.58 at 15min and 60min PGN-exposure are presented (right panel). In addition to recognizable groups, the dsRNA screen identified a large number of Drosophila genes with unknown biological functions as modifiers of PGN-induced dJNK phosphorylation. (D) Heat map of z-score values for S2 cells depleted of known Imd pathway components and exposed to PGN for 15min or 60min. Fifteen core IMD pathway components were identified in the screen as either suppressors (z-scores above 1.96) or enhancers (z-scores below −1.96) of PGN-induced dJNK phosphorylation respectively.

(A) Heat map analysis of known Pvr pathway genes compared to known wg pathway genes at 15 min PGN-exposure. 15min PGN-induced dJNK:f-actin z-scores were ordered from highest to lowest and organized according to percentile intervals. Genes colored more red indicate suppressors of PGN-induced dJNK phosphorylation while genes colored more green indicate enhancers of PGN-induced dJNK phosphorylation. Pvr pathway components were consistently identified as suppressors of PGN-induced dJNK phosphorylation. (B) Quantitative real-time PCR analysis of standardized pvf1,2 and 3 expression in S2 cells treated with PGN for the indicated times. The uninduced expression levels for pvf1,2 and 3 were given values of 1 and the remaining pvf1,2 and 3 expression values are reported relative to these values. Data are presented as mean of three independent experiments and error bars indicate the + SEM. Significant differences in expression values are indicated (** = p-value <0.01). (C) Quantitative real-time PCR analysis of standardized pvf1,2 and 3 expression in S2 cells or S2 cells treated with SP600125. S2 cells were incubated with SP600125 and PGN as indicated. The uninduced expression levels for pvf1,2 and 3 were given values of 1 and the remaining pvf1,2 and 3 expression values are reported relative to these values. Data are presented as mean of three independent experiments and error bars indicate the + SEM. Significant differences in P-JNK values are indicated (* = p-value <0.05, ** = p-value <0.01). (D) Quantitative real-time PCR analysis of standardized pvf2 expression in S2 cells incubated with GFP or PGRP-LC dsRNA and treated with PGN as indicated. pvf2 expression levels of unstimulated S2 cells treated with GFP dsRNA were assigned a value of 1 and the remaining pvf2 expression values are reported relative to these values. Data are presented as mean of three independent experiments and error bars indicate the + SEM. Significant differences in P-JNK values are indicated ( ** = p-value <0.01). (E) Quantitative real-time PCR analysis of standardized pvf2 expression in S2 cells incubated with GFP or a combination of dMKK4/7 dsRNA and treated with PGN as indicated. pvf2 expression levels of unstimulated S2 cells treated with GFP dsRNA were assigned a value of 1 and the remaining pvf2 expression values are reported relative to these values. Data are presented as mean of three independent experiments and error bars indicate the + SEM. Significant differences in P-JNK values are indicated ( * = p-value <0.05).

(A) Western blot analysis of lysates from S2 cells incubated with GFP dsRNA (lane 1–2) or Pvr dsRNA (lanes 3–4). S2 cells were exposed to KC167 cell conditioned media (CM) where indicated to induce MAPK phosphorylation. Lysates were probed with antibodies specific for P-MAPK (top panel), and actin (bottom panel). Data is representative of three individual experiments. (B) Relative quantification of P-MAPK levels from (A). P-MAPK levels were standardized to actin levels for each treatment group. The unstimulated GFP dsRNA treated P-MAPK:actin value was given a value of 1 and the remaining P-MAPK:actin values are reported relative to this value. (C) Quantitative real-time PCR analysis of att expression in S2 cells incubated with GFP dsRNA (columns 1–3) or Pvr dsRNA (columns 4–5). S2 cells were treated with KC167 CM, and PGN as indicated. att expression levels in unstimulated S2 cells treated with GFP dsRNA were assigned a value of 1 and the remaining att expression values are reported relative to these values. Data are presented as the mean of three independent experiments and error bars indicate + SEM. The significance of CM treatment on att expression relative to the untreated samples is indicated (* = p-value <0.05). CM does not suppress att expression in the absence of Pvr. (D) Western blot analysis of lysates from S2 cells treated with the MEK1 inhibitor PD98059 followed by exposure to PGN, and KC167 CM as indicated. Lysates were probed with anti-P-MAPK (top panel), and anti-actin (bottom panel) antibodies. (E) Relative quantification of P-MAPK levels in panel D. P-MAPK levels were standardized to actin levels for each treatment group. The untreated P-MAPK:actin value was given a value of 1 and the remaining P-MAPK:actin values are reported relative to this value. (F) Quantitative real-time PCR analysis of att expression levels in S2 cells (column 1–3) or S2 cells treated with PD98059 (column 4). S2 cells were incubated with KC167 CM, and PGN as indicated. att expression levels in unstimulated S2 cells were assigned a value of 1 and the remaining att expression values are reported relative to these values. Data are presented as the mean of three independent experiments and error bars indicate + SEM. Inhibition of MEK1 activation with PD98059 significantly restored att expression in response to KC167 CM.

