EMBO/SourceData huggingface dataset:
Figure 1Immunoblot of lysates prepared from the indicated SAMHD1-proficient (+/+), deficient (-/-) and rescue (WT, H233A) cell line pairs with the indicated antibodies. Representative of 2 independent experiments.Proliferation inhibition analysis of ara-C and RNRi combination treatment in SAMHD1+/+ or -/- THP-1 cells. Error bars indicate s.e.m. of two (HU and dF-dC) or three (3-AP) independent experiments, each performed in duplicate.Ara-C EC50 values plotted as a function of RNRi concentration in SAMHD1+/+, -/- and rescue (WT, H233A) THP-1 cell line pairs. EC50 values in the absence of RNRi are indicated by the black and red dotted line. Error bars indicate s.e.m. of two (HU and dF-dC) or three (3-AP) independent experiments, each performed in duplicate.Drug synergy plots for ara-C and the indicated RNRi in SAMHD1+/+, -/- and rescue (WT, H233A) cell line pairs. Each data point indicates an average delta score from a single dose-response matrix experiment performed in duplicate. Zero, >0, or <0 corresponds to additive, synergy, or antagonism, respectively, whilst >5 indicates strong synergy. The horizontal line and the error bars indicate the mean and s.d., respectively, statistical significance was determined using a two-tailed unpaired t-test: ns, not significant, P ≥ 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.Spearman correlation of relative SAMHD1 protein abundance and synergy delta scores for ara-C versus HU or dF-dC in a panel (n = 9) of haematological cancer cell lines. Error bars indicate s.e.m. Each data point corresponds to SAMHD1 protein levels determined by immunoblot analysis (n = 4 for each cell line, representative blot shown in Appendix Fig S2) and an average delta score from repeated dose-response matrix experiments each performed in triplicate: THP-1, n =4; HuT-78, n = 2; HL-60/iva, n = 1; KBM-7, n = 2 (HU) and 3 (dF-dC); K562, n = 3 (HU) and 4 (dF-dC); CCRF-CEM, n =3 (HU) and 4 (dF-dC); MV-4-11, n = 2 (HU) and 3 (dF-dC); Jurkat, n = 2 (HU) and 3 (dF-dC); MOLT-4, n = 2 (HU) an 3 (dF-dC).Immunoblot analysis of lysates prepared from SAMHD1+/+ or -/- THP-1 cells treated for 24 h with ara-C and HU, as indicated. Representative of 3 independent experiments.
Figure 2Kaplan-Meier analysis of NOD/SCID mice injected i.v. with luciferase-expressing SAMHD1+/+ or -/- THP-1 cell clones (day 0) and treated with ara-C and/or HU as indicated (day 6). n = 6 per treatment group.Kaplan-Meier analysis of NOD/SCID mice injected i.v. with luciferase-expressing SAMHD1+/+ or -/- HL-60/iva cell clones (day 0) and treated with ara-C and/or HU as indicated (day 6). n = 6 per treatment group.Kaplan-Meier analysis of NOD/SCID mice injected i.v. with luciferase-expressing SAMHD1+/+ THP-1 cell clone (day 0) and treated with ara-C and/or dF-dC as indicated (day 6). n = 7 per treatment group.Kaplan-Meier analysis of CD45.2 C57BL/6J mice injected i.v. with murine MLL-AF9-transformed AML blasts (day 0) and treated with ara-C and/or HU days 20-24. n = 5 per treatment group, except for vehicle (n = 4).
Figure 3Drug synergy plots for ara-C and HU or dF-dC in primary patient-derived AML blasts. Each data point indicates an average delta score from a single patient sample subjected to a dose-response matrix experiment performed in triplicate, n = 16 for HU and n = 9 for dF-dC. Zero, >0, or <0 corresponds to additive, synergy, or antagonism, respectively, whilst >5 indicates strong synergy. Median, upper and lower quartiles, and range of delta scores are indicated by box-and-whisker plots.Pearson correlation of relative SAMHD1 protein abundance and synergy delta scores for ara-C and HU or dF-dC in primary patient-derived AML blasts (n = 23).Immunoblot of primary patient-derived AML blasts treated with control (dX) or Vpx-containing (X) virus-like particles (VLPs): patient A2953 (C), ALG17_001 (E). Accompanying proliferation inhibition analysis of ara-C and indicated RNRi combination in these samples: patient A2953 (D), ALG17_001 (D). Error bars indicate s.d. of single experiment performed in triplicate.Paired drug synergy plot for ara-C and RNRi (HU, n = 7; dF-dC, n = 5) in primary patient-derived AML blasts pre-treated with control (dX) or Vpx-containing (X) VLPs. Zero, >0, or <0 corresponds to additive, synergy, or antagonism, respectively, whilst >5 indicates strong synergy and <5 indicates strong antagonism. Each data point indicates an average delta score from a single patient sample subjected to a dose-response matrix experiment performed in triplicate. Statistical testing was performed using two-way ANOVA.
