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Supplemental Material for Liu, Ruediger, and Shapira, 2018

posted on 2018-10-05, 15:00 authored by Limeng Liu, Cyrus Ruediger, Michael Shapira

Figure S1. Tissue-specific promoters direct expected expression patterns of a KGB-1::GFP fusion protein. Adult worms demonstrating KGB-1::GFP expression controlled by the neuronal rgef-1 promoter (A, blow-up shows localization in neuronal commissures); the intestinal gly-19 promoter (B, showing nuclear localization), and the epidermal wrt-2 promoter (C, with prominent expression in seam cells, and low level expression reported to exist in syncytium cells, but appearing to be below our detection level). Not shown is muscle expression from the myo-3 promoter, which eluded transgenic expression. Orange fluorescence is due to tdTomato expression driven by the pharyngeal myo-2 promoter.

Figure S2. Effects of tissue-specific KGB-1 activation on worm size and pigmentation. Images of day 2 adults of the designated strains following exposure (from the L4 stage) to empty RNAi vector (EV), or vhp-1 RNAi. All images were taken using identical magnification and exposure. Note the effects of vhp-1 RNAi on size and "pigmentation" (representing intestinal lipid granules) of strains expressing neuronal KGB-1 and muscle KGB-1, which is comparable to effects in wildtype animals, but lack of discernable effects in worms expressing KGB-1 in the intestine or epidermis.

Fig S3. KGB-1 expression from its endogenous promoter rescues resistance of kgb-1 mutants. Development of worms (3 days at 20ºC) of designated strains (transgenes expressed from an extrachromosomal array) grown in the presence of 50 μM cadmium (A), or 1 μg/mL tunicamycin (B). Shown are averages ± SDs for an experiment performed in duplicates, N=80-300 worms per groups (panel A is a representative of two experiments with similar results). Asterisks denote significant differences in the fraction of worms of a developmental stage compared to the respective value in wildtype animals (p<0.05, t-test).

Figure S4. Tissue-specific KGB-1 expression has similar outcomes for stress resistance in distinct transgenic integrant lines. Development (3 days at 20°C) of transgenic worms expressing KGB-1 in neurons (A) or in the intestine (B) raised on NGM plates containing 50 μM cadmium. For each tissue, results are shown for two independently-derived transgenic lines (likely with distinct integration sites). Averages ± SDs for 2 independent experiments, each performed in duplicates with >100 worms per strain per experiment. Asterisks denote significant differences in the fraction of worms of a developmental stage compared to the respective value in kgb-1 mutants (* p<0.05, *** p<0.001, paired t-test).

Figure S5. KGB-1 protects larvae form ER stress independently of canonical UPRER signaling. A. Representative images of L3 hsp-4p::gfp transgenics, in a wildtype or kgb-1 genetic background, exposed to tunicamycin for 15 hours before imaging. B. Quantification of GFP signal. Averages ± SDs of two independent experiments (n=25-45 worms per group per experiment, N total is shown on columns). Asterisks indicate significant induction of GFP as compared to respective controls (**p<0.01). C. Development (3 days at 20°C) of worms of the designated strains in the presence of 1 μg/mL tunicamycin. Average ± SDs of two experiments each performed in duplicate with a total of >100 worms per strain per experiment. Asterisks represent significant differences in the fraction of worms of a developmental stage compared to their fraction among wildtype (in black), or compared to kgb-1 single mutants (in purple)(*p<0.05, **p<0.01, paired t-test). Note that disruption of atf-6, thought to be important for regulating constitutive UPRER genes, increases stress resistance as previously reported [45], and that an additional disruption of kgb-1 additively decreases the resistance of these mutants, attesting to lack of epistasis.

Figure S6. Tissue-specific contributions of KGB-1 to target gene expression in larvae are replicated in independent transgene integrant lines. A-C. qRT-PCR measurements of gene induction in L3 larvae of the designated strains and lines, following KGB-1 activation by vhp-1 knock-down from the egg stage. Averages ± SDs for measurements from 2-6 independent experiments, each measured in duplicates. Asterisks mark significant induction following vhp-1 knock-down (*p<0.05, **p<0.01, ***p<0.001, paired t-test).

Figure S7. Cell non-autonomous contributions of neuronal KGB-1 partially require small clear vesicles, but not dense core vesicles. Quantification of GFP signal in L4 cpr-3p::gfp; neuronal kgb-1 B transgenics of the indicated genetic backgrounds, fed control (EV) or vhp-1 RNAi throughout development. Averages ± SDs of average intensity of fluorescence signal in individual worms from a single experiment are shown. N=9-25 for each treatment/group, *** p<0.001, t-test (for fold over EV values).


Article title

Integration of Stress Signaling in Caenorhabditis elegans Through Cell Non-autonomous Contributions of the JNK Homolog KGB-1

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