EMBO/soda-nel huggingface dataset:
Tyrosine kinase inhibition increases functional parkin-Beclin-1 interaction and enhances amyloid clearance and cognitive performance. (2013)
Figure 4

Bosutinib degrades lentiviral Aβ1–42 in a parkin-dependent manner

Staining of 20μm brain sections shows intracellular Aβ1–42 within the (A) hippocampus of lentiviral Aβ1–42 injected WT mice, and (B) Bosutinib clearance of intracellular Aβ1–42. Staining of 20μm brain sections shows intracellular Aβ1–42 within the (C) cortex of WT mice with the lentiviral Aβ1–42, and (D) Bosutinib clearance of intracellular Aβ1–42 (N = 7).

ELISA of human soluble and insoluble Aβ1–42 at 6 weeks post-injection of lentiviral Aβ1–42 and daily treatment with 5 mg/kg Bosutinib (N = 9) for 3 weeks. * Significantly different to control or as indicated, Mean ± SEM, ANOVA with Neumann Keuls multiple comparison.

WB on 4–12% NuPAGE SDS gel of total brain lysates in 1 year old wild type and parkin−/− mice treated with 5 mg/kg Bosutinib for 3 weeks on 4–12% NuPAGE SDS gel (Invitrogen) showing parkin (1st blot)total Abl (2nd blot), T412 Abl (3rd blot), Beclin-1 (4th blot) and LC3 (5th blot) relative to actin (N = 7).

Staining of 20μm brain sections shows plaque formation with 6E10 antibody and DAB 6 weeks post-injection with (G) lentiviral Aβ1–42 + 3 weeks DMSO treatment and (H) IP injection with 5 mg/kg Bosutinib 3 weeks post-lentiviral expression (3 weeks treatment) clears plaques in WT mice (N = 7).(I) Lentiviral Aβ1–42 + DMSO and (J) IP injection with 5 mg/kg Bosutinib 3 weeks post-lentiviral expression (3 weeks treatment) in parkin−/− mice (N = 7).
Tyrosine kinase inhibition increases functional parkin-Beclin-1 interaction and enhances amyloid clearance and cognitive performance. (2013)
Figure 5

Subcellular fractionation suggests impaired autophagy in the absence of parkin*Significantly different to control or as indicated, Mean ± SEM, ANOVA with Neumann Keuls multiple comparison.

Graphs represent ELISA (N = 5) inautophagic vacuoles (AVs) in the brain of 4 and 8 months old Tg-APP mice treated with 10 mg/kg Nilotinib or 5 mg/kg Bosutinib (N = 5) for 3 weeks showing (A)human Aβ1–42 (Insert is WB analysis of AVs showing LC3-B in AV10 and AV20 (1st blot) and LAMP2a in Lys (2nd blot) fraction) and (B)Aβ1-40,(C) p-Tau and (D) parkin.

ELISA in autophagic vacuoles of 1 year old WT and parkin−/− mice (N = 5) injected with lentiviralAβ1–42 for 3 weeks and treated with 10 mg/kg Nilotinib and 5 mg/kg Bosutinib for 3 additional weeks showing (E) human Aβ1–42 and (F) p-Tau levels.
Tyrosine kinase inhibition increases functional parkin-Beclin-1 interaction and enhances amyloid clearance and cognitive performance. (2013)
Figure 6

Bosutinib improves cognitive performance in a parkin-dependent manner*Significantly different to DMSO or as indicated, Mean ± SEM, ANOVA with Neumann Keuls multiple comparison.

(A) Represents results of Morris water maze test in lentiviralAβ1–42-injected ± Bosutinib WT (N = 12) and parkin−/− (N = 10) mice, and (B) heat maps for each group.

Graphs represent total number of entry into platform area and distance travelled.

Represents results of Morris water maze test in Tg-APP ± Bosutinib (N = 12) mice.

