EMBO/SourceData huggingface dataset:
Figure 7Telomere length analysis of ApaI‐digested DNA from cells harvested at early (left) and late generations (right) after expression of YFP‐tagged proteins.Northern blot analysis of ARIA, ARRET, and 18S rRNA (loading control) at early generations after expression of YFP‐tagged proteins. Numbers at the bottom are ratios between ARRET and 18S rRNA signals, expressed as fold increase over wt.ChIP analysis of subtelomeric H3K9ac, H3K9me3, and total H3 in the indicated strains at early generations. Immunoprecipitated DNA is normalized to input DNA and expressed as fold increase over wt after subtraction of values obtained for negative control immunoprecipitations performed with beads only. Bars and error bars are averages and s.d. from 3 independent experiments. Statistical significance was assayed using the unpaired, two‐tailed Student's t‐test. *P 0.05, **P 0.01, ***P 0.001 relative to wt.Serial dilutions of early generation cay1+ and cay1∆ cells expressing Cay1‐YFP and Rap1‐YFP were spotted on complete medium and grown at 30°C and 20°C.Quantification of cell length in strains grown for 24 h in complete medium at 30°C or 20°C. Each dot represents a single cell and median cell length (m) is indicated for each sample. Data are from 3 independent experiments and at least 100 cells were scored per sample in each experiment.Quantification of chromosome bridges of strains grown for 24 h in complete medium at 30°C or 20°C. Bars and error bars are averages and s.d. from three independent experiments and at least 200 cells were scored per sample in each experiment.
Figure 8Northern blot analysis of Tf2 retrotransposons, U6 snRNA, and act1+ mRNA (loading control) in wt, cay1Δ, and prp1‐1 cells. prp1‐1 cells were grown at 36°C for 4 h before harvesting. Black arrowhead indicates unprocessed Tf2 transcripts. The asterisk indicates unspliced U6. Numbers at the bottom are the ratios between Tf2 and act1+ signals (Tf2/Act1) and expressed as fold increase over wt.Northern blot analysis as in (A) in mpn1+ and mpn1∆ strains carrying a stably integrated extra copy of the snu6+ gene or empty vector control plasmids (ev).
Figure 1A Dose-response viability curves in presence of increasing concentrations of IMT1 for one-week, viable cells were counted and normalized to DMSO-treated controls. Data are expressed as mean ± SD of n=3 independent experiments with four technical replicates. IC50 values were calculated with non-linear least square fit, RKO: 521.8 nM, MiaPaCa-2: 291.4 nM and HeLa: 29.9 nM.B Mitochondrial transcript levels measured by qRT-PCR in RKO cells after 96 hours of 1 μM IMT1 treatment. Data are relative to DMSO-treated controls and are expressed as mean ± SD of n=4 independent experiments. Ordinary one-way ANOVA was used for comparisons to DMSO-treated controls (RNR1, RNR2, MT-COX2, MT-ATP6, MT-ND1: ***p<0,0001).D Volcano plot showing proteomic changes of RKO cells treated with 1 μM IMT1 for 96 hours. Data are plotted as average log2-fold change versus log10 of adjusted p-value of DMSO-treated controls; mitochondrial protein are highlighted in red.F CRISPR guides enriched (positive hits) in IMT1-treated RKO cells plotted as log2 fold change (LFC) versus log10 of adjusted p-value. mTORC1 and VHL pathway-related genes are reported in green and blue, respectively.
Figure 2B-D Viable cells count in presence of DMSO, IMT1 alone or in combination with rapamycin in RKO (B), MiaPaCa-2 (C) and HeLa (D) cells. Data are expressed as mean values ± SD of n=6 independent experiments each including four technical intra-plate replicates. Statistical significance was calculated with one-way ANOVA test. Paired IMT1 vs. IMT1 + RAPA comparisons: RKO: ***p<0,0001, MiaPaCa-2: **p= 0,0014, HeLa: *p= 0,0238.F-H Viability assessment of RKO (F), MiaPaCa-2 (G) and HeLa (H) cells treated with DMSO, IMT1 alone and in combination with FG4592. Data are expressed as mean values ± SD of n=6, 5, 6 independent experiments for RKO, HeLa and MiaPaCa-2, respectively. Statistical significance was calculated with one-way ANOVA. Paired IMT1 vs IMT1 + FG4592 comparisons: RKO: **p<0,0028, MiaPaCa-2: **p= 0,0011, HeLa: *p= 0,0029.
