EMBO/soda-nel huggingface dataset:
A secreted factor NimrodB4 promotes the elimination of apoptotic corpses
by phagocytes in Drosophila (2021)
Figure 4

Figure 4. Phagocytosis of apoptotic cells is reduced in NimB4sk2 mutant

(A) Representative confocal imaging of wild type (w1118), NimB4 sk2 and draper macrophages incubated with fluorescently-labeled apoptotic cells (red, CellTraceTM Red) on ice for 1h and stained with AlexaFluorTM 488 phalloidin (green). Scale bar: 10 μm.

(B) Quantification of the number of apoptotic cells (red) binding to the macrophages (green). Values from three independent experiments are represented as mean ± SD (*** P<0.001 by ANOVA test followed by post hoc Dunnett's multiple comparison tests. ns: not significant).

(C) Representative Scanning Electron Microscopy images of spread macrophages extracted from wild type (w1118), NimB4sk2 and draper L3 wandering larvae and incubated 30 min with apoptotic cells (artificially colored in red) at room temperature. The arrows show the binding of apoptotic cells to the macrophages. Scale bar: 1μm.

(D) Ex vivo phagocytosis assay using Alexa555 fluorescent apoptotic bodies. Wild type, NimB4sk2, NimB4 genomic rescue (NimB4sk2, [NimB4]), draper and NimC11, eater1 macrophages from L3 wandering were incubated with Alexa555 fluorescent apoptotic bodies for 30, 60 or 120 min at room temperature. Phagocytosis was quantified by flow cytometry. Data are represented as mean ± SD from three independent experiments (* P<0.05,** P<0.01, **** P<0.0001 by ANOVA test followed by post hoc Dunnett's multiple comparison tests ns: not significant).

(E) Ex vivo phagocytosis assay using AlexFluor488 S.aureus Bioparticles. Wild type, NimB4sk2 and NimC11, eater1 macrophages from L3 wandering were incubated with bioparticles for 30, 60 or 120 min at room temperature. Phagocytosis was quantified by flow cytometry. Data are represented as mean ± SD from three independent experiments (**** P<0.0001 by ANOVA test followed by post hoc Dunnett's multiple comparison tests. ns: not significant).
A secreted factor NimrodB4 promotes the elimination of apoptotic corpses
by phagocytes in Drosophila (2021)
Figure 5

Figure 5. NimB4sk2 mutant macrophages have an increased number of vesicles

(A) Representative fluorescence microscopy images of wild type (w1118), NimB4sk2, draper, eater1 and croquemort△ third instar larvae macrophages stained with LysoTracker Red (live imaging). Overlay of fluorescence and differential interference contrast microscopy (DIC). Scale bar = 10 μm.

(B) Mean fluorescence intensity after staining with LysoTracker Red (live confocal imaging). Values from at least three independent experiments are represented as mean ± SD ( ** P<0.01, **** P<0.0001 by ANOVA test followed by post hoc Dunnett's multiple comparison tests).

(C) Representative transmission electron micrographs of macrophages from wild type (top, w1118) and NimB4sk2 (bottom) L3 wandering larvae. Scale bar: 2 μm

(D) Quantification of the number of vesicles per macrophages in the electron micrographs. Values from at least three independent experiments are represented as mean ± SD (**** P<0.0001 by Mann-Whitney test).

(E) Representative confocal imaging of Rab7EYFP immunostaining in wild type (w1118) and NimB4 sk2 macrophages. Tissues were stained with anti-GFP (red, Rab7), counterstained with phalloidin (gray) and DAPI (blue). The arrows indicate the enlarged vesicles are decorated with Rab7EYFP in the NimB4sk2 macrophages. Scale bar = 10 μm.

(F) Quantification of the diameter of the vesicles. Values from at least three independent experiments are represented as mean ± SD (**** P<0.0001 by Mann-Whitney test).
A secreted factor NimrodB4 promotes the elimination of apoptotic corpses
by phagocytes in Drosophila (2021)
Figure 6

Figure 6. Loss of NimB4 blocks the phagosome maturation process by impairing the phagosome-lysosome fusion

(A) Representative confocal imaging of localization of Lamp1-mcherry and LysoTracker Green in wild type (w1118), NimB4 sk2 and draper△5 macrophages (live imaging). The arrows indicate the colocalization (w1118) or the clustering (NimB4sk2 and draper△5) of Lamp1-mcherry and LysoTracker Green signals. Scale bar: 10 μm.

