Extended Data Fig. 5: AMBRA1 deficiency causes replication stress.

a, Analysis of DEA genes (from n = 3 independent RNA-seq experiments) predicting the transcription factor activated after depletion of AMBRA1. b, qRT–PCR analyses of the indicated genes in control or AMBRA1-silenced U2OS-FUCCI cells sorted for the different cell-cycle phases (n = 3) c, Immunoblot for the indicated proteins in U2OS cells interfered for the indicated autophagy regulators (n = 3). d, Left, violin plot of γH2AX nuclear mean intensity in control and AMBRA1-silenced BJ-hTERT cells that were untreated or treated with 0.1 μM abemaciclib for 48 h. Right, representative scatter plot of single-cell γH2AX nuclear mean intensity versus Hoechst, and cell cycle phase gating strategies from control and AMBRA1-silenced BJ-hTERT cells treated with abemaciclib (n = 643 cells). e, Cell count of control U87-MG cells or U87-MG cells with inducible cyclin D1 expression, three days after stimulation with dox (n = 3). f, Cell count of control BJ-hTERT cells or BJ-hTERT cells with inducible cyclin D1 expression at the indicated time points after stimulation with dox, normalized over non-induced cells (1-d V15+, n = 6; 3-d V15+, 3-d E30+, n = 4; 1-d E30+, 4-d V15+, 4-d E30+, 6-d V15+ and 6-d E30+, n = 5). V15+: dox-treated control cells; E30+: dox-treated cyclin D1-inducible cells. g, h, Percentage of cells in each cell-cycle phase in U87-MG (g) and BJ-hTERT (h) cells, control or with inducible cyclin D1 expression, untreated or 48 h after doxycycline stimulation (n = 3). i, Immunoblot for the indicated proteins in control U87-MG cells or U87-MG cells with inducible cyclin D1 expression at the indicated time points with or without dox stimulation (n = 3). j, k, Mean fork speed (j) (kb min⁻¹) and fork symmetry analysis (k) of DNA fibres from control BJ-hTERT cells and BJ-hTERT cells with inducible cyclin D1 expression treated as in Fig. 2d (scored forks: −dox, n = 312; 3-d dox, n = 449; 4-d dox, n = 429; 6-d dox, n = 426). Data are mean ± s.d. l, Quantification of immunohistochemistry staining in Fig. 2g (n = 3 mice). m, Immunoblot for the indicated proteins in wild-type or Ambra1 cKO NSCs (n = 3). Unless otherwise stated, n refers to biologically independent samples; data are mean ± s.e.m. Data were analysed using a two-tailed unpaired t-test (b, l), two-tailed Mann–Whitney test (d, j, k), two-way ANOVA followed by Sidak’s multiple comparisons test (e, g, h) or one-way ANOVA followed by Sidak’s multiple comparisons test (f). Exact P values are provided in the ‘Statistical analysis and data reproducibility’ section of the Supplementary Methods.

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