Figure 3C Western blot analyses of OXPHOS protein steady state levels of RKO cells after treatment with DMSO, IMT1, rapamycin, rapamycin with IMT1, FG4592 and FG4592 with IMT1. ACTINB is shown as loading control.F, G Cellular OCR measured with Seahorse extracellular flux analyzer after sequential addition of different modulators of mitochondrial function. RKO cells were treated with DMSO, rapamycin (F) or FG4592 (G) in presence or absence of IMT1. Data are expressed as the mean ± SEM of n=3 independent experiments with six technical replicates. Statistical significance was calculated with one-way ANOVA test. DMSO-treated controls vs: IMT1: p=0,0008; RAPA: p=0,1578; RAPA + IMT1: p= 0,0034; FG4592: p=0,872; FG4592 + IMT1: p=0,0029. Paired t-test of IMT1 vs RAPA + IMT1: p= 0,034; IMT1 vs FG4592 + IMT1: non-significant.
Figure 4A Differences in sensitivity to increasing IMT1 doses between RKO cells and resistant RKO cells. The graph shows mean values ± SEM of n=3 independent experiments.B Western blot analyses of OXPHOS protein steady state levels at increasing concentrations of IMT1 of RKO (left) and IMT1-resistant RKO cells (right). The samples were collected and analyzed after eight weeks of chronic IMT1 treatment of the resistant line; GAPDH is shown as loading control.G Relative mtDNA copy number measured in RKO and IMT1-resistant cells treated with IMT1 for 96 hours. Data are expressed as average from n=3 experiments ± SEM. Statistical significance was calculated with one-way ANOVA test. RKO + IMT1 vs Resistant + IMT1: MT-ND1 **p=0,0035, MT-ATP6 ***p=0,0002, MT-CYTB **p=0,0028.H Cellular OCR measured by Seahorse extracellular flux analysis after sequential addition of different modulators of mitochondrial function in RKO and IMT1-resistant RKO cells treated with DMSO or IMT1. Data are expressed as the mean ± SEM of n=3 independent experiments with six technical replicates. Statistical significance was calculated with one-way ANOVA test. RKO DMSO vs RKO + IMT1: p=0,0008; RKO DMSO vs resistant DMSO: p=0,0343; RKO DMSO vs resistant + IMT1: p= 0,0092. Paired t-test of RKO + IMT1 vs resistant + IMT1: p= 0,135.
Figure 5A ECAR measured by Seahorse extracellular flux analysis in DMSO and IMT1-treated RKO and IMT1-resistant cells. Data are expressed as the mean ± SEM of n=3 independent experiments with six technical replicates. Statistical significance was calculated with one-way ANOVA test for multiple comparisons. RKO DMSO vs RKO + IMT1: **p=0,0019; RKO + IMT1 vs resistant + IMT: *p=0,0214; RKO DMSO vs resistant DMSO: non-significant.D, E IMT1 extracellular (D) and extracellular (E) concentrations measured in the medium at 0, 2 and 24 hours of IMT1 treatment in RKO and IMT1-resistant RKO cells. Mean values ± SEM of n=3 experiments.
Figure 6B Comparison between proteomic analyses of parental RKO and resistant cells treated with 1 μM IMT1 for 96 hours (r: sample correlation coefficient). Data are expressed as average log2-fold change of controls (DMSO-treated RKO cells). Mitochondrial proteins are reported in red.
Figure 7A Depleted CRISPR guides (negative hits) in IMT1-treated RKO cells plotted as log2 fold change (LFC) versus log10 of adjusted p-value.C Representative images and related plot of IMT1-resistant RKO spheroid areas in presence of the aforementioned compounds for two weeks (IMT1: 1 μM, CAP: 1 μg/ml); Scale bar: 1mm. Data show the mean values ± SD of n=4 experiments. Statistical significance was calculated with paired t-test: IMT1 vs IMT1+ CAP: *p=0,0242.G Viable cells counts of IMT1-resistant RKO cells treated for one-week with either DMSO or IMT1 in presence of controls (control #1, #2) or TFAM (TFAM #1, TFAM #2) siRNAs. Data are expressed as mean values ± SD of n=5 independent experiments each including six technical intra-plate replicates. Statistical significance was calculated with one-way ANOVA test. Controls + IMT1 vs TFAM #1 + IMT1: p= 0,2148; Controls + IMT1 vs TFAM #2 + IMT1: *p=0,0415.
