Download Supplementary Figure Legends Supplementary Figure S1. Both

Supplementary Figure Legends
Supplementary Figure S1. Both LIMD2 (rAb) and LIMD2 (mAb) detected the exogenous and
endogenous LIMD2 protein in cell lines and human cancer specimens. A, The His-tagged and
GST-tagged full-length LIMD2 proteins were used to produce polyclonal and monoclonal
antibodies, respectively. B & C, The LIMD2 polyclonal antibody (rAb) and monoclonal
antibody (mAb) are highly specific to detect the LIMD2 protein. The HEK293 cells were
transient transfected with expression vector, FLAG-LIMD1, FLAG-LIMD2, or FLAG-AJUBA
plasmids. The expression of LIMD2 was determined by western blot (WB) with FLAG or
LIMD2 (mAb), respectively. D, Endogenous LIMD2 was detected in HEK293 cells and U2OS
cells by western blot using LIMD2 (mAb). E, The LIMD2 protein is cytoplasmic and is
detectable in paraffin-fixed HEK293 cells or human bladder cancer specimens.
Supplementary Figure S2. Total mRNA was extracted from cancer cell lines using RNeasy Mini
Kit (Qiagen). The cDNA was synthesized from total RNA using random primers with High
Capacity cDNA Reverse Transcription Kit (Applied Biosystems), and gene-specific primers with
Ampli Taq Gold (Applied Biosystem). Real-time PCR was performed using an SYBR Green
technology on the CPTICON2 (MJ Research Inc.). The relative expression level was calculated
from a relative standard curve obtained by using two-fold dilutions of cDNA containing the gene
of interest. The average of two independent analyses for each gene and sample was calculated
and was normalized to the endogenous reference control gene GAPDH. A, breast cancer; B,
melanoma; C, bladder cancer; and D, thyroid cancer cell lines.
Supplementary Figure S3. Restoration of LIMD2 in siRNA knockdown cells gained the function
and phenotype of parental cells. A, Restoration of LIMD2 in siRNA knockdown cells. B,
Migration assays of cells with restored LIMD2 expression. T-test was performed and P-value is
indicated. C, The white arrows indicate the enlarged and flattened LIMD2 knockdown cells. The
black arrows indicate the spindle shape of cells wherein LIMD2 is restored. The bar scale is
equal to 100 m.
Supplementary Figure S4. The cell surface area was measured for breast cancer cell lines MDAMB231 (parental, LIMD2-Si5, restoration with vector and LIMD2) using Image-Pro Plus 7.0
program. P-value was indicated.
Supplementary Figure S5. Asymmetry in electrostatic surface of PINCH LIM1 and LIMD2
domains. PINCH-LIM1 (top) is negatively charged whereas LIMD2 (below) has a net positive
charge. Asymmetrical charge distribution is evident in both LIM proteins. This asymmetry may
play an instrumental role in providing different interaction interfaces.
Supplementary Figure S6. Ectopic expression or knockdown of LIMD2 stimulates or abrogates
cell migration and anchorage-independent growth. A, Expression of LIMD2 in bladder cancer
cells analyzed by western blot. B, The bladder cancer cell lines were subjected to the migration
assay. C, The images of RT4 and T24 bladder cancer cell lines were taken from the migration
assay. The bar scale =100 m. D, Ectopic LIMD2 expression promotes bladder cancer cell
migration. E, LIMD2 knockdown siRNA in aggressive T24 cells inhibits soft agar colony
formation. All data (B, C, D, E) are presented as mean  standard deviation. P-value as indicated.
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