Polar distribution of signaling molecules in premitotic HSCs. (A–D) Representative immunofluorescence images of the distribution of (A) tubulin, (B) tubulin and pERK ½, or ERK, (C) tubulin and pmTOR S2448 or tmTOR, and (D) tubulin, CDC42, and NUMB in premitotic HSCs (bar = 3 µm). Images were acquired on a Zeiss Axio Observer 7 inverse microscope using a Plan-Apochromat 63× oil objective (NA 1.4). The stacked bar charts show the percentage of cells with polar tubulin and respective signaling molecules distribution in CTRL (black) and MEK1 cKO (red) HSCs. (E) Representative images showing the mitotic segregation of CDC42 and NUMB. The charts show percentage of cells with ACD from three independent experiments (bar = 5 µm; mean percentage ± SD; ≈300–400 cells total for polarity and ≈140–170 total cells for paired daughters) conducted in CTRL (black) and in MEK1 cKO (red) HSCs isolated from mice recovering from 5-FU treatment. Images were acquired on a Zeiss LSM 980 inverse confocal microscope using a Plan-Apochromat 63× oil objective (NA 1.4). *P < 0.05, **P < 0.01, and ***P < 0.001.