RT-qPCR analysis. HeLa cells were infected with ATG4C-specific shRNA lentiviral particles, total RNA was extracted from wild-type and knockdown cells, RT-qPCR was performed using gene-specific primers. ∆Ct (CtKD-CtWT) was used to calculate mRNA reduction (%) between wild-type and knockdown cells using the following formula: (1-1/2∆Ct) x 100%.

Western blotting analysis. ATG4C protein expression in wild-type (WT) and shRNA knockdown (KD) HeLa cells was detected using Western blotting. GAPDH served as a loading control. The blots were incubated with primary antibodies (Cat#61741, 1:10,000) against ATG4C and GAPDH, respectively, followed by incubating with HRP-conjugated goat anti-rabbit secondary antibody (Cat#201, 1:20,000). Images were developed using FeQ™ ECL Substrate Kit (Cat#226).