JCB
Supplemental material
THE JOURNAL OF CELL BIOLOGY
Yasuda et al., https​://doi​.org​/10​.1083​/jcb​.201608022
Figure S1. FRAP experiments of GFP-FUS(R521C) inclusions and the effect of mutant FUS inclusions on stress response markers and on general cellular
architecture. (A) GFP-FUS(R521C) granules within a fibroblast cell and representative images of FRAP experiments at the indicated time points. White circles
indicate photobleached regions. The graph shows fluorescence recovery. (B) Representative WB detecting phospho- or total eIF2α levels in cells transfected
with the indicated constructs or treated with NaAsO2 to induce oxidative stress. Phospho-eIF2α was normalized to total eIF2α levels and expressed relative
to the wild-type (wt) or GFP control. Note that NaAsO2 treatment increases phospho-eIF2α levels, whereas expression of different FUS variants does not.
(C) Immunostaining of phospho-eIF2α in cells expressing GFP or GFP-FUS(P525L) with or without NaAsO2 treatment. Phospho-eIF2α intensity was expressed
relative to the GFP− sample. Total numbers of cells observed in two independent trials are indicated within each bar. (B and C) P = 0.17 between wildtype and arsenite-treated groups; paired t test; P > 0.9999 between GFP- and GFP-FUS–expressing cells; one-way analysis of variance using Bonferroni’s
multiple comparisons test (B); and *, P < 0.0001; Mann–Whitney U test (C). (D) RT-PCR detection of spliced and unspliced XBP1 RNA. Thapsigargin (TG)
treatment induces the unfolded protein response and leads to splicing of XBP1 RNA, whereas GFP-FUS mutants do not. Similar results were observed in
two independent trials. (E) RT–quantitative PCR quantitation of spliced XBP1 levels, normalized to GAP​DH. There were no statistical differences between
GFP- and GFP-FUS–expressing cells by one-way analysis of variance. Note that for B, D, and E, transfection conditions were optimized to increase the
number of inclusion-containing cells and thus ensure that any effects would be detectable. For the stronger mutants, P525L and R495X, ∼50% of cells
exhibited visible cytoplasmic FUS inclusions. n = 3 for all experiments unless otherwise indicated. (F) Representative images showing the distribution of
mitochondria (left, MitoTracker), Golgi (middle, GM130), and ER (right, ER tracker) in cells expressing GFP or GFP-FUS(P525L) with cytoplasmic inclusions.
Error bars show SEM. Bars, 10 µm.
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Figure S2. Effect of mutant FUS inclusions on the distributions of Kank2 and Arpc3 RNAs and the effect of inclusions formed by the wild-type FUS protein.
(A) Kank2 RNA FISH and Kank2 RNA edge ratio quantitation of cells expressing GFP or GFP-FUS(R521C). Arrows indicate RNA signals in protrusive areas.
*, P = 0.0008; Mann–Whitney U test compared with the granule− group of GFP-FUS(R521C). (B) Arpc3 RNA edge ratio of cell populations analyzed in
Fig. 1 B. *, P < 0.0001 Mann–Whitney U test. (C) Percentage of Glu-MT–positive cells expressing GFP or wild-type GFP-FUS (GFP-FUS(wt)) exhibiting or
not exhibiting cytoplasmic FUS granules. *, P < 0.01; Student’s t test. (D) Representative immunostaining of Glu-tubulin and total α-tubulin of cells expressing
GFP or wild-type GFP-FUS analyzed in C. (E) Representative images of Ddr2 RNA FISH of GFP- or wild-type GFP-FUS–expressing cells exhibiting or not
exhibiting cytoplasmic FUS granules. Edge ratio quantitations are shown to the right. *, P < 0.0001; compared with granule− group by Mann–Whitney U
test. (F) Total GFP-FUS intensity of cell populations analyzed in Fig. 1 B. n = 20–30. The effects on RNA localization in the presence of FUS granules (shown
in Fig. 1 B) do not simply correlate with differences in expression level of the mutant protein. Horizontal bars indicate mean values. (G) Cells expressing
GFP or GFP-FUS(P525L) were immunostained to detect total FUS, and the level of nuclear FUS intensity was quantified. The presence of cytoplasmic FUS
inclusions does not significantly affect the levels of nuclear FUS. (A–C and E) Numbers in bars indicate the total cell numbers observed in three (A) or two
(B, C, E, and G) independent trials. Error bars show SEM. Bars, 10 µm.
