Egfp lc3

mRFP-LC3 (21075), tfLC3 (21074), and LAMP1-mGFP(34831) were obtained from Addgene (Cambridge, MA, USA). EGFP-LC3, BNIP3, and SNAP29 plasmids were constructed by Gene Chem Co. Ltd (Shanghai, China). Cells were transfected with plasmids using Lipofectamine 3000 Transfection Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. After 24 h incubation, the transfection mixture was removed and replaced with fresh complete medium. The target sequence of BNIP3 shRNA (5′-ACTGCACTTCAGCAATAAT-3′) was purchased Gene Chem Co. Ltd. To establish BNIP3 knockdown stable cell lines, 293FT cells were co-transfected with lentiviral packaging vectors pLP1, pLP2, and pLP/VSVG (Invitrogen, K4975) along with shBNIP3 or shCon plasmid using Lipofectamine 3000 (Invitrogen, L3000015) according to the manufacturer’s protocols. Forty-eight hours later, supernatant containing the lentivirus was harvested and infected with MCF-7 cells. Cells were subsequently selected with 4 μg/mL puromycin (Sigma, P9620) to establish stable cell lines.

mRFP-LC3 (21075) and LAMP1-mGFP (34831) were obtained from Addgene. EGFP-LC3 and ATG7-siRNA plasmids were constructed by GeneChem Co. LTD (Shanghai, China). Plasmids were transfected into MDA-MB-231 and MCF-7 cells using Lipofectamine 3000 transfection reagent (Invitrogen, L3000). The transfection mixture was removed and replaced with fresh complete medium after 24 h incubation. The sequences of UBC9 shRNA were 5′-TTGGCAGTAAATCGTGTAGGCC-3′ (sh-UBC9–1#) and 5′-ATTTAGAAGTTCCTGTATTCCT-3′ (sh-UBC9–3#).
293FT cells were transfected with pLP1, pLP2, pLP/VSVG (Invitrogen, K4975) and sh-UBC9 or sh-Con plasmid. Supernatants containing the lentivirus were harvested after 48 h incubation and used to infect target cells. MDA-MB-231 and MCF-7 cells were selected with puromycin (4–8 μg/mL) (Sigma, P9620) to establish stable cell lines.

Transfection was performed using Lipofectamine 3000 transfection reagent following the manufacturer's protocol. The plasmids EGFP-LC3 and RFP-MITO were constructed by GeneChem Co. (Shanghai, China). After 24-48 h of incubation, the transfection solution was replaced with a fresh complete culture medium. For RNA interference, the FASN-siRNA plasmid was constructed by Suzhou GenePharma Co., Ltd. (Suzhou, China), and FASN-siRNA was introduced into A2780 and OV90 cells according to the manufacturer's specifications.