Piwil1

Cells were fixed in 4% PFA for 15 min at room temperature, permeabilized with 0.5% Triton X-100 for 30 min, blocked with 5% BSA for 1 h, and incubated with primary antibodies. The primary antibodies included GPR125 (Santa Cruz,1:200), GFRA1 (Santa Cruz,1:200), UCHL1 (AbD Serotec,1:200), PLZF (Abcam, 1:200), MAGEA4 (a kind gift from Professor Giulio C. Spagnoli, University Hospital of Basel, Switzerland, 1:25), SOX9 (Millipore, 1:200), WT1 (Santa Cruz, 1:200), SCF (Sigma, 1:200), BMP4 (Abcam, 1:200), PIWIL1 (Abcam, 1:200), PIWIL2 (Abcam, 1:200), γH2AX (Millipore, 1:100), Protamine 2 (PRM2, Santa Cruz, 1:200), Acrosin (ACR, Santa Cruz, 1:200), SYCP3 (Abcam, 1:100), H3K9 trimethylation (H3K9me3, Abcam, 1:200), and Human nuclear antigen (HumNuc, Millipore, 1:200). After incubation at 4 °C overnight, cells were washed three times in PBS (Sigma) and followed by incubation with secondary antibodies for 1 h at room temperature. Secondary antibodies were conjugated to Alexa Fluor 488 or Alexa Fluor 594 (Invitrogen). DAPI was used to label cell nuclei, and images were captured with a fluorescence microscope (Leica). Replacement of primary antibodies with isotype IgGs served as negative controls.