Ribosomal profiling assay

The polysome assay was carried out using cell lysate from HeLa or A549 cells (1 × 10⁸) treated with Acr (0–100 μM, 3 h) as described previously [53]. The lysate was prepared in buffer A containing 25 mM HEPES (pH 7.5), 400 mM KOAc, 5 mM Mg (OAc)₂, 2% TritonX-100, 0.2 mM cycloheximide, and 40 U/ml RNasin (Invitrogen). A 12-ml 10–50% sucrose density gradient containing buffer (20 mM Tris–HCl, pH7.5; 50 mM KCl; 3 mM MgCl₂) was used for the analysis. The polysome profile was monitored by absorbance at 254 nm using an ISCO auto fractionation instrument and starting with the free material followed by 40S ribosomal subunit detection and continuing through to polyribosome complexes. Aliquots were taken from each fraction and subjected to the phenol–chloroform extraction to allow rRNA analysis.