EMBO/SourceData huggingface dataset:
Figure 1(A) Western blot analysis of Caspase 8, FLIP and FADD recruitment to the TRAIL-R2 DISC in A549, HCT116 and DU145 cell lines following incubation with increasing concentrations (1x, 2x, or 4x) of anti-TRAIL-R2 (AMG655)-conjugated magnetic beads for 90 minutes.(B) Western blot analysis of the unbound soluble fraction An untreated input of each cell line is included for comparison.(C) Quantification of Western blot analysis for FLIP, FADD, Procaspase 8 and their respective cleavage fragments at the TRAIL-R2 DISC. Proteins were quantified by densitometry and normalised to known protein standards(D) Caspase-8-like (IETDase) activity assay in the unbound soluble fraction from the DISC IP An untreated control (0) was used for each cell line to show basal caspase-8 activity.(E) Ratios of DED proteins quantified at the TRAIL-R2 DISC in A549, HCT116 and DU145 cells; increasing concentrations of AMG655 are indicated by colour intensity.(F) Correlation between caspase-8 activity and ratio of FADD:FLIP(L).
Figure 2(A) Western blot analysis of FADD, FLIP and Caspase 8 at the TRAIL-R2 DISC in HCT116 cells treated with escalating doses (0-30nM) of caspase-8 siRNA for 48 hours. Samples were incubated with anti-TRAIL-R2 (AMG655) beads (4x) for 90 minutes prior to collection. A scrambled control siRNA (SC) was transfected (30nM) for comparison. (B) Quantification of Caspase 8 (p55, p43/41 and p26/24) and FLIP (FLIP(L), FLIP(S) and p43-FLIP) at the TRAIL-R2 DISC by densitometry and normalised to known protein standards (C) Ratio of Caspase-8:FLIP calculated from values presented in (B).(D) Caspase-8 (IETDase) and -3/7 (DEVDase) activity assay in the unbound soluble fraction(E) Western blot analysis of FLIP, Caspase 8, and FADD recruitment to the TRAIL-R2 DISC in PC3 cells stably expressing an empty vector (EV) or flag tagged FLIP L (FL) treated with 2.5µM MS-275 for 48 hours, followed by a 90 minute DISC IP. * modified form of FLIP, potentially mono-ubiquitinated p43-FLIP(L).(G) Caspase-8 (IETDase) activity assays of the unbound soluble fraction
Figure 3(A) Western Blot analysis of Caspase 8, Caspase 10, FLIP and FADD recruitment to the TRAIL-R2 DISC in procaspase 8 (WT) or procaspase 8 deficient (Null) A549 cells incubated with AMG655 beads (4x) for either 30, 60 or 180 minutes.(B) Western blot analysis of the recruitment of FLIP, Caspase 8, Caspase 10 and FADD to the TRAIL-R2 DISC in HCT116 Parental, caspase 8 null (CASP8), caspase 10 null (CASP10) or CASP 8/10 cells treated with 4x AMG655 conjugated beads for 90 minutes.(C) AlphaScreen® assessment of the interaction of recombinant FLIP DED1/2 and recombinant FADD DED.(D) AlphaScreen® assessment of the impact on the FLIP-FADD interaction of unstapled and stapled peptides corresponding to the FADD α1 helix.(E) NanoBiT® assay to quantify FLIP/FADD interactions in a U2OS cell line stably expressing the NanoBiT® constructs, FLIP (LgBiT) and FADD (smBiT) following 48 hour silencing of FLIP, FADD, or caspase 8. Western blot analysis was used to confirm successful knockdown of target proteins by siRNA.(F) NanoBiT® assay of HCT116 and U20S wild type (WT) and procaspase 8 null (Null) cells transiently co-transfected with FLIP (LgBiT) and FADD (SmBiT) NanoBiT® constructs for 24 hours. Construct expression was analysed by Western blotting.