Figure 4Intracellular dNTP measurements using a primer-extension assay in SAMHD1+/+ THP-1 cells treated for 4 or 24 h with either 50 µM HU (middle panel) or 2.5 nM 3-AP (right panel), ratios of dCTP-to-dATP were calculated. Bars indicate mean values of three independent experiments, error bars indicate s.e.m. Statistical analyses were done using unpaired two-tailed t-tests: **, P < 0.01.Intracellular ara-CTP (upper panel) and dCTP:dATP ratio (lower panel) in the indicated cell lines following the indicated treatment determined using HPLC-MS/MS. SAMHD1-/- THP-1 cells were treated with 500 nM ara-C and SAMHD1+/+ THP-1 cells were treated with either solvent, 500 nM ara-C or a combination of 500 nM ara-C and an RNRi (HU, 50 µM; dF-dC, 10 nM; 3-AP, 150 nM) for 24 h prior. Values relative to mean ara-CTP amounts in ara-C treated SAMHD1+/+ THP-1 cells shown (indicated by dashed line). Circles, columns and error bars correspond to individual values, means and s.e.m. of at least three experiments performed independently.Quantificaiton of dCK phosphorylation at serine-74 (S74) with respect to total dCK in SAMHD1+/+ THP-1 cells treated with either solvent, 500 nM ara-C or a combination of 500 nM ara-C and an RNRi (HU, 50 µM; dF-dC, 10 nM; 3-AP, 150 nM) for 24 h. Circles and squares, columns and error bars correspond to individual measurements, means and s.e.m. of one representative out of two independent experiments performed in triplicates.Measurement of released inorganic triphosphate (PPPi) from hydrolysis of ara-CTP (200 µM) by recombinant SAMHD1 (0.35 µM) in the presence of GTP (200 µM) and a titration of different non-hydrolysable dNTP analogues (dNTPαS) in the enzyme-coupled malachite green assay. Error bars indicate s.e.m. of two independent experiments performed in triplicate and quadruplet.
Figure 2In vitro pull-down assays. GST or GST-PSKR1JK fusion proteins coupled with glutathione beads were used to pull down His-CPK28. The amount of fusion proteins loaded on the gel was indicated by Coomassie brilliant blue (CBB) staining. Asterisks indicate the specific bands of fusion proteins.Co-IP assays. PSKR1-HA was co-expressed with CPK28-GFP or GFP in N. benthamiana leaves. PSK (10 μM) or H2O was injected 2 hours before leaves sampling. The protein was precipitated using GFP-Trap. Both immunoprecipitated and input samples were subjected to western blotting using an anti-HA or anti-GFP antibody.
Figure 5A. Co-IP assay showing that PSK promotes CPK28-GS2 interaction. GS2-HA was co-expressed with CPK28-GFP or GFP in N. benthamiana leaves. PSK (10 μM) or H2O was injected 2 hours before leaves sampling. The protein was precipitated by GFP-Trap. Immunoprecipitated and input samples were subjected to western blotting using an anti-HA and anti-GFP antibody. Data information: Experiments were repeated twice with similar results.B. Co-IP assay showing that CPK28-GS2 interaction was enhanced in the presence of PSKR1. GS2-HA and CPK28-GFP were co-expressed with or without PSKR1-FLAG in N. benthamiana leaves. The protein was precipitated by GFP-Trap. Immunoprecipitated and input samples were subjected to western blotting using an anti-HA, anti-GFP or anti-FLAG antibody. Data information: Experiments were repeated twice with similar results.C. In vitro pull-down assays showing that CPK28-GS2 interaction was enhanced in the presence of PSKR1. Fixed amounts of His-CPK28 and GST-GS2 fusion proteins were incubated with an increasing amount of MBP-PSKR1 fusion protein and MBP protein. Asterisks indicate the specific bands of fusion proteins. Data information: Experiments were repeated twice, with similar results.
