DOI: 10.6084/m9.figshare.12965652.v1
title: Additional file 1 of Genetic modifiers ameliorate endocytic and neuromuscular defects in a model of spinal muscular atrophy

description: Additional file 1: Figure S1. PLS3 overexpression suppressed pharyngeal pumping defects in smn-1(ok355) animals. Homozygous null smn-1(ok355) animals lacking smn-1 survive through early larval stages due to maternal loading of SMN-1 proteins and mRNA by heterozygous smn-1(ok355)/hT2 mothers. hT2 carries a functional copy of endogenous smn-1, overexpression of human PLS3 slightly lowered pumping rates in control smn-1(+) animals, but increased pumping rates in homozygous smn-1(ok355) animals (compared to control smn-1(ok355)). To control for genetic background all animals were derived from mothers heterozygous for hT2. To control for transgene insertion position, control and smn-1(ok355) animals carried rtSi28 [dpy-30p::empty]. PLS3 is overexpressed from rtSi27 [dpy-30p::PLS3], a single copy insertion on chromosome II. n≥30 animals per determination, combined from 3 independent trials that the scorer was blinded to the genotype of animals. ANOVA F(9.9,13.1) = 14.23, p&lt;0.001; post-hoc Mann-Whitney U-test *p&lt;0.05, S.E. indicated. Figure S2. sym-2 knockdown using RNAi suppressed the smn-1 locomotion defect. Exhaustion of cholinergic motor neurons using ChR2 slowed locomotion; smn-1(cb131) animals with decreased SMN-1 function had aberrantly low locomotion rates post-exhaustion. RNAi knockdown of hnRNP F/H ortholog sym-2 ameliorated this defect. empty (RNAi) used as a control as the bacterial strain used for RNAi can alter locomotion rates. n≥30 animals per determination, combined from 3 independent trials. Student’s t-test *p&lt;0.05 S.E.M. indicated. Figure S3. Association of SMN and PLS3 with hnRNPF or hnRNPH1/2, is not dependent on RNA. Pretreatment with RNase had no impact on co-immunoprecipitation of PLS3 or SMN from HEK293T cells using GFP tagged hnRNP F or hnRNP H1/2. SMN was tagged with V5; PLS3 was tagged with Flag. Conditions and procedures as described in Fig. 4a. Figure S4.PLS3 and sym-2 suppress behavioral defects in specific C. elegans models of neurodegenerative disease. (a) Neuronal overexpression of wild type human SOD1 (SOD1-WT) had little impact on C. elegans locomotion, but overexpression of SOD1 carrying the familial ALS patient allele G85R (SOD1-G85R) dramatically decreased locomotion in young adult animals. Introduction of ubiquitously expressed human PLS3 decreased locomotion rates in SOD1-WT animals, but ameliorated defects in SOD1-G85R animals. ANOVA F(320.2,967.5) = 6.78, p&lt;0.05 (b) SOD1-G85R animals had decreased pharyngeal pumping rates in young adult animals. PLS3 expression restored normal pumping rates in SOD1-G85R animals. ANOVA F(1305379,327194) = 235.38, p&lt;0.001 (c) SOD1-G85R mutant animals were resistant to the cholinesterase inhibitor aldicarb, based on slower time to paralysis. Introduction of ubiquitously expressed human PLS3 restored sensitivity to normal levels. (d) RNAi knockdown of sym-2 had no impact on SOD1-WT animals, but increased locomotion rates in SOD1-G85R animals. Empty RNAi was used as a control for sym-2(RNAi) as bacterial strain can alter locomotion and other behaviors. sym-2(RNAi) had no impact on SOD1-G85R pumping rates (not shown). In this figure PLS3 was ubiquitously expressed from rtSi27 [dpy-30p::PLS3], a single copy insertion on chromosome II. In each assay an n≥30 animals per determination, combined from 3 independent trials. In all trials scorers were blinded to the genotype of the animals when collecting and scoring the data. Mann-Whitney U-test (panels a,b,d,e and f) or Log Rank test (panels c and g) **p&lt;0.01 S.E.M. indicated. Figure S5. PLS3 does not generically suppress locomotion. (a) C. elegans lacking glutamic acid decarboxylase encoded by unc-25 had decreased locomotion in liquid due to defective GABA synthesis (Jin et al., 1999). The ubiquitous expression of PLS3 did not suppress locomotion defects in unc-25;PLS3oe double mutant animals. PLS3 was overexpressed from rtSi27 [dpy-30p::PLS3], a single copy insertion on chromosome II. The endogenous C. elegans plastin ortholog, plst-1, was intact. Control single copy insertion rtSi28 did not express PLS3, but was inserted at the same location on II, confirming that insertion into this locus had no impact on unc-25 locomotion in this assay. n≥30 animals per determination, combined from 3 independent trials. ANOVA F(7.37,7.45) = 20.09, p&lt;0.001 Mann-Whitney U-test: **p&lt;0.01, S.E.M indicated. b) PLS3 overexpression is unable to rescue the unc phenotype in unc-13 animals when assessing dispersal on food. Animals are reported as the percentage of animals that escaped the center ring after 1, 2 or 3 hours. Mean percent of animals escaped is calculated from three independent trials of at least 24 animals per genotype, per trial. Each trial is shown as a grey circle. Figure S6. Uncropped western blots. Panels a, d and e correspond to Fig. 5 panels a, b and c, respectively. Panels b and c correspond to supplementary figure 3. Table S1. Strains.