(B) Quantification of the colocalization of Lamp1-mcherry with LysoTracker Green, as measured by Pearson's correlation coefficient between the two signals. Values from at least five independent experiments are represented as mean ± SD (* P<0.05 by ANOVA test followed by post hoc Dunnett's multiple comparison tests).

(C) Ex vivo phagocytosis assay using apoptotic cells labelled with pHrodo™ Red. Wild type (w1118), NimB4sk2 and draper△5 macrophages from L3 wandering larvae were incubated with pHrodo™ Red apoptotic cells for 60 min at room temperature. Phagocytosis was quantified by flow cytometry. Data are represented as mean ± SD from three independent experiments (* P<0.05 by ANOVA test followed by post hoc Dunnett's multiple comparison tests. ns: not significant).

(D) Ex vivo phagocytosis assay using pHrodoTM Red S.aureus BioparticlesTM conjugates. Wild type, NimB4sk2 and NimC11, eater1 Hml∆-Gal4>UAS-GFP macrophages from L3 wandering larvae were incubated with pHrodoTM Red S. aureus BioparticlesTM for 30, 60 or 120 min at room temperature. Phagocytosis was quantified by flow cytometry. Data are represented as mean ± SD from three independent experiments (* P<0.05by ANOVA test followed by post hoc Dunnett's multiple comparison tests. ns: not significant).
A secreted factor NimrodB4 promotes the elimination of apoptotic corpses
by phagocytes in Drosophila (2021)
Figure 7

Figure 7. The increased expression of RAB7 rescues the phagosomes accumulation in

NimB4sk2 macrophages

(A-B) Representative fluorescence microscopy images of wild type (A) or NimB4sk2 (B) macrophages expressing Rab5 (middle panel) or Rab7 (right panel) driven by Hml∆-Gal4 and stained with the LysoTracker Red (live imaging). Overlay of fluorescence and DIC. Scale bar = 10 μm.

(C) Quantification of the mean fluorescence intensity of LysoTracker in macrophages from larvae (confocal live imaging). Values from at least three independent experiments are represented as mean ± SD (** P<0.01, **** P<0.0001 by ANOVA test followed by post hoc Dunnett's multiple comparison tests. ns: not significant).

Expanded View Figure legends
CLUH granules coordinate translation of mitochondrial proteins with mTORC1 signaling and mitophagy (2020)
Figure 1

Figure 1. CLUH forms specific RNA granules with its targets.

A-C Confocal images of primary hepatocytes grown under indicated conditions and stained with anti-CLUH antibody and Hadha (A), Pcx (B) and Actb (C) mRNA in situ hybridization. Right panels show 5x magnified boxed areas. Scale bar, 10 μm.

D-F Manders' colocalization coefficient between Hadha (D) Pcx (E) and Actb (F) mRNA molecules and CLUH signal (n ≥ 50 cells isolated from 3-6 mice).

G, H Confocal images of primary hepatocytes grown in HBSS and stained with anti-CLUH and anti-TIA-1 (G) or anti-G3BP1 (H) antibodies. Right panels show 5x magnified boxed areas. Scale bar, 10 μm.

Data information: In (D-F), data are presented as boxplots showing the median, the first and the third quartile. Error bars show minimum and maximum values. ***, P≤0.001 (Student's t-test).
CLUH granules coordinate translation of mitochondrial proteins with mTORC1 signaling and mitophagy (2020)
Figure 2

Figure 2. CLUH granules contain stalled and active translation sites.

A Confocal images of primary hepatocytes grown under indicated conditions and treatments after ribopuromycylation assay stained with anti-puromycin antibody. Scale bar, 10 μm.