Graphs represent total number of entry into platform area and distance travelled.
The plant-specific CDKB1-CYCB1 complex mediates homologous recombination repair in Arabidopsis (2016)
Figure 2

Figure 2 – Mutants of cdkb1 and cycb1 show increased number of DSBs and delayed DNA repair upon cisplatin treatment

A. Representative examples of comets of 21 days old wild-type plants, cdkb1;1 cdkb1;2 and cycb1;1 cycb1;3 double mutant seedlings in full spectrum view of the TriTek Comet Score software. Shown are comets of plants incubated with 50µM cisplatin for one hour and then transferred to medium without cisplatin for 30 min (recovery) and plants incubated without cisplatin for one hour (control), respectively.

B. Box plot of percentage of tail DNA of wild-type cells, cdkb1;1 cdkb1;2 and cycb1;1 cycb1;3 under cisplatin treatment. Plots are based on analyses of 200 cells per sample from random microscopic fields of three independent biological replications. The percentage of DNA fragments in the comet tail was calculated by the TriTek Comet Score software. The box represents the interquartile range, the line across the box indicates the median values and whiskers represent 5-95 percentile values. Brackets connect plots of sample groups that are significantly different with a confidence level higher than 99.99% calculated with Student‘s t-test.

C. Immunostaining of γ-H2AX foci in wild-type plants and mutant cells after two hours of treatment with 50µM cisplatin.

D. Counted number of γ-H2AX foci per cell detected after two hours of treatment with 50µM cisplatin in wild-type and mutant plants. For each sample the γ-H2AX foci of 100 cells were counted and grouped into six categories: cells with no, 1-2, 3-5, 6-10, 11-20 and more than 20 foci per cell.
The plant-specific CDKB1-CYCB1 complex mediates homologous recombination repair in Arabidopsis (2016)
Figure 3

Figure 3 – Homologous recombination frequencies is strongly reduced in cycb1 mutants

A. Schematic drawing of homologous recombination assay. Restoring the functional GUS gene from two disrupted parts (GU’ and ‘US) is restricted to an intermolecular homologous recombination event. Homologous events occur only when a sister chromatid or homolog is available as a template, i.e. in G2 phase of the cell cycle.

B. Wild-type plants show blue spots on the leaves after three days of incubation on 30 μM cisplatin. Arrows indicate representative blue sectors.

C. cycb1;1 plants show blue spots on the leaves after three days of incubation on 30 μM cisplatin. Arrows indicate representative blue sectors.

D. Graphs show numbers of blue sectors per plant grown without drug treatment.

E. Graphs show numbers of blue sectors per plant grown after incubation on 15 μM cisplatin for three days.

F. Graphs show numbers of blue sectors per plant grown after incubation on 30 μM cisplatin for three days.

One or two asterisks indicate significant differences within a 5% and 1% confidence interval, respectively (Student’s t-test).
The plant-specific CDKB1-CYCB1 complex mediates homologous recombination repair in Arabidopsis (2016)
Figure 4

Figure 4 – Mutants of cdkb1 but not cdka;1 are hypersensitive to cisplatin

A. The wildtype and CDKA;1-DE mutants were grown on control plates for 10 days. Root lengths were measured 10 days after germination.

B. The wildtype and CDKA;1-DE mutants were grown on plates containing 1 mM hydroxy urea for 10 days. The mutant wee1 was used as a positive control for hydroxy urea sensitivity. Root lengths were measured 10 days after germination.

C. The wildtype and CDKA;1-DE mutants were grown on plates containing 0,6 μg/mL bleomycin for 10 days. The mutant ku70 was used as a positive control for bleomycin sensitivity. Root lengths were measured 10 days after germination.

D. The wildtype, cdkb1;1, cdkb1;2 and the double mutant cdkb1;1 cdkb1;2 were grown on control plates for 10 days. Root lengths were measured 10 days after germination.

E. The wildtype, cdkb1;1, cdkb1;2 and the double mutant cdkb1;1 cdkb1;2 were grown on plates containing 1 mM hydroxy urea for 10 days The mutant wee1 was used as a positive control for hydroxy urea sensitivity. Root lengths were measured 10 days after germination.

F. The wildtype, cdkb1;1, cdkb1;2 and the double mutant cdkb1;1 cdkb1;2 were grown on plates containing 0,6 μg/mL bleomycin for 10 days. The mutant ku70 was used as a positive control for bleomycin sensitivity. Root lengths were measured 10 days after germination.

