EMBO/SourceData huggingface dataset:
Figure 1Dose-dependent viability assays of BRCA2-proficient (+BRCA2) or -deficient (-BRCA2) human DLD1 cells treated with drugs at the indicated concentrations for six days.Human spheroids established from BRCA2-proficient (+BRCA2) or -deficient (-BRCA2) DLD1 cells, were incubated with 1.25 µM olaparib or 0.5 µM chlorambucil over the indicated period of time.
Figure 2Dose-dependent viability assays of BRCA1-proficient (+BRCA1) or -deficient (-BRCA1) human RPE1-hTERT cells treated with drugs at the indicated concentrations for six days.Dose-dependent viability assays of BRCA1-proficient (+BRCA1) or -deficient (-BRCA1) TP53-deleted cells treated with drugs at the indicated concentrations for six days.Dose-dependent viability assays of Brca1+/+ and Brca1-/- mouse mammary tumour-derived cell lines treated with drugs at the indicated concentrations for six days.
Figure 3Dose-dependent viability assays of BRCA2-deficient (Capan-1) or -proficient (MIA PaCa-2) human pancreatic carcinoma-derived cells treated with drugs at the indicated concentrations for six days.Dose-dependent viability assays of BRCA2-deficient (PEO1) or -proficient (C4-2) human ovarian tumour-derived cells treated with drugs at the indicated concentrations for six days.BRCA2-deficient (PEO1) or -proficient (C4-2) human ovarian tumour-derived cells were infected with lentiviruses expressing control or CHD4 shRNAs, followed by selection with puromycin for 72 hours. Dose-dependent viability assays were performed on cells treated with drugs at the indicated concentrations for six days.
Figure 4BRCA2-proficient (+BRCA2) or -deficient (-BRCA2) human DLD1 cells were incubated with 1 µM chlorambucil (Chl). Whole cell extracts prepared at the indicated time points during treatment were immunoblotted as shown. GAPDH was used as a loading control.Quantification of chromosome aberrations and chromatid/chromosome break frequencies in BRCA2-proficient (+BRCA2) or -deficient (-BRCA2) human DLD1 cells incubated with 1 μM chlorambucil or 1 µM cisplatin for 72 h. Data were obtained from three independent experiments and normalised to untreated controls. A minimum of 60 Giemsa-stained metaphases were analysed for each sample. Cis, cisplatin; Chl, chlorambucil.Quantification of G2/M cell frequency relative to solvent control, using FACS analyses of cells incubated with 1 μM chlorambucil or 1 µM cisplatin for 48 hours. Cis, cisplatin; Chl, chlorambucil.Human H1299 cells were treated with control (CTR) or indicated siRNAs two days before drugs were added to the media for dose-dependent viability assays. Cell extracts prepared at the time of drug addition were immunoblotted as indicated. SMC1 was used as a loading control.Human H1299 cells were treated with control (CTR) or indicated siRNAs two days before drugs were added to the media for dose-dependent viability assays. Cell extracts prepared at the time of drug addition were immunoblotted as indicated. SMC1 was used as a loading control.
Figure 5Nude mice (nu/nu) were injected subcutaneously with 5 x 106 human DLD1 cells, BRCA2-proficient (A) or BRCA2-deficient (B). Tumour-bearing mice were treated with 3 mg/kg daily chlorambucil administered intraperitoneally (i.p.) for a total of ten days. Tumour weight was determined on the indicated days after initiation of the treatment.PDTCs derived from breast cancer samples as previously described (Bruna et al, 2016) were treated with chlorambucil at the indicated doses. Cell survival is represented relative to DMSO control. AB521, ER-negative tumour, no BRCA1 alteration; STG201, tumour with BRCA1 promoter methylation and loss of BRCA1 expression; VHIO179, tumour with BRCA1 germline mutation and MAD2L2 inactivating mutation (olaparib-resistant)CB17/SCID mice were injected intramuscularly with 5 x 106 human BRCA2-deficient HCT116 cells. Tumour-bearing mice were treated on the indicated days with chlorambucil or cisplatin administered intraperitoneally (i.p.), or talazoparib administered orally (o.s.) Tumour volume was measured on the indicated days after treatment initiation and was expressed relative to tumour volume at the beginning of treatment (day 1). Scale bar, 40 µm. Each experimental group included n = 5 mice. Error bars represent SEM. Tumour sections were assessed at the end of each treatment using immunohistochemistry of γH2AX staining.
