JAK2V617F⁺ hematopoietic cells induce DNA damage response in normal cells via paracrine signaling. (A) Schematic representation of the following experiments. EV-32D, JAK2WT-32D, and JAK2V617F-32D cells were cultured at a concentration of 1 × 10⁶ cells per milliliter for 24 hours, and parental 32D cells were incubated at a concentration of 1 × 10⁵ cells per milliliter with CM. (B) Immunofluorescence staining of γH2AX foci formation in parental 32D cells incubated with EV-32D CM, JAK2WT-32D CM, and JAK2V617F-32D CM with or without 1 mM of N-acetylcysteine (NAC). Distribution of the numbers of γH2AX foci per nucleus is shown. More than 100 cells were counted in each specimen. (C) Intracellular levels of ROS were measured by 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) assay following incubation of parental 32D cells with each CM with or without 1 mM NAC for 24 hours. Average mean fluorescein isothiocyanate (FITC) fluorescence intensity is shown (n = 3 each). (D) Relative cell numbers of parental 32D cells 36 hours after incubation with each CM with or without 1 mM NAC (n = 4 each). (E) Immunofluorescence staining of 8-hydroxy-2-deoxyguanosine (8-OHdG) in parental 32D cells incubated with EV-32D CM, JAK2WT-32D CM, and JAK2V617F-32D CM with or without 1 mM of NAC. Average fluorescence intensity of intranuclear 8-OHdG is shown. More than 100 cells were counted in each specimen. Error bars indicate standard deviation (SD).