Figure 4(A) Western blot analysis of FLIP, Caspase 8 and FADD recruitment to the TRAIL-R2 DISC in U20S parental cells (EV) and 3 independent caspase 8 null clones (#1, #2, #3) after incubation with AMG655 conjugated beads for 30, 60 or 180 minutes.(B) Western blot analysis of FLIP, Caspase 8 and FADD in the soluble unbound fraction(C) Quantification of FADD recruitment to the TRAIL-R2 DISCs FADD levels were normalised to TRAIL-R2 levels in the pull-downs. Densitometry was performed using ImageJ®.(E) Western blot analysis of FADD recruitment to the TRAIL-R2 DISC in HCT116 cells transiently transfected with an empty vector (EV), Flag-tagged FADD (WT), or Flag tagged FADD with point mutations at either Phe25 (F25A) or His9 (H9G). Western blotting analysis of the soluble unbound fraction was used to monitor transfection efficiency.(F) NanoBiT® assay of U20S cells transiently co-transfected with caspase 8 (LgBiT) and either WT FADD smBiT or FADD (smBiT) constructs with a point mutation at either phenylalanine 25 (F25A) or histidine 9 (H9G) for 48 hours. Western blot analysis was used to assess the expression level of each construct.
Figure 5(A) Western blot analysis of FLIP, caspase 8 and FADD recruitment to the TRAIL-R2 DISC in parental (WT) and CFLAR deficient (KO) HAP-1 cells treated with AMG655 conjugated beads for either 45 minutes (45'), 90 minutes (90'), 180 minutes (180'), 3 hours (3h), 6 hours (6h) or 9 hours (9h).(B) Caspase activity assays of the unbound soluble fraction(C) Caspase-8 (i) and -3/7 (ii) activity assays and high content microscopy assessment of cell death (iii) in Parental (PAR) and FLIP knockout (CFLAR CRISPR) cells treated with escalating doses of isoleucine zipper TRAIL (IZ-TRAIL) for 6 hours.(D) Caspase-8 (i) and -3/7 (ii) activity assays in Parental (PAR) and FLIP knockout (CFLAR CRISPR) cells treated with escalating doses of isoleucine zipper TRAIL (IZ-TRAIL) for 24 hours. (iii) Flow cytometry assessment of cell death induction in Parental (PAR) and FLIP knockout (CFLAR CRISPR) cells treated with IZ-TRAIL for 24h.(E) Western Blot analysis of Parental (WT) and FLIP knockout (KO) HAP-1 cells treated with 10ng/mL IZ-TRAIL for 0, 3, 6 or 24 hours.(F) Cell viability assay of Parental (PAR) and FLIP knockout (CFLAR CRISPR cells) treated with an escalating dose (0-1000ng/mL) of IZ-TRAIL for 72 hours.
Figure 6(B) NanoBiT® assay performed on TRAIL-R2 DISC IPs (90') carried out in U20S Caspase-8 null cells co-transfected with wild-type or F114A FLIP-LgBiT fusion proteins and wild-type or F122A caspase-8-SmBiT fusion proteins.(E) Western blot analysis of size exclusion chromatography experiments in which HCT116 cells transfected with Flag-tagged wild-type (WT) FLIP(S) were treated with 100ng/mL IZ-TRAIL for 3h prior to cell lysis and separation of low and high MW complexes using a size exclusion column. The fractions eluted at 2MDa and >2MDa are presented.
Figure 3MST analysis of the binding interactions between Sir4 H‑BRCT and Cy5-labeled phospho (filled circles) and non-phospho peptides (empty circles) from Esc1, Ty5 and Ubp10. KD = equilibrium dissociation constant. ΔFnorm values were divided by the amplitude of the saturation level, resulting in the fraction bound (from 0 to 1) for each data point. Data are represented as the mean ± SEM from ≥3 independent measurements.
Figure 4Example images of Nup49-RFP Sir4-GFP in wild-type (wt) cells (GA-10372; row 1); sir4 RKR cells (GA-10373; row 2); wt cells transformed with either empty plasmid (row 3), plasmid overexpressing wt Sir4 H‑BRCT (row 4), or plasmid overexpressing RKR mutant Sir4 H‑BRCT (row 5). The bar indicates 2 μm.Quantification of mean fluorescence intensity of Sir4-GFP using >100 cells. Error bars show the SD from 3 independent experiments. Quantification of cells containing Sir4 clusters at the nuclear envelope (NE). Data are represented as mean ± SD from 3 independent experiments where >100 cells were counted.Top: Schematic representation of the TEL7L::URA3 reporter used to assay silencing at telomeres. Bottom: wt cells (GA-503) transformed with either an empty plasmid (row 1), plasmid overexpressing wt Sir4 H‑BRCT (row 2), or plasmid overexpressing RKR mutant Sir4 H‑BRCT (row 3). Cells lacking endogenous sir4 (GA-5822) (row 4). Cells were grown in the absence of uracil and plated as 1:5 serial dilutions onto control YPAD or YPAD lacking uracil plates and imaged after 2 days. Top: Schematic representation of the HMR-EΔB::TRP1 reporter gene locus used for assaying silencing at the HMR locus. Bottom: wt cells (GA-485) transformed with plasmids as in panel D. Cells lacking endogenous sir4 (GA-6888) (row 4). Cells were grown in the absence of tryptophan and plated as 1:5 serial dilutions onto YPAD or YPAD lacking tryptophan plates and imaged after 2 days.