Figure 6Wild type Balb/c mice were injected intra-peritoneally with solvent (daily) or 3 mg/kg chlorambucil (daily for 5 days) or 3.3 mg/kg cisplatin (daily for 3 days). Uptake of the apoptosis tracer 99mTc-Duramycin 2 h after intra-venous injection was quantified in selected organs using SPECT imaging in the indicated organs. Representative maximum intensity partial projections showing tracer distribution are shown.Immunohistochemical analyses of γH2AX staining in organs from mice treated as in A. Scale bar, 25 µm.
Figure 1(A-D) Images of a representative wild-type adult eye (A) or adult eyes containing mtm mutant clones (B and C). Quantification of relative eye sizes (n=10) (D).(F-G) A pupal eye disc containing mtm mutant clones (marked by the absence of GFP) was stained for Dlg. Note the increased interommatidial cells in the mtm mutant clone. Quantification of numbers of interommatidial cells per ommatidium (n=10) (G).(K) RNAi of Mtm reduces Yki phosphorylation. Kc167 cells were incubated with dsRNA of gfp or mtm for three days before western blot analysis. Total cell lysates were probed with anti-phospho-Yki (p-Yki) antibody. Note that RNAi of mtm decreased Yki phosphorylation.
Figure 4PI(3)P expression levels detected by a 2xFYVE-GFP reporter in a third instar larval wing disc (A) and eye disc (B) containing mtm mutant clones (marked by the absence of RFP). Ten different mtm clones and surrounding regions in three biological replicates were used for the analysis. Note the strong increases of PI(3)P levels in the mtm mutant clones.Quantification of the mean FYVE-GFP fluorescence intensity of mtm mutant clones and surrounding wild-type (WT) cells are shown (C). Ten different mtm clones and surrounding regions in three biological replicates were used for the analysis. Note the strong increases of PI(3)P levels in the mtm mutant clones.
Figure 1(A) Following transfection of given siRNAs and synchronization in G2-phase, Hek293 cells were DRB- or mock-treated and then subjected to IFM using the indicated antibodies. Lower panels display a 3-fold magnification of the boxed area shown above. Scales bars correspond to 5 and 1 μm, respectively (B) Hek293 cells were thymidine-arrested for 20h, mock- or OHT-treated to induce DSBs by nuclear accumulation of ER-AsiSI and then subjected to ChIP-multiplex PCR  (C) ChIP samples from (B) were analysed by qPCR. Shown are averages (bars) of 3 independent experiments (dots) Separase interacts with γH2AX in DSB-containing G2 but not G1 cells. Transgenic Hek293 cells treated with Dox to induce expression of Myc-separase-WT and with OHT to induce nuclear accumulation of ER-AsiSI and infliction of DSBs were synchronized in G1- or G2-phase and analysed by IP-WesternSeparase interacts with γH2AX in DSB-containing G2 but not G1 cells. Transgenic Hek293 cells treated with Dox to induce expression of Myc-separase-WT and with OHT to induce nuclear accumulation of ER-AsiSI and infliction of DSBs were synchronized in G1- or G2-phase and analysed b IFM for γH2AX- and Myc-separase-positive foc The quantification of the IFM in (E) shows averages (bars) of 3 independent experiments (dots) counting ≥ 100 cells each
Figure 2Separase is required for proper HDR but dispensable for NHEJ. U2OS DR-GFP (HDR reporter) and U2OS EJ5-GFP (NHEJ reporter) cells were separase-depleted by RNAi (SEP-1) or control-treated with GL2 siRNA, transfected to express HA-tagged ER-I-SceI, and then supplemented with OHT in G2-phase to induce nuclear accumulation of the homing endonuclease. Ethanol-supplemented samples served as negative controls. Two days later, cells were subjected to immunoblotting (ASeparase is required for proper HDR but dispensable for NHEJ. U2OS DR-GFP (HDR reporter) and U2OS EJ5-GFP (NHEJ reporter) cells were separase-depleted by RNAi (SEP-1) or control-treated with GL2 siRNA, transfected