Figure 6A. PSK induces the phosphorylation of GS2 by CPK28 in vivo. GS2-GFP was co-expressed with CPK28-HA (empty vector as a negative control) in N. benthamiana leaves. PSK (10 μM) or H2O was injected 2 hours before leaves sampling. The phosphorylated GS2 protein was precipitated by GFP-Trap, and then immunoblotted with anti-pSer/pThr and anti-pSer antibodies.C. Site-directed mutation of S334 and S360 residues in GS2 inhibited its phosphorylation by CPK28.D. Identification of OE-GS2 transgenic lines expressing phospho-mutated versions of GS2 in the gs2 background by western blotting using an anti-GLN1/GLN2 antibody. Ponceau S staining was used as the loading control.
Figure 2B) Volcano plot for the F test on differential expression between WT and mdx mice. C) Volcano plot for the F test on differential expression between WT and mdx++ mice. D) Volcano plot for the F test on differential expression between WT and mdx+- mice.E, F) Boxplot comparing the distribution of the Dmd and Prune2 genes in the WT, mdx, mdx++ and mdx+- mice groups. The central band of the boxplot denotes the median; the box denotes the first and third quartile; the whiskers are computed as min(max(x), Q3 + 1.5 IQR) and max(min(x), Q1 - 1.5 * IQR), where min(x) and max(x) denote the minimum and maximum of the distribution, Q1 and Q3 the first and third quartile, and IQR the interquartile range.G) Volcano plot for the F tests on differential expression between mdx++ and mdx+- mice.Scatter plots comparing the estimated logFCs across mice groups. Panel H: mdx vs mdx++.Scatter plots comparing the estimated logFCs across mice groups. ++. Panel I: mdx vs mdx+-. Panel J: mdx++ vs mdx+-.
Figure 3A) Histogram of the likelihood ratio p-values for the test on differences between WT and mdx at any time point.B) Distribution of the estimated logFCs for mdx versus WT mice at weeks 6, 12, 18, 24 and 30 for the 1532 genes with significant differences between WT and mdx.D) Trajectory plots comparing the trajectories of Chordc1 and Psat1 in WT and mdx mice.E) Trajectory plots comparing the trajectories of Cd59a, Chordc1, Hpgd, Ldhb, Lincred1, Myo6, Slc39a11 and Tlr1 in WT, mdx++ and mdx+- mice.F) Scatter plots comparing the estimated logFC of mdx++ vs WT (x axis) and of mdx+- vs WT (y axis) at weeks 6, 12, 18, 24 and 30.H) upstream regulator analysis (H) performed by Ingenuity Pathway Analysis of the comparison between mdx and wt mice.
Figure 5A) RT-PCR (30 cycles) showing skipping of exon 23 for PMO and PS49 oligos. No skipping was visible in saline treated mice.B) Western blot showing the percentage of restoration of dystrophin protein in mdx mice following antisense oligonucleotides administration. M is molecular ladder. WT gastrocnemius represents the standard curve using protein extracts from gastrocnemius muscle. NT is saline treated mdx mice.C) Barplot comparing the estimated logFC for the PS49 and PMO treatments in the MoTreat dataset to the desired drug effect obtained from the MoLong dataset (logFC WT vs mdx at week 12).D, E) Histograms comparing the distribution of selected genes across different mice groups (PMO, PS49 and saline in the MoTreat dataset, WT and mdx at week 12 in the MoLong dataset).
Figure 6B) Volcano plot for the F test on differential expression between healthy individuals and DMD patients.C) Scatter plot showing the group effect at baseline (x-axis) and the effect of the interaction between age and group (y-axis). Black dots represent non differentially expressed genes, while blue dots represent differentially expressed genes.E) Volcano plot for the F test on differential expression between treated and untreated DMD patients.F) Barplot comparing the logFC of the genes associated with steroid treatment estimated in this study to the estimates of Liu et al. (2012).G) Scatter plot showing genes for which a significant effect of treatment with steroids. The group effects at baseline are plotted on the x-axis, while the effects of steroids are plotted on the y-axis.