Figure 5Volcano plot showing proteins that interact with wt Sir4 H‑BRCT but not with the RKR mutant (upper right quadrant).MST analysis of the binding interactions between Sir4 H‑BRCT and Cy5-labeled phospho (filled circles) and non-phospho peptides (empty circles) from Tom1 and Cbf1. KD represents the equilibrium dissociation constant. The KD for interactions with the non-phospho peptides could not be determined (n.d.). ΔFnorm [0/00] represents the change in fluorescence during thermophoresis normalized to the initial fluorescence. Data are represented as the mean ± SEM from at least 3 independent measurements.Extracts from asynchronous cultures of strains Sir4-Myc Tom1-HA (GA-10645), Sir4-RKR-Myc Tom1-HA (GA-10646), Tom1-HA (GA-10647) and Sir4-Myc (GA-10199) were subjected to anti-Myc IP and western blotting with indicated antibodies in the presence and absence of lambda phosphatase.
Figure 6MST analysis of the binding interactions between Dbf4 H‑BRCT and Cy5-labeled phospho (filled circles) and non-phospho peptides (empty circles) from Esc1 and Ubp10. KD = equilibrium dissociation constant, which could not be determined (n.d.) for non-phospho peptides. ΔFnorm [0/00] represents the change in fluorescence during thermophoresis normalized to the initial fluorescence. Data are the mean ± SEM from ≥3 independent measurements.
Figure 1Trajectory plots of siRNA-transfected HUVECs under VEGF and/or Sema3E gradients for 12 h. Red trajectories indicate cells with displacement to the negative y-axis at the end of analyses.Rose plots of cell trajectories in (A). Leaflets are grouped into 10° segments. The radius of each leaflet represents the cumulative number of cells migrating in the corresponding angular bin. P values for the Rayleigh test represent non-random distributions of cell endpoints.Forward migration index (FMI) along the y- and x-axes during cell migration. n = 90 cells per group. Data represent mean ± SEM. **p < 0.01; ***p < 0.001; NS, not significant, by Tukey-Kramer test.
Figure 2Confocal (left three columns) and super-resolution (right-most columns) images of subcellular distributions of ectopic RhoJ (red), endogenous VEGFR2 (green) and PlexinD1 (left, white; right, blue) in cultured HUVECs 6 h after transfection with the indicated RhoJ constructs. Note the colocalization of WT-RhoJ and CA-RhoJ (Q79L) with PlexinD1 in perinuclear endosomes and DN-RhoJ (T35N) with VEGFR2 in cytoplasmic and nuclear puncta. Light-blue asterisks in the PlexinD1 panels indicate nuclei. See also Appendix Fig S2 for the full images of super-resolution microscopy. Scale bar, 5 µm (left); 1 µm (right).Proportion of ectopic RhoJ that colocalized with endogenous VEGFR2 and/or PlexinD1. n = 10 cells per group. Data represent mean.Co-immunoprecipitation (co-IP) from 293T cells 48 h after transfection. WT- and CA-RhoJ associated with PlexinD1, while DN-RhoJ associated with VEGFR2.
Figure 3Co-IP from 293T cells 48 h after transfection. RhoJ-PlexinD1 binding was disrupted by deleting the RBD or ECD of PlexinD1 and by 30-min stimulation with Sema3E. Arrows indicate PD1 Full (upper, 250 kDa) and PD1△RBD (lower, 1.7 kDa smaller than PD1 Full).PlexinD1 internalization in HUVECs 30 min after Sema3E stimulation. Scale bar, 10 µm.Super-resolution images of subcellular distributions of ectopic WT-RhoJ (red) and endogenous PlexinD1 (green) in HUVECs 24 h after transfection. RhoJ dissociated from internalized PlexinD1 30 min after Sema3E stimulation. Scale bar, 1 µm.Labeling of siRNA-transfected HUVECs with phalloidin (white) 30 min after Sema3E stimulation. Scale bar, 50 µm.Quantification of areas of individual HUVECs. n > 100 cells per group. Data represent mean ± SEM. ***p < 0.001; NS, not significant, by Tukey-Kramer test.
