EMBO/soda-nel huggingface dataset:
Bit-by-bit autophagic removal of parkin-labelled mitochondria (2013)
f1

(a) Mitochondrial tubules in HeLa cells (top, TMRE stained) exceeded the dimensions of that for autophagosomes (labelled by EGFP-LC3B) under either normal (middle, same field as TMRE image) or starvation conditions (right). (b) Diagram of light-induced mitophagy. Single mitochondrial tubules containing KR-dMito, when illuminated by 559 nm light, are selectively impaired, leading to surface recruitment of parkin and subsequent autophagic turnover. (c) Illuminating a selective region (white dotted circle, top) within a KR-dMito/EBFP2-parkin-expressing HeLa cell led to the immediate loss of KR fluorescence (top two panels). Third to fifth row: magnified view of the photo-impaired region, EBFP2-parkin translocated onto impaired mitochondrial tubules synchronously (38–60 min after 559 illumination, white arrows). Scale bars (all panels), 10 μm.
Bit-by-bit autophagic removal of parkin-labelled mitochondria (2013)
f2

(a) A HeLa cell expressing KR-dMito, EGFP-LC3B and EBFP2-parkin (cyan) were 559 nm illuminated (white dotted circle region; immediate loss of KR fluorescence). (b–d) Magnified view of the photo-inactivated region in a 34–93 min after 559 nm illumination. (b) Induction of parkin-mediated mitophagy on a single, long mitochondria tubule (white arrows). (c) LC3B-coated structures were recruited onto distinct spots on the photo-impaired mitochondrial tubule (white arrows: the location of the original impaired single mitochondrial tubule). (d) White dotted line region outlined in c, LC3 structures segregated from the main parkin-labelled cluster, carrying away bits of the parkin-labelled mitochondria with them. Two individual events are labelled in the image sequence with white and yellow arrows, respectively. Scale bars (all panels), 5 μm.
Bit-by-bit autophagic removal of parkin-labelled mitochondria (2013)
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(a) A HeLa cell expressing KR-dMito (red), EGFP-DFCP1 (green) and EBFP2-parkin (cyan) were illuminated with 559 nm at the white dotted circle region, leading to the generation of a long parkin-labelled mitochondria tubule (right, magnified view of the light-activated region) for autophagic turnover (white arrow). Scale bars, 10 μm. (b) After the generation of parkin-labelled mitochondrial tubules following treatments in a, discrete DFCP1 foci (green, middle panel, ER-associated omegasomes) formed along the parkin-labelled mitochondria (white arrows indicate DFCP1 spots that colocalized with parkin). Bottom panel: merged image of parkin and DFCP1. Scale bar, 10 μm. (c) Time-lapse imaging showing DCFP1 puncta formation on parkin-labelled mitochondria. A HeLa cell expressing KR-dMito, EGFP-DFCP1 (green) and EBFP2-parkin (pseudo-coloured red) were 559 nm illuminated, leading to the generation of parkin-labelled mitochondria tubules. Time-lapse imaging (1 frame per min) revealed that DFCP1 puncta (green) formed transiently at discrete sites on parkin-labelled mitochondrial tubules (red). White arrows mark two DFCP1 puncta (ER-associated omegasomes) formation/disassembly events directly on parkin-labelled tubules. Scale bar, 5 μm. (d) A COS-7 cell expressing EBFP2-parkin (cyan), AcGFP-sec61β (green) and TagRFP-DFCP1 (red) were treated with 10 μM CCCP for 4 h and imaged. Omegasomes (DFCP1) appeared where ER (sec61β) and impaired mitochondria (parkin) overlap. Scale bar, 10 μm. (e) Histogram of the lifetime of individual DFCP1 foci on parkin-labelled mitochondria in CCCP-treated HeLa cells expressing TagRFP-DFCP1, EGFP-LC3B and EBFP2-parkin. (f) Wortmannin treatments delayed the initial LC3 recruitment times onto parkin-labelled mitochondria in HeLa cells expressing KR-dMito, EGFP-LC3B and EBFP2-parkin. Each ‘+’ marks the times measured in individual HeLa cells (the red ‘□’ (square) denotes the average from five cells). P=0.0172 (Student’s t-test).
Bit-by-bit autophagic removal of parkin-labelled mitochondria (2013)
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(a) A COS-7 cell expressing EBFP2-Ub (red), AcGFP-sec61β (green) and mcherry-parkin (cyan) was treated with 10 μM CCCP for 1 h and imaged. Impaired mitochondria (parkin) displayed preferentially ubiquitinated foci (white arrows) at selected regions that overlap with the ER (sec61β). (b) HeLa cells expressing KR-dMito (red), EGFP-LC3 (green) and EBFP2-parkin (cyan) were illuminated with 559 nm at the white dotted square regions, generating parkin-labelled mitochondria (bottom right, magnified view of the light-activated regions). (c) Magnified view of the photo-inactivated region in b 80 min after illumination (post-stained with anti-ubiquitin antibody). LC3 and ubiquitin colocalized at discrete sites along parkin-labelled mitochondria (white arrows). (d) A MG-132 treated HeLa cell expressing KR-dMito, EBFP2-parkin (green) and EGFP-LC3B (red) was 559 nm illuminated, leading to the appearance of parkin-labelled mitochondria (white arrows one to three indicated structures, top panels). The parkin-labelled structures did not fragment nor became engulfed by LC3 structures (bottom panels) in the presence MG-132. Scale bars (all panels), 5 μm.
Abnormal TDP-43 function impairs activity-dependent BDNF secretion, synaptic plasticity and... (2018)
Figure 1

