(A) RT-PCR analysis of the GDF3 message in F1/F10 cells. F1/F10 cells were transfected with the plasmid for expression of GDF3 (upper panel). Cells just before inoculation (indicated as 0 day) and cells isolated from tumors on day 10 after inoculation (indicated as day 10) were prepared and adjust the cell numbers. These cells were lysed and total RNA was extracted from the lysates. RT-PCR was performed to detect GDF3 as well as Soxl5 (nagative control, center panel) and β-actin (positive control, lower panel). PCR cycles are 32 rounds, 3 times less in those shown in Fig. 2C,D (B) Cell lysate (day 0) was subjected to SDS-PAGE (left 10% gel, right 8% gel) followed by immunoblotting. Lower panel- Commassie brilliant blue (CBB) staining of the blot. Upper panel- blots GDF3 band visualized by treating with anti-GDF3 mAb and then HRP-labeled 2nd Ab. No relevant band of GDF3 was detected by CBB staining.