EMBO/soda-nel huggingface dataset:
Dynein mutations impair autophagic clearance of aggregate-prone proteins (2005)
figf2

(a) Stable PC12 cells expressing A53T α-synuclein were induced for 24 h, after which expression was switched off and cells were simultaneously transfected with p50 or dynamitin (p50), CC1, CC2, motor domain–deleted kinesin heavy chain (ΔM-KHC) or the tail region of kinesin heavy chain (DN-KHC) for the next 24 h. Dominant-negative constructs along with pEGFP-C1 (3:1 ratio of dominant-negative construct:pEGFP-C1) were used to transfect cells, and FACS was used to select transfected cells, which were analyzed by western blotting as in Figure 1a. Quantification of the band intensity from multiple experiments (three independent experiments for dynein mutants and two for kinesin mutants) is shown. ***P 0.0001; *P 0.01; NS, not significant. (b) COS-7 cells were cotransfected with HA-Q74 or GFP-Q74 with empty vector (pEGFP or pcDNA3.1), p50 or dynamitin (p50), p62 subunit of dynactin complex (p62), the tail region of kinesin heavy chain (DN-KHC), CC1 or CC2 for 48 h, after which the cells were fixed for immunofluorescence and immunocytochemistry (using to antibody to HA). The percentage of GFP-positive cells with aggregates and cell death is shown. ***P 0.0001; **P 0.001; NS, not significant. (c) Immunocytochemistry analysis (using antibody to HA) of COS-7 cells cotransfected for 48 h with HA-Q74 (red) and pEGFP or p50 (green). Panels on left are magnifications of boxed areas in second panels. The boxes are the same size. ***P 0.0001; **P 0.001. Examples of fragmented nuclei in cells overexpressing p50 and nuclei in cells overexpressing p50 but treated with Z-VAD-fmk (broad-spectrum caspase inhibitor) are shown in Supplementary Figure 4 online.
Dynein mutations impair autophagic clearance of aggregate-prone proteins (2005)
figf3

(a) Immunofluorescence analysis of COS-7 cells cotransfected for 15 h with either empty vector (control) or CC1 and GFP-LC3 (green), in a 3:1 ratio. Panels on right are magnifications of boxed areas. (b) Western-blot analysis of COS-7 cells treated with EHNA or left untreated using antibodies to LC3 and to actin. Quantification of the band intensity from two independent experiments is shown. (c) NRK cells transiently transfected as in a were immunostained with antibody to lgp120, and the numbers of GFP-LC3 and lgp120 doubly stained vesicles in individual cells were scored.
Dynein mutations impair autophagic clearance of aggregate-prone proteins (2005)
figf4

Frequencies of ommatidia with different numbers of rhabdomeres are shown at 3 days after eclosion in progeny of flies expressing mutant huntingtin exon 1 (gmrQ120) crossed to either a control stock (w1118; W) or to Dhc64C (dynein heavy chain mutant; Dhc; a) or Roblk (dynein light chain mutant; Robl; b) flies. P ≤ 0.0001 (Mann-Whitney U test). (c) Ommatidia from the progeny of flies expressing mutant huntingtin exon 1 (Q120) crossed with either control stock (w1118; W) or with Dhc64C (Dhc) flies. All fly experiments were repeated three times independently with similar significance levels. One representative experiment is shown.
Dynein mutations impair autophagic clearance of aggregate-prone proteins (2005)
figf5