Figure 1. Tardbp knockdown induces production of a soluble form of Sortilin that impairs BDNF sorting to the regulated pathway and activity-dependent secretion in mouse hippocampal neurons

Expression of Tardbp, total Sort1 and exon 17b Sort1 mRNAs quantified by Q-PCR in primary hippocampal neurons infected with either scrambled or Tardbp shRNA lentiviruses. Values were first normalized to Gapdh mRNA levels, then plotted as average ± SEM relative to the value of scrambled shRNA (N=3 independent experiments; n=3 wells per condition in each experiment). ***, p<0.001.

Western blots of cell lysates and culture supernatants showing TDP-43 and Sortilin expression in primary hippocampal neurons infected with scrambled or Tardbp shRNA lentiviruses. Uninfected cultures were used as an additional control. β-actin immunoblotting and Coomasise Blue staining were used to control equal loading of lysates and supernatants, respectively. Molecular weights are indicated in kDa.

Representative photomicrographs of cultured hippocampal neurons infected with scrambled or Tardbp shRNA lentiviruses, immunolabled for BDNF (green), SCG2 (red) and counterstained with DAPI (blue) in orthogonal views (dashed lines) showing colocalization of BDNF and SCG2 in the soma (white arrows).

Colocalization between BDNF and SCG2-positive vesicles expressed as percentage of total BDNF immunoreactivity ±SEM in cultured hippocampal neurons infected with scrambled or Tardbp shRNA lentiviruses (N=3 independent experiments, n>50 individual cells per condition in each experiment). **, p<0.01.

Colocalization between BDNF and SCG2-positive vesicles in hippocampal neurons infected with scrambled or Tardbp shRNA lentiviruses together with either EGFP or Sortilin rescue viruses. Histogram bars show average ± SEM BDNF/SCG2 colocalization expressed as percentage of total BDNF immunoreactivity (N=3 independent experiments, n>50 individual cells per condition in each experiment). **, p<0.01.

BDNF secretion induced by KCl treatment assessed by in-situ BDNF ELISA in cultured hippocampal neurons infected with scrambled or Tardbp shRNA lentiviruses. Histogram bars show average ± SEM BDNF levels normalized to a standard curve (N=3 independent experiments; n=3 wells per condition in each experiment). *, p<0.05; ***, p<0.001.

BDNF secretion in cultured hippocampal neurons infected with scrambled (left) or Tardbp (right) shRNA lentiviruses together with either EGFP, Sort1 shRNA or Sortilin rescue viruses. Histogram bars show average ± SEM BDNF levels normalized to a standard curve (N=3 independent experiments; n=3 wells per condition in each experiment). ***, p<0.001.

Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.
Abnormal TDP-43 function impairs activity-dependent BDNF secretion, synaptic plasticity and... (2018)
Figure 2

Figure 2. CA1-specific knockout of TDP-43 increases levels of exon 17b Sort1 mRNA and soluble Sortilin protein

Representative photomicrographs of TDP-43 (green) and NeuN (red) immunohistochemistry in hippocampal CA1 of Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice. Arrows denote areas of depleted TDP-43 immunoreactivity in the mutant.

Percentage of TDP-43 positive cells relative to NeuN cells in CA1 and the remaining hippocampus (HC [-CA1]) of Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice. Results are presented as average ± SEM (N=3 mice per condition; 5 sections per mouse). **, p<0.01.

