Luciferase activity assay

To generate miR-30a vector, a 335 bp genomic fragment encompassing primary miR-30a gene was PCR amplified and cloned into BamHI/XhoI site of pcDNA3.1 vector. To generate pGL3-miR-217 luciferase construct, 420 bp genomic fragment encompassing primary miR-217 gene was PCR amplified and cloned into XbaI site of pGL3 luciferase plasmid. Luciferase activity assay was performed according to the instruction manual (Promega Company). To generate miR-22 vector, a 438 bp genomic fragment encompassing primary miR-22 gene was PCR amplified and cloned into BamHI/XhoI site of pcDNA3.1 vector. To generate the pGL3 3′-UTR-p53 construct, a 492 bp human p53 gene segment encompassing 3′-UTR was PCR amplified and subcloned into the XbaI site of pGL3 luciferase plasmid. The mutant pGL3 3′-UTR-p53 construct and mutant miR-30a construct were made with the QuikChange site-directed mutagenesis kit (Stratagene). The pGL3 3′-UTR-p53 contains binding sequences for miR-30a. The pGL3 p53 del-3′-UTR construct lacks binding sequences for miR-30a. Luciferase activity assay was performed according to the instruction manual (Promega). Wild type miR-30a^cy3 sequences used in this study are 5′-CUUUCAGUCGGAUG UU UGCAGC-3′. Mutant miR-30a^cy3 sequences used in this study are 5′-CUGGACUGAUU AUGUUUGCAGC-3′. Wild type miR-217^cy5 sequences used in this study are 5′-UACUGCAUCAGGAA CUGAUUGGA-3′.

U87MG cells with luciferase reporter gene under the control of HIV‐1 LTR promoter were incubated with 2 μg/ml HIV‐1 Tat (ABL Inc and NIH AIDS program) in the presence of 100 μM chloroquine. Forty‐eight hours post‐incubation, luciferase activity assays (Promega) were performed and relative luminescence units were quantified using a fluorometer/luminometer plate reader (Spectra MAX GEMINI EM).