VPS45 depletion results in prolonged association of G-CSFR with early endosomes and disrupted transport to late endosomes. (A) Serum-starved control and VPS45 KO HeLa clones stably expressing G-CSFR–green fluorescent protein fusion proteins were stimulated with 100 ng/mL G-CSF for 10 minutes, followed by a chase for the indicated periods. Stimulated cells were fixed and stained with anti-EEA1 and anti-LAMP2 antibodies. DAPI was used as a nuclear stain. Enlarged images represent magnified views of boxed areas (10 × 10 µm). One representative experiment of 2 independent experiments is shown. Scale bars, 10 µm. (B) Quantification of G-CSFR-EEA1 and G-CSFR-LAMP2 colocalization of data shown in panel A. Pearson correlation coefficient is plotted on the y-axis. For each genotype, ≥100 cells were analyzed. Horizontal lines indicate the means, and each symbol represents an individual cell. Statistical analysis was performed using 2-way ANOVA with Tukey multiple comparisons test. ****P < .0001.