Ctr4000b

To assess cell cycle, cells were cultured (1 × 10⁶ cells/well) for 48 h, then collected and analyzed using CELL Quest software (BD Biosciences, San Jose, CA), as previously described. For apoptosis analyses, cells were stained with acridine orange/ethidium bromide (AO/EB) and visualized under a fluorescence microscope (LEICA CTR4000B; Leica Microsystems, Buffalo Grove, IL, USA). Apoptotic rate (%) = (apoptotic cells/total cells) × 100%.

The treated cells were fixed with formaldehyde for 10mins and incubated in 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h to permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with the primary anti-COL2A1 (1:50, Santacruz biotechnology, sc-52658) and ACAN (1:200, proteintech, 13880-1-AP) antibody overnight at 4°C. The secondary antibody (green and red) was goat anti rabbit and mouse (proteintech, SA00003-2 and proteintech, SA00009-1) Ig G(H+L) which were used at a dilution of 1 to 50 for 1h. DAPI was used to stain the cell nuclei(blue) and its concentration was 1.43μM. The cells were observed by fluorescence microscope (CTR4000B, Leica). Experiments were repeated three times independently.

Transfected MCF7 and MB231 cells were plated in six-well plates at 1 × 10⁵ cells/well. After 24 h, the cells were washed three times with phosphate-buffered saline (PBS) and then stained with AO/EB for 5 min. Cells were visualized using a fluorescence microscope (CTR4000B, Leica). The percentages of apoptotic cells were calculated using the formula: percentage of apoptotic cells (%) = (amount of apoptotic cells/total cells examined) × 100%.

Nucleus pulposus cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.5% Triton X-100 for 30 min. After blocking with 5% bovine serum albumin (BSA) for 30 min, slides were incubated with primary antibodies against P62 (1:200) overnight at 4°C. Secondary antibody (1:100; Invitrogen, Carlsbad, CA, United States) was added the next day. The nuclei were stained with 4′,6-diamino-2-phenylindole (DAPI) for 1 min. The cells were observed using a fluorescence microscope (CTR4000B, Leica, Wetzlar, Germany).

To assess cell cycle status, MB231 cells and MCF7 cells were seeded in six-well plates and transfected with 4 μg of DACT2 or empty vector using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) following the manufacturer's protocol. After 48 h, cells were harvested using 0.1% trypsin and washed with PBS, then fixed in ice-cold 70% ethanol for 1 h, and treated with 100 μl of 50 mg/l propidium iodide for 30 min at 4°C in the dark. Data were analyzed with the CELL Quest software (BD Biosciences, San Jose, CA). Acridine orange/ethidium bromide (AO/EB) staining was used for apoptosis analysis. Briefly, the transfected cells were replated in six-well plates containing glass coverslips. After 24 h, cells were washed with phosphate-buffered saline (PBS) then stained with AO/EB for 5 min and visualized immediately under a fluorescence microscope (LEICA CTR4000B). The percentage of apoptotic cells was then calculated.

MCF7 cells were cultured in 6-well plates at a density of 2×10⁵ cells/well and grown overnight. Cultures were then transiently transfected with 4 µg pcDNA3.1(+)-Flag- ZNF545 plasmid or the control vector using Lipofectamine-2000 (Invitrogen, Carlsbad, CA) following the manufacturer's protocol. After 48 h, cells were collected and centrifuged at 800 rpm for 5 min. Cells were then washed with PBS twice and fixed in ice-cold 70% ethanol for 1 h, and treated with 100 µl of 50 mg/l propidium iodide for 30 min at 4°C in the dark. Data were analyzed by CELL Quest software (BD Biosciences, San Jose, CA, USA). Apoptosis was examined using acridine orange/ethidium bromide (AO/EB) fluorescence staining. Transfectants (1×10⁵ cells) were replated in six-well plates. After 24 hours, cells were washed three times with phosphate-buffered saline (PBS) then stained with AO/EB for 5 min and visualized immediately under a fluorescence microscope (LEICA CTR4000B). The percentage of apoptotic cells was then calculated by the formula: percentage of apoptotic cell (%)  =  (amount of apoptotic cell/total cell examined) ×100%.