Lipomax

Transfections were achieved using LipoMAX (32011; SUDGEN Biotech, Nanjing, China) according to the manufacturer's protocols. Cells were transfected with 3 μL LipoMAX and 3 μg plasmids encoding galectin-3-mcherry (#85662), LC3-GFP (#11546) and LC3-GFP-mcherry (#123235, Addgene, Cambridge, MA, USA) in 500 μL serum-free medium. After 72 h incubation, the transfection mixture was removed and replaced with fresh complete medium. For RNA interference by lentiviral vectors, autophagy related 7 (ATG7) short hairpin RNA (shRNA), transient receptor potential mucolipin 1 (TRPML1) shRNA, CTSD shRNA, CTSB shRNA constructs and a negative control construct created in the same vector system (pLKO.1) were purchased from Corues Biotechnology. Before transfection, 293T cells were plated in 12-well dishes. Cells were co-transfected with shRNA constructs (10 μg) together with Lentiviral Mix (10 μL) and HG Transgene™ Reagent (60 μL) according to the manufacturer's instructions of Lentiviral Packaging Kit (YEASEN, 41102ES20) for 48 h. Viral stocks were harvested from the culture medium and filtered to remove non-adherent 293T cells. To select for the Jurkat cells that were stably expressing shRNA constructs, cells were incubated in RPMI-1640 medium with 10% FBS and 2 μg/mL of puromycin for 48 h after lentivirus infection. After selection cells, stably infected pooled clones were harvested for use.