EMBO/soda-nel huggingface dataset:
The RNA exosome complex degrades expanded hexanucleotide repeat RNA in C9orf72 FTLD/ALS (2020)
Figure 7

Figure 7: Arginine-rich DPRs have preferential interaction with endogenous EXOSC10

A. Schematic diagrams of codon optimized (lacking extensive G4C2 repeat) plasmids expressing N-terminally EGFP fused 89 repeats of either poly-GR, poly-PR or poly-GA under the control of CMV promoter.

B. Immunoblot validation of successful fractionation using antibodies against HDAC1 (nuclear marker) and GAPDH (cytoplasmic marker).

C. Co-immunoprecipitation of GFP-DPR interacting proteins in cytoplasmic fraction using GFP-trap beads. Immunoprecipitants were served for immunoblot with anti-EXOSC10 antibody. The same membrane was reprobed with anti-GFP antibody.

D. Quantification of the relative intensities of "EXOSC10 in co-IP fraction" / "EXOSC10 in input". 3 independent experiments.

E. Co-immunoprecipitation of GFP-DPR interacting proteins in nuclear fraction using GFP-trap beads followed by immunoblot using anti-EXOSC10 antibody. The same membrane was reprobed with anti-GFP antibody.

F. Quantification of the relative intensities of "EXOSC10 in co-IP fraction" / "EXOSC10 in input". 3 independent experiments.

Data information: All graphs are shown as mean ± SEM. *p < 0.05, ***p < 0.001; ANOVA with Dunnett's post-test vs GFP (D, F). A dot in graph represents a data point from single well of cell culture plate.
The RNA exosome complex degrades expanded hexanucleotide repeat RNA in C9orf72 FTLD/ALS (2020)
Figure 8

Figure 8: Arginine-rich DPR causes functional impairment of EXOSC10/the RNA exosome complex

A. Schematic diagrams of codon optimized (lacking extensive G4C2 repeat) plasmids expressing N-terminally EGFP fused 177 repeats of poly-GR or 166 repeats of poly-PR under the control of CMV promoter.

B, C. Increased expression of 3'extended snoRA48 precursor upon EXOSC10 knockdown (B) or GFP-poly-GR177/ GFP-poly-PR166 expression (C) on RT-qPCR. 6 independent experiments. Each experiment performed in duplicates.

D, E. Increased expression of 3'extended snoRA68 precursor upon EXOSC10 knockdown (D) or GFP-poly-GR177 /GFP-poly-PR166 expressions (E) on RT-qPCR. 6 independent experiments. Each experiment performed in duplicates.

F. RT-qPCR analysis showed increased cellular G4C2 repeat RNA expression from co-transfected EF1 (G4C2)80 repeat construct (Figure 1A) in the presence of GFP, GFP-poly-GR177 or GFP-poly-PR166. 3 independent experiments. Each experiment performed in duplicates.

Data information: All graphs are shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001; two-tailed unpaired t-test (B, D) or ANOVA with Dunnett's post-test vs GFP (C, E, F). A dot in graph represents a data point from single well of cell culture plate
The centrosome linker protein C-Nap1 maintains stem cells in mouse testes (2022)
Figure 1

Figure 1. CEP250 null mice show defect in germ line development in the testes and ovaries.

A. Size of adult (9-14 week) CEP250+/+ and CEP250-/- mice.

B. Body weight of CEP250+/+ and CEP250-/- mice (9-14 week). CEP250+/+ n=5 mice, CEP250-/- n=5 mice.

C. Brains from adult CEP250+/+ and CEP250-/- mice (9-14 week). Scale bars: 1000 µm.

D. Brain weight of 9-14-week-old mice. CEP250+/+ n=5 mice, CEP250-/- n=5 mice.

E. Adult male reproductive tract (9-14 week) CEP250+/+ and CEP250-/-.

F. Adult testis (9-14 week) of CEP250+/+ and CEP250-/- mice. Scale bars: 1000 µm.

G. Adult testis (9-14 week) weight of CEP250+/+ and CEP250-/- mice. CEP250+/+ n=10 mice, CEP250-/- n=10 mice.

H. Litter size (denoted by average number of offspring/litter) produced by mating pairs of the indicated genotypes of mice (9-14 week). Mating pairs, CEP250+/+ x CEP250+/+ n=19, CEP250-/- (male) x CEP250+/+ (female) n=9 matings, CEP250-/- (female) x CEP250+/+ (male) n=12 matings.

