Figure 1.

ULK1 deficiency inhibits nonselective autophagy and Mycobacterium tuberculosis–selective autophagy in U937-derived monocytes and macrophages. A and B, Nonselective autophagy is inhibited without ULK1. ULK1-deficient (ULK1-knockout [KO]) or empty-vector (EV) U937 cells were incubated in nutrient-rich medium or starvation medium for 4 hours and then fixed, permeabilized, and stained for LC3. Images were obtained via fluorescent microscopy, and the number of LC3-positive puncta were measured per cell for each condition. A, Tukey plot of the number of puncta/cell in each condition. B, Deconvolved images of LC3 staining from individual cells in each condition, representative of each. Green images indicate LC3, and blue images indicate DAPI nuclear staining. Each experiment was performed with at least 100 individual observations and performed 3 times to ensure reproducibility. C and D, Selective autophagy of M. tuberculosis is inhibited in cells lacking ULK1. C, Bar graph of the proportion of intracellular bacteria colocalized with LC3-positive puncta. Bars indicate mean values, with error bars indicating standards error of the mean. D, Deconvolved images of intracellular M. tuberculosis colocalized with autophagosomes in an EV cell and intracellular M. tuberculosis not colocalized with LC3 in an ULK1-KO cell. Green images indicate LC3, red images indicate mycobacteria, and blue images indicate DAPI nuclear staining. To calculate statistics, at least 100 cells were measured for each condition, and each experiment was performed 3 times to ensure reproducibility. *P < .05 by the Wilcoxon rank sum test.