Garcia_Figure S1

1
Supplementary figures
Garcia_Figure S1
A
B
Q0
Q24
Q40
Q35
Q0
Q24
Q40
Q35
wt rm7
wt
wt rm7
wt rm7
wt rm7
wt
wt rm7
wt rm7
YFP (27KDa 33KDa)
?-Tubulin (50KDa)
16 mg total protein
20 mg total protein
C
D
Figure S1. The unc-30(rm7) mutation in the polyQ
background leads to premature protein
aggregation, with no significant changes in
polyQYFP protein levels. (A,B) Protein extracts
of animals expressing polyQYFP in wt and
unc-30(rm-7) backgrounds were analyzed by
SDS-PAGE and immunoblotted with an anti-GFP
antibody and anti-?-tubulin antibody. (C,D)
Protein band intensities (arbitrary units) were
determined using the program Adobe Photoshop 6.0
and the ratios between YFP band intensity and
tubulin band intensity calculated. No
significant relative changes in polyQYFP levels
were observed between the wt and unc-30(rm-7)
backgrounds. Protein extracts were collected at
the ages at which premature aggregation was
observed 4 days old for Q0, Q24 and Q35 animals
and 2 days old for Q40 animals.
2
Garcia_Figure S2
B
A
unc-30 (rm7)
N2
Figure S2. Characterization of unc-30(rm7)
allele. (A) Amplification by PCR was performed
for the unc-30 genomic region containing the rm7
mutation. N2 samples display the expected band
size, approximately 2Kb, and unc-30(rm7) samples
exhibit a smaller PCR product, approximately
1.7Kb, indicating the presence of a deletion.
(B) RT-PCR amplification of unc-30 for N2,
unc-30(e2327), unc-30(rm7) and Q35 RNA samples.
Amplification revealed the presence of a smaller
transcript for the unc-30(rm7) strain. No
transcript was detected for the null
unc-30(e2327). Amplification of actin (act-4)
was used as a control.
3
Garcia_Figure S3A
Garcia_Figure S3B
Figure S3 The rm7 mutation causes a in frame
deletion in the gene unc-30. Represented in
Fig.S3A is the unc-30 DNA sequence from exon 4
(position 1,914) up to beginning of intron 7.
Exons are highlighted in orange and introns in
yellow. Box area indicates the rm7 deletion of
347bp. Represented in Fig.S3B is the amino acid
sequence of UNC-30. The rm7 deletion region is
indicated in bold, corresponding to the 54 amino
acids coded by exon 5. Stripped box indicates
homeobox domain.
4
Garcia_Table S1
Table S1 Q35 and Q40 animals in a GABA- and
egl-19-mutant backgrounds display a premature
aggregation onset. Numbers represent t1/2, which
indicates the average age at which animals reach
50 maximal aggregation, calculated using a
sigmoidal time response curve with a non-linear
slope. t1/2 was calculated using data set from
figures 2F, 2G and 3B, respectively. Values
indicate average ? SEM.
5
Garcia_Figure S4
A
Q35egl-19(n582)
Q35

Number of aggregates

Days old
B
Q35unc-93(e1500)
Q35
Number of aggregates

Days old
Figure S4 Decreases in excitation or in general
Ca2 influx into body wall muscle cells causes a
decrease in Q35 aggregation. Animals were
synchronized and age-dependent accumulation of
aggregates determined by fluorescent microscopy.
(A) Decrease in Ca2 entry in body wall muscle
cells leads to a decrease in Q35 aggregation. (B)
A decrease in body wall muscle excitation leads
to a decrease in Q35 aggregation. The strains
analyzed were Q35, Q35egl-19(n582) (A) and
Q35unc-93(e1500) (B). Q35unc-93(e1500) animals
were not scored beyond day 5 given that the
unc-95(e1500) mutation causes nematodes to bag
and die. Each column represents a minimum of 35
animals (range 35-80) (A) and 55 animals (range
55-71) (B). Error bars indicate SEM, ()
corresponds to a plt0.01.
6
Garcia_Figure S5
Control
Compound
B
A
GABA
C
D
Lindane
Q
GABAR
GABA
F
E
Neuron
Muscle cell
ACh
Lindane
AChR
H
G
Levamisole Nicotine
J
I
Levamisole
K
L
N
M
Nicotine
O
P
Figure S5 One day old (L1s) Q35 animals were
treated with 200mM GABA (B and D), 1mM lindane (F
and H), 25?M levamisole (J and L) or 1mM nicotine
(N and P). Shown in the panels are untreated
control animals (A, E, I and M) and treated
animals (B, F, J and N). The white boxes
indicate magnified areas shown in C, D, G, H, K,
L, O and P. Panels A-D correspond to 6 days old
and panels E-P correspond to 4 days old animals.
Scale bars A, B, E, I and M 200?m, C, D, F, G,
J, K, N and O 100?m, H, L and P 50?m. Shown
in Q is a schematic representation of neuronal
signaling at the neuromuscular junction and how
the compounds tested affect GABA or ACh signaling
in muscle cells.