I. "H and E staining" of adult testis (9-14 week). Cross sections of CEP250+/+ and CEP250-/- testes are shown. Scale bars: 100 µm.

J. "H and E staining" of adult (9-14 week) epididymis caput (left) and epididymis caudal (right) cross sections CEP250+/+ and CEP250-/- mice. Scale bars: 100 µm.

K. Bright-field image of live epididymal sperm from CEP250+/+ and CEP250-/- mice (9-14 week). Scale bars: 10 µm. Arrow heads highlight the sperm heads.

L. Quantification of (K), sperm number per mouse. CEP250+/+ n=3 mice, CEP250-/- n=3 mice.

M. Immunofluorescence for MVH (green) with DAPI staining (blue) of 10 µm testis cryo-sections from adult CEP250+/+ and CEP250-/- mice (9-14 week). In CEP250+/+, numerous MVH+ cells were detected in the seminiferous tubules, whereas CEP250-/- seminiferous tubules lacked MVH+ germ cells. Scale bars: 100 µm.

N. Immunofluorescence for GATA4 (green) with DAPI staining (blue) of 10 µm testis cryo-sections from adult CEP250+/+ and CEP250-/- mice (9-14 week). Scale bars: 100 µm. Dashed lines highlight the STs.

O. Quantification of (N), CEP250+/+ n=3 mice, CEP250-/- n=3 mice. 50 STs were analyzed for each mouse. See Appendix Fig S2 for ST number per section and ST diameter.

Data information: In (B, D, G, H, L, O), data are presented as mean ± SEM. n.s., not significant, **P < 0.01, ****P < 0.0001 (the unpaired Student's t-test). In (B, D, G), whiskers represent the range of the data, with bottom and top represent min and max value of the dataset respectively.
The centrosome linker protein C-Nap1 maintains stem cells in mouse testes (2022)
Figure 2

Figure 2. Early development of male germ cells is affected by CEP250 deletion which is accompanied by increase in apoptosis.

A. Illustration of spermatogenesis after birth (Saitou & Yamaji, 2012).

B. Testis from neonatal mice (P5, P10 and P14). Scale bars: 500 µm.

C. Quantification of testis weight from (B). P5 CEP250+/+ n=5 mice, CEP250-/- n=4 mice, P10 CEP250+/+ n=7 mice, CEP250-/- n=4 mice, P14 CEP250+/+ n=4 mice, CEP250-/- n=4 mice.

D. Immunofluorescence for MVH (green) with DAPI staining (blue) of 10 µm testis cryo-sections from neonatal mice (P5, P10 and P14) CEP250+/+ and CEP250-/-. Scale bars: 20 µm.

E. Quantification of (D). CEP250+/+ n=3 mice, CEP250-/- n=3 mice.

F. Quantification of (G). C-CASP3 positive cells normalized to total cell number per seminiferous tubule (ST). Dots (colors reflect STs from different mice) represent value per ST, CEP250+/+ n=3 mice, CEP250-/- n=3 mice. 30 STs were analyzed for each mouse.

G. C-CASP3 (green) and MVH (magenta) staining with DAPI (blue) of 10 µm testis cryo-sections from neonatal mice (P5, P10 and P14) CEP250+/+ and CEP250-/-, scale bars: 20 µm.

H. Quantification of (G). Percentage of C-CASP3 and MVH double positive cells in relation to total C-CASP3 positive cells per ST from P5, P10 and P14 mice. Dots (colors reflect STs from different mice) represent value per ST, CEP250+/+ n=3 mice, CEP250-/- n=3 mice. 30 STs were analyzed for each mouse.

I. Quantification of (G). Percentage of C-CASP3 and MVH double positive cells in relation to total MVH positive cells (germ cells) per ST from P5, P10 and P14 mice. Dots (colors reflect STs from different mice) represent value per ST, CEP250+/+ n=3 mice, CEP250-/- n=3 mice. 30 STs were analyzed for each mouse.