(a) Tremor onset and severity in a mouse model of Huntington disease are enhanced by the Loa mutation. At 7 weeks: HdhHD/+ Dnchc1Loa/+ n = 9, HdhHD/+ Dnchc1+/+ n = 9, P = 0.14. At 9 weeks: HdhHD/+ Dnchc1Loa/+ n = 8, HdhHD/+ Dnchc1+/+ n = 9, P = 0.027. At 11 weeks: HdhHD/+ Dnchc1Loa/+ n = 6, HdhHD/+ Dnchc1+/+ n = 9, P = 0.001. At 13 weeks: HdhHD/+ Dnchc1Loa/+ n = 5, HdhHD/+ Dnchc1+/+ n = 8, P = 0.021. At 15 weeks: HdhHD/+ Dnchc1Loa/+ n = 3, HdhHD/+ Dnchc1+/+ n = 5, P = 0.021. (b,c) Dynein mutation reduces motor coordination and muscle function in a mouse model of Huntington disease. HdhHD/+ Dnchc1Loa/+, HdhHD/+ Dnchc1+/+ and Hdh+/+ Dnchc1Loa/+ mice are shown. An average of six mice were used at every time point, with at least three mice per group after 13 weeks. (b) Grip strength. Overall effect for all time points between the three genotypes: P = 0.0024. Overall pair comparison between HdhHD/+ Dnchc1Loa/+ and HdhHD/+ Dnchc1+/+: P = 0.001. Overall pair comparison between HdhHD/+ Dnchc1Loa/+ and Hdh+/+ Dnchc1Loa/+: P = 0.0157. Effect at each time point between the three genotypes: 7 weeks, P = 0.023; 9 weeks, P = 0.001; 11 weeks, P 0.0001; 13 weeks, P = 0.004; 15 weeks, P = 0.336 (most of the double mutants were dead by 15 weeks). (c) Accelerating rotarod. Overall effect for all time points between the three genotypes: P = 0.0367. Overall pair comparison between HdhHD/+ Dnchc1Loa/+ and HdhHD/+ Dnchc1+/+: P = 0.0245. Overall pair comparison between HdhHD/+ Dnchc1Loa/+ and Hdh+/+ Dnchc1Loa/+: P = 0.0553. Effect at each time point between the three genotypes: 8 weeks, P 0.001; 10 weeks, P = 0.005; 12 weeks, P = 0.001; 14 weeks, P = 0.009; 16 weeks, P = 0.039. (d) Reduced lifespan of HdhHD/+ Dnchc1Loa/+ mice. HdhHD/+ Dnchc1Loa/+ (red line) n = 13, HdhHD/+ Dnchc1+/+ (green line) n = 9, P = 0.038. Mice were subjected to humane end points, and so the survival curve reflects the onset of the end points rather than the complete lifespan. Asterisks (*) represent mice that were found dead without reaching humane end points. No mortality of Hdh+/+ Dnchc1Loa/+ mice was observed.
Dynein mutations impair autophagic clearance of aggregate-prone proteins (2005)
figf6

(a) Brain sections from HdhHD/+ Dnchc1+/+ or HdhHD/+ Dnchc1Loa/+ mice were stained with antibody to huntingtin (EM48; green). No EM48-stained aggregates were detected in brains of Hdh+/+ Dnchc1Loa/+ mice. Arrows indicate cells with aggregates. (b) Western-blot analysis of mouse brain lysates from wild-type (WT) or Loa mutant mice using antibodies to LC3 and to actin.
Mitochondrial translation deficiency impairs NAD+-mediated lysosomal acidification (2020)
Figure 1

Figure 1 Autophagy is suppressed in the heart of p32cKO mice.

(A) Electron microscopy of abnormal mitochondria in p32cKO hearts of 6 months old mice. These two figures are also presented in Figure EV1A along with other EM figures.　Scale bars, 1 µm.

(B) Western blot analysis of autophagy marker proteins (LC3-II and p62) in 6 months old WT and p32cKO hearts. The bands of LC3-II and p62 are increased in the p32cKO heart. GAPDH was used as an internal control. Quantification is shown on the bottom. These values are presented as mean ± SD (n = 6 mice per group). Student's t-test was performed on WT vs p32cKO, **p < 0.01.

(C) Immunostaining of LC3, p62 and multi-ubiquitin in heart tissues of 6 months old. The magnified image (right panel) shows the ring-like staining pattern in the p32cKO heart (indicated arrows). Scale bar, 20 µm. The panel related to LC3 immunostaining in p32cKO is also presented in Figure EV1B (top left) along with other immunostaining pictures.

(D) In the 6 months old p32cKO heart, p62 and multi-ubiquitin staining patterns were co-localized. Scale bar, 5 µm.

(E) In the 6 months old p32cKO heart, the staining patterns of p62 and the mitochondrial marker protein, pyruvate dehydrogenase (PDH), were co-localized. Scale bar, 5 µm.

(F) Western blot analysis of ubiquitinated proteins using an anti-multi-ubiquitin antibody in the 6 months old WT and p32cKO heart (n = 4 mice per group). Error bars are presented as mean ± SD. Student's t-test was performed on WT vs p32cKO, ***p < 0.005.