Figure 1(B) Detection of soluble, cleaved NrCAM (sNrCAM) and full-length, mature NrCAM (mNrCAM) and soluble APPα (sAPPα) in neuronal supernatants and lysates (prepared from E16 neurons), treated with GI254023x (5 µM), or solvent for 48h. Densitometric quantifications of the Western Blots are shown (** p < 0.01; *** p < 0.001; **** p < 0.0001, two-sided students t-test n = 6-8).(C) Detection of sAPPα, sNrCAM and mNrCAM in ADAM10 cKO neuronal supernatants and lysates at 7 days in vitro (DIV7). The neurons prepared from floxed ADAM10 (ADAM10 fl/fl) mice were infected with a lentivirus encoding improved Cre recombinase (iCre) or a control GFP lentivirus at DIV2. Conditioned media were collected for 48 h. Densitometric quantifications of the Western Blots are shown (** p < 0.01; *** p < 0.001; **** p < 0.0001, two-sided students t-test n = 6-8).
Figure 2HEK 293 cells were transfected with a C-terminally VSV-tagged NrCAM construct or empty vector. Cells were then treated with DAPT (1 µM), GI254023x (5 µM), either substances or solvent for 24 h. The C-terminal NrCAM-fragment was detected with an antibody against the VSV-tag. Representative Western Blots are shown.
Figure 3(A) ADAM10 fl/fl neurons were treated with an iCre (to induce an ADAM10 KO), or a control lentivirus at DIV2. At DIV7, cell surface proteins were labeled with biotin and enriched by streptavidin pull-down. The biotinylated proteins were detected by immunoblotting. Total lysates were analyzed to compare mNrCAM's surface/lysate levels. Densitometric quantifications of the Western Blots are shown. Two-sided students t-test (**** p < 0.0001, n = 7). Given are mean +/- the standard error of them mean. The mean levels of solvent treated cells were set to 1.(B) Primary murine neurons were treated with GI254023x (5 µM) or solvent, for 48h. The cell surface proteins were enriched like in A. Densitometric quantifications of the Western Blots are shown. Two-sided students t-test (**** p < 0.0001, n = 11). Given are mean +/- the standard error of them mean. The mean levels of solvent treated cells were set to 1. Representative Western Blots are shown.(C) Workflow of the neurite outgrowth assay. (D) Representative pictures of single neurites at time-point 0h (DIV3) and 24h (DIV4). Neurons were infected 4h after plating with lentiviruses encoding GFP (to visualize the neurons) together with shRNA expression cassettes (sh1 and sh2) targeting either NrCAM or carrying a scrambled (scr) control construct. Images of neurites were taken at three days in vitro (DIV3) and 24h later at DIV4. In order to study the effect of ADAM10 on neurite outgrowth, neurons were treated with the ADAM10 inhibitor GI254023x, or vehicle (control), at DIV3, after taking the first pictures with an epifluorescent microscope. The differences in neurite length were calculated as absolute values (neurite length at 24h minus neurite length of 0h) for individual neurites passing through the middle channels of the chambers. Only neurites that had already entered the main channel at 0h and had not yet left those channels at 0h were considered. The red arrows indicate the start and the end of the respective length measurements. The scale bar indicates 40 µm. (E) One-way ANOVA with post hoc Dunnett's test. Given are mean +/- the standard error of them mean (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, n-numbers for each condition are shown in the graph in 3E).
Figure 4(A) Wt neurons were kept in culture until DIV5, then the cells were treated with acitretin (4 µM) or vehicle control for 48h, as described earlier (Tippmann et al, 2009). (B) Densitometric quantifications of the Western Blots are shown. Given are mean +/- the standard error of the mean (*** p < 0.001; **** p < 0. 0001, two-sided students t-test n = 9-20). The mean levels of solvent treated cells were set to 1.(C) Detection of sNrCAM and human serum albumin (hSA) in AD patients CSF that had been treated with acitretin (30 mg/d) or placebo for 30 days (n = 9) (Endres et al, 2014). (D) Densitometric quantification of the Western Blots in (C). sNrCAM/hSA ratios were calculated, and then the treatment to baseline ratios (t/b) for every patient were calculated. Two-sided student's t-test (n = 9). Given are mean +/- the standard error of them mean. Representative Western Blots are shown.
