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Supplemental Methods
Cell Culture. Cells were obtained from ATCC (Manassas, VA, USA) and maintained as
recommended. HMECs were a kind gift from Dr. Jing Yang (UCSD).
Plasmids and Transfections
For all transient transfections, MCF10-A, Sk-Br-3, and BT-20 were transfected with
FuGENE HD (Roche, Indianapolis, IN, USA). MCF-7 and ZR-75-1 were transfected with
ExGen500 (Fermentas, Glen Burnie, MD, USA). HCC1428 was transfected with Lipofectamine
LTX (Invitrogen, Carlsbad, CA, USA). For shRNA knockdown of tau in ZR-75-1, MAPT
knockdown sequences were: 5’-aagtgtggctcaaaggataat-3’, 5’-tccacggcatctcagcaatgt-3’, 5’-gacgct
ggcctgaaagaatct-3’, and 5’-gattgggtccctggacaatat-3’.
ggaatctcattcgatgcatac-3’.
The NSC insert sequence was 5’-
The NSC plasmid or all four MAPT shRNA plasmids were
cotransfected into ZR-75-1 using 2.0 μg total DNA (0.5 μg of each MAPT shRNA). Clones were
selected in 1.0 μg/ml puromycin (Sigma-Aldrich, St. Louis, MO, USA). Clones were screened
for tau expression by Western blotting. MCF-7 cells stably expressing GFP-tau or GFP-N1 were
selected in 1.0 mg/ml G418 (Invitrogen). Clones were selected visually by GFP-excitation
(488nm). Western blotting as described below confirmed expression.
For MCF-7 luciferase clones, pMSCV-Luciferase PGK-hygro was transiently cotransfected with the viral envelope-encoding vectors for gag-pol and vsv into Phoenix cells using
TransIT-LT1 transfection reagent according to manufacturer recommendations (Mirus). After
24h, Phoenix cell media (viral supernatant) was removed and filtered to remove cell debris. 4 ml
of viral supernatant was mixed with 6 ml of fresh MCF-7 media containing 1.0 mg/ml G418.
This media was added to MCF-7 cells stably expressing GFP-tau and GFP alone. 24h post-
infection, media was replaced with fresh media. At 48h post-infection, cells were trypsinized
and aliquoted into 96 well dishes at ~5 cells/ml into MCF-7 media containing 1.0 mg/ml G418
and 0.1 mg/ml hygromycin B (Calbiochem).
Tumor microarrays (TMA)
TMAs were stained for vimentin using mouse anti-vimentin (V9, 1:1000, Ventana, Tuscon, AZ)
on a Ventana Benchmark XT auto-stainer. A McNemar’s test assessed the co-expression of tau
and vimentin within patient tumor tissue.
Supplemental Figure Legends
Fig. S1. Tau localizes within the McTNs of suspended breast tumor cells. The tumor cell
lines Sk-Br-3, MCF-7, BT-20, ZR-75-1 and HCC1428 were subjected to indirect
immunofluorescence for tau followed by direct immunofluorescence for -tubulin.
As tau
expression increases (Figure 1A), so does localization of tau within McTNs (white arrows). This
is most evident in the McTNs of ZR-75-1 and HCC1428. Cells and McTNs displayed are
representative of those under conditions of actin depolymerization with LA. Scale bar = 10μm.
Fig. S2. Tau localizes to microtubules in attached breast tumor cells. To confirm that the
tau and tubulin colocalization observed in suspended cell images was not a product of nonspecific overlap due to maximum intensity projection, attached tumor cell lines, ZR-75-1 and
HCC1428, were subjected to indirect immunofluorescence for tau followed by direct
immunofluorescence for -tubulin.
Unstacked, single plane confocal sections reveal
characteristically speckled and robust tau colocalization to microtubules (white arrows).
Fig. S3. Tau within McTNs localizes at foci along microtubules. Confocal image datasets of
Tau and -tubulin immunofluorescence in HCC1428 and Zr-75-1 cells were magnified to isolate
an inset containing McTNs (left panels, white box). Maximum intensity projections combining
all z-section images are displayed to capture the full 3-dimensional length of the McTNs.
Magnification of the isolated McTNs in the inset regions demonstrates that while tubulin stains
along the entire length of the McTN, Tau is found predominantly in foci of higher concentration
(white arrows). This punctate staining of Tau along the McTNs recapitulates the staining pattern
observed for Tau colocalization along microtubules in attached cells (Figure S2).
Fig. S4. Tau and vimentin are not co-expressed. Western blotting confirmed a lack of
vimentin (Vim) expression in the cell line panel utilized in this study, for the exception of MCF10A, which we’ve previously shown to express vimentin (Whipple et al., 2008). Likewise, in a
panel of vimentin-expressing cell lines, tau is not co-expressed to levels significantly above that
of the normal cell lines, HMEC, 184A1, 184B5, and MCF-10A.
Fig. S5. An untagged tau expression vector encodes a 50kDa 3R tau protein. MCF-10A
cells were transiently transfected with an untagged tau protein expression vector. 24hr posttransfection, cells were lysed and probed for tau against lysates from the cell line panel. Western
blotting confirmed expression of tau protein with the expected molecular weight ranged (45-70
kDa).
Fig. S6. MCF-7tau1 and MCF-7tau2 stably express GFP-tau. MCF-7 cells were stably
transfected with GFP-tau (~75kDa). Two clones were chosen for homogenous expression,
MCF-7tau1 and MCF-7tau2. Western blotting confirmed stable expression of GFP-tau versus
the untransfected parent line (MCF-7) and transiently-transfected GFP-tau (MCF-7 GFP-tau).
Expression level was compared to the highest tau expressing cell line, HCC1428.
Fig. S7. Tau and vimentin are not co-expressed in patient tumors. Primary tumors and
patient matched lymph node metastases were evaluated for tau and vimentin expression.
Representative photomicrograph for both tissue types are shown, in which pathological score of
cytoplasmic tumor cell staining for vimentin was compared to that for tau. In Matched Patient 1,
tau is heavily expressed with both primary and metastatic tumor (black arrows). Vimentin is not
tumorally expressed , but localized to surrounding tissue. For Matched Patient 2, vimentin is
tumorally expressed (black arrowheads) whereas tau is not.
Tau was significantly more
expressed than that of vimentin within both primary and metastatic tumor, indicating that the two
proteins are not co-expressed (Table S2). Scale bar = 100m.