EMBO/soda-nel huggingface dataset:
Inhibition of CPAP-tubulin interaction prevents proliferation 
of centrosome amplified cancer cells (2018)
Figure 6

Figure 6: CCB02 enhances microtubule nucleation activities of centrosomes

(A) Experimental scheme of microtubule (MT) regrowth assay.

(B-D) MT-regrowth assays at 0, 1.5 and 3.0 min using MCF10A (-Dox, two centrosomes), MCF10A (+Dox, extra centrosomes) and MDA-MB-231 cells. MT nucleation panel is shown in grey scale (inset images are inverted) and γ-tubulin is shown in red. Note that in contrast to vehicle treatment, CCB02 treatment caused centrosomes to nucleate an enhanced level of microtubules already at 1.5 min after induction of regrowth with simultaneous increase in γ-tubulin recruitment. All these cells were stained with γ-tubulin (red), microtubules (α-tubulin, grey) and DNA (DAPI blue). Scale bar, 2µm and insets, 0.5 µm. Bar graphs show MT and γ-tubulin intensities at 3 min after induction of MT regrowth. (N)=3. At least 80 centrosomes were considered to calculate intensities from each cell line. Error bars, mean ± SEM. Unpaired t-test **P < 0.001, ***P < 0.0001.
CDC7 kinase promotes MRE11 fork processing, modulating fork speed and chromosomal breakage (2020)
Figure 1

Fig 1 CDC7 inhibition suppresses Histone H2AX and RPA2 phosphorylation and DNA double strand breaks formation upon fork stalling.

A. U2OS cells were either mock or treated with 10 μM XL413 for 30 minutes, at which point 4 mM HU was added and cells further incubated for the indicated times. Whole cell extracts were then analysed by western blotting with the indicated antibodies. Reduction of phosphorylation of Ser40/41 on MCM2 is indicative of CDC7 inhibition by XL413. Representative of two independent experiments.

B. U2OS cells were either mock treated or treated with 10 μM XL413, 4 mM HU or both for 24 hours before performing neutral comet assays. Representative images of comets are shown. Scale bar = 100 μM. In the dot plots ~800 comets per each condition were analysed, means are indicated with a red line and their value shown above the plot. Data are from two independent experiments.

C. U2OS cells were either untreated or treated with 10 μM XL413 or with 5 µM AZD6738 alone or in combination and, where indicated, after 30 minutes 4 mM HU was added for a further 2 hours. Whole cell extracts were analysed by western blotting with the indicated antibodies. Total protein stain (TPS) as a loading control. Representative of two independent experiments.

D. U2OS cells were either untreated or treated with 4 mM HU for 5 hours in the presence or absence of 10 μM XL413 which had either been added to cells 30 minutes prior (pre-treatment) or at the same time (co-treatment) to the addition of HU. Whole cell extracts were analysed by western blotting with the indicated antibodies and total protein stain (TPS) is displayed as a loading control. Representative of two independent experiments.
CDC7 kinase promotes MRE11 fork processing, modulating fork speed and chromosomal breakage (2020)
Figure 2

Figure 2: CDC7 inhibition suppresses Histone H2AX phosphorylation independently from origin firing

A. Outline of experimental procedure. U2OS cells were either untreated, treated with 10 μM XL413 or with 4 mM HU in the presence or absence of XL413 for 24 hours. Cells were EdU labelled for 30 minutes before harvesting. Cells that had been treated with both XL413 and HU for 24 hours, were washed with equilibrated media and retreated for a further 2 hours. All onward treatments included 4 mM HU, to prevent further DNA synthesis either in the absence or presence of CDC7 (XL413), MRE11 (Mirin) or ATR (AZD6738) inhibitors.

B. CSK soluble and chromatin-enriched protein fractions were analysed by western blotting with the indicated antibodies. Representative of two independent experiments.