B Quantification of fluorescence intensity per cell of experiment shown in (A). AU, arbitrary units. (n= 90-110 cells per treatment isolated from 4 mice).

C Quantification of the number of puromycin granules under indicated conditions (n ≥ 50 cells isolated from 4 mice).

D Confocal images of primary hepatocytes stained with anti-puromycin and anti-CLUH antibodies. The cells from which the enlarged areas (400 μm2) have been magnified are shown in Appendix Figure S1B for each individual channel. Scale bar, 4 μm. Cells analyzed were isolated from 4 different mice with similar results. Arrows point to colocalizing particles.

E Manders' colocalization coefficient between puromycin and CLUH from experiment shown in (D) (n ≥ 50 cells isolated from 4 mice).

F Quantification of puromycin granules containing CLUH signal (n ≥ 80 cells isolated from 4 mice).

Data information: In (B, C, E, F), data are presented as boxplots showing the median, the first and the third quartile. Error bars show minimum and maximum values. (C, F) ***, P≤0.001; **, P≤0.01 (Student's t-test). (E) ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test).
CLUH granules coordinate translation of mitochondrial proteins with mTORC1 signaling and mitophagy (2020)
Figure 3

Figure 3. CLUH granules are translationally active or dormant depending on the mRNA.

A-C Confocal images of primary hepatocytes after ribopuromycylation experiment combined with mRNA in situ hybridization for (A) Hadha, (B) Pcx and (C) Hmgcs2. Scale bar, 4 µm.

D-F Fluorescence profile of 100-pixel line from the merged channel shown in (A-C). The cells from which the enlarged areas (400 μm2) have been magnified are shown in Appendix Figure S2 for each individual channel. Cells analyzed were isolated from 6 different mice with similar results.
CLUH granules coordinate translation of mitochondrial proteins with mTORC1 signaling and mitophagy (2020)
Figure 4

Figure 4. CLUH granules form in absence of G3BPs and are distinct from SGs.

A Representative western blot of HeLa cells downregulated for G3BPs and overexpressing untagged CLUH. Asterisks indicates signal of previous incubation with anti-G3BP1 antibody. Pan-actin was used as loading control.

B Confocal images of anti-CLUH staining in HeLa cells downregulated for G3BPs and overexpressing untagged CLUH. Insets show 2.6x enlarged area. Scale bar, 10 µm.

C Quantification of cells with CLUH-granules from experiments shown in (B) and Figure EV2B (n=3 independent experiments, >50 cells per experiment per condition).

D Life imaging of WT and CLUH KO HeLa cells transfected with G3BP1-GFP plasmid and incubated in HBSS medium with or without CHX. Cells were recorded for a maximum of 2 h. Insets show 2.5x enlarged areas. Scale bar, 10 µm.

E Total number of cells analyzed by life imaging for the indicated experiments shown in (D). "Positive" relates to a cell which formed G3BP1 granules at the end of the recording.

F Confocal images of anti-G3BP1 staining in primary hepatocytes derived from Li-CluhWT and Li-CluhKO mice grown in indicated media. Arrows point to G3BP1-granules. Scale bar, 10 µm.

G Quantification of percentage of cells with G3BP1-positive granules of experiment shown in F. Cells were analyzed in a blind fashion (n=3 mice per genotype; number of cells in total: WT Basal, 449; KO Basal, 303; WT HBSS, 146; KO HBSS, 161).

H Western blot of primary WT and KO hepatocytes stained with indicated antibodies. Asterisks indicates unspecific signal. Pan-actin was used as loading control.

I Quantification of western blot shown in (H). (n=3 per genotype per condition).

J mRNA levels of G3bp1 in primary hepatocytes grown under indicated conditions. (n=4 per genotype per condition).

Data information: In (C, G, I, J), data are presented as histograms showing the mean ± SEM.

*, P≤0.05; **, P≤0.01; ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test).
CLUH granules coordinate translation of mitochondrial proteins with mTORC1 signaling and mitophagy (2020)
Figure 6

Figure 6. CLUH regulates mTORC1 activation and inhibits apoptosis upon starvation.

