Figure 2

RA sensitizes APL primary blasts to ER stress. (a) APL blasts isolated from three patients’ bone marrow (APL1, APL2 and APL3) were treated in semisolid medium for 8 days. Bone marrow mononucleated cells isolated from two healthy donors (BM1 and BM2) were treated in semisolid medium for 4 days. The box plots report the distribution of the number of cells forming the colonies (APL1 n⩾40, APL2 and APL3 n⩾200 for each treatment; Student’s T-test *P-value<0.05, ***P-value<0.005; BM1 n=200; BM2 n⩾120; analysis of variance (ANOVA) presented in Supplementary table 1). (b) Morphological analysis of patient APL4 cells and of healthy donor BM1 cells isolated from 8 days colonies obtained as in a. The black arrows indicate cell vacuoles, MΦ macrophages, the inset in BM1 RA panel delimits cells from another field. Quantification of the different cell types and of vacuolation is shown in Supplementary Figure 2c. (c) Cell cycle analysis of the same cells to evaluate cell death as subG1 DNA content. (d) Protein extracts of cells isolated from patient APL4, treated in semisolid medium for 4 days, were analyzed by western blot analysis for the expression of p53 protein. GAPDH was used as loading control. (e) Total RNA from cells isolated from patient APL5, treated as in d, with the addition of ATO (see below, Figure 6), for 4 days were analyzed by quantitative reverse transcriptase-PCR (qRT-PCR) for the expression of the p53 targets PIG3, NOXA and p53AIP1.