C-D. Flow cytometry analysis to assess levels of pS139 Histone H2AX (c). Histograms show the mono-parametric analysis of cell count against pS139 Histone H2AX intensity. Histograms are overlaid to appreciate changes in pS139 Histone H2AX intensity upon treatment (Red lines) relative to appropriate experimental baseline controls (Grey lines). Data are representative of two independent experiments.

E. Graphical concept: cells treated with HU and XL413 for 24 hours would have stalled forks, which exist in a stable state due to inhibition of CDC7 and with an active ATR-dependent origin firing checkpoint. Upon washing and retreating of cells in the presence of HU the late origin checkpoint is maintained, in an ATR-dependent manner, and accumulation of DNA damage (pS139 H2AX) can be monitored independently from origin firing in the absence or presence of CDC7 (XL413) or MRE11 (Mirin) inhibitors. AZD6738 was used as a control to investigate molecular events occurring upon loss of checkpoint caused by both fork collapse and loss of the inhibition of origin firing. The increase in chromatin binding of CDC45 was used as a surrogate marker for origin activation, pS139 Histone H2AX was used to monitor fork stability, pS345 CHK1 was used to monitor the ATR-checkpoint signalling and pS40/41 MCM2 was used to monitor CDC7 kinase activity.
CDC7 kinase promotes MRE11 fork processing, modulating fork speed and chromosomal breakage (2020)
Figure 3

Figure 3: CDC7 is a replisome associated protein and its inhibition affects the localization of MRE11 at replication factories.

A. U2OS cells were labelled with EdU for 30 minutes, then EdU was washed and cells were further incubated for either 1 or 2 hours in presence of thymidine. At the indicated time points cells were fixed and proteins binding to EdU-labelled DNA captured by DNA mediated chromatin Pull down technique (DmChP). Graphical experimental outline is shown above the analysis by western blot of relevant proteins in both input and captured material.

B. U2OS cells were labelled with EdU for 30 minutes and then treated with 4 mM HU for 2 hours in the presence or absence of 10 μM XL413. Proteins binding to EdU-labelled DNA captured by DmChP were then analysed by western blot as above.

C. As in panel B but incubation with HU was extended to 24 hours. Black arrow indicates MRE11 electrophoretic-mobility shift.

D. U2OS cells were either mock or treated with 10 μM XL413 for 30 minutes, at which point 4 mM HU was added and cells incubated for further 24 hours. Extracts prepared from HU treated cells were then incubated in presence or absence of λ-phosphatase. Proteins were analysed by western blotting with anti-MRE11 antibodies. Total protein stain (TPS) is used as a loading control. Black arrow indicates MRE11 electrophoretic-mobility shift.

E. U2OS cells were treated with 4 mM HU in the presence or absence of 10 μM XL413 for 24 hours. PCNA (green) and MRE11 (red) were detected by immunofluorescence. Inset I-II represent enlargements of selected region of the merged images.

F. Quantification of PCNA and MRE11 colocalization was assessed with ImageJ in ~70 randomly selected cells for each condition from four biological replicates and expressed as Pearson's correlation coefficient. Error bars represent SEM. Statistical significance was assessed by student t test (*p˂0.05).
CDC7 kinase promotes MRE11 fork processing, modulating fork speed and chromosomal breakage (2020)
Figure 5

Figure 5: CDC7 promotes the nucleolytic degradation of reversed forks in BRCA2 deficient cells.

A. U2OS cells were transfected with control (siLuc) or BRCA2 targeting siRNA (siBRCA2). After 48 hours cells were either mock treated or treated with 10 μM XL413 for 30 minutes and labelled consecutively with CldU (red) and IdU (green), followed by treatment with 4 mM HU for further 5 hours.

B. Representative western blot of indicated proteins obtained from whole cell extracts. pS40/41 on MCM2 indicates effective CDC7 inhibition by XL413.

C. Ratios of IdU/CldU tracts length is plotted. The box extends from the 25th to the 75th percentile with the line in the box representing the median. Whiskers indicate the 10-90 percentiles with data outside this range for individual outliers being plotted as dots. Mean values of IdU/CldU ratios are indicated above the plot. At least 100 replication forks were analyzed for each condition.