A Representative western blot of primary hepatocytes starved in HBSS for the indicated time points and probed with indicated antibodies. Pan-actin was used as loading control.

B, C Quantification of western blots as shown in (A). Antibody signal was normalized to pan-actin signal and signal of phospho-protein was normalized to signal of total protein (n=4 independent experiments).

D Representative western blot of total protein extracts from livers of starved Li-CluhWT and Li-CluhKO mice probed with indicated antibodies. GAPDH was used as loading control.

E, F Quantification of western blots as shown in (D). Antibody signal was normalized to GAPDH signal and signal of phospho-protein was normalized to signal of the total protein (n=8 mice per genotype).

G Confocal images of primary hepatocytes grown in HBSS for 16 h and stained with anti-cleaved caspase 3 antibody. Scale bar, 25 µm.

H Quantification of cleaved caspase 3 positive hepatocytes treated with or without rapamycin. More than 100 cells per replicate were analyzed in a blind fashion (n=3 independent experiments).

I IHC staining of cleaved caspase 3 in liver sections of starved Li-CluhWT and Li-CluhKO mice. Arrows point to positive apoptotic foci. Scale bar, 200 µm.

J Quantification of positive apoptotic foci of pictures shown in (I). The number of positive spots was relativized to the area analyzed (n=5 mice per genotype).

K. Confocal images of primary hepatocytes grown in indicated media and stained with anti-G3BP1 and anti-mTOR. Scale bar, 10 µm.

L Manders' colocalization coefficient between G3BP1 and mTOR staining (n >50 cells isolated from 3 mice).

Data information: In (B, C), data shows the mean ± SEM. **, P≤0.01 for genotype and ***, P≤0.001 for time (Two-way ANOVA). In (E, F, H, J), data are presented as histograms showing the mean ± SEM. In (L), data are presented as boxplots showing the median, the first and the third quartile. Error bars show minimum and maximum values. (E, F, L) ***, P≤0.001 (Student's t-test). (H, J) *, P≤0.05; ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test).
CLUH granules coordinate translation of mitochondrial proteins with mTORC1 signaling and mitophagy (2020)
Figure 7

Figure 7. Lack of CLUH promotes bulk autophagy.

A Confocal images of liver cryosections from Li-CluhWT and Li-CluhKO-LC3-GFP mice. Scale bar, 25 µm.

B Quantification of LC3 signal morphology of images shown in (A). Graphs show relative number of LC3 autophagosomes and the relative area occupied per nuclei. 4 images were quantified per animal and averaged. Nuclear staining was excluded for the analysis (n=4 mice per genotype).

C Confocal images of primary hepatocytes derived from Li-CluhWT and Li-CluhKO-LC3-GFP mice. Hepatocytes were starved for 2 h in HBSS. Scale bar, 10μm.

D-E Quantification of LC3 puncta number (D) and area (E) per cell (n ≥100 cells isolated from 3 mice).

F Western blot of total protein extracts from livers of Li-CluhWT and Li-CluhKO mice probed with LAMP1. GAPDH was used as loading control.

G Quantification of western blot shown in (F). WT: n=4; KO: n=3.

H Representative western blot of primary hepatocytes treated for 2 h with bafilomycin A in the indicated media and probed with indicated antibodies. Two different exposure times are shown for LC3. I refers to total LC3 and II indicates lipidated form of LC3. Asterisk indicates signal of previous incubation.

I Quantification of LC3 levels in western blots as shown in (H) (n=4 independent experiments).

J Quantification of autophagic flux of western blot as shown in (H). The flux was calculated as the increase in LC3II upon bafilomycin A treatment in each condition. The flux in KO cells was normalized to flux in WT cells. (n=4 independent experiments).

K, L Quantification of p62 and CLUH levels in western blots as shown in (H) (n=4 independent experiments).

Data information: In (B, G, I-L), data are presented as histograms showing the mean ± SEM. In (D, E), data are presented as boxplots showing the median, the first and the third quartile. Error bars show minimum and maximum values. (B, G, J) *, P≤0.05; **, P≤0.01; ***, P≤0.001 (Student's t-test). (D, E, I, K, L) *, P≤0.05; **, P≤0.01; ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test).
CLUH granules coordinate translation of mitochondrial proteins with mTORC1 signaling and mitophagy (2020)
Figure 8

Figure 8. CLUH promotes mitophagy.

