Promoter luciferase assay

After PCR, the fragment of gene promoter of ATG4A (2175 bp) or LC3B (1631 bp) was cloned into pGL3-basic vector at KpnI/HindIII site; the fragment of gene promoter of ATG9B (2058 bp) or NPC2 (1803 bp) was cloned into pGL3-basic vector at NheI/XhoI (Promega, #E1751). Promoter construct DNA (100 ng) and renilla plasmid (20 ng) (Promega) were transfected into U251 by using X-tremeGENE HP DNA Transfection Reagent (Sigma) in a 12-well plate with 5% FBS full DMEM medium then infected with adenovirus expression null, N-terminal SREBP-1a, −1c or −2. GBM30 cells were seeded in Geltrex (Gibco) coated 12-well plate, and performed same procedure as done in U251. Luciferase activity was measured 24 h post-transfection by using firefly luciferase assay reagent (Promega) and renilla luciferase assay reagent (Promega) according to the kit instruction, and the signal was detected by Promega GloMax Plate Reader.

Primers used to clone gene promoters are shown in Table S1.