D. The mean values of IdU/CldU tract length ratios from three independent experiments are plotted, with the standard deviation (blue lines) and mean (red line). Statistical analysis: student t-test; ns, not significant; **P<0.01, ***P < 0.001

E. Electron micrographs of representative replication forks from U2OS cells:

A regressed arm is labelled: P=parental strand; D=daughter strand; and R=reversed fork; the four-way junction at the reversed fork is magnified in the inset. The red arrows indicates single stranded DNA at the fork. Scale bar is 200 nm ( = 460 bp), 100 nm ( = 230 bp) in the inset.

F. Frequency of reversed replication forks isolated from mock-depleted (siLuc) and BRCA2-depleted (siBRCA2) U2OS cells upon 5 hour treatment with 4 mM HU in the presence and absence of 10 μM XL413. The number of replication intermediates analyzed is indicated in parentheses. Similar results were obtained in two independent experiments (Appendix Table S1).

G. Amount of ssDNA length at the junction (Red arrows in Figure 4D) in siLuc and siBRCA2 U2OS cells treated with 4 mM of HU for 5 hours in the presence or absence of XL413. N indicates the number of forks observed, and only molecules with detectable ssDNA stretches are included in the analysis. The lines show the mean length of ssDNA regions at the fork and the value is displayed above the plot .
CDC7 kinase promotes MRE11 fork processing, modulating fork speed and chromosomal breakage (2020)
Figure 6

Figure 6: CDC7 contributes to replication dependent chromosomal breakages.

U2OS or AS-CDC7 cells were transfected with control (siCon) or BRCA2 (siBRCA2) targeting siRNAs, and then either mock-treated or treated with 4 mM HU for 5 hours in the presence or absence of 10 μM XL413 or 3MB-PP1 respectively. HU was then washed off and Nocodozalole was added to the medium in order to complete one round of DNA synthesis. Metaphase spreads were prepared and analysed.

A. Representative metaphase spread from BRCA2 depleted U2OS cell treated with HU. Scale bar = 5 μM. Arrows indicate the chromosomes enlarged in the insets, two of which show chromatid breaks (*).

B-C. The graphs show the average number of chromatid breaks per spread. In each experiment 30 chromosome spreads for each condition were analysed and three independent experiments were performed. Error bars represent SEM. Statistical significance assessed by student t test (** p˂0.01, *** p<0.001).

D-E. U2OS and AS-CDC7 cells were transfected with control (siCon) or BRCA2 (siBRCA2) targeting siRNAs. 48 hours post transfection cells were either mock-treated or treated with 10 μM XL413 or 10 μM 3MB-PP1 respectively for 24 hours with Nocodazole added for the last 16 hours of the experiment. The graph shows the average number of chromatid breaks per spread. In each experiment 30 chromosome spreads for each condition were analysed and three independent experiments were performed. Error bars represent SEM. Statistical significance assessed by student t test (*p<0.05, *** p<0.001).

EXPANDED VIEW FUGURE LEGENDS
Novel targets for Huntington's disease in an mTOR-independent autophagy pathway (2008)
figf1

