In order to evaluate how ibrutinib and nutlin-3 affect gene and protein expression of several components of p53 and BTK pathways, and to understand possible common or interrelated molecular targets, leukemic cells were harvested 24 h after treatments and processed for RNA and protein extraction as follows.

Total RNA was purified from lymphocytes by using the QIAGEN miRNeasy Mini kit (QIAGEN, Hilden, Germany), according to the supplier’s instructions. Total RNA (300 ng) was retro-transcribed and amplified by using the Express One-Step Superscript qRT-PCR Kit universal. Analysis of human c-MYC, TP53, MDM2 and CDKN1A gene expression was performed with specific primers sets (TaqMan Gene Expression Assays; c-MYC assay: Hs00153408_m1; TP53 assay: Hs01034249_m1; MDM2 assay: Hs00242813_m1; CDKN1A assay: Hs00355782_m1). Samples were run in duplicate by using the QuantStudio® 3 Real-Time PCR System, and expression values were normalized to the housekeeping gene POLR2A (assay Hs00172187_m1). These values were used to perform statistical analyses. All material was from Thermo Fisher Scientific (Rockford, IL, USA).

For Western blotting analysis, cells were processed as previously described [24] and equal amounts of protein were loaded for each sample. The following antibodies were used for immunoblotting onto nitrocellulose filters: anti c-MYC, anti-phospho p53-Ser15, anti-phospho p53-Ser392, BAD, phospho BAD-Ser136 and p14ARF from Cell Signaling (Danvers, MA, USA); anti-p53, anti-MDM2, anti-p21, anti-PUMA, anti-procaspase 9 and anti-procaspase 3 from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-BAX and anti-Akt from BD (BD Biosciences); phospho Akt-Ser473 from Merck Millipore (KGaA, Darmstadt, Germany) and anti β-actin, from Sigma-Aldrich (Merck KGaA). After incubation with secondary antibodies (anti- mouse or -rabbit IgG HRP-conjugated; Sigma-Aldrich), specific band detection was performed with the ECL Lightning kit (Perkin Elmer, Waltham, MA, USA). Images’ acquisition and analysis was performed using the ImageQuant™ LAS 4000 imager and TL software (GE Healthcare, Buckinghamshire, UK).

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