TDP-43 intensity in TDP-43/NeuN double-positive cells in CA1 and the remaining hippocampus of Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice. Results are presented as average ± SEM (N=3 mice per condition; 5 sections per mouse). *, p<0.05; **, p<0.01.

Expression of Tardbp mRNA in micro-dissected CA1 and the remaining hippocampus of Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice quantified by Q-PCR. Results are presented as average ± SEM relative to levels in Tardbpfx/fx mice, after normalization to Gapdh mRNA. (N=3 mice per condition; **, p<0.01).

Western blots of micro-dissected CA1 and the remaining hippocampus showing expression of TDP-43, full-length and truncated (soluble) Sortilin, and GAPDH in Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice. Bands corresponding to soluble Sortilin are indicated (black arrows). GAPDH immunoblotting was used to control for equal loading. Molecular weights are indicated in kDa.

TDP-43 expression in micro-dissected CA1 and the remaining hippocampus of Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice. Results are presented as average ± SD of densitometry relative to GAPDH (N=3 mice per condition; **, p<0.01).

Expression of exon 17b Sort1 and total Sort1 mRNAs in micro-dissected CA1 and the remaining hippocampus of Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice quantified by Q-PCR. Results are presented as average ± SD relative to levels in Tardbpfx/fx mice, after normalization to Gapdh mRNA. (N=3 mice per condition; **, p<0.01).

Expression of full-length (left) and truncated (soluble, right) Sortilin protein in micro-dissected CA1 and the remaining hippocampus of Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice. Results are presented as average ± SD of densitometry relative to GAPDH (N=3 mice per condition; **, p<0.01).

Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.
Abnormal TDP-43 function impairs activity-dependent BDNF secretion, synaptic plasticity and... (2018)
Figure 3

Figure 3. CA1-specific knockout of TDP-43 reduces dendrite complexity and spine number through altered Sortilin splicing

Representative photomicrograph of Golgi staining in hippocampus of a Tardbpfx/fx mouse.

Sholl analysis of apical and basal dendritic arbors of CA1 pyramidal neurons from Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice. Results are presented as average ± SEM (N=3 mice per condition, 20 neurons per mouse). *, p<0.05; **, p<0.01.

High-magnification representative photomicrograph of Golgi staining of the dendritic arbour of a CA1 pyramidal neuron in a Tardbpfx/fx mouse. Black arrows denote selected dendritic spines.

Spine density in apical and basal dendrites of CA1 pyramidal neurons of Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice. Results are presented as average ± SD (N=3 mice per condition, 15 sections per mouse; *, p<0.05; **, p<0.01).

Representative photomicrographs of a CamKIIaCRE;Tardbpfx/fx hippocampus after stereotaxic injection of EGFP-expressing lentivirus immunostained for EGFP (green) and NeuN (red), and counterstained with DAPI (blue). The injected area within CA1 is indicated in the merged panel.

Expression of Tardbp (left), total Sort1 (center) and exon 17b Sort1 (right) mRNAs quantified by Q-PCR in micro-dissected CA1 of Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice after injection of EGFP-expressing lentivirus or Sortilin rescue virus. Results are presented as average ± SEM relative to levels in Tardbpfx/fx mice injected with EGFP virus, after normalization to Gapdh mRNA. (N=3 mice per condition). **, p<0.01.

Spine density in apical dendrites of CA1 pyramidal neurons of Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice after injection of EGFP-expressing lentivirus or Sortilin rescue virus. Results are presented as average ± SEM (N=3 mice per condition, 15 sections per mouse). **, p<0.01.

Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.
Abnormal TDP-43 function impairs activity-dependent BDNF secretion, synaptic plasticity and... (2018)
Figure 4

Figure 4. CA1-specific knockout of TDP-43 impairs spatial memory and abolishes BDNF-dependent TBS-LTP through altered Sortilin splicing

Percentage of change in field excitatory postsynaptic potential (fEPSP) recorded from hippocampal area CA1 after theta-burst stimulation (TBS) in Schaffer collaterals of hippocampal slices from Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice. Results are presented as average normalised to t=0 ± SEM (N=7 slices from 3 mice per condition). *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Representative traces are shown in Figure S4A.