to express HA-tagged ER-I-SceI, and then supplemented with OHT in G2-phase to induce nuclear accumulation of the homing endonuclease. Ethanol-supplemented samples served as negative controls. Two days later, cells were subjected t flow cytometry to quantify the percentage of GFP positive cells (B The GFP quantification in (B) displays averages (bars) of 3 independent experiments (dots)Separase is required for proper HDR but dispensable for NHEJ. U2OS DR-GFP (HDR reporter) and U2OS EJ5-GFP (NHEJ reporter) cells were separase-depleted by RNAi (SEP-1) or control-treated with GL2 siRNA, transfected to express HA-tagged ER-I-SceI, and then supplemented with OHT in G2-phase to induce nuclear accumulation of the homing endonuclease. Ethanol-supplemented samples served as negative controls Two days later PI-stained cellular DNA (C)Co-depletion of Wapl and separase has a synergistic effect on HDR. U2OS DR-GFP cells were transfected with the indicated siRNAs, treated as in (A+B) and analysed by GFP-flow cytometry (D Shown in (D) are averages (bars) of 2-3 independent experiments (dots)Co-depletio of Wapl and separase has a synergistic effect on HDR. U2OS DR-GFP cells were transfected with the indicated siRNAs, treated as in (A+B) and analysed b immunoblotting (E)
Figure 3(A) Transgenic Hek293 cells constitutively expressing a separase sensor (cartoon below) and inducibly expressing Myc-separase were transiently transfected to express Flag-AsiSI-ER, Dox- and/or OHT-treated in G2-phase and analysed by (IP-)Western (B) Cells from (A) were analysed by IP-Western for cleavage of endogenous Rad21. As a control, in vitro-translated (IVT) Rad21 was incubated with hyperactive separase-SA or a protease-dead (PD) varian  (C) Sensor expressing cells were treated in G2-phase with DRB and nocodazole for 6 and 2 h, respectively, prior to chromosome spreading and IFM using Hec1- and γH2AX-antibodies. The separase sensor was detected based on autofluorescence of eGFP and mCherry, while Hec1- and γH2AX-antibodies were detected with corresponding Cy5- and marina blue-labeled secondary antibodies, respectively. Note that sizes of spread chromosomes vary greatly with buffer conditions, which is why no scale bar is shown
Figure 4Ser1660 is phosphorylated in response to DNA damage and required for the interaction of separase with γH2AX. Hek293 cells were arrested in G2 phase by sequential thymidine- and RO-3306 treatment, DRB- (+) or mock treated (-), and then analysed as indicated Myc-separase-WT or -S1660A expressing cells were subjected to (IP-)Western using, amongst others, a pan-specific antibody against phosphorylated serine (A, pan-pSSer1660 is phosphorylated in response to DNA damage and required for the interaction of separase with γH2AX. Hek293 cells were arrested in G2 phase by sequential thymidine- and RO-3306 treatment, DRB- (+) or mock treated (-), and then analysed as indicated Myc-separase-WT or -S1660A expressing cells were subjected to (IP-)Western using, amongst other a separase antibody specific for phosphorylated Ser1660 (B, pS1660)Ser1660 is phosphorylated in response to DNA damage and required for the interaction of separase with γH2AX. Hek293 cells were arrested in G2 phase by sequential thymidine- and RO-3306 treatment, DRB- (+) or mock treated (-), and then analysed as indicated (C) DNA damage-induced Ser1660 phosphorylation of endogenous separase is largely blocked by ATM inhibition. G2-enriched Hek293 cells were treated with KU-55933 (0.3 μM) and/or DRB and analyzed by (IP-)Western 12 h thereafter using the indicated antibodies (D) Preventing NES phosphorylation spoils nuclear localization of separase in response to DSBs. HeLaK cells expressing N-terminally NLS-eGFP-tagged separase variants were treated with DRB or carrier solvent (− DRB) for 4 h and then subjected to IFM using anti-γH2AX and anti-Nup153 