4 6 diamidino 2 phenylindole (dapi)

Huh7 and SNU387 cells (1 × 10³) were seeded in 24-well plates, fixed with paraformaldehyde, and then incubated with 0.5% Triton X 100. The cells were then pre-hybridized using a fluorescent in situ hybridization kit (RiboBio, Guangzhou, China). Subsequently, a Cy3-labeled circLIFR probe (RiboBio, Guangzhou, China) and a FAM-labeled miR-624-5p probe (IGE Biotechnology, Guangzhou, China) were hybridized with HCC cells at 37 °C overnight. Finally, nuclei were stained with DAPI (CWBio, Beijing, China) and photographed. Probe sequences are listed in Table S3.

To identify the purity of immature and mature SCs, cells were fixed with 0.4% paraformaldehyde (PFA) for 30 min at room temperature and washed with cold PBS for three times. Following permeabilizing using 0.4% triton-X 100, the cells were incubated with 1% BSA for 30 min to block the nonspecific reaction. Then, the mature SCs were incubated with rabbit anti-SOX9 (Abcam, Catologo numberi: ab76997) at a dilution with 1:400 and the immature SCs were incubated with rabbit anti-SOX9 at a dilution with 1:400 and mouse anti-AMH (Santa Cruz, Catologo numberi: sc-377,140) at a dilution with 1:200 overnight at 4 °C. Next day, the cells were washed with DPBS for three times and incubated with Alexa flour 488/594 conjugated donkey anti-rabbit IgG or Alexa flour 488 conjugated donkey anti-mouse IgG at 1:400 for 1 h at room temperature. After washed with DPBS, cell nucleus was labeled by DAPI (CWBIO, China) at a dilution with 1:400. The cell fluorescence was observed and photographed by a fluorescence microscope (Leica, Germany).

Sperm samples were washed with PBS, fixed with 4% paraformaldehyde for 10 min, spread onto the poly-L-lysine slides, and air dried at room temperature. The samples were permeabilized with 0.5% Triton X-100 in PBS for 10 min. Nonspecific binding was blocked with PBS in the presence of 10% BSA (Sigma-Aldrich) for 30 min at room temperature. Samples were then incubated with anti-CPT1(sc-514555; 1:25) and anti-ACADVL (sc-271225; 1:50) overnight at 4 °C. The samples were rinsed with PBS 3 times and then incubated with biotinylated goat anti-mouse FITC-IgG (1:200) the next day. Sperm were washed and counterstained with DAPI (CWBIO). Fluorescent images were captured with fluorescence microscopy (80i, Nikon, Japan). Negative control immunostaining was also performed at the same time without the primary antibody.

The cultured cells were plated on coverslips and transfected with indicated plasmids. After transfection for 48 h, the cells were washed three times with ice-cold PBS, fixed in 4% paraformaldehyde (PFA) at room temperature for 30 min. Cells were then washed two times with 0.1% PBS-T. For permeabilization, cells incubated with 0.25% Triton X-100 in PBS for 15 min and washed two times with 0.1% PBS-T. Cells were incubated in blocking solutions (normal goat serum), to block nonspecific binding of the antibody for 30 min, and incubated in primary antibodies diluted in PBS. After four washes with 0.1% PBS-T, cells were incubated in secondary antibodies for 1 h. After washed three times, nuclear staining was performed with DAPI (cwbiotech, Beijing, China). Coverslips were mounted and imaged by fluorescence microscope.