(G) Western blot analysis of phosphorylated p62 (Se351 and Ser403) and non-phosphorylated p62 in the 6 months old WT and p32cKO heart. Relative expression of phosphorylated p62 is shown on the right. Error bars are presented as mean ± SD. Student's t-test was performed on eight WT mice vs seven p32cKO mice, ***p < 0.005.
Mitochondrial translation deficiency impairs NAD+-mediated lysosomal acidification (2020)
Figure 2

Figure 2 Cardiomyocyte-specific p32cKO causes lysosomal enlargement in mice.

(A) Electron micrographs showing lysosomes in cardiac myofibrils of 2 and 6 months old mice. Lysosome size and density were different in WT (black dot) and p32cKO (red dot) heart tissues. Scale bars, 1 µm. The quantification of the average lysosomal length is presented in the plot in the right panel as mean ± SEM (two months old mice, n = 14-16; 6 months old mice, n = 35-60). Statistical significance was assessed by Student's t-test, ***p < 0.005.

(B) Autofluorescence showing lipofuscin localization around the nucleus in the 6 months old p32cKO heart. Tissues were excited at a wavelength of 540 (upper panel) or 470 (lower panel) and emission spectra were collected with a confocal microscope at wavelengths (band path) of 580-630 nm (upper panel) or 510-560 nm (lower panel). Enlarged view of the white squares is shown right. Scale bars, 20 µm. The quantification of the ratio of autofluorescence of DAPI staining is presented in the plot in the right panel as mean ± SEM (three months old mice, n = 11; 6 months old mice, n = 9-14, 9 months old mice, n = 9-15, 12 months old mice, n = 11-21). Statistical significance was assessed by Student's t-test, ***p < 0.005.

(C) Immunostaining of Lamp2 in the heart using TrueBlackTM to quench lipofuscin autofluorescence in the 6 months old WT and p32cKO heart. Enlarged view of the white squares is shown below. Scale bars, 20 µm.

(D) Western blot analysis of Lamp2 in the 6 months old WT and p32cKO hearts. Quantification is shown in the lower panel (n = 4 mice per group). GAPDH was used as an internal control. Error bars are presented as mean ± SD. Student's t-test was performed on WT vs p32cKO, ***p < 0.005.
Mitochondrial translation deficiency impairs NAD+-mediated lysosomal acidification (2020)
Figure 3

Figure 3 Reduced NAD synthesis gene expression and reduced NAD levels in the p32cKO heart.

(A) LC-MS/MS metabolomic analysis of NAD+, NADH, NADP+, nicotinamide and NAAD in the 6 months old WT and p32cKO hearts. NAD+ and NADP+ showed significantly different levels between WT and p32cKO hearts (n = 12). Error bars are presented as mean ± SD. Student's t-test was performed on WT vs p32cKO, **p < 0.001, ***p < 0.0002.

(B) Real-time PCR analysis of RNA expression of NAD-synthesizing enzymes in the 6 months old WT and p32cKO hearts. Error bars are presented as mean ± SD. Student's t-test was performed on WT mice vs p32cKO mice (n = 6), ***p < 0.005, *p < 0.05. The right panel shows the NAD synthesis pathway and mRNA expression levels are indicated by red arrows. NAM: nicotinamide, NA: nicotinic acid, QA: quinolic acid, NMN: nicotinamide mononucleotide.

(C) Reduced Nmnat3 expression in 9 months old p32cKO heart. Quantification is shown on the right (n = 4 mice per group). Error bars are presented as mean ± SD. Student's t-test was performed on WT vs p32cKO, **p < 0.01.

(D) NAD+ levels without or with NMN addition in 9 months old WT and p32cKO heart lysates (n = 3). Error bars are presented as mean ± SD. Student's t-test was performed on WT vs p32cKO, **p < 0.002.

(E) Western blot analysis of HIF1α in 9 months old WT and p32cKO heart. Quantifications is shown on the right (n = 4 mice per group). Error bars are presented as mean ± SD. Student's t-test was performed on WT vs p32cKO, **p < 0.01. GAPDH was used as an internal control.