Figure 4Co-IP from HUVECs. Blots for β-actin shown in (A) were used to confirm equal loading in (B), as the same cell lysates were analyzed in both experiments.Immunoblots in HUVECs.Immunoblots in siRNA-transfected HUVECs.Co-IP from siRNA-transfected HUVECs. The graph shows relative levels of PlexinD1-VEGFR2 association, in which the values at 0 min are set to 1. n = 3 per group.Super-resolution images of receptor colocalization in siRNA-transfected HUVECs 30 min after Sema3E stimulation. Left panels show immunolabeling of VEGFR2 (green), PlexinD1 (red), and Nrp1 (white), with light-blue asterisks indicating nuclei. Middle panels show magnified views of white boxes in the left panels. Right panels represent fluorescence intensity profiles along 5 μm arbitrary lines in the middle panels. Scale bar, 10 µm. The graph shows the proportion of receptor complexes in PlexinD1-positive vesicles. n = 10 cells per group.Trajectory plots (left) and rose plots (right) of HUVECs pre-treated with the p38 MAPK inhibitor SB203580 under Sema3E gradient for 12 h. Red trajectories indicate cells with displacement to the negative y-axis at the end of analyses. P values for the Rayleigh test represent non-random distributions of cell endpoints. The graphs show FMI along the y- and x-axes during cell migration. n = 90 cells per group.
Figure 5Super-resolution images of receptor colocalization in siRNA-transfected HUVECs 30 min after VEGF stimulation. VEGFR2 (green), PlexinD1 (red), and Nrp1 (white) are shown as in Fig 4E. Scale bar, 10 µm. The graph shows the proportion of receptor complexes in the PlexinD1-positive vesicles. Data of "no stimuli" are the same from Fig 4E. n = 10 cells per group.Co-IP from siRNA-transfected HUVECs. The graph shows relative levels of PlexinD1-VEGFR2 association, in which the values at 0 min are set to 1. n = 3 per group.Immunoblots in siRNA-transfected HUVECs. Blots for β-actin shown in (C) were used to confirm equal loading in (D), as the same cell lysates were analyzed in these experiments. The graph shows changes of total VEGFR2 protein levels as a percentage of the values at 0 min. n = 3 per group.Immunoblots in siRNA-transfected HUVECs. Note the early and transient activation of PLCγ, Erk1/2, and Akt, but not p38 MAPK, in RhoJ-knockdown ECs.Immunoblots for ectopic RhoJ and endogenous VEGFR2 in HUVECs 12 h after transfection of RhoJ-expressing vectors. The graph shows protein levels of VEGFR2 relative to those of the control set as 100%. n = 3 per group.
Figure 6Kinetic analyses of EC migration in an ex vivo aortic ring angiogenesis model. Upper panels are snapshots of time-lapse imaging in which EC nuclei are labeled by SYTO dye (green). In analyzed regions framed by dotted lines, the centroids of the first five EC nuclei from the tip at 0 min are pseudocolored in magenta and those of the remaining EC nuclei are shown in cyan. Initial discrete labeling becomes intermixed over time (see also Movie EV1). Lower panels show trajectory analyses representing normalized positional changes of individual ECs during vessel elongation. Scale bar, 100 µQuantification of the kinetics of EC migration. Values of forward and reverse migration for RhojWT/WT are normalized to 1. n = 33 vessel branches per group.Quantification of vessel elongation (left graph). n = 33 vessel branches per group. Scatter plot shows relationship between "vessel elongation" and "reverse migration" in RhojGFP/GFP aortic rings as evaluated by a Pearson correlation coefficient.Labeling for YFP/GFP (green), CD31 (red), and ETS transcription factor ERG (blue) in retinal vascular fronts of P5 CAG-MerCreMer:R26R-EYFPflox/WT and CAG-MerCreMer:Rhojfloxflox mice after intraperitoneal (i.p.) injections of 10 µg of 4-hydroxytamoxifen (4OHT) at P1. Scale bar, 50 µm.   Relative contribution of YFP/GFP-positive ECs to the tip positions. n = 16 per group. Values for CAG-MerCreMer:R26R-EYPflox/WT are normalized to 1.
