EMBO/soda-nel huggingface dataset:
TAK1 activates AMPK‐dependent cytoprotective autophagy in TRAIL‐treated epithelial cells (2009)
Figure 4

TAK1 mediates TRAIL‐induced AMPK activation and autophagy. (A) The lysates of MCF10A cells treated with 500 ng/ml TRAIL for the indicated times were analysed by immunoblotting. The control lane (0 min TRAIL) originates from the same blot and has been treated and exposed identically. Similar results were obtained in three independent experiments. (B, C) The lysates of MCF10A–eGFP–LC3 cells transfected with indicated siRNAs for 48 h and left untreated (−) or treated with 500 ng/ml TRAIL for 2 h were analysed by immunoblotting. (C, lower panel) MCF10A–eGFP–LC3 cells were transfected with indicated siRNAs for 48 h and analysed for the indicated mRNAs by RT–PCR. Similar results were obtained in two independent experiments. (D, E) MCF10A–eGFP–LC3 cells were transfected with indicated siRNAs for 48 h, treated as in Figure 3C and analysed for LC3 translocation (left) and sub‐G1 DNA content (right). The values represent mean±s.d. of a minimum of three independent experiments except for the right panel in E (two experiments). *P‐value 0.05 and **P‐value 0.01 as compared with cells treated in a same way, but transfected without siRNA.
TAK1 activates AMPK‐dependent cytoprotective autophagy in TRAIL‐treated epithelial cells (2009)
Figure 5

IL‐1β activates AMPK and autophagy. (A) Protein lysates from MCF10A cells left untreated (0) or treated with 50 ng/ml TNF, 500 ng/ml TRAIL (positive control) or 10 ng/ml IL‐1β for indicated times were analysed by immunoblotting for phosphorylated ACC (P‐ACC), phosphorylated IκB (P‐IκB) and GAPDH (loading control). The values show the P‐ACC/GAPDH and P‐IkB/GAPDH ratios as percentages of the ratios in untreated cells (left lanes) and are representative of two independent experiments. (B, C) MCF10A–eGFP–LC3 cells left untreated or treated with 500 ng/ml TRAIL, 50 ng/ml TNF or 10 ng/ml IL‐1β for 24 h were analysed for LC3 translocation (B) and the DNA content (C). The values represent mean±s.d. for two independent experiments. **P‐value 0.01 and ***P‐value 0.001 as compared with untreated cells.
TAK1 activates AMPK‐dependent cytoprotective autophagy in TRAIL‐treated epithelial cells (2009)
Figure 6

TRAIL induces TAK1‐dependent AMPK activation and autophagy in retinal pigment epithelial cells. (A) The lysates of hTERT–RPE1 cells transfected with indicated siRNAs for 48 h and left untreated or treated with 500 ng/ml TRAIL for 2 h were analysed by immunoblotting. (B) hTERT–RPE1–eGFP–LC3 cells transfected with indicated siRNAs for 48 h were left untreated or treated with 500 ng/ml TRAIL for 24 h and analysed for LC3 translocation (left) and sub‐G1 DNA content (middle) and AMPKα expression (right). (C) hTERT–RPE1–eGFP–LC3 cells were transfected with indicated siRNAs for 48 h and analysed for the indicated mRNAs (top). After 48 h, cells were left untreated or treated with 500 ng/ml TRAIL for 2 h, and analysed for P‐ACC and GAPDH (loading control) expression by immunoblotting. Similar results were obtained in two independent experiments. (D) Cells transfected as described in (C) and left untreated or treated with 500 ng/ml TRAIL, 10 μM ionomycin or starved for amino acids and glucose for 24 h were analysed for LC3 translocation. (E) Lysates of wild‐type (WT) MEFs and MEFs with an inactive TAK1 knock‐in (TAK1Δ) treated with 500 ng/ml TRAIL were analysed by immunoblotting. Similar results were obtained in two independent experiments. (F) WT and TAK1Δ MEFs left untreated and treated with 250 or 500 ng/ml TRAIL were analysed for cell death by the LDH release assay. Similar results were obtained in two independent experiments (A, C and E). The values represent mean±s.d. for two (D) or a minimum of three independent experiments (B, F). *P‐value 0.05, **P‐value 0.01 and ***P‐value 0.001 as compared with cells treated in the same way, but transfected without siRNA (B, D) or the wild‐type cells (F).
Muscle-directed gene therapy corrects Pompe disease and uncovers species-specific GAA immunogenicity (2021)
Figure 1

Figure 1. AAV-mediated gene transfer of an engineered GAA transgene to Gaa-/- mice exhibits dose-dependent transduction efficiency, increases in GAA activity, and clearance of accumulated glycogen in cardiac and skeletal muscle.

