RT-qPCR analysis. HeLa cells were infected with TIMELESS-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. TIMELESS protein expression in wild-type (WT) and shRNA knockdown (KD) HeLa cells was detected using Western blotting. Hsp90 α served as a loading control. The blots were incubated with primary antibodies against TIMELESS and Hsp90 α, respectively, followed by incubating with HRP-conjugated goat anti-rabbit secondary antibody. Images were developed using FeQ™ ECL Substrate Kit.