1. Volume 5, Issue 3, Pages 237-250.e8 (September 2017)
Cell-Cycle Position of Single MYC-Driven Cancer Cells Dictates Their Susceptibility to a Chemotherapeutic Drug 
Tatsiana Ryl, Erika E. Kuchen, Emma Bell, Chunxuan Shao, Andrés F. Flórez, Gregor Mönke, Sina Gogolin, Mona Friedrich, Florian Lamprecht, Frank Westermann, Thomas Höfer 
Cell Systems 
Volume 5, Issue 3, Pages e8 (September 2017)
DOI: /j.cels
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2. Cell Systems 2017 5, 237-250.e8DOI: (10.1016/j.cels.2017.07.005)
Copyright © 2017 The Authors Terms and Conditions

3. Figure 1
MYCN Drives Death and Clonal Regrowth after Treatment with a Chemotherapeutic Drug
(A) Colony assay of MYCN-regulatable neuroblastoma cell lines, high- and low-MYCN (IMR5/75 and IMR32, tetracycline-inducible MYCN knockdown; TET21N, inducible MYCN overexpression), treated with doxorubicin (Dox) for 72 hr. Washed cells were reseeded at low density and colonies stained with methylene blue 14 days later; n = 3.
(B) Relative TET21N cell numbers captured continuously by live-cell imaging. Cells were treated for 72 hr with Dox and then incubated in drug-free medium for 11 days; mean ± SEM, n = 2.
(C) Snapshots from movies in (B). Cells express the FUCCI mCherry-hCdt1 degron, which marks G1 phase cells and, upon Dox treatment, accumulates in cells arrested in G1 and G2. Scale bars, 20 μm.
(D) Growth curve of untreated and re-growing high-MYCN cells after a single Dox treatment; mean ± SEM, n = 3.
(E) Clonogenic regrowth after repeated treatment. Cells treated with Dox for 72 hr were reseeded at low density, colonies counted 14 days later, treated again for 72 hr, reseeded and counted 14 days later; mean ± SEM, n = 3. Colony numbers after one and two Dox treatments were not significantly different in paired t test, p = Representative experiments shown in (A and E).
See also Figure S1.
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4. Figure 2 Cell-Cycle-Resolved MYCN-Related Gene Expression Changes
(A) Cell-cycle distributions of primary neuroblastoma tumors measured by flow cytometry; n = 493. Differences between MYCN-amplified (AMP, 43 samples) and MYCN single-copy (SC, 450 samples) tumors were assessed by Wilcoxon rank sum tests.
(B) High- and low-MYCN IMR5/75 cells were synchronized using a thymidine block for 18 hr. After thymidine removal, cell-cycle profile (flow cytometry) and transcriptomes (RNA-seq) were measured. The dominant cell-cycle phase of the synchronized populations at each time point is shown by shaded areas; phases reached synchronously by high- and low-MYCN cells are S (white), G2/(M) (dark gray), and G0/1 (light gray); arrows indicate subsequent S phase entry of high-MYCN (red) and prolonged G0/1 of low-MYCN cells (blue).
(C) Distribution of mRNA fold changes high-/low-MYCN.
(D) k-Means clustering of differentially expressed genes in (C) detected by likelihood ratio testing, showing mean mRNA fold changes high-/low-MYCN of each cluster.
(E) GO-enrichment terms of genes within clusters in (D). The cell-cycle-dependent MYCN-downregulated cluster was not functionally enriched.
(F–I) Examples of genes clustered in (D), showing expression kinetics (read counts) in high- and low-MYCN cells (F) constitutive upregulation, (G) constitutive downregulation, and cell-cycle-dependent upregulation upon divergence at (H) G1/S and (I) G2/M; mean ± SD, n = 2. Boxplots show relative expression of these genes in MYCN-amplified (AMP) versus MYCN single-copy (SC) tumors.
See also Figure S2 and Table S1.
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5. Figure 3
High-MYCN Expression Disables the Bistable Switch Governing the Restriction Point
(A) Scheme of the regulatory network. CKI, CDK inhibitors p21, p27, and p57; Cyc, cyclin.
(B) Visualization of the feedback loops in (A) (purple and green).
(C) Parameters were identified by fitting the model (lines) to normalized mRNA and protein time course measurements of thymidine block-synchronized cell populations (dots, mean, n = 2). Shaded areas show estimated data SD.
(D) FUCCI Cdt1 degron intensity was measured in unsynchronized IMR5/75 cells by time-lapse imaging. Average Cdt1-trace of six low-MYCN cells (dots) was used for model fitting (lines). A representative high-MYCN cell is shown for comparison (triangles). Shaded areas as in (C).
