Journal: iScience

Article Title: TORC1 inactivation promotes APC/C-dependent mitotic slippage in yeast and human cells

doi: 10.1016/j.isci.2021.103675

Figure Lengend Snippet: APC/C-Cdh1 mediates TORC1 inactivation-induced anaphase onset (A) Cells of strain SCU1712 (CLB5-TAP) were treated as described in Figure 1 A. TAP-tagged cyclin B5 (Clb5) was detected by western blotting analysis using the anti-TAP antibody (PAP). (B–D) Cells of strains SCU1485 (BUB1-GFP) and SCU1337 (MAD2-GFP) harboring plasmid pSCU1701 (pMTW1-RFP) were treated as described in Figure 1 C. Bub1-GFP and Mad2-GFP signals were captured with Mtw1-RFP (a kinetochore marker) signals. Scale bar, 2.5 μm. Percentages of cells with Bub1-GFP or Mad2-GFP on the kinetochore were determined, and averages and error bars from two independent experiments are shown. Statistical analyses were carried out using the two-tailed Fisher's exact test. ns, not statistically significant. (E) Cells of strains SCU2755 (PDS1-3HA) and SCU2693 (PDS1-3HA cdc20-3) precultured at 25°C were arrested in metaphase by nocodazole treatment for 3 h and transferred to 37°C for 30 min. Thereafter, cells were treated with rapamycin (+Rap) or released into nocodazole-free media (-Noc) (Time 0). Cells of strain SCU2282 (PDS1-3HA cdh1Δ) were treated as described in Figure 1 A. For comparison, see Figure 1 A, WT. 3HA-tagged securin Pds1 was detected by western blotting analysis using the anti-HA antibody. (F) Cells of strains SCU69 (CEN4-GFP) and SCU2114 (CEN4-GFP cdh1Δ) were treated as described in Figure 1 C. Scale bar, 2.5 μm. Percentages of cells with separated sister chromatids were determined and averages and error bars from two independent experiments are shown. Statistical analyses were carried out using the two-tailed Fisher's exact test. ∗, p <0.05. (G) Five-fold serial dilutions of cells were spotted in 1-μL drops onto YPAD plates with or without 5 ng/mL rapamycin. The plates were incubated at 30°C for 1 day for YPAD plates and 2 days for rapamycin-containing YPAD plates. The yeast strains SCU893 (wild type) and SCU1228 (cdh1Δ) were used. (H) Model of APC/C-Cdh1 mediating TORC1 inactivation-induced anaphase onset. The inactivation of TORC1 sequentially causes APC/C-Cdh1 activation, degradation of securin and cyclin B5, and anaphase onset.

Article Snippet: Rabbit polyclonal anti-Cdh1 , Novus Biologicals , Cat#NBP2-15840; RRID: AB_2895137 , .

Techniques: Western Blot, Plasmid Preparation, Marker, Two Tailed Test, Incubation, Activation Assay

Journal: iScience

Article Title: TORC1 inactivation promotes APC/C-dependent mitotic slippage in yeast and human cells

doi: 10.1016/j.isci.2021.103675

Figure Lengend Snippet: Cdc14 mediates TORC1 inactivation-induced anaphase onset (A) Cells of strain SCU3250 (PDS1-3HA cdc14-1) were treated as described in Figure 2 E. 3HA-tagged securin Pds1 was detected by western blotting analysis using the anti-HA antibody. (B and C) Cells of strains SCU69 (CEN4-GFP) and SCU3204 (CEN4-GFP cdc14-1) preincubated at 25°C were arrested in metaphase by nocodazole treatment for 3 h and then transferred to 37°C for 30 min. Thereafter, the cells were further incubated with or without rapamycin for 1 h. Scale bar, 2.5 μm. Percentages of cells with separated sister chromatids are shown in (C). For the control, cells were observed 30 min after nocodazole removal. Averages and error bars from two independent experiments are shown. Statistical analyses were carried out using the two-tailed Fisher's exact test. ∗, p < 0.05; ∗∗; p < 0.0001. (D and E) Cells of strain SCU1000 (CDC14-5GFP) were treated as described in Figure 1 C. Scale bar, 2.5 μm. Percentages of cells with diffused Cdc14 were determined and averages and SD from three independent experiments are shown in (E). For the control, cells were observed 30 min after nocodazole removal. Statistical analyses were carried out using the two-tailed Fisher's exact test. ∗∗∗∗, p <0.000001. (F) Five-fold serial dilutions of cells of strains SCU893 (wild type) and SCU1001 (cdc14-1) were spotted in 1-μL drops onto YPAD plates with or without 10 ng/mL rapamycin. The plates were incubated at 25°C for 3 days. An unrelated atg1Δ strain (SCU4067) defective in autophagy was used as the control. (G) Model of Cdc14 mediating TORC1 inactivation-induced anaphase onset. The inactivation of TORC1 sequentially causes Cdc14 activation, APC/C-Cdh1 activation, securin degradation, and anaphase onset.

Article Snippet: Rabbit polyclonal anti-Cdh1 , Novus Biologicals , Cat#NBP2-15840; RRID: AB_2895137 , .

Techniques: Western Blot, Incubation, Two Tailed Test, Activation Assay

Journal: iScience

Article Title: TORC1 inactivation promotes APC/C-dependent mitotic slippage in yeast and human cells

doi: 10.1016/j.isci.2021.103675

Figure Lengend Snippet: mTORC1 inactivation elicits mitotic slippage (A) A schematic of the experimental procedure. (B) Phase-contrast time-lapse imaging of nocodazole-treated A549 cells. An example representing each category of cell fate to classify cells in Figure S4 A is shown. Numbers show elapsed time (hour:minute) relative to the start of imaging. Scale bar, 50 μm. (C) Fate of nocodazole-treated A549 cells with or without rapamycin treatment after 60-h imaging. Proportion of cells classified into each category, obtained by averaging the results of four independent experiments shown in Figure S4 A, is presented. Error bars represent standard errors. (D) Rate of mitotic slippage in nocodazole-treated A549 cells with or without rapamycin treatment and/or Cdh1 depletion. Percentage of cells that underwent mitotic slippage among the cells that underwent mitotic cell death or mitotic slippage, obtained by averaging the results of four independent experiments that are shown as dots, is presented. Error bars represent standard errors. ∗∗, p <0.01; ∗∗∗, p <0.001 (Tukey's multiple comparison test). ns, not statistically significant. (E) Duration of mitosis in nocodazole-treated A549 cells that underwent mitotic cell death or mitotic slippage. The results are average of four independent experiments. The results of rare mitotic slippage events in cells depleted of Cdc20 with or without Cdh1 were not shown. Error bars represent standard errors. ∗∗∗, p <0.001 (Tukey's multiple comparison test, comparison with Mock -Rap, mitotic cell death). (F) Duration of interphase after mitotic slippage in nocodazole-treated A549 cells with or without rapamycin treatment and/or Cdh1 depletion. The results are average of four independent experiments, which are shown as dots. Error bars represent standard errors. ∗∗, p <0.01 (Tukey's multiple comparison test). ns, not statistically significant.

Article Snippet: Rabbit polyclonal anti-Cdh1 , Novus Biologicals , Cat#NBP2-15840; RRID: AB_2895137 , .

Techniques: Imaging

Journal: iScience

Article Title: TORC1 inactivation promotes APC/C-dependent mitotic slippage in yeast and human cells

doi: 10.1016/j.isci.2021.103675

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-Cdh1 , Novus Biologicals , Cat#NBP2-15840; RRID: AB_2895137 , .

Techniques: Recombinant, Western Blot, Sequencing, Plasmid Preparation, Software