Figure 1(A) Still images of time-lapse imaging of H3.3-mCherry and H3.2-EGFP proteins in wild-type zygotes (n=17).(B) IF of DAXX and ATRX in zygotes at different pronuclear (PN) stages (PN0, n=8; PN2, n=6; PN5, n= 33). Data information: All panels: images of paternal and maternal pronuclei at given stage/condition are from same zygote.(C) Boxplot displaying signal intensity of DAXX at pat- and mat-PCH in mouse zygotes at PN5 stage, relative to mean signal at pat-PCH. In boxplots, the enter lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Datapoints are indicated by circles. Experiments were replicated 3 times. ***P<0.001, n= 28, paired t-test.(D) IF of DAXX and ATRX in PN5 zygotes upon siRNA-mediated knockdown of Daxx (n=14) or with control siRNAs (n=8). Data information: All panels: images of paternal and maternal pronuclei at given stage/condition are from same zygote. Yellow dashed circles represent the contours of the pronuclei. All scale bars, 10µm.(E) IF of DAXX and ATRX in control (n=15) and Atrxm-z+ (n=14) PN5 zygotes. Data information: All panels: images of paternal and maternal pronuclei at given stage/condition are from same zygote. Yellow dashed circles represent the contours of the pronuclei. All scale bars, 10µm.
Figure 2(A) IF of DAXX and EZH2 in paternal pronuclei of Ezh1m-z+ (n=5), Ezh2m-z+ (n=3) and Ezh1m-z+; Ezh2m-z+ (n=4) late zygotes. Yellow dashed circles represent the contours of the pronuclei. Data information: All scale bars, 10µm.(B) IF of DAXX and RNF2 in PN0 zygotes (n=10). Data information: All scale bars, 10µm.(C) IF of DAXX in wild type (n=30), Ring1m-z+ (n=12), Rnf2m-z+ (n=11) and Ring1m-z+; Rnf2m-z+ (n=12) paternal pronuclei of PN5 zygotes. Data information: All scale bars, 10µm.(D) Anti-Flag co-IP of DAXXMycHis, RNF2 and H3K27me3 with CBX23×Flag from extracts of HEK293 cells in presence or absence of benzonase, followed by immuno-blot analyses for indicated antigens.
Figure 3(C) Occupancy (% of IP-ed DNA / input DNA) of GAL4DBDCBX2, RNF2, DAXXMycHis, H3.3, and H3 proteins at 5×UAS site as measured by ChIP-qPCR of chromatin from HEK2935xUAS cells. Data are means ± SEM (n = 3).(D) Immunoblots for the indicated proteins in HEK2935xUAS cells(E) ChIP-seq analysis of HEK2935xUAS cells expressing full length GAL4DBDCBX2.1 and DaxxMycHis showing CBX2.1 dependent recruitment of DAXX to promoters of CCND2 and RUNX1 genes.(F) ChIP-seq analysis of GAL4DBDCBX2.1 occupancy in HEK2935xUAS cells expressing DAXXMycHis (X-axis) or GAL4DBDCBX2.1 and DAXXMycHis (Y-axis), showing (log2) enrichment for GAL4DBDCBX2.1 at many TSSs (± 1 kb) in cells expressing the CBX2.1 protein (Y-axis).(G) ChIP-seq analysis of GAL4DBDCBX2.1 versus DAXXMycHis occupancy in HEK2935xUAS cells expressing GAL4DBDCBX2.1 and DAXXMycHis, revealing very comparable enrichment levels (log2) of both proteins at TSSs. TSSs highlighted in red show enrichment with anti-Myc antibody independently of GAL4DBDCBX2.1 occupancy.(H) ChIP-seq analysis of DAXXMycHis occupancy in HEK2935xUAS cells expressing DAXXMycHis (X-axis) versus GAL4DBDCBX2.1 and DAXXMycHis (Y-axis) showing strong enrichment (log2) for DAXXMycHis at many TSSs of genes, but only when both proteins are expressed (Y-axis), indicating that CBX2.1 targets DAXX to chromatin. TSSs highlighted in red show enrichment with anti-Myc antibody independently of GAL4DBDCBX2.1 occupancy.