(A to D) 10- to 11-week-old mice were treated with vehicle control or AT845 at the vector doses indicated and followed for 12 weeks (N = 7 or 8 per cohort). (A) Vector copy number, (B) GAA levels, (C) GAA activity, and (D) glycogen content in cardiac and skeletal muscles. (C to D) Statistical analysis: two-way ANOVA, Dunnett's test. Data are presented as box-and-whisker plots with Tukey whiskers that show minimum, median, and maximum. Asterisks (*) indicate significant differences compared with untreated Gaa-/- mice (black) or wild-type mice (red). *, P<0.05; **, P<0.01; ***, P<0.001; ****, P <0.0001. See also Appendix Table S1.
Muscle-directed gene therapy corrects Pompe disease and uncovers species-specific GAA immunogenicity (2021)
Figure 2

Figure 2. AAV-mediated transfer of an engineered GAA transgene improves histopathology and exhibits a reduction in glycogen content in Gaa-/- mice.

Representative images of H&E (top) and PAS (bottom) staining of the quadriceps in wild-type and Gaa-/- mice untreated or treated with escalating doses of AT845 (3×1013, 1×1014, and 3×1014 vg/kg). Scale bar, 80 µm. (See also Appendix Table S1)
Muscle-directed gene therapy corrects Pompe disease and uncovers species-specific GAA immunogenicity (2021)
Figure 3

Figure 3. Dose-dependent functional correction in Gaa-/- mice.

A-D (A, B) Body weight curves and (C, D) grip response - ability to grip onto an inverted wire screen for a 60-second period - in wild-type and Gaa-/- mice untreated or treated with escalating doses of AT845 (3×1013, 1×1014, and 3×1014 vg/kg). All groups had n=9 at all timepoints, except the 6 week timepoint (females) and the 12 week timepoint (males and females) for the Gaa-/- mice, which had n=8 per group. Female mice dosed with 3×1013, 1×1014, and 3×1014 vg/kg had a 22%, 11%, and 0% failure rate at week 12, respectively; in comparison, the female control WT mice had a 22% failure rate and the control Gaa-/- mice had a 38% failure. Male mice dosed with 3×1013, 1×1014, and 3×1014 vg/kg had a 56%, 33%, and 11% failure rate at week 12, respectively; in comparison, the male control WT mice had a 11% failure rate and the control Gaa-/- mice had a 100% failure. Bars represent median. Data show a dose-dependent improvement in grip response following treatment with AT845. Statistical analysis: two-way ANOVA, Dunnett's test. Asterisks (*) indicate significant differences compared with untreated Gaa-/- mice at each respective time point. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.
Muscle-directed gene therapy corrects Pompe disease and uncovers species-specific GAA immunogenicity (2021)
Figure 4

Figure 4. Dose-dependent increase in transduction level and GAA activity in NHPs.

A, B Equal numbers of male and female cynomolgus monkeys were treated with vehicle control (n=4) or escalating doses AT845 (n=6) and followed for approximately 12 weeks. (A) Vector copy number and (B) GAA activity in heart, quadriceps, triceps, diaphragm, and liver. Statistical analysis: mixed-effects analysis with Dunnett's test. Data are presented as box-and-whisker plots with Tukey whiskers that show minimum, median, and maximum values. Asterisks (*) indicate significant differences compared with vehicle control NHPs. *, P<0.05; **, P<0.01.
Muscle-directed gene therapy corrects Pompe disease and uncovers species-specific GAA immunogenicity (2021)
Figure 5

Figure 5. Anti-GAA activity and cardiac biomarkers in male NHPs.

