Table S1. Designation and sequences of siRNAs used for syncytin-1 knockdown.
SsiRNA: Syncytin-1-specific siRNA
Ctl siRNA: Control siRNA with scrambled sequences
Table S2. Designations, sequences, and the sizes of amplicons of primers used for real-time PCR.
Fig. S1. Final products of real-time PCR resolved by agarose gel electrophoresis. 4 µl of the final products of real-time PCR was resolved in agarose gels. DNA bands were visualized under UV lights following ethidium bromide staining. The single band pattern with the predicted size indicated the accomplishment of specific amplification by real-time PCR. The left lane shows the DNA markers for molecular sizes.
Fig. S2. Screening for the most effective siRNA for syncytin-1 knockdown. BeWo cells were transfected with each of the four siRNAs (SsiRNA1, SsiRNA2, SsiRNA3, SsiRNA4) designed for syncytin-1 knockdown. Transfection with the non-specific siRNA (Ctl siRNA) was included as a control. At 48 hours post-transfection time the cells were harvested and total RNA isolated. Real-time PCR was performed to determine syncytin-1 mRNA levels. The results were normalized by those of the GAPDH internal reference. The data is expressed as relative folds of changes over the control siRNA group. Student t test was performed to compare the results from syncytin-1 siRNAs and control siRNA. Statistical significance is indicated by asterisks (**, P < 0.01).
Fig. S3. Time-response of BeWo cell apoptosis to syncytin-1 knockdown. BeWo cells were transfected with 100 nM of control siRNA (left panels) or syncytin-1-specific siRNA (right panels). TUNEL assays were performed at 48(A), 72 (B), and 96 (C) hours post-transfection time, respectively. Nuclei were counter stained with hematoxylin. Brown color staining in the nuclei indicated cell apoptosis (marked by arrows). D. Apoptotic and total cells were counted in 10 randomly selected microscopic areas and the percentage of apoptotic cells was calculated for the control (Ctl siRNA) and syncytin-1 knockdown (SsiRNA1) groups. Although significant cell apoptosis were found at all the three time points (**, P < 0.01), most apoptotic cells was observed at 48 and 72 hours post-transfection time
Fig. S4. Dose-response of BeWo cell apoptosis to syncytin-1 knockdown. BeWo cells were transfected with 20 (panel A), 50 (panel B), and 100 (panel C) nM of control siRNA (left panels) or syncytin-1-specific siRNA (right panels). TUNEL assays were performed at 48 hours post-transfection time. Nuclei were counter stained with hematoxylin. Brown color staining in the nuclei indicated cell apoptosis (marked by arrows). D. Apoptotic and total cells were counted in 10 randomly selected microscopic areas and the percentage of apoptotic cells was calculated for the control (Ctl siRNA) and syncytin-1 knockdown groups (SsiRNA1). More apoptotic cells were observed when cells were transfected with higher concentration of syncytin-1 siRNA. A significant cell apoptosis was observed when 100 nM of syncytin-1 siRNA was applied. (**, P < 0.01).