EMBO/soda-nel huggingface dataset:
Astrogenesis in the murine dentate gyrus is a life-long and dynamic process (2022)
Figure 2

Figure 2: Simultaneous occurrence of astrogenesis and neurogenesis in the developing DG.

A Experimental scheme of birth-dating paradigm used in B-D. NestinCreERT2; GFP mice were injected with the thymidine analogue 5-Bromo-2-desoxyuridine (BrdU, pink arrows) once at distinct postnatal timepoints of DG development (P3, P7, P14, P21) and killed at P28.

B Representative pictures of P28 animals that received BrdU at P3, P7, P14 or P21. For cell type identification of generated progeny, immunostainings against NESTIN (white), GFAP (green), and BrdU (magenta) were performed. rNSCs were identified as NESTIN+/GFAP+/BrdU+ with a radial process (as indicated with arrows), neurons as NESTIN-/GFAP-/BrdU+ cells, and astrocytes as NESTIN-/GFAP+/BrdU+ cells in the hilus, GZ, and ML (arrowheads).

C Quantification of total numbers of generated cell types (rNSCs, neurons, astrocytes) per area at distinct timepoints as indicated by different colours.

D Quantification of total numbers of newly generated astrocytes in the hilus, GZ, and ML at P3, P7, P14, P21.

E Experimental scheme of fate-mapping paradigm used in E-H. Perinatal NestinCreERT2; GFP mice were injected with tamoxifen (twice, 12 h apart) and killed at P3, P7, P10, or P14.

F Scheme illustrating that recombined Nestin+ rNSCs can self-renew and give rise to neurons and/or astrocytes.

G Representative pictures of immunostainings against cell type-specific proteins (all depicted in white). Recombined cells were identified by GFP, rNSCs by their radial process labelled with NESTIN (P3, P7) or GFAP (P10, P14), astrocytes by ACSBG1, immature neurons expressed DCX, and proliferating cells the cell cycle markers KI67 (P3, P7) or MCM2 (P10, P14).

H Quantification of the percentages of GFP+ recombined cell types out of all recombined cells at distinct timepoints. Intermediate precursor cells (IPCs) identified by TBR2 expression (Fig EV1H) were added to DCX+ neuroblasts.

I Quantification of the proportions of proliferating cell types (rNSCs, IPCs/neuroblasts, astrocytes) out of all proliferating cells at P3, P7, P10, P14.

Data information: Values represent mean ± SEM; number of experimental animals: (C,D) n = 5 (P3, P21), n = 4 (P7, P14); (H,I) n = 5 (P3), = 6 (P7, P10, P14); all numbers are indicated in Tables EV1, 2; dotted lines label GZ borders; scale bars = 50 µm.
Astrogenesis in the murine dentate gyrus is a life-long and dynamic process (2022)
Figure 4

Figure 4: Dynamics of adult astrogenesis under proneurogenic conditions.

A Experimental scheme of pro-neurogenic BrdU birth-dating paradigm used in B-D. 8 week-old hGFAPeGFP mice had access to running wheels, controls were housed under standard conditions. All mice were administered with BrdU via drinking water for 12 days and BrdU- incorporation was analysed at day 12.

B Representative images of BrdU-incorporating newly-generated cells in control and running hGFAPeGFP animals. BrdU+ cells (magenta) were assigned to distinct cell types: rNSCs [identified by their SOX2+ (cyan) cell bodies residing in the SGZ with a GFAP+ radial process (green)]; IPCs/neuroblasts [SOX2+ (cyan) nuclei located to the SGZ, DCX+ cells (white) in the SGZ and GZ, respectively], and astrocytes [SOX2+ cells (cyan) in the hilus, GZ, and ML]. Arrows mark newly generated neuronally committed cells (BrdU+/DCX+), while arrowheads indicate BrdU+ astrocytes (SOX2+).

C Quantification of total numbers of generated cell types (BrdU+) per area in the DGs of control and runner animals. rNSCs: **** p < 0.0001; IPCs/neuroblasts: *** p = 0.0007; astrocytes: ** p = 0.0032; unpaired t-test.

D Quantification of the percentages of generated cell types out of all generated cells (rNSCs, IPCs + neuroblasts, astrocytes) in the DGs of controls and runners; not significant, unpaired t-test.