A-C Male cynomolgus monkeys were treated with vehicle control (n=2), AT845 (n=3) at 6×1013, 2×1014, or 5×1014 vg/kg, or Cyno-AT845 (n=3) at 2×1014 vg/kg and followed for approximately 12 weeks. (A) Anti-GAA antibody titer, (B) cardiac troponin I (cTnI), and (C) N-terminal pro b-type natriuretic peptide (BNP). Bars show mean and SEM.
Muscle-directed gene therapy corrects Pompe disease and uncovers species-specific GAA immunogenicity (2021)
Figure 6

Figure 6. Human and cynomolgus GAA activity in NHPs.

A, B Male cynomolgus monkeys were treated with vehicle control (n=2), AT845 (n=3), or Cyno-AT845 (n=3) at 2×1014 vg/kg and followed for approximately 12 weeks. GAA activity in (A) heart, quadriceps, triceps, and diaphragm, and (B) liver, brain, spinal cord, and testes. Data are presented as box-and-whisker plots with Tukey whiskers that show minimum, median, and maximum values.
Muscle-directed gene therapy corrects Pompe disease and uncovers species-specific GAA immunogenicity (2021)
Figure 7

Figure 7. Species-specific processing efficiency of GAA in cardiac/skeletal muscles.

A, B Western blot analysis of heart and quadriceps lysates using anti-GAA detects both the precursor GAA (110 kDa) and the mature lysosomal GAA (70 to 76 kDa) forms in muscle of NHPs treated with (A) AT845 (n=3) or (B) Cyno-AT845 (n=3) at 2×1014 vg/kg. Each tissue was analyzed in triplicate, and each respective symbol indicates use of a vehicle control heart tissue (^) or a vehicle control quadriceps tissue (*). (See also Appendix Table S3)

Expanded View Figure Legends
Hair cell synaptic dysfunction, auditory fatigue and thermal sensitivity in otoferlin Ile515Thr mutants (2016)
Figure 1

Figure 1. Otoferlin levels and cellular distribution are differentially affected in OtofI515T/I515T and OtofPgaPpga IHCs.

A-C, Immunofluorescence images (inverted intensity) of P14-P15 IHCs from indicated genotypes, revealing differences in otoferlin fluorescence intensity and distribution. Maximum projection of confocal stacks, scale bar 10µm.

D-F, Upper panels, examples for IHCs, co-labelled for otoferlin (magenta) and Vglut3 (green) and position of the line for line scans; maximum projection of few optical sections, scale bars 5µm. Lower panels, for quantification of membrane staining, the fluorescence was normalized to the cellular fluorescence for each fluorophore, then the average of five parallel line scans through the middle of the cells for the sum of both fluorescence values (black line) was used to determine the position of the basal membrane. At the most basal cellular point along this line which exceeds the threshold value of 2 (yellow diamond), the otoferlin-Vglut3 fluorescence difference (blue line) gave the value for relative otoferlin plasma membrane levels (orange diamond). Insets: enlargements of basal regions.

G, Otoferlin protein levels were reduced in OtofI515T/I515T IHCs (indicates numbers represent numbers of cells) and OtofPga/Pga IHCs compared to wild-type (Otof+/+) controls (mean±SEM).

H, ratio of apical/basal protein levels (above/below nuclear midline depicted as green line in A) indicates an apical shift of otoferlin in OtofPga/Pga IHCs.

I, Relative levels of membrane-bound otoferlin at the basal pole of IHCs.

K, Absolute amount of otoferlin at the basal IHC plasma membrane, gained by multiplication of relative plasma membrane levels (I) x total cellular otoferlin protein levels (G). Kruskal-Wallis test; *** indicates p<0.001; **, p<0.01; *, p<0.05.
Hair cell synaptic dysfunction, auditory fatigue and thermal sensitivity in otoferlin Ile515Thr mutants (2016)
Figure 2

Figure 2. Hearing, assessed by ABR, is impaired due to the Ile515Thr mutation in otoferlin.