Data information: In (C, E), data are presented as mean ± SEM. n.s., not significant, **P < 0.01, ****P < 0.0001 (the unpaired Student's t-test). In (F, H, I), data are presented as mean ± SEM. n.s., not significant, ***P < 0.005 , ****P < 0.0001 (the Mann-Whitney test).
The centrosome linker protein C-Nap1 maintains stem cells in mouse testes (2022)
Figure 3

Figure 3. Loss of CEP250 leads to premature differentiation.

A. Immunofluorescence staining for PLZF (red) with DAPI staining (blue) of 10 µm cryo-sections of testes from CEP250+/+ and CEP250-/- mice at P5, P10 and P14. Scale bars: 20 μm.

B. Quantification of (A). Quantification of PLZF+ cells per seminiferous (ST) section of CEP250+/+ and CEP250-/-, testes at P5, P10 and P14. Dots (colors reflect STs from different mice) represent value per ST, CEP250+/+ (P5) n=3 mice, CEP250-/- (P5) n=3 mice, 50 STs were analyzed for each mouse; CEP250+/+ (P10) n=3 mice, CEP250-/- (P10) n=3 mice, at least 30 STs were analyzed for each mouse; CEP250+/+ (P14) n=3 mice, CEP250-/- (P14) n=3 mice, at least 50 ST were analyzed for each mouse.

C. Immunofluorescence staining for SCP1 (green), PLZF (red) and DAPI (blue) of 10 µm cryo-sections of testes from CEP250+/+ and CEP250-/- mice at P2, P3, P5 and P7. Arrowheads highlight PLZF- SCP1+ (green) cells, which represent differentiated spermatogonia. Scale bars: 20 μm.

D. Illustration of sequential spermatogonia differentiation and formation of seminiferous tubules.

E-H. Quantification of (C). The number of PLZF+ SCP1-, PLZF+ SCP1+, PLZF- SCP1+ cells normalized to total cell number per ST section in CEP250+/+ and CEP250-/- testes at P2, P3, P5 and P7 respectively. Dots (colors reflect STs from different mice) represent value per ST, CEP250+/+ (P2-P7) n=3 mice, CEP250-/- (P2-P7) n=3 mice, at least 40 STs were analyzed for each mouse.

I. Summary of proportion of PLZF+ SCP1-, PLZF+ SCP1+, PLZF-, SCP1- cells per ST section in CEP250+/+ and CEP250-/- testes from P2-P7.

Data information: In (B, E-H), data are presented as mean ± SEM. n.s., not significant, *P<0.05, ***P < 0.005, ****P < 0.0001 (the Mann-Whitney test).
The centrosome linker protein C-Nap1 maintains stem cells in mouse testes (2022)
Figure 4

Figure 4. CEP250-/- mice show premature apical translocation of GSCs into the seminiferous tubule lumen.

A. Schematic diagram showing translocation of differentiating spermatogonia.

B. Representative images of mouse seminiferous tubule (ST) sections stained for PLZF (red), SCP1 (green) and DAPI (blue) at P4, P6 and P7. Scale bars: 20 μm. Arrowheads highlight PLZF- SCP1+ (green) cells localize to the middle of the ST lumen, which represent differentiated spermatogonia that translocated to the middle of the ST lumen.

C. Quantification of (B). Shown is the number of PLZF- SCP1+ cells which translocated inwards the seminiferous tubule lumen per seminiferous tubule section, as normalized to the total cell number per ST section. Dots (colors reflect STs from different mice) represent value per ST, CEP250+/+ n=3 mice, CEP250-/- n=3 mice, at least 30 STs were analyzed for each mouse.

D. Quantification of (B). Shown is the total number of SCP1+ cells which translocated inwards the seminiferous tubule lumen per ST section, as normalized to the total cell number per seminiferous tubule section. Dots (colors reflect STs from different mice) represent value per ST, CEP250+/+ n=3 mice, CEP250-/- n=3 mice, at least 30 STs were analyzed for each mouse.

E. Quantification of (B). Shown is the number of PLZF+ SCP1+ cells which translocated inwards the seminiferous tubule lumen per seminiferous tubule section, as normalized to the total cell number per seminiferous tubule section. Dots (colors reflect STs from different mice) represent value per ST, CEP250+/+ n=3 mice, CEP250-/- n=3 mice, at least 30 STs were analyzed for each mouse.

