Extended Data Fig. 4: Cyclin B1 is not a direct substrate for APC/CPIWIL1 E3 complex in PDAC cells.
a, PIWIL1 was persistently associated with APC/C complex in AsPC-1 cells during the cell cycle. Both HeLa (lanes 1–4) and AsPC-1 cells (lanes 5–8) were synchronized in G1/S phase, then released and harvested at indicated time points for anti-APC3 co-IP assay of the association of Cdc20, Cdh1, or PIWIL1 with APC/C (top), and cell cycles were examined by FACS (bottom). The phosphorylated APC3 was indicated by “*”. b, c, Piwil1 knockdown (c) or overexpression (d) barely affected Cyclin B1 ubiquitination in PDAC cells. BxPC-3 and AsPC-1 cells were co-transfected with pEF-HA-Ub and scramble siRNA (lanes 1, 4, 7 and 10), Cdc20 siRNA (lanes 2, 3, 8 and 9) or Piwil1 siRNAs (lanes 5, 6, 11 and 12), respectively. PANC-1, Capan-1 and MIA PaCa-2 cells were co-transfected with pEF-HA-Ub and p3xFlag control vector (lanes 1, 3, 5, 7, 9 and 11) or p3xFlag-PIWIL1 (lanes 2, 4, 6, 8, 10 and 12), respectively. Top, anti-Cyclin B1 IP pellets were immunoblotted by anti-Ub for Cyclin B1-Ub conjugates (upper) or anti-Cyclin B1 to normalize the loading amounts (lower). Bottom, cell lysates were immunoblotted for PIWIL1, Cdc20, and Cyclin B1, with β-actin serving as a loading control. d, PIWIL1 depletion did not alter the expression pattern of Cyclins and progression of cell cycle in BxPC-3 cells. 24 h post-transfection with Piwil1 siRNA (lanes 7–12) or control scramble siRNA (lanes 1–6), BxPC-3 cells were synchronized in G1/S phase, then released and harvested at indicated time points for immunoblotting of indicated proteins in cell lysates (left), or FACS analyses of cell cycles (right). e, Gating strategy for analysis of synchronized cells. Results shown in a–d are representative of three independent experiments. Unprocessed blots are provided in Source Data Extended Data Fig. 4.