(a) Densitometric analysis relative to actin of A53T α-synuclein clearance in stable inducible PC12 cell line expressing A53T α-synuclein. Transgene expression was induced with doxycycline for 48 h, and then switched off (by removing doxycycline), with drug (all 1 μM) or DMSO (control) treatment for 24 h. Control condition is set to 100%. Error bars show s.e.m. (b) Densitometric analysis relative to actin of soluble EGFP-HDQ74 clearance in stable inducible PC12 cell line expressing EGFP-HDQ74. Transgene expression was induced with doxycycline for 8 h, and then switched off (by removing doxycycline) with drug (all 1 μM) or DMSO (control) treatment for 96 h. Control condition is set to 100%. Error bars show s.e.m. (c) SK-N-SH cells transfected with EGFP-HDQ74 construct for 4 h were treated with drugs (all 1 μM) or DMSO (control) for 48 h post-transfection. The proportions of EGFP-positive cells with aggregates or cell death were expressed as odds ratios and the control was taken as 1. Error bars show 95% confidence interval. (d) PC12 cells were treated with clonidine, minoxidil and verapamil (all 1 μM) or DMSO (control) for 24 h. Endogenous LC3-II levels were detected with anti-LC3 antibody and quantified relative to actin. Rapamycin (0.2 μM) was positive control. Error bars show s.e.m. (e) The proportions of EGFP-positive cells with EGFP-HDQ74 aggregates in wild-type (Atg5+/+) and knockout (Atg5−/−) Atg5 MEFs, transfected with EGFP-HDQ74 for 4 h and then treated for 48 h with drugs (all 1 μM) or DMSO (control). Error bars show 95% confidence interval. ***P 0.001; **P 0.01; *P 0.05; NS, nonsignificant.
Novel targets for Huntington's disease in an mTOR-independent autophagy pathway (2008)
figf2

(a) A53T α-synuclein clearance in stable PC12 cells as in Figure 1a, treated with or without 1 μM rilmenidine, 1 mM dibutyl-cAMP (db-cAMP), 24 μM forskolin or 500 μM 2′5′-dideoxyadenosine (2′5′ddA) for 24 h. Note that control clearance levels for a given substrate may vary from figure to figure, due to different exposures of blots, which aid illustration of agents that either increase or retard clearance. (b) The proportions of EGFP-positive cells with EGFP-HDQ74 aggregates in SK-N-SH (for rilmenidine) and COS-7 (for db-cAMP, forskolin and 2′5′ddA) cells as in Figure 1c, treated with concentrations used in a for 48 h post-transfection. Error bars show 95% confidence interval. (c) Endogenous LC3-II levels in PC12 cells treated with 1 μM rilmenidine or 500 μM 2′5′ddA for 24 h. (d) A53T α-synuclein clearance in stable PC12 cells as in Figure 1a, treated with or without 8-CPT-2Me-cAMP or 6-Bnz-cAMP (1 μM or 10 μM). (e) The proportions of EGFP-positive COS-7 cells with aggregates as in Figure 1c, treated with or without 10 μM 8-CPT-2Me-cAMP or 6-Bnz-cAMP for 48 h post-transfection. Error bars show 95% confidence interval. (f) COS-7 cells, transfected for 4 h with EGFP-HDQ74 along with empty vector (pcDNA3.1), dominant-negative S17N Rap2B (DN Rap2B) or wild-type PLC-ε (1:3 ratio), were assessed for the proportions of EGFP-positive cells with aggregates after 48 h. Error bars show 95% confidence interval. (g) Endogenous LC3-II levels in COS-7 cells transfected with a dominant-negative Rap2B (DN Rap2B) for 4 h were assessed after 48 h expression. (h) The proportions of EGFP-positive COS-7 cells with aggregates, transfected with EGFP-HDQ74 along with empty vector (pcDNA3.1) or dominant-negative Rap2B (1:3 ratio) for 4 h, were treated with or without 10 μM 8-CPT-2Me-cAMP for 48 h. Error bars show 95% confidence interval. (i) COS-7 cells, transfected with EGFP-HDQ74 and dsRed2 or cytosolic dsRed2-IP3 kinase A (1:3 ratio) for 4 h, were assessed at 48 h post-transfection for aggregation and cell death (as in Fig. 1c). Error bars show 95% confidence interval. ***P 0.001; **P 0.01; *P 0.05; NS, nonsignificant.
Novel targets for Huntington's disease in an mTOR-independent autophagy pathway (2008)
figf3