F. Summary of proportion of PLZF+ SCP1+ cells, which translocated inside the seminiferous tubule per section against which remained at the basal membrane.

G. Summary of proportion of PLZF- SCP1+ cells, which translocated inside the seminiferous tubule per section against which remained at the basal membrane.

Data information: In (C-E), data are presented as mean ± SEM. n.s., not significant, *P<0.05, **P < 0.01, ***P < 0.005 (the Mann-Whitney test).
The centrosome linker protein C-Nap1 maintains stem cells in mouse testes (2022)
Figure 5

Figure 5. CEP250 regulates the oriented division of male GCs.

A. Representative images of P4 mouse seminiferous tubule (ST) 10 µm cryo-sections stained for PLZF (red), γ-Tubulin (green), PH3 (magenta) and DAPI (blue). Arrowheads highlight the centrosomes. Dashed yellow line highlights the basement membrane. Semi-transparent white line, which bisects both spindle poles was used to determine spindle angle Ø orientation in relation to the basement membrane. Anaphase cells were analyzed. Scale bars: 5 µm.

B. Representative images of P5 mouse seminiferous tubule 10 µm cryo-sections stained for PLZF (red), γ-Tubulin (green), PH3 (magenta) and DAPI (blue). Arrowheads highlight the centrosomes. Dashed yellow line highlights the basement membrane. Semi-transparent white line, which bisects both spindle poles was used to determine spindle angle Ø orientation in relation to the basement membrane. Anaphase cells were analyzed. Scale bars: 5 µm.

C-F. (C, E) Quantitation of spindle angle of anaphase GSCs in P4 ST sections (from A). Spindle angle relative to basement membrane for CEP250+/+ and CEP250-/-). In (C), dots (colors reflect different mice) represent spindle angle of each GSC. In (E), the three dots represent averaged spindle angle per mouse. CEP250+/+ n=3 mice, CEP250-/- n=3 mice. At least 10 cells were analyzed for each mouse. (D, F) Quantitation of spindle angle of anaphase GSCs in P5 ST sections (from B). As (C, E) but for P5. In (D), dots (colors reflect different mice) represent spindle angle of each GSC. In (F), the three dots represent averaged spindle angle per mouse. CEP250+/+ n=3 mice, CEP250-/- n=3 mice. At least 10 cells were analyzed in each mouse.

G. Schematic diagram showing spindle angles relative to basement membrane were identified for CEP250+/+ and CEP250-/- GSCs. The spindle angle of CEP250+/+ mitotic GSCs mainly fall into two distinct populations. First, between 0° and 10°, indicating self-expansion. Second, between 75° and 90°, indicating differentiation. In contrast, CEP250-/- mitotic GSCs display more randomized distribution of spindle angles.

Data information: In (E, F), data are presented as mean ± SEM. n.s., not significant, *P<0.05, ***P < 0.005 (the unpaired Student's t-test).
The centrosome linker protein C-Nap1 maintains stem cells in mouse testes (2022)
Figure 6

Figure 6. CEP250-/- mice show defects in E-cadherin polarity change during cell cycle progression.

A. Illustration showing distinctive polarity patterns regarding E-cadherin in GSCs. The patterns were classified using following criteria. "Non-polarized: nearly continuous signal of E-cadherin patches across the cell cortex; basal polarized: preferable localization of E-cadherin to the side of cell cortex close to the basement membrane; apical polarized: preferable localization of E-cadherin to the side of cell cortex pointing away from the basement membrane; side polarized: preferable localization of E-cadherin to the side of cell cortex, which is nearly perpendicular to the basement membrane; null: no E-cadherin signal on the cell cortex." The representative immunofluorescence images for each pattern were shown below the illustrations. 10 µm cryo-sections stained for PLZF (red), E-cadherin (green) and DAPI (blue) showing non-polarized, basal polarized, apical polarized pattern respectively. Note, the non-polarized cell example in (A) is identical to the CEP250+/+ cell shown in (B). The basal polarized cell example in (A) is identical to the CEP250+/+ cell shown in Fig EV3C.