G. The wildtype, CDKA;1-DE and the double mutant cdkb1;1 cdkb1;2 were grown on control plates and were transferred to plates containing 15 μM or 30 μM cisplatin three days after germination. Root lengths were measured three days after transfer and the net root growth of three days is shown in the graphs.

H. Graphs represent the ratio of the mean growth rate on 15 μM cisplatin compared to control experiments on plates lacking cisplatin for the wildtype, CDKA;1-DE and cdkb1;1 cdkb1;2.

I. Graphs represent the ratio of the mean growth rate on 30 μM cisplatin compared to control experiments on plates lacking cisplatin for the wildtype, CDKA;1-DE and cdkb1;1 cdkb1;2.

J-N. Images show a wild-type plant, CDKA;1-DE and the double mutant cdkb1;1 cdkb1;2 (from left to right) on the indicated day after germination and the indicated drug treatment.

One or two asterisks indicate significant differences within a 5% and 1% confidence interval, respectively (Student’s t-test). Scale bars: 1 cm.
The plant-specific CDKB1-CYCB1 complex mediates homologous recombination repair in Arabidopsis (2016)
Figure 5

Figure 5 – The CDKB1;1-CYCB1;1 complex phosphorylates RAD51

A. WT and rad51 mutants were grown on control plates or transferred to plates supplemented with 15 μM or 30 μM cisplatin, respectively, three days after germination. Root lengths were measured three days after transfer and the net root growth of three days is shown in the graph. Asterisk indicates significant differences within a 5% confidence interval (Student’s t-test).

B. Image shows a wild-type plant (left) and rad51 mutant (right) grown on control plates. Images were taken 6 days after germination.

C. Image shows a wild-type plant (left) and rad51 mutant (right) germinated on control plates and transferred to plates supplemented with 15 μM three days after germination. Images were taken 6 days after germination, i.e. 3 days after transfer to cisplatin.

D. Immunostaining of RAD51 foci in the wildtype and indicated mutant cells after two hours of treatment with 50µM cisplatin.

E. Counted number of RAD51 foci per cell detected after two hours of treatment with 50µM cisplatin in wild-type and mutant plants. For each sample the RAD51 foci of 100 cells were counted and grouped into six categories: cells with 0, 1, 2, 3, 4 or >5 foci per cell.

F. In vitro kinase assay of purified CDK complexes phosphorylating RAD51. RAD51 and histone H1 kinase assays were performed with [γ-32P]ATP as a phosphate donor. Proteins were subjected to SDS-PAGE after the kinase reaction and stained with coomassie brilliant blue demonstrating the equal loading of the substrates (lower panels). Phosphorylated proteins were detected by autoradiograph (upper panels). The reactions were normalized by using equal amounts of CDKs assuring equal levels of active CDK-cyclin complexes, protein blots indicate the relative amounts of the CDKs in the reaction (bottom panel).

Abbreviation: p-RAD51 and p-Histone H1 for [32P]-phosphorylated MBP-RAD51-His6 and recombinant human histone H1, respectively, resulting from kinase assays with radio-labeled ATP. Asterisks indicate varying amounts of cyclins that can be in the reaction due to purification procedure. 1: without kinase, 2: CDKA;1-CYCA2;3, 3: CDKB1;1-CYCA2;3, 4: CDKB1;1-CYCD2;1, 5: CDKA;1-CYCB1;1, 6: CDKB1;1-CYCB1;1.

Scale bar: 1 cm.
The plant-specific CDKB1-CYCB1 complex mediates homologous recombination repair in Arabidopsis (2016)
Figure 6

Figure 6 – CYCB1;1 is upregulated after cisplatin treatment

A. Transgenic Arabidopsis plants harboring the CYCB1;1 promoter and a GFP fused to the N-terminal part of CYCB1;1 including the destruction box grown on control plates and imaged five days after germination.

B. Transgenic Arabidopsis plants harboring the CYCB1;2 promoter and a GFP fused to the N-terminal part of CYCB1;2 including the destruction box grown on control plates and imaged five days after germination.

C. Transgenic Arabidopsis plants harboring CDKB1;1 promoter fused to GUS grown on control plates, were stained and imaged five days after germination.