A, ABR thresholds in OtofI515T/I515T (red, n=5) and Otof -/I515T (blue, n=7) mice were elevated compared to Otof+/+ mice (black, n=5) at an age of 3-4 weeks. The grey dotted line indicates the maximum loudspeaker output of 90dB; thresholds exceeding this value were set to 100dB for calculation of the mean ± SEM. At 12kHz, only the threshold increase for Otof-/I515T vs Otof+/+ is significant (Kruskal-Wallis test with Dunn’s multiple comparisons test between all 3 groups).

B, Grand averages of ABR waveforms ± SEM in response to 80dB click stimulation of the mice analyzed in A: The small wave preceding ABR wave I probably represents the summating potential (SP, hair cell receptor potential), which is intact in Otof mutants. ABR wave I is reduced in amplitude while subsequent peaks are better preserved in OtofI515T/I515T mice (Fig EV1).

C, Mean ABR wave I amplitude ± SEM for different stimulus intensities (all differences between genotypes are significant; 2-way ANOVA with Tukey’s multiple comparison test).

D, At 8 weeks (circles) and 25 weeks (open squares), OtofI515T/I515T mice showed highly elevated ABR thresholds compared to Otof+/+ mice (n=7-8 each; p<0.001 at 12kHz, Mann-Whitney-U-test). Grey dotted line as in A.

E, Grand averages of ABR waveforms ± SEM in response to 80dB Click stimulation in mice aged 8 weeks. OtofI515T/I515T (n=8) have drastically reduced ABR amplitudes compared to Otof+/+ mice (n=7). Otof-/- mice have no ABR (green, n=9).

F, Mean ABR wave I amplitude ± SEM for different stimulus intensities for 8 week and 25 week old OtofI515T/I515T and Otof+/+ mice (p<0.001, 2-way ANOVA).
Hair cell synaptic dysfunction, auditory fatigue and thermal sensitivity in otoferlin Ile515Thr mutants (2016)
Figure 3

Figure 3. Sustained exocytosis is impaired in OtofI515T/I515T hair cells.

A,B, No difference in voltage dependent Ca2+-currents (A) and fractional activation of ICa-channels (B) between OtofI515T/I515T IHCs (n=16) and IHCs of Otof+/+ littermates (n=13).

C, Exocytosis was recorded by measures of changes in membrane capacitance (ΔCm, lower panel) in response to depolarization (left, 20ms; right, 100ms) to the voltage where maximum Ca2+-currents were elicited (upper panel), typically -14mV.

D, Upper panel, while for stimuli up to 20ms exocytosis from OtofI515T/I515T (n=13) and Otof+/+ (n=11) IHCs was similar, sustained exocytosis, representing most likely the release of replenished vesicles, was significantly reduced in OtofI515T/I515T IHCs (t-test). Lower panel, Ca2+-current integrals were of similar size.

E, Flash photolysis of caged Ca2+ elicited a smaller exocytic response in OtofI515T/I515T IHCs.

F, The kinetics of the fast component from E was comparable between OtofI515T/I515T IHCs and Otof+/+ littermates (b). Open circles represent previously published data on IHCs of hearing wild-type mice (a; Beutner et al, 2001; Pangrsic et al, 2010).
Hair cell synaptic dysfunction, auditory fatigue and thermal sensitivity in otoferlin Ile515Thr mutants (2016)
Figure 4

Figure 4. Enhanced adaptation and slowed recovery of SGN spiking in OtofI515T/I515T.

A, Spontaneous rates of SGNs from OtofI515T/I515T (red, n=35) and Otof+/+ littermates (black, n=39) were not significantly different (p=0.83, Kolmogorov-Smirnov test).

B-D, Averaged poststimulus time histograms ±SEM from OtofI515T/I515T mice (n=25-32) and Otof+/+ littermate SGNs (n=13-27) to stimulation with 50ms tone bursts at the characteristic frequency (CF) of each fiber, 30dB above threshold at indicated stimulus rates.

E, Quantification of onset (largest 0.5ms bin, Mann-Whitney U test) and adapted responses (averaged 35-45ms from response onset, t-test) from data in B-D.

F, The jitter of the first sound-evoked spike was significantly increased (p<0.001, Mann-Whitney U test) but retained its inverse correlation with spike rates.

