10.25386/genetics.7159487.v1
Limeng Liu
Limeng
Liu
Cyrus Ruediger
Cyrus
Ruediger
Michael Shapira
Michael
Shapira
Supplemental Material for Liu, Ruediger, and Shapira, 2018
GSA Journals
2018
Caenorhabditis elegans
KGB-1
autonomous
non-autonomous
DAF_16
stress
Genetics
2018-10-05 15:00:22
Dataset
https://gsajournals.figshare.com/articles/dataset/Supplemental_Material_for_Liu_Ruediger_and_Shapira_2018/7159487
<p><b>Figure S1. Tissue-specific promoters
direct expected expression patterns of a KGB-1::GFP fusion protein.</b>
Adult worms demonstrating KGB-1::GFP expression controlled by the neuronal <i>rgef-1</i> promoter (<b>A</b>,
blow-up shows localization in neuronal commissures); the intestinal <i>gly-19</i>
promoter (<b>B,</b> showing nuclear localization), and the epidermal <i>wrt-2</i>
promoter (<b>C</b>, 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 <i>myo-3</i>
promoter, which eluded transgenic expression. Orange fluorescence is due to
tdTomato expression driven by the pharyngeal <i>myo-2</i> promoter. </p>
<p> </p>
<p><b>Figure S2. Effects of tissue-specific
KGB-1 activation on worm size and pigmentation.</b>
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.</p>
<p> </p>
<p><b>Fig S3. KGB-1 expression from its
endogenous promoter rescues resistance of kgb-1 mutants. </b>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).</p>
<p> </p>
<p><b>Figure S4. Tissue-specific KGB-1
expression has similar outcomes for stress resistance in distinct transgenic
integrant lines. </b>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 <i>kgb-1</i> mutants (* p<0.05, ***
p<0.001, paired t-test).</p>
<p> </p>
<p><b>Figure S5. KGB-1 protects larvae form ER
stress independently of canonical UPR<sup>ER</sup> signaling. A. </b>Representative
images of L3 <i>hsp-4p::gfp </i>transgenics, in a wildtype or <i>kgb-1 </i>genetic
background, exposed to tunicamycin for 15 hours before imaging. <b>B. </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). <b>C.</b>
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 <i>kgb-1</i>
single mutants (in purple)(*p<0.05, **p<0.01, paired <i>t</i>-test). Note
that disruption of <i>atf-6</i>, thought to be important for regulating
constitutive UPR<sup>ER</sup> genes, increases stress resistance as previously
reported [45], and that an additional disruption of <i>kgb-1</i> additively
decreases the resistance of these mutants, attesting to lack of epistasis.</p>
<p> </p>
<p><b>Figure S6. Tissue-specific contributions
of KGB-1 to target gene expression in larvae are replicated in independent
transgene integrant lines. A-C.</b> qRT-PCR measurements of gene
induction in L3 larvae of the designated strains and lines, following KGB-1
activation by <i>vhp-1</i> knock-down from the egg stage. Averages ± SDs for
measurements from 2-6 independent experiments, each measured in duplicates.
Asterisks mark significant induction following <i>vhp-1</i> knock-down
(*p<0.05, **p<0.01, ***p<0.001, paired t-test).</p>
<p> </p>
<p><b>Figure S7. Cell non-autonomous
contributions of neuronal KGB-1 partially require small clear vesicles, but not
dense core vesicles. </b>Quantification of GFP signal in L4 <i>cpr-3p::gfp;
neuronal kgb-1</i> 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).</p>