PML acts as a ROS sensor in ELANE-I60F HPCs. (A) Representative immunofluorescent image showing complete loss of PML-NBs after introduction of a stop codon in exon 3 of the PML gene and subsequent PML-NB quantification. The red line indicates the median number of PML-NBs/cell for the control HPCs. Number of cells analyzed (n): control, n = 441; control PML^−/−, n = 175; ELANE-I60F, n = 353; ELANE-I60F PML^−/−, n = 172. (B,D) Quantifications of immunofluorescent images showing increased nuclear NRF2 levels in ELANE-I60F PML-deficient HPCs (B), but not in control (D). Data are pooled from 3 independent experiments (ELANE-I60F, n = 365; ELANE(-I60F) PML^−/−, n = 370; control, n = 631; control PML^−/−, n = 644, where n = the number of cells). The red line indicates the median number of NRF2 nuclear intensity for the PML-proficient HPCs. Microscope settings (laser power) differed between figures. (C) Relative quantification of the mean fluorescent intensity (MFI) showing no increased CellROX Deep Red levels after PML knockout, where PML-proficient cells (ELANE-I60F or control) were used as baseline. Data are from 3 independent experiments. ***P < .001.