Immunofluorescence Staining of piPSCs

After twice washing with PBS, the piPSCs (cultured for 5 days) were fixed in 4% paraformaldehyde (pH 7.4) at room temperature for 15 min. We used 0.1% Triton-100 to perforate the membranes at room temperature for 10 min. Then, 10% FBS was used to block the membranes at room temperature for 1 h. The membranes were incubated with primary antibodies, including LIN28A (1∶200; Santa Cruz Biotechnology, USA) and FLAG (1∶1 000; Sigma-Aldrich, USA), for 12 h at 4 °C and then washed three times with PBS. The membranes were then incubated with goat anti-mouse IgG (H+L) secondary antibody Alexa Fluor 488 conjugate (1∶500; ZSGB-BIO, China) at room temperature for 1 h and nuclei were stained with Hoechst33342 (1∶1 000) at room temperature for 5 min (Ma et al., 2019 (link); Wei et al., 2021 (link)).