Supplemental Material

This article contains the following supporting material:

• Figure S1 - Knockdown of PICT-1 by siRNAs directed to different target sites comparably enhances proliferation of HeLa cells. Cells were transfected with GFP5 (open circles; Control), PIC247 (open squares), PIC318 (open triangles), PIC749 (closed triangles), or PIC1235 (closed circles) siRNA. Target sequences of PIC318 and PIC1235 corresponded to nucleotides 318–338 and 1235–1255 of human PICT-1, respectively. The proliferation at indicated time was monitored as described in Materials and Methods. Mean±SD from sextuplicated experiment is represented.

• Figure S2 - PICT-1 expression suppresses proliferation of HeLa cells. Cells were transfected with pEF1 empty vector (open circles; Control) or PICT-1/pEF1 (closed circles; PICT-1). The expression vector, PICT-1/pEF1, was described previously (Okahara et al., 2005). The proliferation at indicated time was monitored as described in Materials and Methods. Mean±SD from sextuplicated experiment is represented.

• Figure S3 - Introduction of PTEN restores the sensitivity towards PICT-1 knockdown on cell proliferation. PC3-Ec-PTEN cells were cultured in the presence of 5 μM ponasterone A to induce PTEN expression (Right Panel) or left untreated (Left Panel). After 2-day incubation, cells were transfected with GFP5 siRNA for control knockdown (open symbols; Control) or PIC749 siRNA for PICT-1 knockdown (closed symbols; ΔPICT-1). (A) The proliferation at indicated time was monitored as described in Materials and Methods. Mean±SD from sextuplicated experiment is represented. (B) Cell lysates at 0 h were prepared and subjected to immunoblot analyses by indicated antibodies. PC3-Ec-PTEN cell was derived from PTEN-null human prostate carcinoma PC3 cell and kindly provided by Dr. Jack E. Dixon (UCSD).

• Figure S4 - PICT-1 knockdown promotes anchorage-independent growth of MCF7 cells. After the transfection with pSilencer (Control), GLT318SH/pSilencer (ΔPICT-1), or PTEN/pSilencer (ΔPTEN), MCF7 cells were cultured and expanded for 7–14 days in the presence of 150 μg/ml of hygromycin B. Then the cells (4.0×10⁵ cells) were suspended in 3 ml of top agar (DMEM containing 2% fetal bovine serum, 150 μg/ml of hygromycin B and 0.3% SeaPlaque GTG agarose) and added onto prelayered bottom agar (3 ml of DMEM containing 2% fetal bovine serum, 150 μg/ml of hygromycin B and 0.5% SeaPlaque GTG agarose) in a ø60-mm dish. After the incubation at 37ºC with 5% CO₂ for 16 days, the diameter of individual colonies was measured. Distribution of the diameter is represented as a histogram. Average diameters of Control, ΔPICT-1, and ΔPTEN cells were 66, 122, and 133 μm, respectively.
• Table S1 - Summary of neuroblastoma patient data.