to visualize sites of DNA damage and nuclear pore complexes, respectively. Transgenic separase was detected based on the eGFP autofluorescence. Note that due to their relatively high nuclear concentration co-localization of separase-WT and -S1660D with γH2AX-foci is not discernable. Scale bar = 5 μm  (E) In vitro phosphorylation of separase on Ser1660 by ATM kinase. Incubation of GST-p53 (amino acids 9-22), GST, separase-WT, or separase-S1660 with recombinant ATM-WT, ATM-KD (kinase dead), and/or KU-55933 in presence of [γ-33P]-ATP was followed by immunoblotting and autoradiography (F) Arg-methylation of RG-repeats mediates recruitment of separase to DSB-containing chromatin. Myc-separase-WT or -KG expressing cells were treated with DRB as indicated and analysed by (IP-)Western and Coomassie staining  (G) In vitro Arg-methylation of separase's RG-repeats by PRMT1. Incubation of histone H4, separase-WT, or separase-KG with recombinant PRMT1 or reference buffer in presence of S-adenosyl-L-[methyl-3H]-methionine was followed by Coomassie staining and autoradiography
Figure 5(A) Persistence of γH2AX- and MDC1-positive foci in absence of NES-phosphorylation or RG-methylation of separase. Transfected Hek293T cells were siRNA- and Dox-treated to deplete endogenous separase and induce expression of the indicated, siRNA resistant Myc-tagged separase variants, respectively. Then, they were constitutively (− washout) or transiently exposed to DRB (+ washout) and finally quantitatively assessed by IFM for γH2AX- and MDC1-positive foci. Shown are averages (bars) of 3 independent experiments (dots) counting ≥ 100 cells each. Scale bar = 5 μm (C) Recruitment of separase-WT and -S1660D but not separase-superNES, -S1660A, and -KG to DSBs. Cells from (A) were treated as indicated and analysed by IFM for co-localization of Myc-separase with γH2AX-foci. Scale bar = 5 μm  (D) Interaction of separase-WT and -S1660D but not separase-superNES, -S1660A, and -KG with γH2AX. Cells from (A) were subjected to (IP-)Western analysis as indicated. Lanes that illustrate cellular levels of cyclin A2 and γH2AX after DRB-washout and those that analyse interaction of Myc-separase variants with γH2AX in presence of DRB are labeled by red arrows and blue arrow heads, respectively
Figure 6Separase is sumoylated in response to DSBs. Hek293T cells transfected to express His6-separase (A were treated with DRB or carrier solvent (− DRB) and then subjected to denaturing IMAC followed by immunoblotting of input samples and eluates using the indicated antibodiesSeparase is sumoylated in response to DSBs. Hek293T cells transfected to expres His6-Sumo2 (B) were treated with DRB or carrier solvent (− DRB) and then subjected to denaturing IMAC followed by immunoblotting of input samples and eluates using the indicated antibodiesLys-1034 is a major target of DSB-induced sumoylation of separase. Hek293T cells expressing His6-Sumo2 (C or -K1034R (D) were DRB- or mock-treated and subjected to denaturing IMA Input samples and eluates were immunoblotted using the indicated antibodiesLys-1034 is a major target of DSB-induced sumoylation of separase. Hek293T cells expressin Myc-separase-WT or -K1034R (D) were DRB- or mock-treated and subjected t Myc-I Input samples and eluates were immunoblotted using the indicated antibodies (E) Mms21-dependent in vitro sumoylation of separase at Lys-1034. Myc beads were loaded with separase-WT or -K1034R or left empty (last lane), combined with recombinant Ubc9 and/or Mms21, as indicated, and incubated in presence of His6-Sumo2, Sae1-Sae2 and ATP. Supernatant and washed beads were analysed by Coomassie staining (lower panels) and immunoblotting (upper panels), respectively  (F) Cells expressing the indicated separase variants were DRB- or mock-treated and then lysed. Lysates and chromatin pelleted therefrom were assessed by immunoblotting and Coomassie staining, as indicated. Note that the lanes shown in the upper panels, although not directly juxtaposed, nevertheless originate from the same gel  (G) Preventing sumoylation of Lys-1034 compromises separase's ability to support DSB repair. The indicated variants were probed for their ability to functionally replace endogenous separase in HDR as described in figures 6A and 6B. Shown are averages (bars) of 3 independent experiments (dots) counting ≥ 100 cells each