(F) Immunoblot analysis of HIF1α; the expression in 3T3-L1 cells was increased after treatment with 1 mM chloramphenicol (CAP), which inhibits mitochondrial translation. β-actin was used as an internal control. One representative experiment out of three shown.

(G) Nmnat3 mRNA expression in 3T3-L1 cells after 1 mM CAP treatment for 72 h. The HIF1α inhibitor (20 or 50 µM) was added 2 h before the CAP treatment. Error bars are presented as mean ± SD of three independent experiments. Student's t-test was performed on WT cells vs WT cells treated CAP and (or) HIF1α inhibitor,**p < 0.01, *p < 0.05.

(H) Immunoblot analysis of HIF1α after treatment with 150 µM CoCl2 in 3T3-L1 cells, which stabilized HIF1α. β-actin was used as an internal control.

(I) Nmnat3 mRNA expression in 3T3-L1 cells after 150 µM CoCl2 treatment for 72 h (n = 3). Error bars are presented as mean ± SD of three independent experiments. Statistical significance was assessed by Student's t-test, **p < 0.01.
Mitochondrial translation deficiency impairs NAD+-mediated lysosomal acidification (2020)
Figure 4

Figure 4 NMN addition and Nmnat overexpression rescue lysosomal function.

(A) Lysosomal acidification is impaired in p32KO MEFs. Representative images of WT and p32KO MEFs, stained with dextran-Oregon Green (488) and dextran-TMRM. Scale bar, 5 µm. Addition of 1 mM NMN for 48 h to p32KO MEFs rescued lysosomal acidification, and 1 mM CAP treatment of WT MEFs for 48 h decreased lysosomal acidification. Dextran-Oregon Green is quenched under acidic pH, and thus an increased green:red ratio denotes impaired lysosomal acidification. The average ± SEM of the green:red ratio in at least 30 cells in two independent experiments is presented in the plot (normalized to WT green/red = 1). Statistical significance was assessed by Student's t-test, *p < 0.05, ***p < 0.001.

(B) Two Nmnat3 constructs: Nmnat3 (full) with a mitochondrial-targeting sequence (MTS), and Nmnat3(v1) without MTS.

(C) Both these constructs were effectively expressed in p32KO MEFs. After transfection, lysates were subjected to western blot analysis with anti-Flag and anti-Nmnat3 antibodies. GAPDH was used as an internal control. One representative experiment out of three shown.

(D) Nmnat3 (full) was localized in mitochondria, but Nmnat3(v1) did not co-stain with MitoTracker red in p32KO MEFs. Scale bars, 10 µm. The magnified image is shown on the right.

(E) Nmnat3(v1) overexpression in p32KO MEFs rescued lysosomal function. LysoTracker Red-stained p32KO MEFs were quantified by flow cytometry. Error bars are presented as mean ± SEM of 4 independent experiments. Statistical significance was assessed by Student's t-test, ***p < 0.001.
CRYPTOCHROMES promote daily protein homeostasis (2021)
Figure 5

Figure 5. Time-dependent differences in cellular composition and ion transport between WT and CRY-deficient cells.

A-C) Gene ontology analysis for rhythmic proteins was carried out using Gene ontology enrichment analysis and visualisation (GOrilla) (Eden et al, 2007, 2009). Significantly rhythmic WT proteins were compared against background (all proteins identified in the experiment), and the top non-overlapping GO Biological Process terms shown, sorted according to FDR q-value. Fold enrichment is annotated on each bar (A). The same GO analysis was carried out comparing proteins rhythmic that were rhythmic in CKO cells (B) and for proteins that were rhythmic across both genotypes (C).

D-E) From one time-course experiment, ions, cytosolic proteins and total protein were extracted in parallel samples. The presented experiment is representative of 3 separate time-course experiments that were carried out (N=3). Representative experiment shown (n=4 technical replicates for [K+], n=3 technical replicates for the other traces). Blue lines highlight the antiphasic relationship between oscillations in cytosolic protein and potassium concentration. Mean±SEM, p-values from RAIN, red lines are fits by a damped cosine compared with a straight line (null hypothesis). Parallel PER2::LUC recordings were also performed and plotted as a phase marker.

F) Examples of key ion transporters are shown, as detected in the proteomics experiment. P values show the results of an F test comparing fits of damped cosine against straight line. All proteins except WT NKCC1 had RAIN p-values <0.05.