Figure 5(A) To ensure proper synapse formation, primary neurons were kept in culture for ten days prior to the treatment (Wan et al, 2012). Then, cells were treated with NMDA (50 µM), NMDA (50 µM) and GI254023x (5 µM), GI254023x (5 µM) alone or vehicle for 30 minutes. Total cellular mADAM10 levels remained unchanged by the treatment, which is in line with NMDA altering mainly the intracellular localization of ADAM10 by driving the protein to the synaptic membranes (Marcello et al, 2007). Densitometric quantifications of the Western Blots are shown. One-way ANOVA with post hoc Dunnett's test (*** p < 0.001, n = 7). Given are mean +/- the standard error of them mean. The mean levels of solvent treated cells were set to 1. The membrane was reprobed with the different indicated antibodies.(B) Wt neurons were kept in culture until DIV10, then the cells were treated with NMDA (50 µM), or vehicle for 30 minutes. Cell surface proteins were labeled with biotin and enriched by streptavidin pull-down. The biotinylated proteins were detected by immunoblotting. Total lysates were analyzed to compare mNrCAMs surface/lysate levels. Densitometric quantifications of the Western Blots are shown. Two-sided students t-test (** p < 0.01, n = 6). Given are mean +/- the standard error of them mean. The mean levels of solvent treated cells were set to 1. Representative Western Blots are shown.
Figure 6Volcano plot of the proteomic analysis of CSF from AD patients treated with acitretin or placebo for 30 days. Every circle represents one protein. The log10 transformed t-test p-values (again treatment/baseline for every patient) are plotted against the log2 transformed label-free quantification intensity ratios of acitretin and placebo CSF. ADAM10 substrates are indicated in bold writing. In total, more than 50 ADAM10 substrates were identified, but only the significantly altered substrates as well as NrCAM and APP are indicated.
Figure 7HEK293 cells were transiently transfected with hNrCAM, FLAG-tagged hTSPAN15, either plasmids or the empty vector. Detection of sAPPα and sNrCAM in the conditioned media and ADAM10 in the cell lysate. Densitometric quantification of the Western Blots are shown (** p < 0.01), (*** p < 0.001), (**** p < 0.0001), two-sided students t-test, or one-way ANOVA with post hoc Dunnett's test (n = 6). Given are mean +/- the standard error of them mean. The mean levels of solvent treated cells were set to 1. Representative Western Blots are shown.
Figure 1(A) Time series of TIRF microscopyimages showing GFP-dynein (green in merge) localising to the plus ends of dynamic Alexa568-microtubules (Alexa568-MT, magenta in merge). Protein concentrations were 10 nM GFP-dynein, 20 nM human dynactin, 20 nM EB1 and 17.5 µM Alexa568-tubulin (5% labelling ratio).(B) individual and dual colour kymographs showing GFP-dynein (green in merge) localising to the plus ends of dynamic Alexa568-microtubules (Alexa568-MT, magenta in merge). Protein concentrations were 10 nM GFP-dynein, 20 nM human dynactin, 20 nM EB1 and 17.5 µM Alexa568-tubulin (5% labelling ratio).(C) Merged triple colour and single fluorescence channel kymographs showing microtubule end tracking of GFP-dynein (green in merge) and Alexa647-EB3 (red in merge) on dynamic Alexa568-microtubules (blue in merge). Concentrations were 10 nM GFP-dynein, 10 nM human dynactin, 10 nM Alexa647-EB3, and 17.5 µM tubulin.(D, E) Dual and single colour kymographs of GFP-dynein (green) on dynamic Alexa568-microtubules (magenta) in the absence of either dynactin (D) . Concentrations of the proteins present as in A.(D, E) Dual and single colour kymographs of GFP-dynein (green) on dynamic Alexa568-microtubules (magenta) in the absence of EB proteins (E). Concentrations of the proteins present as in A.(F) Averaged fluorescence intensity profiles of GFP-dynein at growing microtubule plus ends in the presence of both human dynactin and EB1 (blue squares, as in A), in the absence of dynactin (red circles, as in D), or in the absence of an EB protein (black triangles, as in E). Mean values from at least two separate experiments per condition (with a total of at least 30 kymographs) are shown; error bars are s. e.(G) Kymographs of GFP-dyneinmicrotubule end tracking in the absence of ATP (in contrast to all other experiments that contain ATP). Other conditions as in A. For merged kymographs, microtubule plus and minus ends are labelled by (+) and (−). Experiments were performed at 30°C.