E Representative images of proliferating (MCM2+; magenta) cells of adult control and running hGFAPeGFP animals; rNSCs, IPCs (arrows) and astrocytes (arrowheads) were identified by criteria described in (B). Single channel images are represented in Fig EV3D.

F Quantification of total numbers of proliferating cell types (MCM2+) per area in the DG of controls and runners. rNSCs: ** p = 0.0052; IPCs: *** p = 0.0008; astrocytes: * p = 0.0210; unpaired t-test.

G Experimental scheme of pro-neurogenic fate-mapping paradigm used in H, I. 8 week-old NestinCreERT2; GFP mice were intraperitonially injected with tamoxifen for five days (10 times, every 12 h). After tamoxifen induction, runners had access to running wheels, controls were housed in standard conditions for 12 days after tamoxifen injections.

H Representative images of recombined (GFP+; green) cells of distinct cellular identities: rNSCs [SOX2+ (cyan) with a NESTIN+ radial process (white)], IPCs (SOX2+ nuclei in the SGZ; cyan), DCX+ neuroblasts in the SGZ and GZ (magenta), and astrocytes (SOX2+ cells in the hilus, GZ, and ML; cyan).

I Quantification of the total numbers of recombined cell types (GFP+) per area in the DG of controls and runners; rNSC: p = 0.074; IPCs/neuroblasts: * p = 0.0460; astrocytes: p = 0.2314; unpaired t-test.

Data information: All data are represented as mean ± SEM; number of experimental animals (indicated by red dots): (C,D) n = 5 for controls, n = 6 for runners; (F) n = 4 for controls, n = 5 for runners; (I) n = 3 for both controls and runners; numbers are depicted in Table EV3; dotted lines border SGZ and GZ; scale bars = 50 µm.
Drug-like sphingolipid SH-BC-893 opposes ceramide-induced mitochondrial fission and corrects diet-induced obesity (2021)
Figure 1

Figure 1: SH-BC-893 protects from ceramide-induced mitochondrial network fragmentation by disrupting intracellular trafficking.

A) Citrate synthase staining in MEFs treated for 3 h with BSA or palmitate (250 μM) after a 3 h pre-treatment with DMSO (control), YM201636 (YM, 800 nM), NAV2729 (NAV, 12.5 μM) or SH-BC-893 (893, 5 μM).

B-D) ImageJ was used to calculate aspect ratio (B), branch length (C), and roundness (D) of mitochondria in the cells in (A) as described in Fig. EV1. PA, palmitate.

E) MEFs were treated with BSA, palmitate (250 μM), or C16:0 CER (100 μM) for 3 h after a 1 h pre-treatment with vehicle or SH-BC-893 (5 μM) and mitochondria stained as in (A).

F-H) aspect ratio (F), branch length (G), and roundness (H) of mitochondria in the cells in (E).

I) Mean C16:0 ceramide levels in MEFs pre-treated for 3 h with vehicle (n=7), SH-BC-893 (5 μM, n=7), myriocin (myr, 10 μM, n=5), or fumonisin B1 (FB1, 30 μM, n=3) then treated with BSA or palmitate (250 μM) for 3 h. Cells treated with C16:0 CER (100 μM, n=2) for 3 h shown as a positive control. Error bars, SEM.

J) MEFs treated as in (A) but evaluated for DRP1 (red) and citrate synthase (green) co-localization (yellow) using confocal immunofluorescence microscopy. Nuclei are labeled with DAPI (blue).

K) Mander's overlap coefficient for DRP1 and citrate synthase (CS) for the cells in (J) calculated on a per cell basis.

L,M) Representative DRP1 western blot (L) or quantification of DRP1 levels (M) using cells treated as in (J); n=3. Mean ± SD.

N) MEFs expressing vector, TRPML-1, or GRP1-DD were treated with BSA or palmitate (250 μM) for 3 h and stained as in (A).

O-Q) aspect ratio (O), branch length (P), and roundness (Q) of mitochondria in the cells in (N).