G-H) Relative amplitudes of cytosolic protein (G) and potassium concentrations oscillations (H) in D) and E) were greater in CKO compared to WT (Student's t test with Welch correction, mean±SD, *p≤0.05, **p≤0.01).
CRYPTOCHROMES promote daily protein homeostasis (2021)
Figure 6

Figure 6. CRY-deficient cells and tissues are more sensitive to stress.

A) WT and CKO cells were treated with 500 nM tunicamycin (TUN) and lysed in RIPA buffer at time points between 0-24 hours afterwards. Western blots were carried out, probing for phosphorylated eIF2α, total eIF2α and actin. Representative blots shown, n=4.

B) Quantification of all replicates represented in (A), where phosphorylated eIF2α is normalised to total eIF2α. Mean±SD. 2-way ANOVA (TWA) with Holm-Sidak's multiple comparisons.

C) Mice were housed in 12h:12h light-dark cycles, with unrestricted feeding. Mouse lungs were collected 7 hours after transition to the light phase, 5 hours after intraperitoneal injection of mice with bortezomib (BTZ, 2.5 mg/kg) or vehicle (Veh, 1% DMSO in sterile PBS). Tissues were lysed in RIPA buffer and western blots were performed, probing for phosphorylated STAT3, total STAT3 and actin. Male (left) and female (right) replicates shown, N=2 mice for each condition.

D) Quantification of the blots shown in (C), where phosphorylated STAT3 is normalised to total STAT3. Mean±SD. Multiple t-tests, corrected for multiple comparisons with the Holm-Sidak method, *p≤0.05, ***p≤0.001.
CRYPTOCHROMES promote daily protein homeostasis (2021)
Figure 7

Figure 7. WT cells recapitulate the CRY-deficient phenotypes upon induced chronic stress.

A) WT PER2::LUC cells were treated with epoxomicin or vehicle control. Blue arrows show the time points where drug was washed off. Mean (solid lines) ± SD (dotted lines).

B) Quantification of damping rate of the PER2::LUC recordings shown in A), for the duration of drug treatment. Mean±SD. One-way ANOVA with Holm-Sidak's multiple comparisons, **p≤0.01, ****p≤0.0001.

C) WT PER2::LUC cells were treated with tunicamycin or vehicle control. Mean (solid lines) ± SD (dotted lines).

D) Quantification of damping rate of the PER2::LUC recordings shown in C), for the duration of drug treatment. Mean±SD. One-way ANOVA with Holm-Sidak's multiple comparisons, ****p≤0.0001.

E) WT PER2::LUC cells were treated with radicicol or vehicle control. Blue arrows show the time points where drug was washed off. Mean (solid lines) ± SD (dotted lines).

F) Quantification of damping rate of the PER2::LUC recordings shown in E), for the duration of drug treatment. Mean±SD. One-way ANOVA with Holm-Sidak's multiple comparisons, ****p≤0.0001.

G-H) WT and CKO cells were treated with 0.3 µM cycloheximide (CHX) or DMSO vehicle control, and kept in constant conditions, n=4, Mean±SD. Panels above show baseline-subtracted raw data, and panels below show data detrended with a 24-hour moving average.

I-J) Representative double-plotted actograms of WT mice receiving drinking water with DMSO vehicle control (N=7 mice) or 35 µg/mL ixazomib (N=8 mice). Two representative actograms are shown per condition. For the first 7 days the mice were in 12:12h light-dark cycles (LD), and had drinking water supplemented with blackcurrant squash. Ixazomib/DMSO was added to this, and the lighting was changed to constant darkness (DD). After 7 days the drug/vehicle was removed, and the mice remained in DD until the end of the experiment.

K-L) Quantification of the amplitude of wheel-running activity and period length from the experiment shown in I), J), including pre-treatment, during treatment and post-drug treatment. Mean±SEM. 2-way ANOVA with Holm-Sidak's multiple comparisons, *p≤0.05, ***p≤0.001, ****p≤0.0001, TWAINT = two-way ANOVA interaction.

M) Schematic diagram: our data suggests that CRY deficiency is associated with proteome imbalance, which in turn disrupts circadian rhythmicity of physiology.
SARS-CoV-2 -host proteome interactions for antiviral drug discovery (2021)
Figure 6

Figure 6. The antiviral activities of selected drugs against SARS-CoV-2 in vitro.

