Supplementarymaterials for hPuf-A mediated tumorigenesis

Materials and methods

Antibodies

The mouse anti-hPuf-A monoclonal antibody was described previously [9]. Rabbit anti-Akt, anti-phospho-Akt (S473), anti-RPA70 and anti--catenin antibodies were from Cell Signaling (Danvers, MA).Mouse anti-p16 and anti-cyclin E antibodies were from Thermo Scientific (Fermont, CA).Rabbit anti-cyclin A, anti-p107, anti-p130 antibodies, mouse anti-p53 and anti-vimentin antibodies were from Santa Cruz Biotech (Santa Cruz, CA).

Colony formation
A total of 1x106 of T47D cells were seeded in a 6 cm tissue culture dish, incubated for 16-20h at 37°C in a 5% CO2 incubator and subjected to hPuf-A or luciferase siRNAs(20 nM) treatment for 72 h. The cells were trypsinized and 5x103 ofcells were plated in complete growth medium and allowed to grow until visible colonies formed (12 days in T47D case). Cell colonies were washed with PBS twice, stained with 0.25% of crystal violet in ddH2O for 5 min, washedwith PBS twice, and air dried.

Cell cycle analysis

Cells were harvested, washed with PBS and fixed with ice-cold 70% ethanol overnight at -20°C. After removing the ethanol, the cell pellets were washed and subjected to the staining with20µg/ml propidium iodide, 200 µg/mlRNaseA and 0.1% Triton X-100 for 30 min and followed byflow cytometry analysis.

Fig.S1Quantification of hPuf-A IHC stainingin breast cancer specimenswitha T classification, b N classification,cmetastasis, ER, PR, HER2 and TNBC and d histological grade

Table S1

Correlation between clinicopathologic characteristics of patient samples and expression of hPuf-A in breast cancer
No. of cases (%) with hPuf-A staining
SI<4 (None and Low) / SI≥4 (High) / p
Age (y)
≤ 45 / 9 (34.6) / 17 (65.4) / 0.998
> 45 / 55 (34.6) / 104 (65.4)
Clinical stage
DCIS / 15 (65.2) / 8 (34.8) / 0.005
I / 16 (43.2) / 21 (56.8)
II / 17 (28.3) / 43 (71.7)
III / 11 (25.0) / 33 (75.0)
IV / 5 (23.8) / 16 (76.2)
T classification
T1 / 43 (41.3) / 61 (58.7) / 0.137
T2 / 19 (26.0) / 54 (74.0)
T3 / 1 (16.7) / 5 (83.3)
T4 / 1 (50.0) / 1 (50.0)
N classification
N0 / 50 (37.6) / 83 (62.4) / 0.253
N1 / 11 (33.3) / 22 (66.7)
N2 / 3 (20.0) / 12 (80.0)
N3 / 0 (0) / 4 (100)
Distant metastasis
Yes / 24 (29.3) / 58 (70.7) / 0.174
No / 40 (38.8) / 63 (61.2)
Estrogen receptor
Positive / 47 (36.2) / 83 (63.8) / 0.493
Negative / 17 (30.9) / 38 (69.1)
Progesterone receptor
Positive / 44 (37.3) / 74 (62.7) / 0.307
Negative / 20 (29.9) / 47 (70.1)
HER2 receptor
Positive / 41 (32.5) / 85 (67.5) / 0.391
Negative / 23 (39.0) / 36 (61.0)
Histological grade
I / 16 (43.2) / 21 (56.8) / 0.238
II / 19 (28.8) / 47 (71.2)
III / 12 (24.0) / 38 (76.0)
n.d. / 2 (22.2) / 7 (77.8)

Fig.S2Colony formation inhPuf-A silenced T47D cells.aT47D cells were transfected with siRNA duplexes and immunoblotting analysis was conducted. b 5,000 cells of siRNA transfected cells were plated on 6 cm dish and allowed to grow for 12 days until colonies appeared. (**P ≤ 0.01 with compared to control)

Fig.S3Cell cycle progression in hPuf-A knockdown and overexpressing cells.aDNA contents were determined by flow cytometry in hPuf-A silenced and overexpressing MDA-MB-231 cells. bMDA-MB-231 cells were transfected with siRNA duplexes. Immunoblottinganalysis was carried out using indicated antibodies