Figure 7(A, B) SEPARASE+/+ and SEPARASE+/- MEFs were constitutively (− washout) or transiently exposed to DRB (+ washout) and then analysed by IF for cells with γH2AX- and 53BP1-positive foci. Shown are averages (bars) of 3 independent experiments (dots) counting ≥ 100 cells each. Scale bar = 5 μm. (C) Cell lysates from one experiment in (A) were analysed by immunoblotting using the indicated antibodies SEPARASE+/+ and SEPARASE+/- MEFs were treated with DRB (+) or carrier solvent (−) for the indicated times and then analysed by immunoblotting of total cell lysates (DSEPARASE+/+ and SEPARASE+/- MEFs were treated with DRB (+) or carrier solvent (−) for the indicated times and then analysed b IPs using anti-Rad21 or unspecific IgG (E). The lower right two lanes show in vitro-expressed mouse Rad21 treated with hyperactive (SA) separase or a protease-dead (PD) variant
Figure 8(A) Transformation susceptibility of primary MEFs. SEPARASE+/+ versus SEPARASE+/- MEFs were infected to express E1A and hRasV12 and then subjected to a colony formation assay. Shown are representative images (left) and the graphical representation of the number of transformed foci (right) from two independent experiments (bars = standard deviations) (B) Two-stage DMBA (initiation) plus TPA (promotion) skin carcinogenesis. The number and size of skin papillomas are plotted against time for each genotype: SEPARASE+/+ (n=8) versus SEPARASE+/- (n= 11)  (C) Representative hematoxylin- and eosin-stained sections from SEPARASE+/- biopsies. (i) glandular hyperplasia; (ii) benign papilloma; (iii) squamous cell carcinoma. Scale bars are 200 µm in (i) and 500 µm in (ii) and (iii). (D) Quantitative histological assessment of the DMBA-TPA skin cancerogenesis assay. Given are numbers of total, and corresponding percentage in brackets
Figure 1.(A) Flow cytometry analysis of l(2)mbn cells starved for 2 h (2h S) or 4 h (4h S) showed an increase in LTG fluorescence levels (x axis) compared with control cells in full-nutrient medium (C). The gate shown on the histogram represents the LTGhigh population. (B) Representative images of GFP-LC3 puncta in control and 2-h starved l(2)mbn cells. An increase in GFP-LC3 puncta was observed in the starved cells (left). Bar, 10 μm. (C) Flow cytometry analysis of 4-h starved cells were incubated with 3MA (4h S + 3MA) and Baf (4h S + Baf). Both autophagy inhibitors reduced the LTG fluorescence levels compared with starved cells (4h S). Control cells in nutrient-full medium (C) are represented by the brown line. (D) Both autophagy inhibitors, 3MA and Baf, reduced the LTGhigh population significantly. (3MA, P = 0.00001; and Baf, P = 0.00006).(E) RNAi of representative Atg genes decreased the LTR fluorescence levels compared with control. (Atg1, P = 0.01; Atg5, P = 0.01; Atg7, P = 0.002; Atg8a, P = 0.008; Atg8b, P = 0.006; and Atg12, P = 0.02).(F) RNAi of all tested genes in the TOR-PI3K pathways had a statistically significant effect on LTG fluorescence levels. Known negative regulators of autophagy are shown with gray bars; positive regulators are shown with white bars. (Pten, P = 0.007; Tsc1, P = 0.027; Tsc2, P = 0.025; RheB, P = 0.005; Tor, P = 0.016; and S6k, P = 0.012). dsRNA corresponding to a human gene (Hs) was used as a negative control in E and F. Results represent the mean value ± SD from at least three independent experiments.
