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. 2021 May 13;10:e65234. doi: 10.7554/eLife.65234

Figure 5. Cell cycle and p53 status at the time of exposure correlates with cell fate outcomes.

(A, B) The fate of asynchronously growing FUCCI expressing A549 individual control (n = 50) and cisplatin pulsed cells (n = 100) was tracked by time-lapse microscopy, with images taken every 30 min for 72 hr. Cisplatin-treated cell analysis was slightly biased for cells that underwent multiple divisions. (C) Violin plots of the number of divisions from cells in (A, B), based on the cell cycle phase at the time of cisplatin exposure. Statistical significance was determined by one-way ANOVA with Tukey’s correction for multiple comparisons (**p<0.01, ****p<0.0001, ns = not significant). (D) Schematic (created with BioRender.com) of optical window-based longitudinal in vivo imaging of FUCCI A549 cells. (E) Representative 3D projection images from mice imaged at days 1 and 7 with carboplatin (Carbo) or control (Saline). (F) Quantification of the proportion of red (G1), yellow (G1/S), and green (S/G2-M) cells found in tumours (n = 3) from day 0 to 7.

Figure 5.

Figure 5—figure supplement 1. Additional replicates of FUCCI expressing A549 cells pulsed with cisplatin.

Figure 5—figure supplement 1.

(A) Representative images of A549 FUCCI expressing cells treated with (Cisplatin) or without (Control) 5 µg/ml cisplatin for 2 hr and then followed by time-lapse microscopy. Images were taken every 20 min for 3 days. Coloured arrows indicate specific mother and daughter cells followed through time. (B) Quantification of cell cycle phase length from data generated in (B) and (C). Statistical significance was determined by one-way ANOVA with Tukey’s correction for multiple comparisons (****p<0.0001, n.s = not significant). (C) The total number of divisions completed by biological repeats of asynchronously growing FUCCI expressing A549 cells pulsed with or without (control) with 5 µg/ml cisplatin for 2 hr. Data was extracted from time-lapse imaging analysis. Statistical significance was determined by one-way ANOVA with Tukey’s correction for multiple comparisons (****p<0.0001, ***p<0.001, **p<0.01, ns = not significant) and an unpaired two-tailed Student’s t-test for summary data (****p<0.0001).
Figure 5—figure supplement 2. Role of p53 in regulating response to pulsed cisplatin.

Figure 5—figure supplement 2.

(A) The total number of divisions completed by asynchronously growing FUCCI expressing A549 (data from Figure 5—figure supplement 1C) and NCI-H1299 cells treated without (control) or with 5 µg/ml cisplatin for 2 hr. Data was extracted from time-lapse imaging analysis. Statistical significance was determined by one-way ANOVA with Tukey’s correction for multiple comparisons (****p<0.0001, ns = not significant). (B) The individual fate maps of NCI-H1299 cells (n = 50 control, 100 = cisplatin) from (A), tracked by time-lapse microscopy, with images taken every 30 min for 72 hr. (C) Quantification of cell fate outcomes from (B), including G1 arrest before mitosis (G1 ABM), G1 arrest after mitosis (G1 AAM), death before mitosis (DBM), and death after mitosis (DAM) and proliferative (Prolif.), that is, cells that divided two or more times. (D) Violin plots of the number of divisions completed by cisplatin-treated NCI-H1299 cells based on the cell cycle phase at the time of cisplatin exposure. Statistical significance was determined by one-way ANOVA with Tukey’s correction for multiple comparisons (ns = not significant). (E) Additional analysis of data from Hastings et al., 2020 showing the total number of divisions completed by (n = 50) asynchronously growing FUCCI expressing A549 cells treated with either scramble (siControl) or one of two separate siRNAs targeting p53. Statistical significance was determined by one-way ANOVA with Tukey’s correction for multiple comparisons (****p<0.0001, ***p<0.001, **p<0.01, ns = not significant).
Figure 5—figure supplement 3. In vivo analysis of FUCCI expressing LUAD cells.

Figure 5—figure supplement 3.

Similar to Figure 5D–F, quantification of the proportion of red (G1), yellow (G1/S), and green (S/G2-M) cells found in tumours (n = 3) from day 0 to 7 of (A) NCI-H1573 and (B) NCI-H1299 placed under optical windows in mice treated with or without carboplatin.
Figure 5—video 1. Asynchronous A549 cells pulsed with or without cisplatin.
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