Fig. 1 | Signal Transduction and Targeted Therapy

Fig. 1

From: Synergistic induction of mitotic pyroptosis and tumor remission by inhibiting proteasome and WEE family kinases

Fig. 1

Inhibition of proteasome induces M-phase arrest, multi-polar spindle formation and mitotic catastrophe. a–c The effects of proteasome inhibition on cell cycle progression. SNU449 cells were treated with the indicated dose of BTZ (a) for 30 h or transfected with the indicated RNA duplexes for 60 h (b, c), then stained for Ser-10-phosphorylated histone H3 (pH3-S10) to indicate M-phase cells and stained with propidium iodide (PI) to indicate DNA content, followed by FACS for phase distribution of the cell cycle. d–i Inhibition of proteasome induced mitotic arrest, multi-polar spindle formation and ballooning bubbles from cell membranes. SNU449 subline that stably expressed histone H2B-EGFP and mCherry-α-tubulin were treated with vehicle or 30 nM BTZ (d, f, h), or transfected with NC or siPSMC5-1/2 (mixture of siPSMC5-1 and siPSMC5-2) for 24 h (e, g, i), followed by live-cell imaging for 46 h (d, f, h) or 70 h (e, g, i). For d (Vehicle, n = 25; BTZ, n = 14) and e (NC, n = 28; siPSMC5-1/2, n = 27), the time from nuclear envelope breakdown (NEBD) to the end of anaphase or cell death was designated as mitotic duration (right panel). White arrows indicate the large bubbles blowing from the plasma membrane. Scale bar, 5 μm. For f and g, cell death was determined by the emergence of pyroptosis characteristics or cell detachment, and the fractions of cells died at interphase or M-phase were quantified based on at least 118 cells in each group. For h (Vehicle, n = 25; BTZ, n = 14) and i (NC, n = 28; siPSMC5-1/2, n = 27), the fates of individual mitotic cell are shown. For d, e, h and i, the time point of NEBD was set as 0. j, k Inhibition of proteasome caused multi-polar spindle formation. SNU449 cells were treated with vehicle or 15 nM BTZ for 30 h (j), or transfected with the indicated RNA duplexes for 60 h (k), then stained for pericentrin (PCNT, red), α-tubulin (TUBA, green) and DAPI (blue) to indicate centrosome, spindle and chromosome, respectively. The proportion of mitotic cells possessing multi-polar spindles was calculated (right panel). Scale bar, 2.5 μm. Error bars: SEM from at least three independent experiments. One-way ANOVA (a–c and k) and Student’s t test (d, e and j) were used. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns not significant

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