Fig. 7. Chlorpromazine-induced inhibition of CME arrests development of thymocytes.

a Thymi-derived double-negative three progenitors (DN3: CD4⁻CD8⁻CD44⁺CD25⁻) were sorted and cultured on OP9-DL1 stroma cells in the presence of Flt-3L, SCF and IL-7. At day 5 of co-culture cells were left untreated (ctrl) or treated with chlorpromazine (15 µM) to partially inhibit CME (chlorpromazine IC₅₀ for CME was established at 17.4 µM³⁵). Subsequently, development of double-positive (DP: CD4⁺CD8⁺) progenitors was assessed by FACS (left) and quantified (right). b Bone marrow-derived LSK progenitors (lineage⁻Sca-1⁺CD117⁺) were sorted and cultured on OP9-DL1 stroma cells in the presence of Flt-3L, SCF and IL-7. Analogous to a, at day 8 of culture cells were left untreated (ctrl) or treated with 15 µM chlorpromazine. Effect of partial CME inhibition on early-thymocyte progenitor development was assessed by FACS. Double-negative (DN) cells were defined as follows: DN1–CD44⁺CD25⁻; DN2–CD44⁺CD25⁺ and DN3–CD44⁻CD25⁺. c, d Bone marrow-derived LSK progenitors (lineage⁻Sca⁺1⁻CD117⁺) were sorted and cultured on OP9 stroma cells in the presence of Flt-3L, SCF and IL-7. At day 8 of culture cells were left untreated (ctrl) or treated with chlorpromazine (15 µM) to partially inhibit CME. Subsequently, development of c B-cell committed progenitors defined as B220⁺CD19⁺ and d granulocytes (CD11b^int−hiGr-1⁺) was assessed by FACS (left) and quantified (right). a–d Density FACS plots and charts are representative of two independent experiments where one to four wells were measured at each indicated time point. Number adjacent or within the gates indicate frequency. Each dot on the chart represents one well. Whiskers indicate the range (min to max). Arrows indicate time points when chlorpromazine was added. Statistical analysis was performed using two-way ANOVA (p-values for effect of chlorpromazine). Source data are provided as a Source Data file.