Pgl3.0 enhancer

HCC (3 × 10⁴) cells were plated in 48-well plates and transfected with pGL3.0-enhancer (Promega) and full or various truncated BECN1 promoter-luciferase constructs together with the pRL-TK in triplicate by X-tremeGENE HP DNA Transfection Reagent (Roche, 06366236001). We treated cells as indicated at 24 h after transfection, and then collected cells for performing the luciferase assays with the Dual Luciferase Reporter Assay System (Promega). Luciferase activities were calculated as fold induction compared with that in pGL3.0-enhancer. All bar diagrams are shown as the means±S.D.

The luciferase reporter plasmids, pGL-hamster-HMGCS1-promoter and pGL-LDLR-promoter were designed and generated (details can be found in the supplementary material). The pGL3.0-enhancer (Promega) and the constructed luciferase reporter plasmids were transfected into HCC (3 × 10⁴) cells in 48-well plates with the pRL-TK in triplicate by X-tremeGENE HP DNA Transfection Reagent. Then the cells were collected after 24 h transfection to measure the luciferase activities with the Dual Luciferase Reporter Assay System (Promega). All data are presented as the means ± S.D. The experiments have been repeated at least three times.

pcDNA3.1-ASPP2 (full, (1–360), (360–925), (925–1128))-V5 was obtained from Dr Xin Lu's Lab at Ludwig Institute for Cancer Research, Oxford, UK. pcDNA3.0-flag-BECN1 was obtained from Prof. Mujun Zhao's Lab at Institute of Biochemistry and Cell Biology, Chinese Academy of Science, Shanghai, People's Republic of China. pCMV-p65/RelA-flag was constructed by PCR and inserted into pCMV-C-Flag vector (Beyotime Biotech. D2632, Shanghai, People's Republic of China). Various truncated BECN1 promoters were generated by PCR and inserted into reporter vector pGL3.0-enhancer (Promega, Madison, WI, USA). p40(phox)PX-EGFP, pFlag-BECN1 ((1–150), (150–241), (242–450)), pCI-neo-HA-hUVRAG and pCI-neo-HA-hATG14 were purchased from Addgene (Cambridge, MA, USA).
Small interfering RNAs targeting BECN1 (siBECN1) were generated by GenePharma (Shanghai, People's Republic of China) and were transfected with DharmaFECT4 (ThermoScientific, T-2004-02). Lentiviral plasmid vectors encoding short hairpin RNAs (shRNAs) targeting ASPP2 or scramble shRNA were generated and designated as LV-shASPP2 and LV-shNon, respectively. Further details are available in the Supplementary Material.