(a) SK-N-SH cells were pretreated with or without 10, 15 or 20 μM calpastatin for 15 min followed by addition of 1 μM (±)-Bay K8644 for 4 h. Calpain activity was detected by immunoblotting with anti-calpain small subunit antibody. Densitometry analysis is relative to actin. Error bars show s.e.m. (b) SK-N-SH cells transfected with EGFP-HDQ74 for 4 h, then pretreated with or without 10, 15 or 20 μM calpastatin for 15 min followed by addition of 1 μM (±)-Bay K8644 for 48 h post-transfection, were assessed for the proportion of EGFP-positive cells with EGFP-HDQ74 aggregates. Error bars show 95% confidence interval. (c) SK-N-SH cells were pretreated with or without 10, 15 or 20 μM calpastatin for 15 min followed by addition of 1 μM (±)-Bay K8644 for 4 h. Endogenous LC3-II levels were detected by immunoblotting with anti-LC3 antibody. (d) Comparison between calpain activity, EGFP-HDQ74 aggregation and autophagosomes as seen in a–c. The control condition for all the assessments was set at 100%. Calpastatin lowered the increased calpain activity and EGFP-HDQ74 aggregation caused by (±)-Bay K8644 in a dose-dependent manner with a simultaneous increase in LC3-II levels (autophagosomes). (e) COS-7 cells transfected with empty vector (pcDNA3.1) or wild-type PLC-ε for 4 h and treated with or without 50 μM calpeptin for 48 h post-transfection were assessed for the proportion of EGFP-positive cells with EGFP-HDQ74 aggregates. Error bars show 95% confidence interval. ***P 0.001; **P 0.01; *P 0.05; NS, nonsignificant.
Novel targets for Huntington's disease in an mTOR-independent autophagy pathway (2008)
figf4

(a) The proportions of EGFP-positive SK-N-SH cells with EGFP-HDQ74 aggregates or cell death as in Figure 1c, treated with or without 10 μM calpastatin, 50 μM N-acetyl-Leu-Leu-methional (ALLM) or 50 μM calpeptin for 48 h post-transfection. DMSO was control for ALLM and calpeptin. Error bars show 95% confidence interval. (b) HeLa cells were transfected with EGFP-HDQ74 construct along with siGLO (control siRNA) in presence or absence of calpain 1 or calpain 2 siRNA (1:3 ratio) for 96 h. EGFP- and siGLO-positive cells were assessed for aggregation and cell death as in Figure 1c. Error bars show 95% confidence interval. (c) HeLa cells, transfected with siGLO or siRNAs for calpain 1 or calpain 2 for 96 h, were analyzed with anti-calpain 1 or anti-calpain 2 domain III antibodies. (d) Clearance of soluble EGFP-HDQ74 in stable PC12 cells as in Figure 1b, treated with drugs (concentrations in a) for 96 h. Densitometry is relative to actin. Error bars show s.e.m. (e) A53T α-synuclein clearance in undifferentiated or nonmitotic neuronally differentiated (with 100 ng ml−1 nerve growth factor for 5 d) stable PC12 cells as in Figure 1a, treated with or without 10 μM calpastatin for 24 h. Densitometry is relative to actin. Error bars show s.e.m. (f) COS-7 cells, transfected with EGFP-HDQ74 along with empty vector (pcDNA3.1) or constitutive active (CA) m-calpain (1:3 ratio) for 48 h, were assessed for aggregation and cell death as in Figure 1c. Error bars show 95% confidence interval. (g) SK-N-SH cells, transfected with EGFP-HDQ74 along with empty vector (pcDNA3.1) or constitutive active m-calpain (1:3 ratio) for 4 h, were subsequently treated with DMSO (control), 1 μM verapamil or 1 μM clonidine for 48 h. The proportions of EGFP-positive cells with aggregates were assessed as in Figure 1c. Error bars show 95% confidence interval. ***P 0.001; **P 0.01; *P 0.05.
Novel targets for Huntington's disease in an mTOR-independent autophagy pathway (2008)
figf5

