Figure 1.
iNeurons as a model to study dynein-mediated transport. (A) Schematic of differentiation of NGN2 hESCs/hiPSCs into iNeurons. hESC/hiPSCs were split and 300,000 cells were plated. 2 d later, differentiation media (Diff.) was added to contain doxycycline. At 2 DPI, cells were split again and plated into microfluidics. At 7 DPI, doxycycline was removed from the media and cells were allowed to grow until 21–23 DPI. (B) Example image of 21–23 DPI iNeurons in microfluidic device. Cells were fixed and stained with an antibody against β-tubulin. (C) Kymographs of endosomes (Endo, CTB AlexaFluor 488), lysosomes (Lyso, Lysotracker Deep Red), and mitochondria (Mito, mitotracker Deep Red FM) in iNeurons at 21–23 DPI. (D) The mean speed of endosomes, lysosomes, and mitochondria in both anterograde (gray) and retrograde (light green) directions (Retrograde: endo versus lyso *P = 0.027, endo versus mito **P = 0.0025, lyso versus mito P = 0.71; Anterograde: endo versus lyso P = 0.99, endo versus mito ***P = 0.0002, lyso versus mito ***P = 0.00027), Kruskal–Wallis, Dunn post hoc test, N = 3, Boxplot shows median, first, and third quartiles. Upper/lower whiskers extend to 1.5× the interquartile range. (E) The directionality of endosomes, lysosomes, and mitochondria movements in iNeurons at 21–23 DPI (Endosomes: 594 cargoes, 21 videos, N = 3; lysosomes: 276 cargoes, 11 videos, N = 3; mitochondria: 285 cargoes, 22 videos, N = 3). Error bars represent the standard error of the mean (SEM).

iNeurons as a model to study dynein-mediated transport. (A) Schematic of differentiation of NGN2 hESCs/hiPSCs into iNeurons. hESC/hiPSCs were split and 300,000 cells were plated. 2 d later, differentiation media (Diff.) was added to contain doxycycline. At 2 DPI, cells were split again and plated into microfluidics. At 7 DPI, doxycycline was removed from the media and cells were allowed to grow until 21–23 DPI. (B) Example image of 21–23 DPI iNeurons in microfluidic device. Cells were fixed and stained with an antibody against β-tubulin. (C) Kymographs of endosomes (Endo, CTB AlexaFluor 488), lysosomes (Lyso, Lysotracker Deep Red), and mitochondria (Mito, mitotracker Deep Red FM) in iNeurons at 21–23 DPI. (D) The mean speed of endosomes, lysosomes, and mitochondria in both anterograde (gray) and retrograde (light green) directions (Retrograde: endo versus lyso *P = 0.027, endo versus mito **P = 0.0025, lyso versus mito P = 0.71; Anterograde: endo versus lyso P = 0.99, endo versus mito ***P = 0.0002, lyso versus mito ***P = 0.00027), Kruskal–Wallis, Dunn post hoc test, N = 3, Boxplot shows median, first, and third quartiles. Upper/lower whiskers extend to 1.5× the interquartile range. (E) The directionality of endosomes, lysosomes, and mitochondria movements in iNeurons at 21–23 DPI (Endosomes: 594 cargoes, 21 videos, N = 3; lysosomes: 276 cargoes, 11 videos, N = 3; mitochondria: 285 cargoes, 22 videos, N = 3). Error bars represent the standard error of the mean (SEM).

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