A For the image-based drug screening, Calu-1 cells were infected with SARS-CoV-2 in the presence of drugs with indicated doses for 48 h, immunostained, imaged and analyzed. The example images of mock-infected cells and virus-infected control cells (no drugs). The viral yield in cells was detected with an antibody for the viral N protein (virus-infected cells; upper image), and nuclear staining (mock-infected cells; lower image) (Scale bar: 200 μm).

B The dose-response curves for six drugs with potential antiviral effects. The drug-induced inhibition of virus infection (count of N protein positive cells) was quantified using the area under the curve (AUC) values.

C The effect of drugs on cell viability in the presence of the virus, e.g., cytotoxicity was evaluated by the count of nuclei.

D Inhibition of interactions formed with DDX39B in the presence of MTX using co-IP assay. DDX39B is fused to the C terminus of a V5 tag, and the interaction partner is fused with a Strep-HA tag. The diagram (upper) shows the interaction pairs formed with DDX39B used for co-IP assay. Dot-blot results (lower) show the interaction pairs (arrangement as upper panel) in presence of different concentration of MTX as indicated on the top of the image. The image displayed is representative of 3 replicates (n = 3).

E Quantification of DDX39B binding to different interaction partners. Quantification of V5-tagged DDX39B protein amount was normalized over HA-tagged interaction partner intensity and displayed as a fold change over the ratio of untreated cells. Data that represent the mean of 3 replicates are highlighted in red. A fold change > 0.5 is considered significant.

Expanded View Figure legends
Nitric oxide‐induced mitochondrial fission is regulated by dynamin‐related GTPases in neurons (2006)
Figure 2

Mitochondrial fission occurs before dendritic injury and without AIF or cytochrome c release from mitochondria. Neurons were co‐transfected with vectors encoding (A) Mito‐DsRed2 and (B) MyrPalm‐mCFP plus (C) AIF‐GFP and the effects of SNOC were recorded using 3D time‐lapse imaging. Projection images zoomed in onto the dendritic arbor of one neuron (representative of n=42). Arrows indicate dendritic spines. The inset shows the soma at appropriate grayscale levels. The apparent brightening of AIF‐GFP fluorescence in the nucleus at 5 h was due to nuclear and cell body shrinkage rather than nuclear translocation of AIF. Scale bars, 10 μm. See also Supplementary Video 2. (D–E) Neurons transfected with Mito‐DsRed2‐ plus cytochrome c‐GFP were exposed to 300 μM SNOC. 3D time‐lapse images were captured by fluorescence deconvolution microscopy and analyzed using Volocity software. (D) 3D time‐lapse image reconstructions of mitochondria and cytochrome c in a dendritic arbor at indicated time points following SNOC addition. Scale bar, 10 μm. Five mitochondria were selected and motion tracked throughout the imaging series. (E) Traces show the ratio of mean GFP to DsRed2 emission intensity from each mitochondrion. Each trace color corresponds to the same colored mitochondrion. (representative of n=9) (F–G). Immunocytochemistry for staining anti‐AIF antibodies (red) anti‐NeuN antibodies (green) and nuclei labeled with Hoechst 33342 dye (blue) after (F) aged SNOC or (G) SNOC (150 μM; 4 h) treatment. Volume rendered 3D reconstruction of confocal image stacks are shown above, and a single confocal plane in grayscale below. Grid, 3.3 μm for (F) and 1.9 μm for (G), scale bars, 5 μm. (H) Ratio of nuclear to somal AIF signal was measured as mean fluorescence intensity in the center of nuclei divided by the mean fluorescence intensity in the cell body, outlined by the NeuN staining (excluding the nucleus). The bar diagram summarizes the mean±s.e.m. of 282 and 262 neurons measured in n=19 and 20 image stacks in three independent experiments for aged SNOC and SNOC, respectively (P∼0.99 not significant by Student's t‐test). (I) DEVDase activity. Purified cortical neurons were exposed to either 200 μM SNOC or 1 μM staurosporine, as positive control. The rate of zDEVD‐AMC caspase substrate cleavage per minute was monitored and expressed in arbitrary fluorescence units. Data show mean±s.e.m. for four independent experiments (*significance at P0.01 by ANOVA).