Fitc annexin 5 apoptosis detection kit with pi

HT (purity ≥98%) was obtained from Extrasynthese, France. Dulbecco’s modified Eagle’s medium (DMEM) and sodium pyruvate were from Capricorn Scientific, Germany, foetal bovine serum (FBS) was from Sigma, USA, and Trypan Blue Solution, 0.4% from Thermo Fisher, USA. Primary antibodies mTOR (2972 S), p-mTOR (2971 S), S6 (2217 S) and p-S6 (2211 S) were purchased from Cell signaling Technology, USA; HIF-1α (A300–286A) from Bethyl, USA; PARP-1 (C-2-10) from Calbiochem, Germany; anti-poly(ADP-ribose) (α-PAR) (4355-MC) from Trevigen, USA; α-Tubulin antibody (T5168) from Sigma, USA and FIH-1 (sc-26219) from Santa Cruz, USA. Apoptosis was quantified using the FITC Annexin V Apoptosis Detection Kit with PI (ANXVKF, Immunostep, Spain). RNA was isolated using the RNeasyPlus Mini kit (Qiagen, Germany). cDNA Synthesis Kit for RT-qPCR and iTaq UniverSYBR for Real-time PCR were from Bio-Rad, USA. Primers were synthesized by Biomedal S.L. (Spain). ARNT siRNA (s1613 and s1615), scramble siRNA (sc-37007) and the transfection reagent jetPRIME were from Ambion, Santa Cruz and Polyplus Transfection, USA, respectively. HIF-1α siRNA was from Sigma (forward 5′-CUGAUGACCAGCAACUUGA-3′, reverse 5′-UCAAGUUGCUGGUCAUCAG-3′).

Cell viability, proliferation, apoptosis, intracellular expression of VDR and CYP24A1 as well as the expression of phospho-ERK (pERK) and phospho-NF-κB p65 (pNF-κB) were determined by flow cytometry. In all experiments, the BD Horizon Fixable viability stain 660 (BD Biosciences, Franklin Lakes, NJ, USA) was used for gating viable cells. Co-cultured CLL cells were further stained for surface CD19 (PE-Cy5 mouse anti-human CD19 antibody, BD Biosciences) in order to be gated against the HS5 cells. Intracellular staining of VDR and CYP24A1 was performed with the Fixation/Permeabilization Solution Kit (BD Biosciences) following the manufacturer’s instructions. Intracellular staining of pERK and pNF-κB was carried out following the phosflow protocol from BD Biosciences. The list of the antibodies used is given in Table S4. Apoptosis was measured using the FITC Annexin V Apoptosis Detection Kit with PI (Immunostep, Salamanca, Spain), whereas cell proliferation was measured using FITC Mouse Anti-Ki-67 Set (BD Biosciences).
Flow cytometric analysis was conducted on a BD FACSCalibur flow cytometer (BD Bioscience). For each sample, 10,000 events were acquired and nonspecific binding was excluded by using appropriate isotypic negative control antibodies. Gating analysis strategy included only viable cells and was performed using FlowJo software (BD Biosciences).

The levels of viable cells, apoptotic, early apoptotic, and necrotic cells were evaluated in the CP0024 cell line, transduced with shHMGA1 or shControl, and treated with 10 nM trabectedin for 24 h. FITC Annexin V Apoptosis Detection Kit with PI was used to determine cell death (Immunostep; Salamanca, Spain), following the manufacturer’s instructions. Three biological replicas were performed. Apoptosis levels were determined by flow cytometry (BD Accuri C6 Plus) and data was analyzed with BD Accuri C6 Plus software.