Figure S1. Analysis of Proliferation upon Knockdown of LSD1. Proliferation assays
of PC-3M-luc (a) and DU145 cells (b) after knockdown of LSD1. (c) Proliferation assay
of PC-3M-luc cells overexpressing LSD1. (a-c) Cell indices and relative velocities are
shown. n ≥ 3. Error bars represent ± SD or +SD. * p ≤ 0.05.
Figure S2. LSD1 directly regulates the expression of FA-associated genes. (a)
DAVID analysis for gene ontology (GO) of cellular compartment of differentially
regulated genes upon knockdown of LSD1. Cellular components enriched with a p-value
≤ 10-10 are shown. (b) mRNA levels of LSD1, PXN, TLN1, ZYX, and PIK3R2 were
analysed by qRT-PCR. (c) Levels of LSD1, TLN1, ZYX, and PIK3R2 were analysed by
Western blots decorated with the indicated antibodies. β-Actin was used as loading
control. (d) Migration assays of PC-3M-luc cells treated either with siRNA against
TLN1, ZYX, PIK3R2, or unrelated control siRNA. Cell indices and relative velocities are
shown. (e) mRNA levels of TLN1, ZYX, or PIK3R2 were analysed by qRT-PCR. n ≥ 3.
Error bars represent ± SD or + SD. *** p ≤ 0.001.
Figure S3. Migration is mediated through a novel LSD1-LPAR6-FA signalling
network. (a) Read coverage of LPAR1-6 in the RNA-Seq of PC-3M-luc cells transfected
with siRNA against LSD1 or unrelated control siRNA. (b) mRNA levels of LPAR1-3
were analysed by qRT-PCR. (c) Migration assay of DU145 cells treated with siRNA
either against LSD1, LPAR6, or both. (e) Migration assay of PC-3M-luc cells
overexpressing LPAR6 after knockdown of PXN. (c, e) Cell indices and relative
velocities are shown. (d, f) mRNA levels of LSD1, LPAR6, and PXN were analysed by
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qRT-PCR. n ≥ 3. Error bars represent ± SD or +SD. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤
0.001.
Figure S4. LPAR6 controls metastasis in vivo. (a) Hematoxylin and eosin (HE)
stainings of the tumours and corresponding axillary lymph nodes from the orthotopic
metastasis model are shown. (b) Equal cell injection into the tail vein of immunodeficient
mice was confirmed by bioluminescent signals after cell application. (c) Increased overall
metastases in mice injected with PC-3M-luc cells overexpressing LPAR6 (LPAR6; n=15)
or control cells (Ctrl; n=16). Statistics were done using Fisher’s exact test. (d, e)
Metastases indicated by bioluminescent signals were confirmed ex vivo by HE staining.
(f) Equal cell injection into the tail vein of immunodeficient mice was confirmed by
bioluminescent signals after cell application. (g) Ex vivo confirmation of the metastases
28 days after tail vein injection of LPAR6 depleted PC-3M-luc cells by HE staining. (h)
Persistence of LPAR6 knockdown was controlled by qRT-PCR in parallel to the in vivo
experiment over 8 days. One experiment was done in triplicate. Error bars represent ± SD
or +SD.
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