(E) Bifurcation analysis with respect to the MYCN-perturbed parameters. Parameter fitting places high- and low-MYCN cells within the state space, showing the MYCN effect (orange arrow).
(F) Model-simulated distributions of E2F1 mRNA and pRb generated by burst-like transcription of E2F target genes and selecting one random time point from each simulated trajectory, corresponding to unsynchronized cells in G1 phase.
(G) G1-gated cells (by DNA content) expressing E2F1-d2GFP to quantify E2F1 promoter activity co-stained with pRb Ser807/811 antibody measured by flow cytometry; n = 4, representative experiment shown.
(H) Examples of simulated single-cell trajectories of high-MYCN (red) and low-MYCN (blue) cells. Black diamonds show S phase entry.
(I) Distribution of simulated G0/1 lengths.
(J) G0/1 phase lengths of TET21N cells expressing the FUCCI geminin degron captured by time-lapse imaging; n = 3.
See also Figure S3.
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6. Figure 4
MYCN-Disabled Restriction Point Facilitates Regrowth Post-chemotherapeutic Treatment
(A) Scheme of the molecular network underlying prolonged cell-cycle arrest after DNA damage.
(B) Expression of p53 phosphorylated at Ser15 and p21 protein during and after Dox. TET21N cells stained with fluorescence-conjugated antibodies and median intensities measured by flow cytometry; mean ± SEM, n = 3.
(C) A 72-hr treatment regime was simulated by increasing the CKI production rate with subsequent reduction post-treatment (lower panel). Fraction of simulated cells that enter S phase after treatment-induced G1 arrest dependent on the CKI expression post-treatment (upper panel).
(D) Single-cell, cell-cycle-resolved p21 protein expression for the data shown in (B).
(E) Conditional MYCN expression switched after treatment. Low- and high-MYCN cells were treated for 72 hr with Dox. During Dox washout MYCN expression was switched by removing or adding doxycycline. Sixteen days later cells were stained with EdU, and DNA synthesis was measured by flow cytometry; n = 2, representative experiment shown.
(F) Principal-component analysis of whole-genome RNA-Seq data of TET21N cells treated with Dox. Arrows highlight the changes in expression; n = 2.
(G) GO-enrichment terms of genes showing significant expression differences between high- and low-MYCN cells in (F).
See also Figures S4 and S5.
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7. Figure 5
Only Cells Newborn at the Start of Chemotherapeutic Treatment Regrow
(A–E) Treatment-induced fates of individual TET21N mCherry-hCdt1 cells imaged continuously before, during, and for 9 days after Dox treatment. (A) Examples of cellular fates. Each row follows one cell (white arrow) and its fate throughout the movie. Scale bars, 20 μm for all images. (B–E) Individual high-MYCN cells were divided into four groups according to their cell-cycle phase at the start of treatment and their phase of cell-cycle arrest during treatment. After treatment, the cumulative percentage of cells undergoing (B) cell death, (C) mitotic entry, and (D) death following mitosis were quantified in each group. Representative experiment shown. (E) Percentage of cells in each group and their fates 8 days after treatment.
(F) Untreated or Dox-treated single cells in G1, G2, or G1+G2 were sorted into 96-well plates using fluorescence-activated cell sorting. Three weeks after sorting, the fraction of regrown single cells was counted; mean ± SEM, n = 3.
See also Figure S6.
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8. Figure 6 Newborn Cells Efficiently Repair DNA Damage during Treatment
(A) Examples of 53BP1 distributions in untreated and Dox-treated cells expressing mCherry-53BP1. 53BP1 foci form after Dox treatment.
(B) Average 53BP1 foci dynamics of high-MYCN cells followed by live-cell imaging and grouped according to their cell-cycle behavior during treatment using GFP-hGeminin (cf. Figure 5).
(C) Examples of cell-cycle-dependent single-cell 53BP1 foci dynamics.
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9. Figure 7
Viable Therapy Survivors Are Eliminated by Combining Doxorubicin with Inhibition of DNA Damage Signaling
(A) Cellular regrowth of cells treated with Dox alone or pretreated with CDK inhibitor for 24 hr and then with Dox and CDK inhibitor for 24 hr. Cell numbers were counted by flow cytometry; mean ± SEM, n = 2.
(B) Average 53BP1 foci number in high-MYCN cells untreated or treated with Dox alone or together with ATM inhibitor (ATMi); n = 3, representative experiment shown.
(C) Relative cell numbers of cells treated for 24 hr with Dox alone or with Dox and ATMi counted by flow cytometry; mean ± SEM, n = 3.
(D) Clonogenic regrowth of other neuroblastoma high-MYCN cell lines treated with Dox alone or together with ATMi.
See also Figure S7.
Cell Systems 2017 5, e8DOI: ( /j.cels )
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