Neutral phenol chloroform isoamyl alcohol

The changes of nuclear morphology for assessing apoptosis were assessed using DAPI staining. Briefly, cells were cultured with or without different concentrations of isorhamnetin for 48 h, and were then fixed with 4% paraformaldehyde (Sigma-Aldrich Chemical Co.) for 10 min at RT. The cells were rinsed with PBS, and incubated with 1 μg/mL DAPI solution (Sigma-Aldrich Chemical Co.) at 37 °C for 10 min. Stained cells were visualized and photographed using fluorescence microscopy (Carl Zeiss, Oberkochen, Germany). For DNA fragmentation assay, the collected cells were lysed in a buffer containing 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM ethylenediaminetetraacetic acid, and 0.5% Triton X-100 for 30 min. The fragmented DNA in the supernatant was extracted using an equal volume of neutral phenol:chloroform:isoamyl alcohol (25:24:1, Sigma-Aldrich Chemical Co.), analyzed electrophoretically on 1% agarose gel containing EtBr (Sigma-Aldrich Chemical Co.), and photographed under a Fusion FX Image system (Vilber Lourmat, Torcy, France).

pcDNA3.1(+) (Thermo Fisher Scientific, Waltham, MA) was modified using Gibson Assembly Master Mix (New England Biolabs, Ipswich, MA) and appropriate DNA fragments according to the Gibson assembly method (Gibson et al., 2009 (link)) to generate pBAO1124, which contains (in order): T7 promoter, 46-nt 5′-UTR lacking any AUG or near-AUG codons (i.e., NUG, ANG, or AUN, where N is any nucleotide), 3xHA-NanoLuc luciferase ORF, 56-nt 3′-UTR, and 50-nt poly(A) sequence. RNAs were generated by run-off transcription with T7 RNA Polymerase using the MEGAscript T7 Transcription Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions using PCR-amplified DNA templates derived from pBAO1124 or its variants (DNA sequences are provided in Supplementary file 2). Transcription reactions were terminated by addition of ammonium acetate stop solution. RNA was extracted with neutral phenol:chloroform:isoamyl alcohol (25:24:1) (Sigma, St. Louis, MO), precipitated with ethanol, and resuspended in nuclease-free water. A 5′−7-methylguanosine cap was added to RNA post-transcriptionally using the Vaccinia Capping System (New England Biolabs). Capping reactions were desalted using Micro Bio-Spin Columns with Bio-Gel P-30 (Bio-Rad, Hercules, CA) before RNA was extracted with phenol, precipitated with ethanol, and resuspended in nuclease-free water.