Data information: In B-D, F-H, K, and O-Q, 40 cells from 2 biological replicates were evaluated and mean ± SD shown. Using a one-way ANOVA with Tukey's correction (C,D,H,M,P and Q), Brown-Forsythe and Welch ANOVA tests with Dunnett's correction for multiple comparisons (B,F,G,K and O), or unpaired, two-tailed t tests (I), ***, P≤0.001; **, P≤0.01; *, P≤0.05; ns, not significant, P>0.05 (key comparisons shown). Scale bars, 20 µm.
Drug-like sphingolipid SH-BC-893 opposes ceramide-induced mitochondrial fission and corrects diet-induced obesity (2021)
Figure 3

Figure 3: SH-BC-893 acutely restores a tubular mitochondrial network in the liver and brain of HFD-fed mice.

A) Body weights of mice that had consumed a SD for 22 weeks or a HFD for 26 weeks; n=4-5. Mean ± SD. Mice are grouped to reflect their randomization to vehicle or SH-BC-893 groups, weight measured prior to treatment.

B) NAD(P)H autofluorescence evaluated by confocal microscopy in freshly resected livers from mice in (A) 4-8 h after treatment with vehicle (n=5) or 120 mg/kg SH-BC-893 (n=4) by gavage at ZT8.5. Mice were sacrificed and imaged in pairs (SD and HFD).

C,D) Aspect ratio (C) and roundness (D) of mitochondria in the livers shown in (B) calculated per field as described in Fig. EV3; 40 fields examined from each treatment group.

E) Body weights of mice that had consumed a chow or HFD diet for 22 weeks; n=3. Mean ± SD. Mice are grouped to reflect their randomization to vehicle or SH-BC-893 groups, weight measured prior to treatment.

F) Coronal brain section showing the areas where mitochondria were imaged in G-L.

G) Citrate synthase staining in the arcuate nucleus of mice in (E) 4 h after treatment with vehicle or 120 mg/kg SH-BC-893 by gavage at ZT8.5; n=3.

H,I) Aspect ratio (H) or roundness (I) of the mitochondria in (G); 18 fields examined from each treatment group.

J-L) As in G-I but imaging was performed in the areas of the cortex outlined in (F). 18 fields examined from each treatment group.

Data information: In A,C,D,E,H, I,K and L mean ± SD shown; 8-12 (C,D) or 6 (H, I, K and L) fields evaluated per mouse. Using a one-way ANOVA with Tukey's correction for multiple comparisons, ***, P≤0.001; **, P≤0.01; *, P≤0.05; ns, not significant, P>0.05 (key comparisons shown). Scale bars, 20 µm (B, G, and J) or 1 mm (F).
Drug-like sphingolipid SH-BC-893 opposes ceramide-induced mitochondrial fission and corrects diet-induced obesity (2021)
Figure 4

Figure 4: SH-BC-893 preserves mitochondrial function in the WAT of mice with HFD-induced obesity.

A) Body weight of mice fed the SD or HFD for 16 weeks; n=5. Mice are grouped to reflect their randomization to vehicle or 120 mg/kg SH-BC-893 groups, weight measured prior to treatment.

B) NAD(P)H autofluorescence was evaluated in epididymal fat (eWAT) freshly resected from the mice in (A) 4 h after treatment with vehicle or 120 mg/kg SH-BC-893 by gavage at ZT8.5; n=5.

C) NAD(P)H intensity from (B) calculated on a per cell basis; 16 - 25 cells from each of the 5 mice per group were quantified (87 - 109 total cells per treatment group).

D) Body weight of mice fed the SD or HFD for 16 weeks; n=5. Mice are grouped to reflect their randomization to vehicle or 120 mg/kg SH-BC-893 groups, weight measured prior to treatment.

E) TMRE (100 nM) and MitoTracker Green staining (200 nM) in eWAT resected from mice in (D) treated as in (B,C); n=5.

F,G) TMRE (F) or MitoTracker (G) intensity for (E) calculated on a per cell basis; 15 - 24 cells from each of the 5 mice in each group were quantified (99 - 104 total cells per treatment group).

H) MitoSOX (5 μM) and MitoTracker Green staining in eWAT resected from the mice in (D) treated as in (B and E); n=5.

I,J) MitoSOX (I) or MitoTracker (J) intensity calculated as in (F and G); 10 - 13 cells from each of the 5 mice in each group were quantified (57 - 58 total cells per treatment group).