Figure 7Labeling for GFP (green) and CD31 (red) in retina of P4 RhojGFP/GFP mouse. Scale bar, 200 µm (left); 20 µm (right).Labeling for EdU (green), ERG (red), and CD31 (gray) in retinas of P4 RhojWT/WT and RhojGFP/GFP mice 2 h after i.p. EdU injections. Scale bar, 20 µm. The graph shows EC proliferation index in retinal capillaries behind the angiogenic fronts. n = 12 per group.Labeling for CD31 in retinas of P4 and P7 RhojWT/WT and RhojGFP/GFP mice. Scale bars, 500 µm.Morphometric analyses of retinal vessels in RhojWT/WT (P4, n = 18; P7, n = 8) and RhojGFP/GFP (P4, n = 16; P7, n = 11) mice.Labeling for CD31 in retinas of P4 Rhojflox/flox and Pdgfb-iCreERT2:Rhojflox/flox (RhojiΔEC) mice after single i.p. injection of 100 µg of 4OHT at P1. Scale bar, 500 µm.Morphometric analyses of retinal vessels in P4 Rhojflox/flox (n = 7) and Rhoji∆EC (n = 6) mice.Relative expression levels of Vegfa and Sema3e mRNA from WT mouse retinas. n = 3 per group.Labeling for GFP (green) and CD31 (red) in retina of P18 OIR-RhojGFP/WT mouse. Scale bar, 200 µLabeling for CD31 in retinas of P18 OIR-Rhojflox/flox and OIR-Rhoji∆EC mice after daily i.p. injection of 200 µg of 4OHT from P12. Scale bar, 500 µm. Quantification of areas of neovascular tufts in P18 OIR-Rhojflox/flox and OIR-Rhoji∆EC mice. n = 6 per group.
Figure 1(A) Western blot of Beclin‐1 and FACS profiles following siRNA treatment in HeLa cells and cells stably expressing a HA‐Beclin‐1 construct resistant to the siRNA targeting endogenous Beclin‐1.(B) FACS profiles of cells treated with indicated siRNA following nocodazole blockage and release.(C,D) Quantification of cells in mitosis (C) and quantification of each step of mitosis (D) by H3Ser10P staining for Control and Beclin‐1 siRNA‐treated cells. Error bars represent standard deviations (s.d.) (n=number of counted cells) calculated on three independent experiments. Two and three asterisks indicate significant results (P=0.0028 and P=0.0006, respectively).(E) Knockdown of Beclin‐1 increases the number of mitotic cells with misaligned chromosomes. siRNA‐treated HeLa cells were fixed in PFA, immunolabelled for α‐tubulin (green) and stained with DAPI (blue). White arrows indicate misaligned chromosomes. Quantification of cells with misaligned chromosomes is shown at the right (n=number of counted cells). Three asterisks indicate significant results (P=0.0001). Scale bar, 10 μm. DAPI, 4′,6‐diamidino‐2‐phenylindole; HA, haemagglutinin; PFA, paraformaldehyde; siRNA, small interfering RNA.
Figure 2Beclin‐1 depletion prolongs prometaphase by delaying chromosome congression. (A-H) Time‐lapse films of HeLa cells stably expressing YFP‐α‐tubulin and H2B‐Cherry and treated with the indicated siRNA. The pictures are acquired every 10 min. Time lapse starts at nuclear envelope breakdown. (A) Mitosis in control cells. (B) Western blot of whole‐cell lysates from Control, Beclin‐1 and Beclin‐1+Mad2 siRNA‐treated cells. (C-E) Three different classes of mitotic progression were observed upon Beclin‐1 depletion. Higher magnification of lagging chromatids in the anaphase of Beclin‐1‐depleted cells is shown on the right of (D).(F) The prometaphase delay is dependent on MAD2 and the spindle checkpoint. Cells treated simultaneously with siRNA targeting MAD2 and Beclin‐1 rapidly exit mitosis. (G) Average duration of mitosis in different classes of Beclin‐1‐depleted cells, compared to control and MAD2 depletions. n=number of counted cells in three independent experiments. Error bars on graphs represent s.d.(H) The frequency of the different Beclin‐1 phenotypes depended on the efficiency of Beclin‐1 knockdown. Mitotic progression in cells treated with increasing concentrations of Beclin‐1 siRNA was followed by video‐microscopy. Left panel: western blot of Beclin‐1 upon RNAi treatment; right panel: quantification of the appearance of the three different mitotic progression patterns, that is, prolonged mitosis with normal anaphase (as C), with lagging chromatids (as D) and mitotic exit without clear anaphase (as E) upon increased Beclin‐1 knockdown experiments. NEB, nuclear envelop breakdown; RNAi, RNA interference; siRNA, small interfering RNA; YFP, yellow fluorescent protein.