Figure 2(A) Merged triple colour and single fluorescence channel TIRF microscopy kymographs of GFP-dynein showing end tracking, processive motility and diffusive and static binding in the presence of all DDB components and EB1. Protein concentrations are 10 nM GFP-dynein (green in merge), 20 nM human dynactin, 20 nM EB1, 200 nM Alexa647-BicD2-N (magenta in merge), and 17.5 µM Alexa568-tubulin (blue in merge). Alexa647-BicD2-N often co-localises with processively moving and occasionally with statically bound GFP-dynein, but not with plus end-tracking GFP-dynein.(B) Averaged fluorescence intensity profiles of GFP-dynein (green squares) and Alexa647-BicD2-N (magenta circles) at growing microtubule plus ends. Mean values from three separate experiments per condition (with a total of at least 75 kymographs) are shown; error bars are s. e.(C, D) Dual and single colour kymographs of an Atto565-microtubule (magenta) in the presence of GFP-dynein (green), human dynactin and EB1, and either (C) in the absence or (D) presence of 5 µM BicD2-N; other conditions as in A. The increased BicD2-N concentration in D compared to A strongly reduces microtubule plus end tracking of GFP-dynein.(E) Averaged fluorescence intensity profiles of GFP-dynein at growing microtubule plus ends for the condition without BicD2-N (red circles, as in C) or with 5 µM BicD2-N (black squares, as in D). Mean values from three separate experiments per condition (with a total of at least 106 kymographs) are shown; error bars are s. e. Microtubule plus and minus ends in merged kymographs are labelled by (+) and (−). Experiments were performed at 30°C.
Figure 3(A) Example dual colour kymograph and a schematic showing how the distance of initiation of processive runs was measured. The protein concentrations were 10 nM GFP-dynein, 20 nM pig dynactin, 5 µM BicD2-N, 20 nM EB1 and 17.5 µM Atto647-tubulin.(B) Histograms of the spatial initiation probabilities of processive runs within the first 7.2 µm from growing microtubule plus ends in the absence (black bars), or presence of 10 nM EB1 (red bars). 199 and 221 initiation positions were determined for the two conditions (from three experiments each).(C) Averaged fluorescence intensity profiles of GFP-dynein at growing microtubule plus ends for the conditions as in B. Mean values from three separate experiments per condition (with a total of at least 69 kymographs) are shown; error bars are s. e. Experiments were performed at 30°C.(D) Histogram of spatial initiation probabilities of DDB on static GMPCPP-microtubules. 294 initiation positions were detemined (from three experiments). Concentrations of DDB components as in B.
Figure 4A, B) Dual and single colour kymographs showing under certain conditions p150 or dynactin-dependent microtubule plus end tracking of dynein in the absence of EB proteins: (A) 10 nM GFP-dynein (green) tracking the growing end of a Atto565-microtubule (magenta) in the presence of pig dynactin at an elevated concentration of 80 nM. Experiments in (A) and (B) were performed in standard assay buffer (BRB20 supplemented with 50 mM KCl, for details see Methods).(A, B) Dual and single colour kymographs showing under certain conditions p150 or dynactin-dependent microtubule plus end tracking of dynein in the absence of EB proteins: (B) 10 nM GFP-dynein (green) tracking the plus end of an Atto565-microtubule (blue) in the presence of 10 nM Alexa647-p150 (a fragment containing the first 517 amino acids of p150; called p150-N in the text) (red). Experiments in (A) and (B) were performed in standard assay buffer (BRB20 supplemented with 50 mM KCl, for details see Methods).(C, D) Dual and single colour kymographs showing that microtubule binding behaviour of p150 depends strongly on ionic strength: (C) In standard assay buffer, 10 nM Alexa647-p150 (green) accumulates at the plus end and on the GMPCPP-stabilised segment of a growing Atto565-microtubule (magenta).(C, D) Dual and single colour kymographs showing that microtubule binding behaviour of p150 depends strongly on ionic strength: (D) At higher ionic strength (BRB80 supplemented with 60 mM KCl, see Methods), Alexa647-p150 (green) binds only weakly to the microtubule without a detectable plus end preference. Protein concentrations as in C. The Atto565-tubulin concentration was always 17.5 µM tubulin. The temperature was 30°C.
