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. 2017 Oct 12;11(5):313–322. doi: 10.1080/19336896.2017.1356559

FIGURE 1.

FIGURE 1

(next page). Aging in combination with mild chronic stress enhances abnormal distribution of RNA granule components in C. elegans pharyngeal muscles. (A) Mild stress by elevation of growth temperature from 20°C to 25°C causes abundant tagRFP::PAB-1 stress granule formation in Pmyo-2::tagRFP::PAB-1 transgenics at day 1. Representative confocal images displayed as single plane. Scale bar 7 µm. (B) Mild chronic stress highly enhances tagRFP::PAB-1 aggregation with age in Pmyo-2::tagRFP::PAB-1 transgenics (Number of animals per group and time point is indicated in the columns; Fisher's exact test 2-tailed comparing low aggregation vs. all other categories at day 2, 4 and 7, 20°C vs. 25°C, ****p < 0.0001). (C) Representative images of tagRFP::PAB-1 aggregation levels in pharyngeal muscles (maximal z-stack projection). Low, up to 10 puncta in posterior bulb; intermediate, more than 10 puncta in posterior bulb; high, more than 10 puncta in anterior bulb; very high, more than 50 puncta in anterior bulb. Scale bar 10 µm. (D) Modest increase in KIN-19::mEOS aggregation in Pkin-19::KIN-19::mEOS animals aged at 25°C compared with 20°C. (Aggregation determined in anterior pharyngeal bulb: low, up to 10 puncta; intermediate, between 10 and 100 puncta; high, over 100 puncta. Fisher's exact test 2-tailed comparing high + intermediate against low aggregation levels at day 7, 20°C vs. 25°C p < 0.0001). (E) Mild chronic stress in combination with aging causes abnormal cytoplasmic tagRFP::HRP-1 localization in aged Pmyo-2::tagRFP::HRP-1 transgenics (Fisher's exact test 2-tailed at day 7, 20°C vs. 25°C, ****p < 0.0001). (F) High-magnification representative confocal images with nuclear Hoechst33342 staining showing cytoplasmic tagRFP::HRP-1 puncta at day 7 but not day 1 of adulthood, in Pmyo-2::tagRFP::HRP-1 transgenics aged at 25°C. Scale bar 5 µm. (G) Representative confocal images showing tagRFP::PAB-1 co-localization with small cytoplasmic FUST-1::Venus puncta in double transgenic animals expressing Pmyo-2::tagRFP::PAB-1; Pkin-19::FUST-1::Venus at day 7. Arrows mark co-localization. Scale bar: 4 µm.