Figure 3(A) Inter‐kinetochore distance is reduced in unaligned chromosomes of Beclin‐1‐depleted cells. The distance between each kinetochore pair was quantified in siRNA‐treated and control cells treated with nocodazole for 3 h before fixation. Cells were stained with anti‐CREST antibody and DAPI. Quantification of the distance between kinetochore pairs (indicated by coloured bars) is reported at the bottom and done on sister kinetochores present in the same focal plane. One asterisk indicates significant result (P=0.0365). Graphs represent three independent experiments (error bars=s.d.).(B) Depletion of Beclin‐1 leads to defects in K‐MT attachment. siRNA‐treated cells were cooled on ice before fixation to reveal stable k‐fibres and then stained with anti‐α‐tubulin (red) and anti‐CREST (green) antibodies. Numbers point to magnified areas and indicate the mode of attachment of k‐fibres to kinetochores (1, bi‐oriented; 2, unattached; 3, mono‐oriented kinetochores). Scale bar, 10 μm. DAPI, 4′,6‐diamidino‐2‐phenylindole; NS, not significant; siRNA, small interfering RNA.
Figure 4(A) CENP‐E and CREST. Two asterisks indicate significant results, P=0.0053.(B) CENP‐F and CREST. Three asterisks indicate significant results, P=0.0006.(C) ZW10 and CREST. Three asterisks indicate significant results, P=0.0007.(D) MAD2 and CREST. Two asterisks indicate significant results, P=0.0032.(E) HEC1 and CREST.(F) KNL‐1 and CREST. On the right, magnified areas and quantification of the fluorescence intensity of the protein of interest normalized to the fluorescence intensity of CREST staining are shown. Graphs represent three independent experiments (error bars=s.d.). Scale bar, 10 μm.(G) Western blot of whole‐cell lysates from control and Beclin‐1 siRNA‐treated cells. siRNA, small interfering RNA.
Figure 5(A) Immunoprecipitation of endogenous Zwint‐1 in HeLa cell extracts expressing HA‐Beclin‐1 or HA‐GFP. Input is 3.5% of the immunoprecipitated material.(B) GST‐Zwint‐1 or GST‐beads were used to pull down recombinant purified His6‐Beclin‐1. (Left) Coomassie‐blue‐stained gel of the purified recombinant proteins (1 μg). (Right) A western blot of GST proteins and His6‐Beclin‐1 in the input and bound fractions. His6‐Beclin‐1 in the input represents 4% of the amount used for the pull‐down.(C) Zwint‐1 levels do not change in Beclin‐1‐depleted cells. Cells were treated with nocodazole before fixation. On the right, magnified individual kinetochore pairs stained for Zwint‐1 (red) and CREST (green), and quantification of Zwint‐1 levels normalized to CREST signal from three independent experiments (error bars=s.d.).(D) Beclin‐1 is associated with both the centrosome and the mitotic spindle during mitosis. HeLa cells were permeabilized before fixation and immunostained for Beclin‐1 (green), α‐tubulin (red) and DNA (DAPI, blue).(E) Beclin‐1 is retained on the k‐fibres. HeLa cells were cooled on ice before permeabilization and fixation, and stained with anti‐α‐tubulin (red) and anti‐Beclin‐1 (green) antibodies.(F) Beclin‐1 forms discrete foci in the vicinity of the outer kinetochore in a microtubule‐dependent manner. Untreated cells (left) and nocodazole‐treated cells (right) were pre‐extracted and fixed before labelling with Beclin‐1 (green) and Zwint‐1 (red) antibodies.(G) The fluorescent‐tagged Cherry‐Beclin‐1 forms foci in the vicinity of the outer kinetochore. HeLa cells transfected with indicated constructs were pre‐extracted and fixed before labelling with Zwint‐1 (green) antibody. Magnified areas are shown on the right.(H) Beclin‐1 localization close to the kinetochore does not depend on the ZW10 protein. Cells treated with Control, Beclin‐1 and ZW10 siRNA were pre‐extracted and fixed before labelling with Beclin‐1 (green), CREST (blue) and ZW10 (red) antibodies. DNA was visualized with DAPI. (C-H) Scale bar, 10 μm. DAPI, 4′,6‐diamidino‐2‐phenylindole; DNA, deoxyribonucleic acid; GFP, green fluorescent protein; GST, glutathione S‐transferase; HA, haemagglutinin.