(A) Representative Western blot analysis of S2 cell lysates treated with PGN for the indicated period. Lysates were probed with anti-P-JNK (top panel) and anti-actin (middle panel). To visualize relative dJNK phosphorylation P-JNK (green) and actin (red) channels were false colored and merged (bottom panel). (B) Quantification of relative dJNK phosphorylation in (A). dJNK phosphorylation levels were quantified and reported relative to actin levels for each of the indicated time points. (C) Schematic representation of a quantitative RNAi screen for modifiers of PGN-induced dJNK phosphorylation. S2 cells were incubated with dsRNA in 96 well plates prior to exposure to PGN. Cells were stained with an antibody specific for P-JNK and were counterstained with fluorescently labled phalloidin to visualize filamentous actin (f-actin). JNK phosphorylation levels were quantified relative to f-actin levels. Loss of activators (enhancers) of JNK phosphorylation decreases dJNK phosphorylation. In contrast, loss of inhibitory gene products (suppressors) increases dJNK phosphorylation. Essential gene products are visible as wells with no P-JNK or f-actin staining. (D) Representative plate from screen. S2 cells incubated with 96 distinct dsRNAs were treated with PGN for 15min. Cells were stained for P-JNK with monoclonal antibodies specific for P-JNK (left) and counterstained for f-actin (center). To visualize relative P-JNK levels, P-JNK (green) and f-actin (red) channels were false colored and merged (right). (E) Quantification of relative dJNK phosphorylation levels from panel D. Raw dJNK phosphorylation values were graphed against raw f-actin values. Red dashed lines indicate + or − 2.58 standard deviations from the median for both P-JNK and f-actin values. dsRNA targeting the established JNK modifiers Dredd and Cka decrease or increase dJNK phosphorylation levels respectively with no effect on f-actin levels. (F) Statistical analysis of PGN-induced dJNK phosphorylation relative to f-actin from panel E. P-JNK values were standardized to f-actin values for each of the 96 dsRNA treatments. The red dashed lines represent z-score values of + or − 2.58. dsRNA that targeted Cka and Dredd were identified as significant modifiers of PGN-induced dJNK phosphorylation.