Percentage fEPSP change in CA1 after TBS in Schaffer collaterals of hippocampal slices from Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice stereotactically injected with EGFP-expressing lentivirus or Sortilin rescue virus (red) superimposed to the same data shown in panel (a) (black). Results are presented as average normalised to t=0 ± SEM (N=7 slices from 3 mice per condition). *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Representative traces are shown in Figure S4B.

Percentage fEPSP change in CA1 after TBS in Schaffer collaterals of hippocampal slices from Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice under control conditions (black) or with BDNF (200ng/ml) perfused from t=0 (green). Results are presented as average normalised to t=0 ± SEM (N=7 slices from 3 mice per condition). *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Representative traces are shown in Figure S4C.

Percentage of total time spent in the target quadrant or in the remaining three quadrants of the Barnes maze for Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice at 3 different probe times: 20-30 min after first training session (short-term memory) and 24h or 14 days after last training session (long-term or remote memory, respectively). Results are presented as average ± SEM (N=8 mice per condition; * p < 0.05, ** p < 0.01, *** p < 0.001).

Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.
Abnormal TDP-43 function impairs activity-dependent BDNF secretion, synaptic plasticity and... (2018)
Figure 5

Figure 5. Aggregation of TDP-43 impairs activity-dependent BDNF secretion through altered Sortilin splicing

Representative photomicrographs of cultured hippocampal neurons immunostained for TDP-43 (green), FLAG (red) and counterstained with DAPI (blue) in control conditions (untransduced) or infected with lentivirus expressing FLAG-tagged human TDP-43 12xQN construct.

TDP-43 intensity in cell nuclei of untransduced or TDP-43 12xQN-infected hippocampal neurons normalized to mean nuclear intensity in untransduced neurons. Results are presented as average ± SEM (N=3 independent experiments; n=3 wells per condition in each experiment). Student's t-test; **, p<0.01.

Expression of Tardbp (left), total Sort1 (center) and exon 17b Sort1 (right) mRNAs quantified by Q-PCR in hippocampal neurons untransduced (control) or infected with lentiviruses expressing Tardbp shRNA or human TDP-43 12xQN constructs. Values were first normalized to Gapdh mRNA levels, then plotted as average ± SEM relative to levels in untransduced neurons (N=3 independent experiments; n=3 wells per condition in each experiment). 1-way ANOVA with Tukey's post-hoc; **, p<0.01.

BDNF secretion induced by KCl treatment as assessed by in-situ BDNF ELISA in cultured hippocampal neurons untransduced (control) or infected with lentiviruses expressing Tardbp shRNA or human TDP-43 12xQN constructs. Histogram bars show average ± SEM BDNF levels normalized to a standard curve (N=5 independent experiments; n=5 wells per condition in each experiment). **, p<0.01; ***, p<0.001.

BDNF secretion in cultured hippocampal neurons untransduced (control) or infected with lentiviruses expressing Tardbp shRNA or human TDP-43 12xQN constructs together with either EGFP or Sortilin rescue viruses. Histogram bars show average ± SEM BDNF levels normalized to a standard curve (N=5 independent experiments; n=5 wells per condition in each experiment). **, p<0.01.

Data information: Results are presented as average ± standard error of the mean (SEM). Statistical tests were performed using 2-way ANOVA with Tukey or Sidak post hoc analysis, as indicated.

Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using Student's t-test (B) or 2-way ANOVA with Sidak post hoc analysis (all other panels).
Abnormal TDP-43 function impairs activity-dependent BDNF secretion, synaptic plasticity and... (2018)
Figure 6

Figure 6. Disease-associated mutations in TDP-43 increase levels of exon 17b Sort1 mRNA and impair activity-dependent BDNF secretion in mouse hippocampal neurons and patient-derived human neurons

Expression of Tardbp (left), total Sort1 (center) and exon 17b Sort1 (right) mRNAs quantified by Q-PCR in hippocampal neurons infected with Tardbp shRNA and mutant human TARDBP lentivirus constructs as indicated. Absolute levels of mouse Tardbp and human TARDBP mRNAs were determined using a standard curve (see Methods) and added together to assess total Tardbp mRNA levels relative to control uninfected cells after normalization to Gapdh mRNA. Results are presented as average ± SEM (N=3 independent experiments; n=3 wells per condition in each experiment). *, p<0.05; **, p<0.01.

Western blots of cell lysates showing total TDP-43 expression in primary hippocampal neurons infected with Tardbp shRNA and mutant human TARDBP lentivirus constructs as indicated. The antibody used here detects both mouse and human TDP-43 proteins. GAPDH immunoblotting was used to control for equal loading. Molecular weights are noted in kDa. Bands corresponding to mouse and human TDP-43 are indicated.

