Supplementary materials and methods
DNA construction
The worm BTBD10 homolog gene was amplified from the N2 genome by PCR using a
sense primer 5’-ACCGCGGTGGCGGCCGCTTTTCACTCGGGAATCGCTC-3’ and an
antisense primer 5’-GCAGCCCGGGGGATCCCCCACGACATCTGCGAATAT-3’. The
amplified worm btbd-10 gene was inserted into the pBluescript cloning vector at the
BamH I/Not I site using Clontech In-FusionTM PCR Cloning System (Mountain View,
CA).
Human
BTBD10
cDNA
was
amplified
using
a
sense
primer
5’-GCATGGACGAGCTGTACAAGATGGCAGGACGGCCTCAT-3’ and an antisense
primer 5’-CGGGATCCTCACAGCATTGGATTCTGTGCATC-3’. The worm btbd-10
cDNA
(R05F9.1a)
was
amplified
using
a
sense
primer
5’-GCATGGACGAGCTGTACAAGATGAGCTCATTTTCTACTTTTCCTCA-3’
and
an antisense primer 5’-TTATTCTTCCTCACGATCATTTC-3’. The promoter region of
btbd-10
gene
was
amplified
using
a
sense
primer
5’-ACCGCGGTGGCGGCCGCTTTTCACTCGGGAATCGCTC-3’ and an antisense
primer
5’-TCCTCGCCCTTGCTCACCATTATAAAACGAGAAAACAGATGGAGCC-3’. The
cDNA of mCherry was a kind gift of Dr. Roger Y. Tsien and amplified using a sense
primer
5’-GGCTCCATCTGTTTTCTCGTTTTATAATGGTGAGCAAGGGCGAGGA-3’
an
antisense
and
primer
5’-TGAGGAAAAGTAGAAAATGAGCTCATCTTGTACAGCTCGTCCATGC-3’. The
promoter region of the btbd-10 gene, mCherry cDNA, worm btbd-10 cDNA, and human
BTBD10 cDNA were amplified by PCR and inserted into pCR-Blunt II-TOPO vector
(Invitrogen). The promoter region of worm akt-1 was amplified using a sense primer
5’-CGCGGATCCGAACAACAGGAAAGTTTGAGAAC-3’ and an antisense primer
5’-AAGGAAAAAAGCGGCCGCTCTTTCACCGGCGCCGT-3’. The amplified akt-1
promoter region was inserted into pFX/mCherry vector at BamH I/Not I site. The human
constitutive active Akt1 (caAkt1) cDNA was amplified using a sense primer
5’-AAGGAAAAAAGCGGCCGCACCATGGGGAGTAGCAAG-3’ and an antisense
primer 5’-AAGGAAAAAAGCGGCCGCCGGATTCGGCCGTGCCG-3’. The caAkt1
cDNA was inserted into pFX/Pakt-1::mCherry vector at Not I site.
Isolation of the mutated alleles
The nested primers used to screen for btbd-10 (tm3335 and tm3607) were
5’-TCAGGACGGTCATTATCGTG-3’ and 5’-CGGGAACCAAACCTAGCTAA-3’ for
the
first-round
amplification
and
5’-TCAATGTCACTTGGAGGTCC-3’
and
5’-TGACGCGAACTAAACTACCA-3’ for the second-round amplification. ced-3
(tm1196) was screened using primers 5’-ACGGCTTATGGTTGGTGCAT-3’ and
5’-TCTGACTACGGGTCAGTAGA-3’
for
the
first-round
amplification
and
5’-GCTTTGCTGAATGAGCGATT-3’ and 5’-CAGCCGAAGATGATGCGTCA-3’ for
the second-round amplification. akt-1 (tm399) was screened using primers
5’-TTTACGACGGCGCCGGTGAA-3’ and 5’-CTTGCTTGGGAACCTTAGAT-3’ for
the
first-round
amplification
and
5’-CGGTGAAAGAATGTCGATGA-3’
and
5’-CCAGCCATGATAAGTTCGAA-3’ for the second-round amplification. akt-2
(tm1075) was screened using primers 5’-TTATGCGGATAGCGCCGACT-3’ and
5’-CTGTCGGAAGTTCTTCCCCT-3’
for
the
first-round
amplification
and
5’-GTCAGATGTGGATTGAAGCA-3’ and 5’-TCTACGTGGAGGCGTCAGTT-3’ for
the second-round amplification. All mutants were backcrossed with N2 at least twice
prior to analysis.