D. Transgenic Arabidopsis plants harboring the CYCB1;1 promoter and a GFP fused to the N-terminal part of CYCB1;1 including the destruction box germinated on control plates and were transferred four days after germination to plates supplemented with 50 μM cisplatin and were imaged 24h after drug application.

E. Transgenic Arabidopsis plants harboring the CYCB1;2 promoter and a GFP fused to the N-terminal part of CYCB1;2 including the destruction box germinated on control plates and were transferred four days after germination to plates supplemented with 50 μM cisplatin and were imaged 24h after drug application.

F. Transgenic Arabidopsis plants harboring CDKB1;1 promoter fused to GUS germinated on control plates and were transferred four days after germination on plates supplemented with 50 μM cisplatin and were stained and imaged 24h after drug application.

G. Chromatin immunoprecipitation (ChIP) of wild-type plants and PROSOG1:SOG1-Myc lines and anti-MYC antibody. The promoter region of CYCB1;1 is enriched in SOG1-MYC after cisplatin treatment.

H. Structure of genes tested by ChIP with an antibody anti-MYC in PROSOG1:SOG1-Myc and wild-type plants. Red arrowheads indicate the primer binding sites for PCR. A total of four regions were tested as indicated by arrowheads. Asterisks indicates significant differences within a 5% confidence interval (Student’s t-test).

Scale bars: 20 μm.
The plant-specific CDKB1-CYCB1 complex mediates homologous recombination repair in Arabidopsis (2016)
Figure 7

Figure 7 – Mutants of cycb1 ku70 are hypersensitive to BLM: Genetic interaction between NHEJ und HR

A. The wildtype, ku70, the double mutants cycb1;1 ku70, cycb1;2 ku70 and cycb1;1 cycb1;2 and the triple mutant cycb1;1 cycb1;2 ku70 were grown on control plates and root lengths were measured 10 days after germination.

B. The wildtype, ku70, the double mutants cycb1;1 ku70, cycb1;2 ku70 and cycb1;1 cycb1;2 and the triple mutant cycb1;1 cycb1;2 ku70 were grown on plates containing 0,6 μg/mL BLM. Root lengths were measured 10 days after germination.

C. Graph represents the ratio of the mean growth rate on 0,6 µg/mL BLM compared to control experiments on plates lacking BLM for the wildtype, ku70, cycb1;1 ku70, cycb1;2 ku70 and cycb1;1 cycb1;2 and the triple mutant cycb1;1 cycb1;2 ku70.

D. Image shows the wildtype, ku70, the double mutants cycb1;1 ku70, cycb1;2 ku70 and cycb1;1 cycb1;2 and the triple mutant cycb1;1 cycb1;2 ku70 (from left to right) grown on control plates 10 days after germination.

E. Images show the wildtype, ku70, the double mutants cycb1;1 ku70, cycb1;2 ku70 and cycb1;1 cycb1;2 and the triple mutant cycb1;1 cycb1;2 ku70 (from left to right) on plates containing 0,6 μg/mL BLM.

One asterisk indicates significant differences within a 5% confidence interval between the wildtype and the mutants (Student’s t-test). Two asterisks represent significant differences within a 5% confidence interval between the wildtype and the triple mutant cycb1;1 cycb1;2 ku70 as well as a significant reduction of root growth between ku70 and the triple mutant cycb1;1 cycb1;2 ku70. Scale bars: 1 cm.
Mechanical forces impair antigen discrimination by reducing differences in T cell receptor/peptide-MHC off-rates (2022)
Figure 4

Figure 4. The ability of adhesion receptors to enhance T cell antigen discrimination and sensitivity can be explained by them shielding the TCR/pMHC interaction from mechanical force.

(A) Schematic of the kinetic proofreading model modified to allow molecular forces to impact the membrane off-rate (kmoff).

(B) The concentration of productively signalling TCRs (CN) over the antigen concentration for antigens with the indicated zero-force off-rates (koff). Shown is the effect of forces at 100 pN (left) and 10 pN (right). The horizontal dashed line is the threshold value of CN required for activation in the model (λ). The vertical dashed line shows the antigen concentration (P) required to elicit activation by the antigen with the largest value of koff.