B. Representative images of P5 mouse ST. 10 µm cryo-sections stained for PLZF (red), E-cadherin (green) and DAPI (blue) showing GSCs in interphase. Scale bars: 5 μm.

C. Quantification of (B). Percentage of GSCs displaying different polarity status are indicated. CEP250+/+ n=5 mice, CEP250-/- n=4 mice. At least 100 cells were analyzed in each mouse and the average was calculated for each mouse.

D. Representative images of P5 mouse ST. 10 µm cryo-sections stained for PLZF (red), E-cadherin (green) and PH3 (magenta) showing GSCs in prophase. PH3 staining marks the mitotic chromatin. Scale bars: 5 μm.

E. Quantification of (D). Percentage of GSCs displaying different polarity status are indicated. CEP250+/+ n=5 mice, CEP250-/- n=4 mice. At least 10 cells were analyzed in each mouse and the average was calculated for each mouse.

F. Representative images of P5 mouse ST 10 µm cryo-sections stained for PLZF (red), E-cadherin (green) and PH3 (magenta) showing GSCs in prometaphase. PH3 staining marks the mitotic chromosome condensation. Scale bars: 5 μm.

G. Quantification of (F), percentage of GSCs displaying different polarity status are indicated. CEP250+/+ n=5 mice, CEP250-/- n=4 mice. At least 30 cells were analyzed in each mouse and the average was calculated for each mouse.

Data information: In A, B, D, and F, white arrowheads highlight basement membrane; yellow arrowhead highlights cortical enrichment of E-cadherin towards the basement membrane. In (C, E, G), data are presented as mean ± SEM. n.s., not significant, *P<0.05, **P < 0.01, ***P < 0.005 (the unpaired Student's t-test).
The centrosome linker protein C-Nap1 maintains stem cells in mouse testes (2022)
Figure 7

Figure 7. CEP250 regulates centrosome cohesion and centrosome positioning in GSCs during mitosis.

A. Representative images of P5 mouse seminiferous tubule (ST) 10 µm cryo-sections stained for PLZF (red), ODF2 (green), NIN (magenta) and PH3 (blue) showing GSCs in prophase. Scale bars: 2 μm. Yellow arrowheads highlight the two centrosomes.

B. Quantification of (A). Centrosome distance in GSCs in prophase at P5. CEP250+/+ n=3 mice, CEP250-/- n=3 mice. At least 50 cells were analyzed in each mouse (color of dot indicates mouse). Dashed line indicates 4 μm as the threshold of well separated centrosomes.

C. Illustration showing methodology to determine the mother centrosome and the daughter centrosome. In brief, the intensity of centrosomal NIN and ODF2 in GSCs in prometaphase was measured. The ratio of centrosomal NIN or ODF2 intensities of centrosomes close v.s. far from the basement membrane in one GSC was calculated. Thresholds were drawn at 85% and 115%, indicating >15% difference. A GSC with both NIN and ODF2 intensity ratio <85% was considered to display mother centrosome close to the basement membrane. A GSC with both NIN and ODF2 intensity ratio >115% was considered to display daughter centrosome close to the basement membrane.

D. Representative images of P5 mouse seminiferous tubule 10 µm cryo-sections stained for PLZF (red), ODF2 (green), NIN (magenta) and PH3 (blue), focusing on PLZF+ germ stem cells (GSCs) in prometaphase. Scale bars: 2 μm. Yellow lines highlight basement membrane. Yellow arrowheads highlight the mother centrosomes and white arrowheads highlight the daughter centrosomes.

E. Quantification of (D). Percentage of prometaphase GSCs which preferentially position their mother centrosome close to the basement membrane in CEP250+/+ and CEP250-/- testis at P5. CEP250+/+ n=3 mice, CEP250-/- n=3 mice. At least 50 cells were analyzed in each mouse and the average was calculated for each mouse.

F. Schematic diagram showing CEP250 regulates the position of centrosomes in GSCs during mitosis. In CEP250+/+ GSCs centrosome linker keeps the centrosomes relatively close until late prophase, which induces the formation of basal enrichment of E-cadherin. On the one hand, such enrichment might be important in determining the horizontal position of centrosome to facilitate the horizontal division and stem cell maintenance. In addition, basal E-cadherin enrichment helps to position the mother centrosome relatively close to the basement membrane, therefore facilitates the inheritance of mother centrosome by the GSCs. In contrast, such regulation of centrosome positioning is lost in CEP250-/- GSCs. Thus CEP250-/- GSCs display randomized spindle orientation and randomly inherit mother or daughter centrosomes.

