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. 2018 Aug 16;13(8):e0202233. doi: 10.1371/journal.pone.0202233

Fig 4. SKN-1 activation is dependent on the p38 MAPK pathway in response to the pathogens.

Fig 4

(A) Representative images of the localization of SKN-1B/C::GFP in worms exposed to the WT, ΔspxB mutant and ΔspxB;spxB+ complement strains of S. gordonii. Close-ups are shown in the upper right-hand corner of each image. (B) The degree of nuclear localization of SKN-1B/C::GFP and the percentage of worms in each category fed on WT, ΔspxB mutant and ΔspxB;spxB+ complement strains of S. gordonii. A total of more than 100 worms exposed to each strain were imaged and the experiment was repeated 3 times. Significantly lower levels of nuclear localization of SKN-1B/C::GFP was observed in the ΔspxB mutant (P<0.0001) compared to the WT and ΔspxB;spxB+ complement strains of S. gordonii. (C) Representative images of the localization of SKN-1B/C::GFP in nsy-1, sek-1, pmk-1, skn-1 knockdown and vector control treated worms on S. gordonii. Close-ups are shown in the upper right-hand corner of each image. (D) The degree of SKN-1B/C::GFP nuclear localization and the percentage of worms in each category fed on nsy-1, sek-1, pmk-1, skn-1 knockdown and vector control treated worms on S. gordonii. A total of more than 100 worms exposed to each strain were imaged and the experiment was repeated 3 times. Significantly lower levels of nuclear localization of SKN-1B/C::GFP was observed in the nsy-1(P<0.01), sek-1 (P<0.001), pmk-1 (P<0.0001), skn-1 knockdown (P<0.0001) compared to the vector control treated worms on S. gordonii. (E) Representative images of gcs-1::gfp expression in nsy-1, sek-1, pmk-1, skn-1 knockdown and vector control treated worms on S. gordonii. (F) The level of gcs-1::gfp expression and the percentage of worms in each category fed on nsy-1, sek-1, pmk-1, skn-1 knockdown and vector control treated worms on S. gordonii. A total of more than 100 worms exposed to each strain was imaged and the experiment was repeated 3 times. Significantly lower levels of gcs-1::gfp expression was observed in the nsy-1(P<0.001), sek-1 (P<0.0001), pmk-1 (P<0.0001), skn-1 knockdown (P<0.0001) compared to the vector control treated worms on S. gordonii. (G) Western blot was used to analyze the level of phosphorylation of PMK-1 in N2, nsy-1, sek-1 and pmk-1 mutant worms exposed to S. gordonii for 2 hours. Phosphorylation of PMK-1 was partial reduced in nsy-1 mutant worms, while in the sek-1 mutant worms phospho-p38 was completely absent. pmk-1 mutant worms was used as a negative control and α-tubulin as a loading control. (H) Survival of N2 and nsy-1(ok593), sek-1(km4) and pmk-1(km25) IV L4 larvae when fed on THY plates containing S. gordonii. The data are representative of experiments repeated two or more times with an n = 60–90 worms for each condition. Kaplan-Meier log rank analysis was used to compare survival curves and to calculate the median survival. P-values <0.05 were considered to be statistically significant.