Figure 5(A-C) Kymographs showing GFP-dynein (green) on dynamic Atto647N-microtubules (magenta) in the presence of all the regulators at different Lis1 concentrations. Protein concentrations were 10 nM GFP-dynein, 20 nM pig dynactin, 20 nM EB1, 5 µM BicD2-N, and (A) either no Lis1, (B) 1 μM mCherry-Lis1, or (C) 5 μM mCherry-Lis1. Together with BicD2, Lis1 increases the number of processive dynein runs and high concentrations of Lis1 restore plus-end localisation of dynein in the presence of BicD2-N. Microtubule orientation as indicated.(D) Averaged intensity profiles of GFP-dynein at growing microtubule plus ends in the presence of EB1, dynactin and 5 µM BicD2-N (black squares, as in A), in the presence of 1 µM mCherry-Lis1 (blue circles, as in B) and 5 µM mCherry-Lis1 (red triangles, as in C). Mean values from three separate experiments per condition (with a total of at least 49 kymographs) are shown; error bars are s. e.(E) The average number of processive DDB runs per µm microtubule length for the three different Lis1 conditions (as in A, B and, C). Error bars are s. d. *p = 0.001, **p = 0.003, ***p = 0.001 (unpaired t-test). 218, 969, and 646 processive DDB events were analysed for the three conditions, respectively (from two experiments each).(F) Bar graphs showing the mean velocity and the mean run length of processive DDB events for the three Lis1 conditions (as in A, B and C). Error bars are the s.e.m. Mean velocities were 0.37 ± 0.02 µm/s (A), 0.38 ± 0.03 µm/s (B), and 0.35 ± 0.03 µm/s (C). Mean run lengths were 3.1 ± 0.4 µm (A), 3.2 ± 0.5 µm (B), and 2.9 ± 0.3 µm (C). For each condition over 300 complexes were analysed from three different data sets.(G) Histogram of spatial initiation probabilities of DDB runs in the presence of EB1 and Lis1. 381 initiation positions were determined (from two experiments). Experiments were performed at 30°C.
Figure 2(C) ChIP-qPCRs at ARAP1, RFX5-GBAT2, WDR74 and HAUS5 promoters for IgG and DVL3 in MDA-MB-468 (NTC, shA DVL3, and shB DVL3).(D) ChIP-qPCRs at RFX5-GBAT2, COX14, HLA-E and PSMB8 promoters for IgG and DVL3 in MCF7 (NTC, shA DVL3, and shB DVL3).(E) RT-qPCR-based analysis of expression changes of RFX5, GBAT2, WDR74, HCG15, POU2F3, SNORD3B, APOC1P1, HLA-A, TAP1, PSMD8, PDG1 and JUNB genes in MDA-MB-468 (NTC, shA DVL3, and shB DVL3) cells. (F) RT-qPCR-based analysis of expression changes of RFX5, GBAT2, PCAT6, ZNF672, SSC5D, APOC1P1, HLA-A, HLA-F and TAP1 genes in MCF7 (NTC, shA DVL3, and shB DVL3) cells. D
Figure 4(C) ChIP-qPCRs at ARAP1, RFX5-GBAT2, KDM5B, APOC1P1, and PSMB8 promoters for IgG and H3K4me3 in MDA-MB-468 (NTC, shA DVL3, and shB DVL3).(D) ChIP-qPCRs at RFX5-GBAT2, PCAT6, APOC1P1 and HAUS5 promoters for IgG and H3K4me3 in MCF7 (NTC, shA DVL3, and shB DVL3).