Figure 2.(A) The percentage of LTGhigh cells was reduced by hid-RNAi (P = 0.006) but not by rpr, grim, and skl-RNAi.(B) Knockdown of Ras, phl, and rl expression by RNAi resulted in an increase in the percentage of LTGhigh cells. (Ras, P = 0.003; phl, P = 0.001; and rl, P = 0.028).(C) th-RNAi treatment (24 h) had no significant effect on LTG levels; in contrast, RNAi of Bruce resulted in an increase in LTG fluorescence levels (P = 0.01).(D) Reduction of debcl, Buffy, or p53 expression by RNAi resulted in a decrease in LTG fluorescence levels. (debcl, P = 0.018; Buffy, P = 0.006; and p53, P = 0.004).(E) RNAi of effector caspase Dcp-1 resulted in a significant decrease in the LTGhigh population (P = 0.001).(F) Representative images of GFP-LC3 puncta in cells treated with the indicated RNAi after a 2-h starvation treatment. Bar, 10 μm.(G) Quantification of cells with GFP-LC3 puncta after RNAi treatment. Cells with more than three GFP-LC3 punctate dots were considered to be GFP-LC3-positive cells. Cells treated with the RNAi indicated here all showed a significant difference (P < 0.05) in the percentage of GFP-LC3-positive cells compared with the human (Hs) RNAi control. (Pten, P = 0.006; Tor, P = 0.034; Buffy, P = 0.005; debcl, P = 0.003; Bruce, P = 0.003; Dcp-1, P = 0.007; hid, P = 0.002; Ras, P = 0.006; and phl, P = 0.050). Results represent the mean value ± SD from three independent experiments.
Figure 3.(A) GFP-LC3 proteins were expressed in nurse cells (NC) but not in follicle cells (FC) by using the UASp/nanos Gal4 system. DAPI staining of nuclei is shown in blue.(B) UASp-GFP-LC3; nanos GAL4 flies were conditioned on yeast paste and had a diffuse GFP-LC3 pattern. Numerous GFP-LC3 puncta (green) at region 2 within germarium were observed in nutrient-deprived flies. Ovaries were stained with LTR in w1118 flies. Germarium of nutrient-deprived w1118 flies had an increase in punctate LTR staining (red) compared with well-fed germarium.(C) Degenerating stage 8 egg chambers (arrows) had numerous GFP-LC3 puncta (green) and an increase in LTR-positive dots (red) compared with healthy egg chambers (arrowheads). DAPI (blue) staining of nuclei is shown in the two panels on the right.(D) Degenerating stage 8 egg chambers (arrows) of nutrient-deprived Atg7 mutants (Atg7d77/Atg7d14) showed a dramatic decrease in LTR staining. DAPI staining of nuclei is shown in blue.
Figure 4.(A) Germaria of the nutrient-deprived Dcp-1Prev flies showed a dramatic decrease in LTR staining compared with nutrient-deprived wild-type flies shown in Fig. 3 B. DAPI staining of nuclei is shown in blue. (B) Degenerating stage 8 egg chambers (arrows) of nutrient-deprived Dcp-1Prev flies showed a dramatic decrease in LTR staining compared with nutrient-deprived wild-type flies shown in Fig. 3 C.(C) Lack of Dcp-1 function (UASp-GFP-LC3 Dcp-1Prev/Dcp-1Prev; nanos-GAL4/+) resulted in uniform diffuse staining of GFP-LC3 rather than the punctate pattern observed in wild-type degenerating stage 8 egg chambers shown in Fig 3 C. (D) Dying egg chambers (arrows) of NGT/+; nanos-GAL4/UASp-fl-Dcp-1 flies that were conditioned on yeast paste showed a significant increase in punctate LTR staining (red) compared with healthy egg chambers (arrowheads). (E) Expression of activated Dcp-1 (a truncated form) and GFP-LC3 in the germ line (UASp-GFP-LC3/+; nanos-GAL4/nanos-GAL4 UASp-tDcp-1) resulted in abundant degenerating stage 8 egg chambers (arrows) with numerous GFP-LC3 puncta (green). DAPI staining of nuclei is shown in blue.
Figure 5.(A) The germarium in well-fed BruceE81 flies showed an increase in LTR staining (red) compared with wild-type well-fed flies shown in Fig. 3 B (top right). DAPI staining (white) of nuclei is shown on the right. (B) In well-fed wild-type flies, mid-oogenesis nurse cell death is a rare event. Lack of Bruce function resulted in an increase in dying stage 8 egg chambers (arrows) in ovaries under well-fed conditions, and these degenerating stage 8 egg chambers had numerous LTR (red) punctate dots. DAPI staining (white) is shown on the right. Bars: (A) 20 μm; (B) 50 μm.