Hmga2

Proteins were extracted using RIPA buffer (CWBIO, China). After being electrophoresed by SDS-PAGE, the samples were transferred to nitrocellulose membranes and incubated with primary antibodies specific for E-cadherin (diluted 1:1000, Proteintech, USA), vimentin (diluted 1:1000, ABclonal, China), N-cadherin (diluted 1:1000, ABclonal, China), HMGA2 (diluted 1:1000, ABclonal, China) and GAPDH (diluted 1:1000, ABclonal, China) at 4 °C overnight. Then the membranes were incubated with secondary antibodies at room temperature for 1 h. Signals were detected with images acquisition using Immobilon ECL substrate (Millipore, Germany) and Optimax X-ray Film Processor (Protec, Germany).

Ethical approval was obtained from the Institutional Research Ethics Committee of Sun Yat-sen University. All animal care and procedures were performed in accordance with institutional guidelines. To assess the effect of hsa_circ_0006948 on tumor growth in xenograft models, TE-1 cells (5×10⁶/0.2 ml PBS) were injected subcutaneously into the right backs of 4-week-old BALB/c nude mice. After 12 days, the mice were treated with intertumoral injection of negative control, si-hsa_circ_0006948 and combined si-hsa_circ_0006948 and agomiR-490-3p every two days, respectively. The tumors were measured every three days and their volumes were calculated according to the following formula: tumor volume = (length × width²)/2. Thirty days later, the mice were sacrificed, and the tumors were excised for further immunohistochemistry. Primary antibodies against E-cadherin (Proteintech, USA), vimentin (ABclonal, China), N-cadherin (ABclonal, China) and HMGA2 (ABclonal, China) were used. We captured images using a Nikon Eclipse 80i system with NIS-Elements software (Nikon, Japan).