G-CSF induces autophagy in HSCs and neutrophils. (A) Representative images of LC3-GFP punctae formation in sorted BM LKS⁺ cells from LC3-GFP mice treated with saline or G-CSF. Mean LC3-GFP punctae/cell ± SEM (n = 6-7, 2 experiments). (B) Total cell lysates from LKS⁺ were immunoblotted with anti-LC3B or anti-β actin. CHO cell lines were used as a control expressing LC3 I. The fold increase of LC3 II band intensities, normalized to β actin, was determined by the ratio of CQ-treated and CQ-untreated cells (representative experiment). (C) LC3-GFP punctae in purified BM neutrophils (CD11b⁺Gr1^hi) culture in duplicate for 6 hours ±G-CSF and ±CQ. LC3-GFP punctae quantification in neutrophils (representative experiment). (D) Blood from human donors were collected before and after 5 days of G-CSF treatment (10 mcg/kg per day). Sorted neutrophils were collected for quantitative real-time polymerase chain reaction analyses and western blot. Relative gene expression of ATG5 and ATG7 from sorted human blood neutrophils before and after G-CSF treatment (n = 10). (E) Total cell lysates were immunoblotted with anti-ATG5, ATG7, LC3, or anti-β actin antibodies. A paired Student t test was performed for statistical analyses (**P < .01, ***P < .001, ****P < .0001). The mean ± SEM is presented. Each dot corresponds to an individual donor. The data before and after G-CSF are color-paired.