Legends to supplementary figures and movies
Figure S1. Sequence alignment of H. sapiens, M. musculus, and C. elegans BTBD10
proteins. Multiple alignment was performed by ClustalW. Identical residues are shown
by red characters while residues that are strongly similar or weakly similar are shown by
green or blue characters, respectively. Gaps are represented by a dash (-).
Figure S2. The structure of the C. elegans btbd-10 gene. Deletion or insertion in the
two btbd-10 deficient lines, tm3335 or tm3607, are indicated as a blue or yellow box,
respectively.
Figure S3. Decreased BTBD10 expression in motor neurons in G93A-SOD1 mice.
(A, B) A frozen section of a 3 months old mouse spinal cord was immunostained with an
anti-BTBD10 antibody (green) and a motor neuron marker, SMI32 (A) or an astrocyte
marker, GFAP (B) (red). Nuclei were visualized with Hoechst 33258 (blue). (C) Frozen
sections of spinal cords from G93A-SOD1 mice or their littermates at 60, 120, or 140
days old were immunostained with an anti-BTBD10 antibody (green). Arrows indicate
motor neurons.
Figure S4. Overexpression of FUS reduces BTBD10 and p-Akt (Ser473) expression.
(A-C) HeLa cells (A, C) or NT-2 neuron progenitor cells (B) were infected with an
adenovirus encoding cre-wt-FUS at an MOI of 400 and an adenovirus encoding
cre-recombinase at an MOI of 40 and harvested at 48 hr (HeLa cells) or 32 hr (NT2 cells)
postinfection. A LacZ-encoding adenovirus was used as a negative control. The results of
two independent identical experiments with densitometrical analysis of immunoblotted
bands were shown side by side in (B). The protein levels of BTBD10, FUS, p-Akt
(Ser473), total Akt, or actin were determined by immunoblot analysis (A, B). Total RNA,
isolated from HeLa cells infected with the indicated adenovirus, was used for quantitative
real-time PCR using primer sets for BTBD10, FUS, or G3PDH. Statistical analysis was
performed by Student’s t-test. (*P< 0.01, error bars indicate SD.) (C). Densitometric
calculation of immunoreactive bands was performed using ImageJ (version 1.32i
software).
Figure S5. (A, B) The low-grade overexpression of TDP-43 does not decrease
BTBD10 expression in human cultured cells. HeLa (A), and NT-2 (B) cells were
infected with an adenovirus encoding wt-TDP-43 at an MOI of 200 and harvested at 48 hr
(HeLa cells) or 32 hr (NT2 cells) postinfection. A LacZ-encoding adenovirus was used as
a negative control. The protein expression level of BTBD10, TDP-43 or actin was
determined by immunoblot analysis with anti-BTBD10, anti-TDP-43 antibody, or
anti-actin antibody, respectively. (C) The high-grade overexpression of TDP-43 does
not decrease BTBD10 expression in NSC34 cells. NSC34 cells were infected with an
adenovirus encoding wt-TDP-43 at an MOI of 400 and harvested at 48 hr postinfection. A
LacZ-encoding adenovirus was used as a negative control. The protein expression level
of BTBD10, TDP-43 or actin was determined by immunoblot analysis with anti-BTBD10,
anti-TDP-43 antibody, or anti-actin antibody, respectively.
Supplementary movie 1.
Body bending of an N2 worm.
Supplementary movie 2.
Body bending of a btbd-10(tm3335) worm.