(C) The antigen concentration required to elicit activation (P) over koff for the indicated molecular forces on the TCR/pMHC interaction.

(D) Antigen sensitivity quantified as the value of P from (C) over the applied force for a higher-affinity antigen with koff = 0.5 s-1.

(E) Antigen discrimination quantified by the discrimination power (α) over the applied force. Discrimination power is defined as the ratio of the logarithm of the fold-change in P over the fold-change in koff for a higher-affinity ligand (koff = 0.5 s-1) and a lower-affinity ligand (koff = 101.5 s-1) from panel (C).

(F) Experimental value of P (symbols) over antigen affinity fitted by the kinetic proofreading model with force (dashed and solid lines). The value of P is defined as the concentration of pMHC required for 15% upregulation of CD69 above baseline and is shown as the mean with SEM from 4 independent experiments. All experimental data is taken from Fig. 4 in (4).

(G) The fitted value of the applied force with SEM.

(H) The fitted value of λ with SEM.

(I) Proposed mechanisms by which adhesion receptors can reduce pulling forces on TCR/pMHC interactions (black arrow). Molecular forces on individual TCR/pMHC interactions can be produced by multiple mechanisms, including a mismatch in size between the short TCR/pMHC interaction (~14 nm) and larger surface molecules, such as CD43 and CD45 that extend 21-45 nm (left panel). These forces may be reduced by force-sharing with the CD2/CD58 adhesion molecules that span the same distance and can therefore closely colocalise (middle panel). Alternatively, the larger LFA-1/ICAM-1 adhesion interaction may reduce forces by acting as a diffusional barriers to maintain CD43 and CD45 further away from TCR/pMHC interactions, and tether and stabilize cellular processes such as microvilli, thereby reducing the forces imposed on all interactions within an area of close contact surrounded by LFA-1/ICAM interactions (right panel)

Data information: An F-test is used to produce a p-value for the null hypothesis that the applied force or λ is the same across the three conditions. All calculations (B-E) and data fitting (F-H) used N = 2.46 and kp = 2.15 s−1 (average of CD58 and ICAM-1 conditions) taken from Fig. 5 in (4).

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MIWI2 targets RNA Transcribed from piRNA-dependent Regions to drive DNA Methylation in Mouse Prospermatogonia (2018)
Figure 2

Figure 2. Hypomethylated regions show features of active promoters.

A. Chromatin marks associated with promoters are observed in hypomethylated regions without young LINE1 retrotransposons (HMR_-L1s). Metaplots show the average ChIP-seq signals across HMR_-L1s, transcription start sites (TSSs), and randomly selected sites. The signals were normalized by either H3 or input signal. The results shown in heat maps were not normalized by H3 or input.

B. The initiation form of Pol2 with an unphosphorylated carboxy terminal domain (Pol2 null-P CTD) is highly enriched in HMR_-L1s.
MIWI2 targets RNA Transcribed from piRNA-dependent Regions to drive DNA Methylation in Mouse Prospermatogonia (2018)
Figure 3

Figure 3. MIWI2 and piRNAs target RNAs from hypomethylated regions.

A. RNA-immunoprecipitation (RIP) analyses of EGFP-MIWI2 and EGFP. EGFP-MIWI2 and EGFP were immunoprecipitated from 16.5 dpc prospermatogonia, and long RNAs bound to these proteins and input RNAs were sequenced. RNAs expressed from HMR_-L1s and piRNA clusters, but not Ensembl and prospermatogonium-expressed mRNAs, are overall enriched by immunoprecipitation (IP) of EGFP-MIWI2 (left). This enrichment is not observed when EGFP is immunoprecipitated (right). The numbers of mRNAs, piRNA clusters, and HMR_-L1s analyzed were shown in parentheses. Prospermatogonium-expressed mRNAs are those that are highly and specifically expressed in prospermatogonia. The bottom and top of boxes represent 25% percentile and 75% percentile, respectively. The line in the box represents the median. Circles represent outliers, and the bottom (top) of whiskers represents the minimum (maximum) values except for outliers.