K) Chop, Xbp1s, Atf6, and Grp78 mRNA measured in eWAT resected from mice maintained on a HFD for 13 weeks and gavaged with vehicle or 120 mg/kg SH-BC-893 on Monday, Wednesday, and Friday for the final 3 weeks; n=7-9.

Data information: In A,C,D,F,G, and I-K mean ± SD shown. Using a one-way ANOVA and Tukey's correction for multiple comparisons (A,D), Brown-Forsythe and Welch ANOVA tests with Dunnett's correction for multiple comparisons (C,F,G,I, and J), or unpaired t tests with Welch's correction (K), ***, P≤0.001; **, P≤0.01; *, P≤0.05; ns, not significant, P>0.05. Scale bars, 20 µm.
Drug-like sphingolipid SH-BC-893 opposes ceramide-induced mitochondrial fission and corrects diet-induced obesity (2021)
Figure 5

Figure 5: SH-BC-893 normalizes adipokine levels in mice consuming a HFD.

A,B) Plasma adiponectin (A) or leptin (B) in mice maintained on a SD or HFD for 8-9 weeks then gavaged with vehicle or 120 mg/kg SH-BC-893 4 h prior to blood collection. Pair-fed mice were provided with the amount of food consumed by SH-BC-893-treated mice in 4 h but left untreated; n=8-10.

C) The adiponectin:leptin ratio was calculated for the mice in A,B.

D,E) As in (A,B) but in mice maintained on the HFD for 10 weeks and then gavaged Mondays, Wednesdays, and Fridays for 3 weeks with vehicle or 120 mg/kg SH-BC-893; n=9. Rx, treatment number of a total of nine doses.

F) The adiponectin:leptin ratio was calculated for the mice in (D,E); n=9.

G,H) Mice were treated as in D-F, sacrificed 4 h after the ninth dose, and the levels of Socs3 (G) or Lepr (H) mRNA measured in the hypothalamus; n=8.

I) Body weights of mice fed the HFD for the indicated interval. Two cohorts were utilized, one measured at 4 and 8 wk and the other at 10 and 16 wk; n=16-20.

J,K) Plasma adiponectin and leptin were measured in the mice in (I) 4 h after a single dose of 120 mg/kg SH-BC-893; n=7-10.

L) The adiponectin:leptin ratio was calculated for the mice in J,K; n=7-10.

Data information: Mean ± SEM shown. Using a one-way ANOVA with Tukey's correction (A), Brown-Forsythe and Welch ANOVA tests with Dunnett's correction for multiple comparisons (B,C), or unpaired two-tailed t tests (D-H and J-L, Welch's correction applied in H), ***, P≤0.001; **, P≤0.01; *, P≤0.05; ns, not significant, P>0.05.
DEAD-box polypeptide 43 facilitates piRNA amplification by actively liberating RNA from Ago3-piRISC (2021)
Figure 1

Figure 1 Identification of RNA helicase DDX43 associated with Ago3

(A) Co-immunoprecipitation of the Siwi−Vasa complex in the presence and absence of EDTA. Isolated proteins were subjected to western blotting. n.i.: non-immune mouse IgG. (B) Co-immunoprecipitation and identification of Ago3-associated proteins. Left: The immunopurified Ago3 complex was resolved by SDS-PAGE and silver-stained. Right: DEAD-box helicases identified by mass spectrometry. Proteins identified by co-immunoprecipitation under EDTA-containing conditions only are listed in red. PSM: peptide sequence match. (C) Co-immunoprecipitation using BmN4 cells transfected with the Flag-tagged DEAD-box helicases identified by shotgun proteomics. The Flag-tagged proteins and Ago3 were detected by western blotting. Full-length Flag-tagged proteins are indicated with red asterisks. h.c.: heavy chain. (D) Co-immunoprecipitation of endogenous Ago3 and DDX43 in the presence or absence of EDTA. These factors were detected by western blotting. (E) Pull-down assay using high salt-washed Ago3-conjugated magnetic beads and purified His-DDX43. All samples were subjected to SDS-PAGE and detected by silver staining. (F, G) Analysis of Siwi-piRNA and Ago3-piRNA levels in Siwi and Ago3 complexes with DDX43, respectively. The WB (western blotting) and NB (northern blotting) signals represent the PIWI protein and piRNA levels, respectively (see also Figs EV1A and EV1B). The relative ratios of piRISC were calculated based on the signal intensities of the PIWI proteins and piRNAs.
DEAD-box polypeptide 43 facilitates piRNA amplification by actively liberating RNA from Ago3-piRISC (2021)
Figure 2