Figure 4(A) IF of CBX2 and SUMO2/3 in PN0 (n=10) and PN5 (n=12) zygotes. Data information: Scale bars, 10µm.(B) ChIP-qPCR analyses of HEK2935xUAS cells co-expressing GAL4DBDCBX2.1 either with DAXXMycHis or DAXXΔSIM1/2MycHis. Data are means ± SEM (n = 3).(C) IF of EGFP and DAXX in Daxxm-z+ PN5 zygotes micro-injected with Daxx-EGFP or DaxxΔSIM1/2-EGFP mRNA (100ng/µl). Data information: Scale bars, 10µm.(D) Left: Boxplot displaying EGFP signal intensities at PCH relative to euchromatin in Daxxm-z+ zygotes micro-injected with Daxx-EGFP or DaxxΔSIM1/2-EGFP mRNA. Right: Boxplot displaying signal intensity of EGFP at euchromatin in same samples. In boxplots, the enter lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Datapoints are indicated by circles. Experiments were replicated 3 times. ***P<0.001, N.S.: no statistical difference, P>0.05, t-test.(F) Ni-pulldown of 6xHisSUMO1/2/3-conjugated proteins from HEK293 extracts expressing 6xHis-tagged SUMO1, SUMO2 or SUMO3 in combination with CBX2-EGFP. CBX2 and SUMO-conjugated CBX2 were detected with an anti-GFP antibody.(G) Ni-pulldown of 6xHis-SUMO2 conjugated proteins from HEK293 extracts transiently co-expressing 6xHis-SUMO2 with CBX2-EGFP or CBX24KR-EGFP. Detection of CBX2(H) IF of DAXX and EGFP in wild-type PN5 zygotes, micro-injected with Cbx2 or Cbx24KR-EGFP mRNA (100ng/µl). Data information: Scale bars, 10µm.(I) Left: Boxplot displaying signal intensity of DAXX at PCH relative to mean intensity at pat-PCH in wild-type zygotes micro-injected with Cbx2 or Cbx24KR-EGFP mRNA. Right: Absolute intensities of EGFP in same samples. In boxplots, the enter lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Datapoints are indicated by circles. Experiments were replicated 2 times. ***P<0.001, t-test.
Figure 5(A) IF of DAXX and ATRX in PN5 stage pronuclei of wild-type (n=19), Hp1βm-z+ (n=10) and Suv39h2m-z+ (n=9) zygotes. Data information: Panels Paternal and maternal pronuclei of a given condition originate from same zygotes. All scale bars 10µm.(B) Boxplots displaying signal intensities of DAXX or ATRX at pat- and mat-PCH in wild-type (n=5) and Hp1βm-z+ (n=10) mouse zygotes at PN5. In boxplots, the enter lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Datapoints are indicated by circles. Experiments were replicated 2 times. *P<0.05, **P<0.01, t-test.(C) IF of DAXX in PN5 stage pronuclei of zygotes expressing CBX2-EGFP (n=9) or CBX2F12A-EGFP (n=17). mRNAs (100ng/µl) were injected into mouse PN2 stage zygotes. Yellow dashed circles represent the contours of the pronuclei.. Data information: Panels C: Paternal and maternal pronuclei of a given condition originate from same zygotes. All scale bars 10µm.