A Western blot analysis of total protein extracts from livers of 8 weeks old Li-CluhWT and Li-CluhKO mice probed with indicated antibodies. GAPDH was used as loading control.

B Quantification of western blot shown in (A). WT: n=4; KO: n=3.

C Western blot analysis of isolated mitochondria from Li-CluhWT and Li-CluhKO mice probed with indicated antibodies. PonceauS staining was used as a loading control.

D Quantification of western blot shown in (C) (n=3 mice per genotype).

E Confocal images of liver cryosections derived from Li-CluhWT and Li-CluhKO-LC3-GFP mice and stained with anti-TOM20 antibody. On the right side, indicated boxes were 3.5x magnified. Arrows point to colocalizing spots. Scale bar, 25 μm.

F Manders' colocalization coefficient between TOM20 and LC3 from experiments shown in (E). 5 images were analyzed per animal to get an averaged value (n=5 mice per genotype).

G Confocal images of primary hepatocytes derived from Li-CluhWT and Li-CluhKO-LC3-GFP mice and stained with anti-TOM20 antibody. When indicated cells were treated with rapamycin (200 nM for 4h). Bottom panels show 2.3x magnified areas for each channel of indicated boxes. Arrows point to colocalizing spots. Scale bar, 10 μm.

H Manders´ colocalization coefficient from experiment shown in (G) (n ≥ 50 cells isolated from 3 mice per genotype).

I Confocal images of WT and Cluh KO MEFs transfected with mCherry-GFP-FIS101-152 and grown for 16 h in galactose medium or in medium containing 10 μM antimycin A/ oligomycin. Arrows indicate mitophagosomes. Scale bar, 10μm.

J Quantification of percentage of cells with mitophagosomes. Cells were considered positive when red signal was clearly recognizable (n= 3-5 independent experiments, >100 cells per experiment).

K Confocal images of WT and Cluh KO MEFs transfected with mCherry-GFP-FIS101-152, incubated with 200 nM rapamycin and grown for 16 h in galactose medium or in medium containing 10 μM antimycin A/ oligomycin. Arrows indicate mitophagosomes. Scale bar, 10 μm.

L Quantification of percentage of cells with mitophagosomes. Cells were considered positive when red signal was clearly recognizable (n= 3-5 independent experiments, >100 cells per experiment).

Data information: In (B, D, F, J, L), data are presented as histograms showing the mean ± SEM. In (H), data are presented as boxplots showing the median, the first and the third quartile. Error bars show minimum and maximum values. (B, D, F) ***, P≤0.001; *, P≤0.05 (Student's t-test). (H, J, L) *, P≤0.05; **, P≤0.01; ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test).
CLUH granules coordinate translation of mitochondrial proteins with mTORC1 signaling and mitophagy (2020)
Figure 9

Figure 9. Inhibition of mTORC1 and downregulation of G3BPs recovers mitochondrial clustering in absence of CLUH.

A Confocal images of liver cryosections from Li-CluhWT and Li-CluhKO mice injected with rapamycin or mock solution and stained with anti-TOM20 antibody. Scale bar, 25 µm.

B Quantification of morphological parameters of experiments shown in (A). 4 images were quantified per animal and averaged. Nuclear staining was excluded for the analysis (n=3 mice per genotype).

C Confocal images of liver cryosections from Li-CluhWT and Li-CluhKO mice injected with rapamycin or mock solution and stained with anti-LAMP1 antibody. Scale bar, 25 µm.

D Quantification of morphological parameters of experiments shown in (C). 4 images were quantified per animal and averaged. Nuclear staining was excluded for the analysis (n=3 mice per genotype).

E Confocal images of COS-7 cells downregulated for the indicated genes and stained with anti-TOM20 antibody. Scale bar, 10 µm.