(a) The proportions of EGFP-positive MEFs with aggregates, transfected with EGFP-HDQ74 and pcDNA3.1 (empty vector) in Atg5+/+ cells, or transfected with EGFP-HDQ74 and pcDNA3.1, WT Atg5 or K130R Atg5 constructs in Atg5−/− cells for 4 h (1:3 ratios), were treated for next 48 h with or without 10 μM calpastatin. All data are from the same experiment, but odds ratios for reference conditions in specific experiments have been set to 1, to facilitate comparisons. Atg5−/− cells had increased EGFP-HDQ74 aggregates compared to Atg5+/+ cells. Error bars show 95% confidence interval. (b) HeLa cells were transfected with siGLO (control) with or without calpain 1 siRNA or calpain 2 siRNA (1:3 ratio) for 96 h, followed by transfection with EGFP-LC3 construct for 4 h. Cells were fixed after a further 2 h. The proportions of transfected cells with >5 EGFP-LC3 vesicles were assessed. Error bars show 95% confidence interval. (c) Endogenous LC3-II levels in SK-N-SH cells treated with or without 10 μM or 30 μM calpastatin for 24 h. (d) HeLa cells stably expressing EGFP-LC3 were treated for 4 h with 400 nM bafilomycin A1 in presence or absence of 10 μM calpastatin, 50 μM ALLM or 50 μM calpeptin. Calpain inhibitors were treated for 24 h before bafilomycin A1 addition. EGFP-LC3-II was detected with anti-EGFP antibody. Data from different calpain inhibitors are from different blots. (e) COS-7 cells, transfected with myc-LC3 and EGFP-C1 with pcDNA3.1 (empty vector) or constitutively active (CA) m-calpain (1:1:3 ratio) for 4 h, were analyzed after 24 h post-transfection for LC3-II levels by immunoblotting with anti-myc antibody. Error bars show s.e.m. (f) COS-7 cells, transfected with EGFP-LC3 and pcDNA3.1 (empty vector) or CA m-calpain (1:3 ratio) for 4 h and fixed 24 h post-transfection, were assessed for proportions of EGFP-positive cells with >5 EGFP-LC3 vesicles. Error bars show 95% confidence interval. ***P 0.001; **P 0.01; *P 0.05; NS, nonsignificant.
Novel targets for Huntington's disease in an mTOR-independent autophagy pathway (2008)
figf6

(a) A53T α-synuclein clearance in stable PC12 cells as in Figure 1a, treated with or without 100 nM PACAP, with or without 10 μM calpastatin or 500 μM 2′5′ddA for 24 h. (b) The proportion of EGFP-positive cells with aggregates in SK-N-SH cells as in Figure 1c, treated for 48 h with 1 μM PACAP or 200 μM NF449. Error bars show 95% confidence interval. (c) Aggregation in COS-7 cells transfected with either pcDNA3:1 (empty vector) or constitutively active (CA) m-calpain and EGFP-HDQ74 (3:1 ratio) for 4 h and then treated with or without 500 μM 2′5′ddA for 48 h. Error bars show 95% confidence interval. (d) HeLa cells transfected with control or Gsα siRNA for 72 h were analyzed for Gsα levels by immunoblotting with anti-Gsα antibody. Aggregation was assessed in HeLa cells transfected with control or Gsα siRNA and EGFP-HDQ74 for 72 h. Error bars show 95% confidence interval. (e) Endogenous LC3-II levels in HeLa cells transfected with control or Gsα siRNA for 72 h. (f) Endogenous LC3-II levels in PC12 cells treated with or without 200 μM NF449 for 24 h. (g) Endogenous LC3-II levels in HeLa cells transfected with control or Gsα siRNA for 72 h and treated with or without 400 nM bafilomycin A1 for the last 4 h. Densitometric analysis is relative to actin. Error bars show s.e.m. (h) The proportions of EGFP-positive HeLa cells with aggregates, transfected with control or Gsα siRNA for 48 h, and then retransfected together with pcDNA 3.1 (empty vector) or CA m-calpain and EGFP-HDQ74 (3:1 ratio) for a further 48 h. Error bars show 95% confidence interval. ***P 0.001; **P 0.01; *P 0.05; NS, nonsignificant.