Figure 7(A) RNA FISH of forward and reverse strand major satellite sequences in Daxxm+z+ and Daxxm-z+ PN5 zygotes. Scale bars, 10µm.(B) Box plots indicating the foci number (left) and total intensity (right) of forward (top) and reverse (bottom) transcription as measured by RNA FISH on 3D paternal pronuclei in Daxxm+z+ and Daxxm-z+ PN5 zygotes. Lower hinge, central line and upper hinge in boxplots represent 25th, 50th (median) and 75th percentiles respectively. Upper/lower whiskers extend to the largest/smallest values no further than 1.5 * IQR from the upper/lower hinge, where IQR is inter-quartile range or distance between 25th and 75th percentiles. Datapoints are indicated by circles. Experiments were replicated 3 times. ***P<0.001; t-test.(C, D) MA-plots showing Fold Change (FC) (in log2) in gene expression between indicated samples as a function of averaged expression across all samples. Dotted lines indicated log2(FC) of 1.(E) Histogram showing classification of genes into 3 groups according to distribution of ChIP enrichment (log2) of H3K27me3 around TSS in MII oocytes(F) MA-plot showing FC (in log2) in gene expression between indicated samples as a function of averaged expression across all samples. Dotted lines indicated log2(FC) of 1.(G - I) Boxplots showing FC (log2) in gene expression between indicated samples for 3 groups of genes classified according to H3K27me3 enrichments (as shown in E). Lower hinge, central line and upper hinge represent 25th, 50th (median) and 75th percentiles respectively. Upper/lower whiskers extend to the largest/smallest values no further than 1.5 * IQR from the upper/lower hinge, where IQR is inter-quartile range or distance between 25th and 75th percentiles. Outliers are not displayed. The notches extend $1.5*IQR/\sqrt{n}$ where n is a number of genes in each H3K27me3 class. Number of genes (n) in "Low", "Medium" and "High" H3K27me3 class used for boxplots are 9824, 2311, 2647 respectively. Expression changes were estimated using 2 or 3 biological replicates for each condition. Statistical significance was estimated using two-sided t-tests.(J) Boxplot showing FC (log2) in gene expression between Daxx deficient and control MII oocytes for five groups of genes classified according to FC (log2) in gene expression between Ring1 Rnf2 deficient and control GV oocytes. Boxplots were created and number of genes used for each boxplot is displayed below. Expression changes were estimated using 3 biological replicates for each condition. Statistical significance was estimated using two-sided t-tests.
figf1a, Western blot analysis of yeast cell lysates with polyclonal anti-flag antibody to detect flag-tagged human Beclin 1 expression. Two-hundred micrograms of protein per lane was loaded. SEY6210, wild-type yeast; JCY3000, SEY6210 disrupted in apg6/vps30; (+) control, lysates of mammalian cells transfected with flag-beclin 1.b, Autophagicbody formation in yeast deprived of nitrogen in the presence (+) and absence (-) of 1 mM PMSF. Arrows denote cells that would be scored as positive in the experiment shown in c.c, Quantitative effects of apg6/vps30 and beclin 1 transformation on autophagicbody formation in apg6/vps30-disrupted yeast in the presence of PMSF. Cells with one or more autophagic bodies within the vacuole were scored as positive (see arrows in b). A minimum of 100 cells was counted for each sample. Results represent the mean (±s.e.m.) percentage of cells with autophagic bodies within the vacuole for triplicate samples. Similar results were obtained in five independent experiments.d, Sorting of vacuolar protein carboxypeptidase Y, (CPY). p2CPY, precursor form; mCPY, sorted mature form. Mr, relative molecular mass.
figf2a-f, Electron micrographs of MCF7.control (clone 38) (a, b) and MCF7.beclin1 (clone 17) cells (c- f) grown in nutrient-rich media (a, c) or subjected to 4 h of serum and amino-acid deprivation (b, d-f). Asterisks in e and f denote autophagic vacuoles that would be counted in the experiment shown in g. Autophagic vacuoles were defined as double-membrane vacuolar structures containing recognizable cytoplasmic contents. Scale bars, 1 µm.g, Quantitative effects of beclin 1 on basal and nutrient-deprivation-induced autophagy of MCF7 cells. Bars indicate mean (±s.e.m.) number of autophagic vacuoles per cell for cells growing in normal media (black), for cells subjected to 4 h of serum and amino-acid deprivation (grey), and for cells pre-treated for 30 min with 10 mM 3-MA and subjected to 4 h of serum and amino-acid deprivation in the presence of 3-MA (open). Mean was determined by counting the total number of autophagic vacuoles in each cell for 100 cells per clone per treatment.h, Comparison of the rates of degradation of long-lived proteins in MCF7.control cells (squares, clone 38), MCF7.beclin1 cells (circles, clone 17) and MCF7.beclin1stop cells (triangles, clone 70), in EBSS + 10% serum and complete amino acids (open symbols, solid lines) in EBSS alone (closed symbols, solid lines), and in EBSS + 10 mM 3-MA (closed symbols, dotted lines). Results are mean (±s.e.m.) of triplicate wells. Similar results were obtained in three independent experiments. Results shown with these clones are representative of results obtained with other clones analysed by electron microscopy in g.