Fig 1: Pharmacological inhibition of SOC channels and CAMKK2 enhanced ATRA-induced cell death. (A) NB4 cells were pre-treated with 5 μg/mL STO609 and then treated with 1 μM ATRA for 72 h. Granulocytic differentiation was determined by cytofluorometric evaluation of CD11c expression. Left panel, representative flow cytometry histograms are shown; right panel, the median fluorescence intensity (MFI) of CD11c was scored for each sample. (B) NB4 cells were pre-treated with 44 μM 2APB and then treated with 1 μM ATRA for 72 h and granulocytic differentiation was determined. Results correspond to the means ± SD of three independent experiments realized in triplicate. p value * < 0.05, p value *** < 0.001.
Fig 2: ATRA induces autophagy through a Ca2+-dependent mechanism. (A) NB4 cells were pre-treated with 5 μM BTP2 for 2 h before treatment with 1 μM ATRA for 24 h. Cell extracts were then analyzed for LC3 levels by western blotting. (B) Immunoblot analysis of LC3 levels in NB4 cells pre-treated with 44 μM 2-APB for 2 h and then treated with 1 μM ATRA for 24 h. (C) NB4 cells were treated with 1 μM ATRA for the indicated time then subjected to immunoblot analysis for phospho-CAMKK2, CAMKK2, p62 and LC3-II. Ponceau S red staining was used to score the protein loading levels. The expression levels of p62 and LC3-II were scored and normalized to the protein loading levels of each sample. The expression levels of phospho-CAMKK2 and CAMKK2 were quantified and given as ratio of phospho-CAMKK2/CAMKK2. The quantification values represent the ratio of treated cells versus untreated cells (fold change, FC) for each time point.
Fig 3: Pharmacological inhibition of SOC channels and CAMKK2 promotes ATRA-induced cell death. NB4 cells were pre-treated with STO609 (5 μg/mL) and then treated with 1 μM ATRA for 5 days. Cell death was assessed by the measurement of (i) the mitochondrial transmembrane potential using TMRM dye and (ii) the plasma membrane permeability using propidium iodide (PI) staining. (A) Left panel, representative flow cytometry histograms for TMRM and PI cell staining. Right panel, percentage of cells exhibiting low TMRM staining and high PI staining were scored for each condition. (B) NB4 cells were pre-treated with 2-APB (44 μM) prior to treatment with 1 μM ATRA for 2 days. At the end of this treatment, cell death was scored as described in (A,B) Results are means ± SD of three and four independent experiments realized in triplicate for STO-609 and 2-APB, respectively. **** p-value < 0.0001.
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