Lsm880 laser confocal microscopy

The transfected cells were washed with cold PBS and then incubated with a medium containing 10% FBS at 37 °C. At given times during the 37 °C incubation (0 h, 6 h or 12 h), the cells were fixed and permeabilized in 4% paraformaldehyde containing 0.2% Triton X-100. The cells were then incubated with anti-FLAG mouse monoclonal antibody (1:1000, Beyotime, Shanghai, China) for overnight, then incubated with goat anti-mouse IgG-RBITC (1:1000, Ricky, Shanghai, China) at 37 °C for 1 h. The co-localization of mitochondria and FLAG at 12 h post-transfection is based on this method, and incubated Mito-Tracker Green (Invitrogen, Carlsbad, CA, USA) was used to incubate the cells at 37 °C for 30 min in the dark according to the manufacturer ’s specified guidelines. The cells were washed twice with a fresh cell culture medium and then added to a fresh cell culture medium at 37 °C. The mitochondria were observed by LSM880 laser confocal microscopy (Carl Zeiss Inc., Oberkocken, Germany).

MDA-MB-231 cells (6 × 10⁵) were first seeded in cover glass chamber (80826, ibidi, Martinsried, Germany) overnight, washed with PBS once, and fixed with 4% paraformaldehyde for 10 min. After washing with PBS twice, cells were dehydrated with 70% EtOH for 2 h, and incubated with RNA probe (125 nM) at 37 °C overnight. The RNA labeling probes conjugated with 5ʹ modification 6-FAM (TTC CAA GGA TAT CAT TCT TCA TCA) were designed to target the back-splicing site of circ-AAGAB. Next, the cells were washed at 37 °C for 30 min, and mounted with Mounting Medium containing DAPI (ab104139, Abcam, Cambridge, UK). Finally, the images were acquired using a ZEISS LSM880 laser confocal microscopy (ZEISS, Heidelberg, Germany).