G, Spike rate increases with rising stimulus intensity were significantly shallower in OtofI515T/I515T SGNs, both for repetitive stimulation with 50ms tone bursts (left, p=0.014, t-test) and for continuous stimulation with amplitude modulated tones (right, p<0.001, Mann-Whitney U test).

H, Phase locking to amplitude modulated tones (assessed as the maximal synchronization index) was typically less precise in OtofI515T/I515T SGNs than in Otof+/+SGNs (p=0.09, Mann-Whitney U test).

I, 100ms masker tone and 15ms probe tones (both at CF, 30dB above threshold) were separated by silent intervals of variable duration. Inter-masker intervals were 500ms for Otof+/+, and 1000ms for OtofI515T/I515T. The ratio of probe and masker onset responses revealed enhanced RRP depletion after stimulation in OtofI515T/I515T (pink; mean ±SEM red) compared to Otof+/+ (grey; mean ±SEM black; for 4ms interval: p=0.001, t-test) and a slowed time course of recovery (x: half time of recovery, taken from normalized recovery functions; p<0.001, Mann-Whitney U test).

K, Mice learned to drink water when continuous noise was present but avoided drinking when the noise was interrupted by silent gaps. 5 OtofI515T/I515T mice (pink, mean ±SEM red) avoided drinking less efficiently than 2 Otof+/+ mice (grey, mean ±SEM black) for shorter gap durations. See also Figs S2/S3.
Hair cell synaptic dysfunction, auditory fatigue and thermal sensitivity in otoferlin Ile515Thr mutants (2016)
Figure 5

Figure 5. Apparently lower thermal sensitivity of hearing in mice than in humans, likely due to a human RXR motif reducing plasma membrane localization of Ile515Thr-otoferlin

A, Exemplary ABR traces (Click 100dB) from one OtofI515T/I515T mouse recorded at indicated bulla temperatures during local heating. Note that ABRs never disappeared completely, but wave I amplitude changed reversibly with temperature.

B,C, ABR wave I amplitudes in OtofI515T/I515T (A, Click 100dB) and Otof+/+ mice (B, Click 80dB) decreased with increasing temperature in the bulla. Each color represents a different experiment, dashed lines are line fits. Open symbols indicate the beginning of the experiments; subsequent recordings are connected by lines. The four indicated data points in B correspond to ABRs in A.

D, Explanted organ of Corti from an Otof+/+ mouse at P4 after two days in vitro (DIV2) at 37°C immunostained against otoferlin (magenta) and Vglut3 (green). Note the intense immunostaining of otoferlin at the plasma membrane. Scale bar 10 µm.

E-J, Explanted organs of Corti from Otof-/- mice at P4 were transfected by GeneGun with otoferlin cDNA and eGFP and immunostained at DIV2.

E, Example cell transfected with wild-type mouse otoferlin cDNA.

F,G, Representative cells transfected with mouse otoferlin including the 20 amino acids of the RXR motif (see Fig EV4). Cells in G were incubated at 38.5°C for 30 min prior to fixation. The RXR-otoferlin transfected cells show a membrane localization of otoferlin surrounding the Vglut3 immunofluorescence/eGFP fluorescence at the basal pole of the cells (arrows), similar as in controls (A,B).

H,J, Both the human RXR motif and the Ile515Thr mutation were introduced in mouse cDNA, and cells were incubated at 37°C (H) or for 30min at 38.5°C (J) before fixation. Here, green fluorescence surrounds the otoferlin immunofluorescence (arrows), suggesting loss of otoferlin from the plasma membrane.

K, Quantification of the relative plasma membrane levels of otoferlin (as in Fig 1) revealed normal plasma membrane abundance when the human RXR motif was present, but a strong reduction for otoferlin with RXR and Ile515Thr (individual cells; mean ±SEM; t-test).
Hair cell synaptic dysfunction, auditory fatigue and thermal sensitivity in otoferlin Ile515Thr mutants (2016)
Figure 6

Figure 6. Temperature alters exocytosis, otoferlin protein levels and plasma membrane abundance

A-E, Capacitance increments recorded from Otof+/+ (black) and OtofI515T/I515T (red) IHCs. Individual cells recorded at indicated temperatures (light circles), mean ±SEM for perforated patch clamp experiments (t-test).