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Figure S3. Changes in Glu-tubulin levels upon different treatments, validation of KIF5B and KIF5C antibody specificity, and the requirement of the IDR
domain of KIF5B for accumulation in FUS inclusions. (A) Representative WBs of Glu-tubulin and α-tubulin from control or parthenolide-treated cell lysates (left)
or control and TTL-knockdown cell lysates (right). Glu-tubulin levels normalized to α-tubulin were quantified from four or nine independent trials, respectively.
(B) Edge ratio of Kank2 mRNA in control and parthenolide-treated cells. Total numbers of cells observed in two independent trials are indicated. (C) KIF5B
immunostaining of cells expressing GFP-FUS(P525L) additionally transfected with control or Kif5b siRNA. KIF5B intensity within FUS cytoplasmic inclusions
or within the cytoplasm was measured and expressed relative to the siControl group. Arrows indicate FUS inclusions showing colocalization with KIF5B.
Total numbers of measured inclusions or cytoplasmic areas collected from two independent trials are indicated within each bar. Note that KIF5B signal
within inclusions appears to be largely specific, whereas signal within the cytoplasm mostly seems to result from nonspecific staining. (D) Relative Kif5c
RNA amounts quantified via quantitative RT-PCR from control or Kif5c siRNA–treated cells. (E) KIF5C immunostaining of cells expressing GFP-FUS(P525L)
additionally transfected with control or Kif5c siRNA. KIF5C intensity within FUS cytoplasmic inclusions was measured and expressed relative to the
siControl group. Arrows indicate FUS inclusions showing colocalization with KIF5C. Total numbers of measured inclusions are indicated within each bar.
(A–E) *, P = 0.0049; **, P < 0.0054; paired Student’s t test (A); *, P < 0.0001; Mann–Whitney U test (B, C, and E); *, P = 0.0002; Student’s t test (D).
(F) Representative images of cells coexpressing RFP-FUS(P525L) and YFP-KIF5B or GFP-KIF5B(ΔIDR). Arrows indicate FUS granules showing colocalization
with YFP-KIF5B. Note that such colocalization is not observed for the ΔIDR mutant. (G) WBs of KIF3A or KIF5B expression in control cells or cells transfected
with the indicated siRNAs. GAP​DH was used as a loading control. Error bars show SEM. Bars, 10 µm.
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Figure S4. Accumulation of YFP-KIF5B in FUS inclusions and rescue of Ddr2 RNA localization upon overexpression of untagged KIF5B, as well as the lack
of accumulation of KIF5B in TDP-43(A315T) inclusions and lack of rescue of RNA localization and Glu-MT structure upon YFP-KIF5B overexpression. (A)
YFP-KIF5B– and RFP-FUS(P525L)–expressing cells showing colocalization of KIF5B in FUS granules (arrows). (B) Representative images of Ddr2 RNA FISH
in cells expressing GFP or GFP-FUS(P525L) with or without overexpression of untagged KIF5B. The graph shows edge ratios of Ddr2 RNA in those cells.
(C) IF detection of KIF5B in cells containing GFP–TDP-43(A315T) inclusions. No appreciable recruitment of KIF5B is detected within TDP-43 inclusions. (D)
Cells expressing RFP or RFP–TDP-43(A315T), with or without YFP-KIF5B, were stained for Glu-tubulin, and cells containing Glu-MTs were scored. (E) Ddr2
RNA edge ratio of cells expressing RFP or RFP–TDP-43(A315T) with or without YFP-KIF5B. (B, D, and E) Total numbers of cells observed in two (B and E)
or three (D) independent trials are indicated. No rescue upon KIF5B expression is observed. *, P < 0.0001; Mann–Whitney U test (B and E); or Student’s
t test (D). Error bars show SEM. Bars, 10 µm.
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Figure S5. WB detection of TTL levels in knockdown experiments and IF quantitations of acetyl-tubulin levels in the presence or absence of FUS inclusions
combined or uncombined with taxol treatment. (A) WBs of TTL expression in control cells or cells transfected with the indicated mutant proteins or siRNAs.
GAP​DH and β-actin were used as loading controls. The asterisk indicates a nonspecific band. (B) Immunostaining of acetylated tubulin and α-tubulin in GFPor GFP-FUS(P525L)–expressing cells treated or not treated with taxol. Arrows indicate cytoplasmic FUS inclusions. Acetyl-tubulin intensity was normalized
to α-tubulin and expressed relative to the GFP sample. Numbers of cells observed in two independent experiments are indicated in each bar. *, P < 0.03;
Mann–Whitney U test. Note that taxol samples are the same as presented in Fig. 2 C. Error bars show SEM. Bars, 10 µm.
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