Figure 2 DDX43 depletion does not affect the formation of the Ago3−Vret complex

(A) Immunofluorescence showing the Vret (red) and Ago3 (green) signals in DDX43-depleted BmN4 cells (siDDX43). DAPI shows the nuclei (blue). Scale bar: 10 µm. The insets show high-magnification images of the part indicated sections. (B) Immunoprecipitation and western blotting showing that DDX43 associated with Vret and Ago3. (C, D) Immunoprecipitation of endogenous Siwi, DDX43, and Vret bound with Ago3 from DDX43-depleted (C) or Vret-depleted (D) cells. The factors were detected by western blotting. Ago3 interacts with Vret and DDX43 independently of the other. (E) Immunofluorescence showing Ago3 (red) and Flag-DDX43 (green) signals in BmN4 cells. DAPI shows the nuclei (blue). Arrowheads indicate colocalization of these factors. Scale bar: 10 µm.
DEAD-box polypeptide 43 facilitates piRNA amplification by actively liberating RNA from Ago3-piRISC (2021)
Figure 3

Figure 3 DDX43 unwinds Ago3-cleaved RNA and drives Siwi-piRISC biogenesis

(A) Analysis of Siwi-piRNA and Ago3-piRNA production in DDX43- or Vasa-knockdown BmN4 cells. Left: The expression levels of DDX43 and Vasa following RNAi treatment. Middle and right: Comparison of Siwi-piRNA and Ago3-piRNA levels following DDX43 or Vasa knockdown by RNAi. The piRNAs were isolated by the immunoprecipitation of Flag-tagged proteins from knockdown cells transfected with Flag-Siwi or Flag-Ago3 and then 32P-labeled. (B) Artificial Siwi-piRNA production from a reporter plasmid encoding the PiggyBac-piRNA target sequence. (C) Analysis of the artificial piRNA levels in DDX43-knockdown BmN4 cells. Left: Artificial piRNA and Flag-Siwi were detected by northern blotting and western blotting, respectively. Right: Quantification of artificial Siwi-piRISC formed by the ping-pong cycle pathway. Data represent the mean+/- standard deviations, n = 3 independent experiments. *p < 0.05 as determined with the t-test. (D) RNA unwinding assays upon Ago3-piRISC-dependent target RNA cleavage (Fig EV3D). Recombinant Vasa or DDX43 was added and incubated in the reaction mixture upon cleavage and the materials were separated using a magnet to supernatant (Sup.) and bead (Beads) fractions. RNAs cleaved by Ago3 are shown as "Cleaved RNA". DDX43 but not Vasa released the cleaved target RNA from Ago3-piRISC. (E) Time dependency of the DDX43 unwinding activity. Sup: supernatant. (F) RNA unwinding assays upon Ago3-piRISC-dependent target RNA cleavage as in (D). Recombinant DDX43 was added and incubated in the reaction mixture upon cleavage for 0 min (i.e., no cleavage), 15 min, 30 min, and 60 min, and the materials were separated using a magnet to supernatant (Sup.) and bead (Beads) fractions. RNAs cleaved by Ago3 are shown as "Cleaved RNA". DDX43 fails to unwind cleaved RNAs from Siwi-piRISC. − DDX43 means no DDX43 added in the reaction mixture.
DEAD-box polypeptide 43 facilitates piRNA amplification by actively liberating RNA from Ago3-piRISC (2021)
Figure 4

