(A) Asynchronous cultures of HeLa mCherry-Tubulin cells were transfected with the specified siRNA. Midbody resolution times were calculated from three separate experiments (mean times ±SD were non-targeting (NT): 96 ± 36 min; CHMP4C: 74 ± 26 min; ULK3: 74 ± 31 min). (B) HeLa cells were stained with Hoechst, α-Tubulin, and α-ULK3 antibody (Santa Cruz). ULK3 was observed at the Flemming body in 79% of observed midbodies (total n = 34). Image shows a representative example of Flemming body localization, with the ULK3 signal in green, Tubulin in red, and nuclei in blue. Bar = 5 μm. Inset shows an expanded view of the midbody. (C and D) HeLa mCherry-Tubulin cells transfected with NT or ULK3 siRNA were transfected with Nucleoporin 153 (NUP153) siRNA to trigger the abscission checkpoint. In (C), cells were fixed and stained with α-Tubulin antibody to visualize midbody-arrested cells. Data are represented as a mean percentage of midbodies ±SD from six separate experiments. NUP153 depletion levels as mean percentages from three independent experiments were lane 1: 100%, 2: 67%, 3: 108%, 4: 70%. In (D), midbody abscission times were analyzed (mean times are 1: 86 ± 22 min; 2: 118 ± 50 min; 3: 70 ± 15 min; 4: 81 ± 25 min). (E) Clonal HeLa cells stably expressing a lentiviral vector containing CRISPR-Cas9 with a guide RNA sequence targeting the ULK3 locus (δULK3-1 or δULK3-2) or without guide RNA (Controls) were transfected with NT or NUP153 siRNA as above and stained with α-Tubulin antibody to visualize midbody-arrested cells. Data are represented as the mean percentage increase in midbody-connected cells compared to the percentage of midbodies in NT-treated cells for each cell line ±SD from four separate experiments. Mean percentages of NUP153 depletion levels compared to HeLa + NT siRNA (lane 1) from four independent experiments were lane 1: 100%, 2: 52%, 3: 85%, 4: 52%, 5: 94%, 6: 51%, 7: 75%, 8: 48%, 9: 96%, 10: 48%. (F) HeLa YFP-lamina-associated polypeptide 2β (LAP2β) expressing cells were transfected with NT and ULK3 siRNA, and resolution times of intercellular chromatin bridges were quantified in three separate experiments (mean times are NT: 708 min; ULK3: 459 min). (G) Analysis of tension-dependent modulation of abscission time, mean times: 1: 120 ± 53 min; 2: 73 ± 19 min; 3: 77 ± 41 min; 4: 69 ± 20 min). Data in (A, D, F–G) are represented as box plots showing median abscission times (A, D, and G) or LAP2β bridge resolution times (F). Here and throughout, whiskers mark 5–95 percentiles, box edges represent the first and third quartiles, and red bars denote the median. n = total number of events counted per sample. Cell lysates in (A, C, E, and F) were examined by Western blot using indicated antibodies. See also Figure 2—figure supplements 1 and 2, and Videos 1–11.