Data information: In (B), data are presented as mean ± SEM. ****P < 0.001 (the Mann-Whitney test). In (E), data are presented as mean ± SEM. ****P < 0.001 (the unpaired Student's t-test).

Expanded View Figure Legends
Resident macrophages restrain pathological adipose tissue remodeling and protect vascular integrity in obese mice (2021)
Figure 3

Fig. 3: ATM-depleted CD169-DTR mice show increased hypertrophic and dysfunctional adipocytes.

A-B. Representative flow cytometric showing the specific depletion efficiency of ATM subsets from eWAT of CD169-DTR ND- (A) or HFD-treated (16 weeks) (B) mice. A representative analysis of three independent experiments is shown. C-D. eWAT was collected from lean (C) and obese (HFD 16 weeks) (D) WT or CD169-DTR mice after 12 days DT-mediated ATM depletion. Representative pictures of dissected eWAT are shown on the left, the corresponding eWAT weights expressed as mean ± SD of more than 12 samples are shown in the right, with statistical significance calculated using the Student's t-test (**P < 0.01 and ****P < 0.0001). The zoomed-in area shows clear, oily lipid accumulation in the absence of ATMs (lower left part). E-F. Adipocyte hypertrophy is visible in the absence of ATMs. Representative images of hematoxylin and eosin staining (H&E) (E) and corresponding adipocyte diameters (F) of lean and obese (HFD 16 weeks) eWAT from WT or CD169-DTR mice treated for 7 or 12 days with DT. Scale bar, 50 μm. Each group comprised 3-5 mice. Statistical significance was determined using one-way ANOVA test. **P < 0.01; ****P < 0.0001; ns: no significant difference. The box and whisker plots show the median value and 10-90 percentiles of adipocyte diameters. G. Relative expression of adipokines (adiponectin, leptin, and resistin) in eWAT obtained from WT or CD169-DTR mice, as measured by qPCR after 4, 7, and 12 days DT treatment. Black circles, ND-fed mice; white circles, HFD-fed (16 weeks) mice (n ≥ 5 mice per group, mean ± SD).
Resident macrophages restrain pathological adipose tissue remodeling and protect vascular integrity in obese mice (2021)
Figure 4

Fig. 4: Perturbed lipid metabolism in eWAT in the absence of CD169+ ATMs.

A-B. 2D PCA plots showing distinct lipid metabolite profiles of lean (square) and obese (round, HFD 16 weeks) WT (light green and light blue) and CD169-DTR (A) or anti-CSF1R antibody-injected (B) (red and orange) mice. Mice were under DT or anti-CSF1R antibody treatment for 12 days. C. Hierarchical clustering heatmap showing the major changes in lipid metabolites in eWAT among the ND or HFD (16 weeks) CD169-DTR or anti-CSF1R antibody-treated groups. D. Dot plots showing the percentage of perturbed lipid categories (fold change >2) in HFD (16 weeks) CD169-DTR mice. Upregulated (left) or downregulated (right) lipid metabolites between HFD (16 weeks) WT and CD169-DTR mice were classified into their lipid categories according to the LIPID MAPS Structure Database (https://www.lipidmaps.org/data/classification/LM_classification_exp.php). E. qPCR showing the relative expression of genes for adipogenesis (Pparγ), lipolysis (Lpl), and lipogenesis (Dgat1), plus other lipid receptor/transporters (Cd36, Abca1) or chaperones (Fabp4) in obese (HFD 16 weeks) eWAT of CD169-DTR (red bars) mice and DT-injected WT controls (blue bars) (upper panel) and obese eWAT of anti-CSF1R antibody-injected mice (green bars) mice and isotype control-injected WT controls (white bars) (lower panel). Mice were under DT or anti-CSF1R antibody treatment for 12 days. (n = 5 pooled mice per group, mean ± SD). Statistical significance was determined using an unpaired Student's t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ns: no significant difference.