B. RIP analyses of EGFP-MIWI2 in newborn testes from WT and Mili KO mice. RNA bound to EGFP-MIWI2 and input RNA were sequenced. All RNAs derived from each class was counted for this analysis.

C. HMR_-L1s preferentially generate piRNAs. X- and Y-axes represent expression levels of long RNAs (16.5 dpc prospermatogonia) as determined by RNA-seq and MIWI2-bound piRNAs (18.5 dpc testes) as determined by small RNA-seq, respectively. Circles in the charts represent individual mRNAs, piRNA clusters and HMR_-L1s. More than half of the HMR_-L1s (right) are located in the area of y > x (upper part of the diagonal), whereas most mRNAs (left) are located in the area of y < x (lower part of the diagonal). RPKM, reads per kilobase per million mapped reads.

D. Frequent 10-nt overlap between sense and antisense piRNAs derived from HMR_-L1s. Top 100 HMR_-L1s that generate the largest number of unique piRNAs were analyzed. Frequencies of 1−18-nt overlaps were analyzed for each HMR_-L1.

E. piRNAs derived from HMR_-L1s are enriched in adenine at their 10th nucleotide. HMR_-L1s generating more than 40 unique piRNAs were analyzed (678 HMR_-L1s). The proportions of piRNAs with adenine (left) and uridine (right) at the indicated positions were analyzed for each HMR_-L1. See A for the explanation of box plots.
MIWI2 targets RNA Transcribed from piRNA-dependent Regions to drive DNA Methylation in Mouse Prospermatogonia (2018)
Figure 4

Figure 4. RMER4B sequence in the Rasgrf1 DMR is essential for de novo DNA methylation of the Rasgrf1 DMR.

A. DNA methylation status of a portion of the Rasgrf1 differentially methylated region (DMR) in 0−2-dpp Mili+/− and Mili−/− prospermatogonia. Regions corresponding to B, C, D, E, and F are demethylated in Mili−/− prospermatogonia. Vertical bars represent DNA methylation levels at CpG sites determined by whole genome bisulfite sequencing (see below). The top dark gray bar represents a portion of the Rasgrf1 gametic DMR (Tomizawa et al, 2011). The CpG cluster is shown. The deleted RMER4B sequence is indicated.

B. Deletion of key sequences (circles labeled as −) in the Rasgrf1 DMR and chr7 piRNA cluster. Shown is our proposed mechanism in which piRNAs generated from RMER4B sequence in chr7 piRNA cluster target RMER4B sequence in pit-RNA for the establishment of DNA methylation of the Rasgrf1 DMR.

C. DNA methylation status of the Rasgrf1 DMR in Rasgrf1ΔRMER4B/ΔRMER4B and control spermatogonia are shown. The regions examined are shown in (A). Black (white) circles represent methylated (unmethylated) CpGs.

D. Rasgrf1 RMER4B functions in cis. Wild-type and mutant alleles of the Rasgrf1 DMR in Rasgrf1+/ΔRMER4B spermatogonia were analyzed separately.
MIWI2 targets RNA Transcribed from piRNA-dependent Regions to drive DNA Methylation in Mouse Prospermatogonia (2018)
Figure 5

Figure 5. chr7 piRNA cluster directs de novo DNA methylation of the Rasgrf1 DMR.

A. Structure of the chr7 piRNA cluster. Top gray bars represent a putative piRNA precursor predicted by H3K4me3 ChIP-seq, RNA-seq, and small RNA-seq. Only uniquely mapped reads were used for displaying the sequencing result. For RNA-seq and small RNA-seq, only the results of minus strand are shown, because plus strand does not express appreciable level of RNA. A promoter region deleted is shown above the putative piRNA precursor. RPM, Read Per Million.

B. Low expression level of piRNAs from chr7 cluster in chr7 piRNA clusterΔpromoter/Δpromoter testes. Unchanged expression of piRNAs from chr10 cluster (bottom) shows chr7 cluster is specifically affected. Only uniquely mapped piRNAs are shown.

C. DNA methylation status of the Rasgrf1 differentially methylated region (DMR) in spermatogonia from chr7 piRNA clusterΔpromoter/Δpromoter. See Figures 4A and B for information about the region examined.