F,G, Summary of the capacitance changes and Ca2+-current integrals for Otof+/+ (F) and OtofI515T/I515T IHCs (G) at the different temperatures illustrates the drastic increase in exocytosis for physiological temperature. *Significant differences compared to RT measurements are indicated with colors of the respective temperature, and between PT and high temperature in violet (t-test).

H-J, Otoferlin immunofluorescence in Otof+/+ IHCs (upper panel) and OtofI515T/I515T IHCs (middle and lower panels) of explanted organs of Corti at P7-P8 after incubation at indicated temperatures for 24h; maximum projections of z-stacks, inverted images, scale bar 10µm. The same imaging settings have been applied in all experiments, and the same lookup table was applied for the upper and middle panels. Images of lower panels are enhanced compared to middle panels (lookup table covering full data range of only this genotype) to visualize otoferlin distribution.

K, Quantification of otoferlin immunofluorescence in Otof+/+ IHCs (black bars, n=98-137 cells, 4-5 experiments) and OtofI515T/I515T IHCs (red bars, n=90-97 cells; Kruskal-Wallis test as in L-N), indicate reduced protein levels with increasing temperature.

L, Apical/basal otoferlin protein distribution, revealing a significant apical shift of otoferlin and Vglut3 for OtofI515T/I515T IHCs (red symbols) at 38.5°C compared to Otof+/+ (grey/black symbols).

M, Relative levels of membrane bound otoferlin were lowered with increasing temperature.

N, Absolute otoferlin membrane immunostaining strongly decreased with temperature.
Hair cell synaptic dysfunction, auditory fatigue and thermal sensitivity in otoferlin Ile515Thr mutants (2016)
Figure 7

Figure 7. The Ile515Thr mutation does not affect the subcellular localization of otoferlin but alters the size of endosomal and synaptic vesicles

A-C, Pre-embedding immunogold labelling for EM visualizes otoferlin localization in random ultrathin sections through the basal part of IHCs in Otof+/+ (A) and OtofI515T/I515T (B) but not in Otof-/- (C) mice. IHCs are highlighted in beige. Scale bar 100nm.

D-G, Magnified synaptic ribbons (R) and postsynaptic afferent boutons of SGNs (Aff), displaying otoferlin immunogold labelling at active zone membranes (pink arrowheads) but not around the synaptic ribbon, using saponin permeabilization (D-F), or Triton-X-100 treatment (G).

H, Vglut3 immunogold labelling adjacent to the ribbon as expected for synaptic vesicle. Scale bar 200nm.

I,J, Some otoferlin labelled structures bear clathrin coats (red arrowheads), suggesting that they originate from bulk endocytosis. Scale bar 100nm.

K, Otoferlin labelled vesicles were on average larger in OtofI515T/I515T IHCs (OtofI515T/I515T, n=86 vesicles, 18 images; Otof+/I+, n=82 vesicles, 6 images; Wilcoxon-rank-sum-test, as in L-M).

L, Quantification of immunogold clusters at the plasma membrane revealed a strong trend towards reduced levels of otoferlin in OtofI515T/I515T (6 images each).

M, Distal from the plasma membrane, immunogold labels were reduced in OtofI515T/I515T IHCs.

N, Representative electron micrographs of IHC ribbon synapses in OtofI515T/I515Tand Otof+/ (conventional embedding without immunogold) after pre-incubation for 10min at the indicated temperature followed by high K+ stimulation or 0 Ca2+ inhibition for 1min 45sec. Scale bars 100nm.

O, Illustration depicting criteria for random section analysis (not drawn to scale). Ribbon-associated synaptic vesicles (SVs, green) in the first row ≤80 nm from ribbon (R) and membrane-proximal vesicles (yellow) in ≤25 nm membrane-to-membrane distance from the plasma membrane (blue) and ≤80 nm from each side of the presynaptic density.

P-Q, Synaptic vesicles with >45 nm diameter appeared at 39°C in random sections; fraction of large membrane-proximal (P; Kruskal-Wallis test) and ribbon-associated (Q; one way ANOVA followed by Tukey’s test) synaptic vesicles (see also Appendix Table S1).