Figure 4 The KH domain in DDX43 is required for enzymatic activation

(A) The domain organization of DDX43 and detail of the mutants. The conserved KH domain and DEAD-box helicase domain are indicated. D399 and E400 are highly conserved residues within the ATPase active site in the helicase domain. (B) Quantification of ATPase activity of DDX43 variants. The graph on the right indicates the ratio of the hydrolyzed ATP produced by DDX43 mutants to that of the WT DDX43. Data represent the mean+/- standard deviations. n=3 independent experiments. *p < 0.002 as determined with the t-test. (C) Immunoprecipitation of Ago3 with Flag-tagged DDX43 WT and its D399A and E400Q mutants. Ago3 and DDX43 were detected by western blotting using anti-Ago3 and anti-Flag-antibodies, respectively. Flag-EGFP was used as a negative control. The E400Q mutation in DDX43 impairs the interaction between Ago3 and DDX43. IgG h.c.: IgG heavy chain. (D) Immunoprecipitation of Ago3 with Flag-tagged DDX43 WT and its ∆KH mutant lacking KH domain. Ago3 and DDX43 were detected by western blotting using anti-Ago3 and anti-Flag-antibodies, respectively. Flag-EGFP was used as a negative control. The ∆KH mutant maintains affinity to Ago3. IgG h.c.: IgG heavy chain. (E) RNA-binding activity of DDX43 variants as analyzed by EMSA. Upper: The GIDN mutant was substituted G90 and I96 conserved within the KH domain to Asp and Asn, respectively. Lower: A 40-nt single-stranded RNA was 5′ end-labeled with 32P and incubated with DDX43 variants in the presence or absence of 0.1 mM ATP. Supershift means non-canonical complex. (F) RNA unwinding assays upon Ago3-piRISC-dependent target RNA cleavage. Unwinding activities of DDX43 WT and its D399A, E400Q, and ∆KH mutants are shown. RNAs cleaved by Ago3 are shown as "Cleaved RNA". (G) RNA unwinding assays upon Ago3-piRISC-dependent target RNA cleavage. Unwinding activities of DDX43 WT and its GIDN mutant are shown. RNA cleaved by Ago3 is shown as "3′ cleaved RNA".
Peroxisomal protein PEX13 functions in selective autophagy (2016)
Figure 1

Figure 1. PEX13 is Required for Sindbis Virophagy but not General Autophagy.

A. Western blot detection of PEX13, ATG7, SMURF1, and FANCC in HeLa/GFP-LC3 cells transfected with the indicated siRNA. Asterisk denotes non-specific band.

B. Representative images of GFP-LC3 colocalization with mCherry-capsid at 10 h after Sindbis virus (strain AO30) infection of HeLa/GFP-LC3 cells treated with the indicated siRNA. siPEX13 oligo #2 is shown; similar results were observed with three other siPEX13 oligos. Arrowheads denote representative colocalized GFP-LC3/mCherry-capsid puncta. Scale bars, 10 μm.

C. Quantification of colocalized GFP-LC3 and mCherry-capsid puncta normalized to the number of mCherry-capsid puncta per cell (% colocalization) in the experiment shown in (B). Bars are mean ± SEM of triplicate samples (~100 cells analyzed per sample). Similar results were observed in more than three independent experiments. ***p<0.001; one-way ANOVA with adjustment for multiple comparisons. See Fig EV1A and B for quantification of mCherry-capsid puncta per cell and GFP-LC3 puncta per cell from the same experiment.

D. Western blot detection of Pex13 in MEFs of indicated genotype. Asterisks denote non-specific bands.

E. Quantification of colocalized GFP-LC3 and mCherry-capsid puncta normalized to the number of mCherry-capsid puncta per cell (% colocalization) at 8 h after Sindbis virus (AO28 strain) infection in MEFs of the indicated genotype. Bars are mean ± SEM of triplicate samples (~100 cells analyzed per sample). Similar results were observed in more than three independent experiments. *p<0.05; two-tailed unpaired t-test. See Fig EV1G and H for quantification of mCherry-capsid puncta per cell and GFP-LC3 puncta per cell from the same experiment.

F. Western blot detection of p62 and LC3 in MEFs of indicated genotype treated with 100 nM Baf A1 or DMSO vehicle and cultured in normal medium (starvation “-”) or EBSS (starvation “+”) for 3 h. Similar results were observed in three independent experiments.

G. Quantification of GFP-LC3 puncta in MEFs of indicated genotype treated with 10 nM Baf A1 or DMSO vehicle and cultured in normal medium or HBSS (starvation “+”) for 3 h. Bars are mean ± SEM of triplicate samples (~100 cells analyzed per sample). *p<0.05; two-tailed unpaired t-test. NC, nonsilencing control.