Extended Data Fig. 3: The AMBRA1–cyclin D1 axis affects the cell cycle.
From: AMBRA1 regulates cyclin D to guard S-phase entry and genomic integrity
a, Immunoblot of control or AMBRA1-silenced U87-MG cells for the indicated proteins (n = 3). b, Immunoblot of cyclin D1 in control or AMBRA1-silenced U87-MG cells treated with cycloheximide and/or MG132 for the indicated times (n = 3). c, Analysis of densitometry for the cyclin D immunoblot in U87-MG cells, silenced for the indicated genes, shown in Fig. 1g (n = 4). P values by one-way ANOVA followed by Dunnett’s multiple comparisons test. d, Left, immunoblot of cyclin D1 in U87-MG cells silenced for AMBRA1 expression and overexpressing empty vector (pcDNA), wild-type AMBRA1 or AMBRA1(ΔWD40). Right, analysis from densitometry (n = 3). P values by one-way ANOVA followed by Tukey’s multiple comparisons test. e, Immunoblot analysis of cyclin D1 immunoprecipitation from protein extracts of control and AMBRA1-silenced U87-MG cells (n = 3). f, Co-immunoprecipitation of AMBRA1 in U87-MG cells transiently overexpressing empty vector, cyclin D1–Flag or cyclin D1(T286A)–Flag. Cells were treated with MG132 for 3 h before lysis (n = 3). g, Fold change in the number of cells in control or AMBRA1-silenced U87-MG cells (n = 11). P value by two-tailed unpaired t-test. h, Immunoblot of the indicated proteins of U87-MG, BJ-hTERT and U2OS cells that were untreated or treated with MLN4924 for 4 h (n = 3). i, j, Cells immunostained with cyclin D1, EdU antibody and counterstained with Hoechst. i, Scatter plots reporting single-cell total nuclear intensities of EdU versus Hoechst (cells examined over three independent experiments: siSCR, n = 3,279; siAMBRA1, n = 3,608 cells). j, Box plots (centre line, median; box limits, 25th and 75th percentile) indicating total cyclin D1 nuclear intensities (siSCR, n = 3,279; siAMBRA1, n = 3,608 cells. median siSCR = 169,654; siAMBRA1 = 429,623). k, l, Immunoblot of cell-cycle markers in control and AMBRA1-silenced BJ-hTERT cells (k) and cell-cycle-sorted U2OS-FUCCI cells (l) (n = 3). m, Immunoblot of the indicated proteins in AMBRA1-silenced BJ-hTERT cells synchronized by 24-h serum starvation. Cells were collected after the indicated starvation recovery time points (n = 3). n, Representative images of live-cell imaging of control and AMBRA1-silenced U2OS-FUCCI cells from 0 to 14 h with a 2-h interval between different images. The length of the G1 phase is shown in Fig. 1k (n = 3). Scale bar, 5 μm. o, Cell proliferation in control or AMBRA1-silenced BJ-hTERT cells (24 h and 48 h n = 6; 72 h siSCR n = 6, siAMBRA1 n = 5). p, q, Control or AMBRA1-silenced U2OS-FUCCI cells. p, Representative contour plot. q, Fold increase of cells present in S–G2 phase in AMBRA1-downregulated cells with respect to control cells (n = 10). r, Box plots (centre line, median; box limits, 25th and 75th percentile; whiskers, minimum and maximum) showing the cell-cycle length of siSCR (n = 65; median = 13) or siAMBRA1 (n = 65; median = 8.5) U2OS-FUCCI cells examined over three independent experiments. 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 (g, o, q) or two-tailed Mann–Whitney test (j, r). For immunoblots, actin or β-tubulin were used as loading control.