F Representative western blot of COS-7 cells downregulated for the indicated genes of experiments shown in (E) and probed with indicated antibodies. Asterisks indicate unspecific signal or signal of previous incubation.

G Quantification of morphological parameters of experiments shown in (E) (n>100 cells from 3 independent experiments).

Data information: In (B, D), data are presented as histograms showing the mean ± SEM. In (G), data presented as boxplots showing the median, the first and the third quartile. Error bars show minimum and maximum values. (B, D) **, P≤0.01; *, P≤0.05 (Student's t-test). (G) ***, P≤0.001; **, P≤0.01 *, P≤0.05 (One-way ANOVA, Tukey's multiple comparisons test).

EXPANDED VIEW FIGURE LEGENDS
RGS9-2 rescues dopamine D2 receptor levels and signaling in DYT1 dystonia mouse models (2018)
Figure 1

Figure 1. Striatal levels of DRD2 and RGS9-2 are reduced in the Tor1a+/- and Tor1a∆gag/+ DYT1 dystonia mouse models.

A1) Representative WBs showing DRD2 and RGS9-2 downregulation in adult (P60-P90) Tor1a+/- (+/-) striata, characterized by a reduced torsinA protein level with respect to Tor1a+/+ (+/+) littermates. The striatal level of RGS7 is unchanged. The dot plot shows Tor1a+/- data, normalized to Tor1a+/+ controls of the same experiment. TorsinA: Tor1a+/+ N=7, Tor1a+/- N=6, t test ***P=0.0003; DRD2: Tor1a+/+ N=18, Tor1a+/- N=16, t test **P=0.0029; RGS9-2: Tor1a+/+ N=32, Tor1a+/- N=33, t test *P=0.0115). RGS7: Tor1a+/+ N=7, Tor1a+/- N=7, t test P=0.8721). WB quantification data, expressed as the ratio of protein vs. loading control intensity level, are normalized to the wild-type samples of the same experiment. A2,A3,A4) Time-course of changes in torsinA, DRD2 and RGS9-2 striatal levels along postnatal development. Summary plot data are normalized to the Tor1a+/+ P60 sample of each independent experiment. A2) TorsinA level is significantly reduced in mutants throughout the developmental period considered (P7 N=3; P14-P60 N=4; one-way ANOVA P<0.0001 and Bonferroni's Multiple Comparison Test **P<0.01 at P7, P14 and P60). A3,A4) DRD2 and RGS9-2 levels show parallel courses, with a similar increase from P7 to P21 in Tor1a+/+ as well as Tor1a+/- striatal lysates (P7: DRD2 N=3, RGS9-2 N=4; P14-P21 N=4; one-way ANOVA with Bonferroni's Multiple Comparison Test P>0.05). At P60, a simultaneous reduction of DRD2 and RGS9-2 levels is observed in Tor1a+/- striatal lysates (N=4; Tor1a+/- one sample t test DRD2: *P=0.0250, RGS9-2: *P= 0.0175). WB quantification data, expressed as the ratio of protein vs. loading control intensity level, are normalized to the wild-type P60 sample of the same experiment. B) Representative WB images and the dot plot show downregulation of torsinA, DRD2 and RGS9-2 striatal levels also in adult P60-P90 Tor1a∆gag/+ (∆gag/+) mice. TorsinA: Tor1a+/+ N=10, Tor1a∆gag/+ N=12, t test ***P= 0.0006; DRD2: Tor1a+/+ N=7, Tor1a∆gag/+ N=9, t test *P= 0.0183; RGS9-2: Tor1a+/+ N=10, Tor1a∆gag/+ N=9, t test *P= 0.0192. WB quantification data, expressed as the ratio of protein vs. loading control intensity level, are normalized to the wild-type samples of the same experiment. Mean ± SEM is represented in the graphs. C) Left. Representative image of coronal striatal slices of fresh frozen brains of wild-type and Tor1a+/- mice probed with the DRD2 radioligand 3H-spiperone. Scale bar 1.5 mm. (Right) Graph showing DRD2 binding density in Tor1a+/- striatal sections, obtained from the densitometric quantification analysis, expressed as percentage variation compared to control animals.