Fig. 2: Autophagy inhibition enhances ruxolitinib efficacy in JAK2^V617F cells in vitro.

A, B HEL and SET-2 cells were treated or not with ruxolitinib (1 µM) in the presence or absence of chloroquine (20 µM) or SAR405 (3 µM). After 3 days, the number of cells was assessed by trypan blue exclusion counting (n = 4 ± sem). A and the percentage of cell death was determined by Annexin-V labeling and flow cytometry analysis (n = 4 ± sem) B. C, D CD34+ cells obtained from JAK2^V617F-positive MPN patient samples were treated or not with ruxolitinib (1 µM) in the presence or absence of chloroquine (10 µM) or SAR405 (3 µM). After 2 days, the number of cells was assessed by trypan blue exclusion counting (n = 6 ± sem) C and the percentage of cell death was determined by Annexin-V labeling and flow cytometry analysis (n = 5 ± sem) D. E CD34+ cells from JAK2^V617F-positive MPN patient samples were plated for colony forming assay in semi-solid medium supplemented or not with ruxolitinib (250 nM) and chloroquine (1 µM) or SAR405 (3 µM). After 14 days, clonogenic potential was assessed by counting the number of BFU-E clones per dish. Data are represented as percentage of control (n = 14 ± sem). F, G. JAK2^WT HL-60 cells were treated or not with ruxolitinib (1 µM) in the presence or absence of chloroquine (20 µM) or SAR405 (3 µM). After 3 days, the number of cells was assessed by trypan blue exclusion counting (n = 3 ± sem) F and the percentage of cell death was determined by Annexin-V labeling and flow cytometry analysis (n = 3 ± sem) G. H, I CD34+ cells from JAK2^WT healthy donor samples treated or not with ruxolitinib (1 µM) in the presence or absence of chloroquine (10 µM) or SAR405 (3 µM). After 2 days, the number of cells was assessed by trypan blue exclusion counting (n = 5 ± sem) H and the percentage of cell death was determined by Annexin-V labeling and flow cytometry analysis (n = 5 ± sem) I. J CD34+ cells from JAK2^WT healthy donor samples were plated for colony forming assay in semi-solid medium supplemented as indicated. After 14 days, clonogenic potential was assessed by counting the number of BFU-E clones per dish. Data are represented as percent of control (n = 10 ± sem).