(A,B) Quantification of PGN-induced dJNK phosphorylation relative to f-actin (A) or total JNK (B). S2 cells were incubated with the indicated dsRNAs and exposed to PGN at 0min, 15min or 60min as indicated. Key and dTak1 dsRNA were used as modifier dsRNA controls, whereas Toll and CG11318 dsRNA were used as non-modifier dsRNA controls. Cells were stained with anti-P-JNK antibody and dJNK phosphorylation was standardized to f-actin (A) and total dJNK (B). Data are presented as the mean of two independent experimental values and error bars indicate + SEM. The grey dashed line represents the mean dJNK phosphorylation value for Toll dsRNA and dsRNAs that significantly modulated dJNK phosphorylation from this value are indicated (* = p-value <0.05, ** = p-value <0.01). Secondary dsRNA analysis recapitulates the dJNK phosphorylation values from the primary screen. (C) A partial genetic and physical interaction network of dJNK phosphorylation modifiers. Modulators of dJNK phosphorylation with z-scores greater than 1.96 or less than −1.96 were grouped in an interaction network using known genetic and physical interaction databases. Within this network, Pvr (black circle) and dJnk (white circle) are connected directly and through a number of intermediate genes (yellow circles). The Pvr and dJnk interaction network also connects to IMD pathway (blue circles). (D) Quantitative Western blot analysis of lysates from S2 cells treated with either Pvr or GFP dsRNA. Lysates were probed with anti-Pvr (top blot) and anti-actin (bottom blot) antibodies. Pvr levels were quantified relative to actin (top graph). Data are presented as mean of three independent experiments and error bars indicate + SEM. Both Pvr dsRNA molecules deplete Pvr in S2 cells. (E) Quantification of PGN-induced dJNK phosphorylation. S2 cells were treated with GFP dsRNA as a control or two independent non-overlapping dsRNA targeting Pvr as indicated. Cells were exposed to PGN and dJNK phosphorylation was monitored relative to total dJNK. P-JNK:JNK values at 0h PGN exposure with GFP dsRNA were assigned a value of 1 and the remaining P-JNK:JNK values are reported relative to these data. Data is expressed as the mean of two independent experiments and the error bars represent +/− SEM. Significant differences in pJNK values are indicated (* = p-value<0.05, ** = p-value<0.01). Depletion of Pvr increases PGN-induced dJNK phosphorylation at 15min, indicating that Pvr negatively regulates dJNK activation in the IMD pathway.

(A) Quantitative real-time PCR analysis of standardized att expression in S2 cells incubated with either GFP or two distinct Pvr dsRNA. S2 cells were treated with PGN where indicated. att expression levels of unstimulated S2 cells treated with GFP dsRNA were assigned a value of 1 and the remaining att expression values are reported relative to these values. Data are presented as mean of two independent experiments and error bars indicate the + SEM. (B) Quantitative real-time PCR analysis of standardized dipt expression in S2 cells incubated with either GFP or two distinct Pvr dsRNA. S2 cells were treated with PGN for 6h where indicated. dipt expression levels of unstimulated S2 cells treated with GFP dsRNA were assigned a value of 1 and the remaining dipt expression values are reported relative to these values. Data are presented as mean of two independent experiments and error bars indicate + SEM. (C) Quantitative real-time PCR analysis of standardized att expression in S2 cells incubated with GFP or Pvr dsRNA in combination with Rel dsRNA. S2 cells were treated with the indicated dsRNAs and unstimulated or stimulated with PGN for 6h as indicated. att expression levels of unstimulated S2 cells treated with GFP dsRNA were assigned a value of 1 and the remaining att expression values are reported relative to these values. Data are presented as mean of three independent experiments and error bars indicate the + SEM. Significant differences in att expression values are indicated with a p-value. (D) Quantitative real-time PCR analysis of standardized att expression in S2 cells incubated with GFP or Ras85D dsRNA and treated with PGN as indicated. att expression levels of unstimulated S2 cells treated with GFP dsRNA were assigned a value of 1 and the remaining att expression values are reported relative to these values. Data are presented as mean of two independent experiments and error bars indicate the + SEM. (E) Western blot analysis of lysates from S2 cells incubated with GFP dsRNA (lanes 1–6) or Pvr dsRNA (lanes 7–12). S2 cells were untreated or treated with PGN in increasing ten-fold gradations of LPS from 5×10⁻⁴µg/ml to 5µg/ml. Lysates were probed with anti-Rel (top panels), anti-P-JNK (middle) and anti-JNK (bottom panel). (F) Western blot analysis of lysates from S2 cells incubated with Pvr dsRNA (lane 1–6) or GFP dsRNA (lanes 7–12) and treated with PGN for the indicated period. Lysates were probed with anti-P-Rel (top panel), anti-P-JNK (middle) and anti-JNK (bottom panel).

Quantitative real-time PCR analysis of att expression in hs-gal4/+ control flies (columns 1–2), hs-gal4/uas-Pvr-IR1 flies (columns 3–4) and hs-gal4/+; uas-Pvr-IR2/+ flies (columns 5–6). Flies were uninfected or infected through septic injury with E. coli as indicated. att expression levels of uninfected controls were assigned a value of 1 and the remaining att expression values are reported relative to these value. Data are presented as mean of three independent experiments and error bars indicate the + SEM. Significant differences in att expression are indicated (** = p-value <0.01).