BDNF secretion induced by KCl treatment assessed by in-situ BDNF ELISA in cultured hippocampal neurons infected with Tardbp shRNA and mutant human TARDBP lentivirus constructs as indicated. Histogram bars show average ± SEM BDNF levels normalized to a standard curve (N=5 independent experiments; n=5 wells per condition in each experiment). *, p<0.05; ***, p<0.001

BDNF secretion induced by KCl treatment in cultured hippocampal neurons infected with Tardbp shRNA and mutant human TARDBP lentivirus constructs together with either EGFP or Sortilin rescue viruses. Histogram bars show average ± SEM BDNF levels normalized to a standard curve (N=5 independent experiments; n=5 wells per condition in each experiment). *, p<0.05; **, p<0.01.

Expression of TARDBP (left), total SORT1 (center) and exon 17b SORT1 (right) mRNAs quantified by Q-PCR in human neurons derived from the indicated stem cells carrying either wild type Met337 (grey bars) or disease-associated Val337 (black bars) TARDBP alleles. Results are presented as average ± SEM (N=3 independent experiments; n=3 wells per condition in each experiment). *, p<0.05; **, p<0.01.

BDNF secretion in stem-cell derived human neurons carrying wild type Met337or Val337 TARDBP alleles, infected with proBDNF lentivirus 5 days prior to depolarization by KCl treatment. Results are presented as average ± SEM (N=3 independent experiments; n=5 wells per condition in each experiment). **, p<0.01; ***, p<0.001.

Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.
Pharmacoproteomic characterisation of human colon and rectal cancer (2017)
Figure 4

Figure 4 - MAP2K1 is a predictive marker for inhibitors targeting EGFR

Effect-size heat maps of six drugs (see titles of panels) targeting EGFR. It is evident that the different drugs showed different profiles but also that high MAP2K1 expression (blue/red gradient across cell lines) was consistently associated with drug resistance (dark blue/yellow gradient across cell lines; AUC: area under the curve; see main text and Appendix for details). See also Figure EV5.
Pharmacoproteomic characterisation of human colon and rectal cancer (2017)
Figure 5

Figure 5 - MERTK is a predictive marker for inhibitors targeting MEK1/2 in CRC cell lines

(A) Effect-size heat maps of two drugs (one from two different drug sensitivity screens) targeting MEK1/2 show consistent association of high MERTK expression with drug resistance. The colour scheme is the same as in Figure 4.

(B) Bar chart visualising the top kinases recurrently associated (absolute effect-size>0) with drug resistance (top seven bars) and sensitivity (bottom seven bars) in the GDSC and CCLE drug sensitivity datasets.

(C) Dose-response curves of two drugs for which high MERTK (left panels) or ACVR2A (right panels) expression was predicted to confer drug resistance. For each drug, three cell lines predicted to be sensitive (dark blue) and three cell lines predicted to be resistant (yellow) were assayed for viability. The experimental data validated that cell lines predicted to be more sensitive to a drug indeed showed this phenotype (data represents the average of three technical replicates; see Appendix for details).

(D) Boxplots summarizing the data shown in panel (C) using the area under the curve (AUC) as a measure for drug sensitivity. High AUC values indicate drug resistance. Again, it is evident that cell lines predicted to be more resistant to a certain drug were in fact significantly (p≤0.05, one-sided Mann-Whitney test) more resistant than cell lines predicted to be more sensitive.

(E) Western blot of and cells, visualising successful knockout by CRISPR/Cas9.

(F) Dose-response curve of RDEA119 (MEK1/2 inhibitor) in and cells. The knockout is more sensitive to MEK1/2 inhibition than the wild type. See also Figure EV6.
Pharmacoproteomic characterisation of human colon and rectal cancer (2017)
Figure 6

Figure 6 - MERTK is a prognostic marker in CRC patients

(A) Western Blot visualising MERTK expression in two negative (RKO, CC07) and two positive control cell lines (CaCo-2; HT55).

(B) IHC staining of MERTK expression in RKO (negative control) and CaCo-2 (positive control) cells.

(C) IHC staining of three representative TMAs from patients enrolled in the QUASAR 2 clinical trial and the corresponding quantification of the signal by pathologists.

(D) Kaplan-Meier plots showing worse overall, disease-free and recurrence-free survival of patients with high cytoplasmic/membranous expression of MERTK.

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