EMBO/soda-nel huggingface dataset:
Telomeric epigenetic response mediated by Gadd45a regulates stem cell aging and lifespan (2018)
Figure 5

Figure 5. APE1 inhibition restores telomeric heterochromatin marks and promotes the function of intestinal stem cell in G3Terc-/- mice.

A-C Representative images of small intestine organoids from WT and G3Terc-/- mice incubated with or without APE1 inhibitor (CRT0044876, 10μM) treatment. Arrows indicate the crypt-like structure in organoid cultures (A). Quantification of organoid number (per well) and size (in 104 μm2) (n=3-4 mice per genotype/treatment) (B-C).

D,E Representative images of γH2AX antibody staining on dissociated organoid cells from WT and G3Terc-/- organoid cultures with or without APE1 inhibitor (CRT0044876, 10μM) (DNAs are stained with DAPI) (D). Quantification of γH2AX positive cells (n=3-4 mice per group) (E).

F-H Telomeric DNA quantification and dot-blot assay of H3K9me3-ChIP (F, up and low panel), H3K9ac-ChIP (G, up and low panel), and HP1a-ChIP (H, up and low panel).

I,J Representative images of Olfm4 staining in small intestine sections from mice treated with or without APE1 inhibitor (CRT0044876, CRT). Arrows indicate Olfm4 positive intestine stem cells (I). Quantification of Olfm4 positive intestine stem cells (n=4 mice per group) (J).

Data information: Scale bar: 60 μm for Fig. 5A, 1 µm for Fig. 5D and 50 µm for Fig. 5I. Data are presented as mean±s.e.m.
Telomeric epigenetic response mediated by Gadd45a regulates stem cell aging and lifespan (2018)
Figure 6

Figure 6. GADD45A is induced in old human colon and its knockdown delays replicative senescence of human cells.

A The knockdown efficiency of shRNA-GADD45A in human WI38 fibroblasts.

B The proliferation curves of human WI38 fibroblasts treated with control shRNA or shRNA against GADD45A.

C,D Representative images of β-gal staining in human fibroblast cultures with control and shRNA-GADD45A treatments at passage 54 (C). Quantification of the percentage of β-gal positive cells in human fibroblasts with control and shRNA-GADD45A treatments (duplicates per group) (D).

E-G Representative images of GADD45A and γH2AX staining in colon sections from young (35-50 y/o) and old (75-85 y/o) people (E). Quantification of the percentage of GADD45A positive cells in young and old human colon tissues (n=8 per age group) (F). Quantification of the percentage of γH2AX positive cells in young and old human colon tissues (n=6 per age group) (G).

H Model for the role of Gadd45a in regulation of DNA damage response initiation. In G3Terc-/- mice, Gadd45a promotes the accessibility of telomeric heterochromatin and enhances the DNA damage response initiation (e.g. pATM activation) through a BER-dependent pathway. The initiated DNA damage response further causes upregulation of p53 and p21, which leads to impairment of intestine stem cell maintenance and function. In G3-dKO mice, the telomeric heterochromatin is condensed and damage response initiation is reduced upon Gadd45a deletion, which further leads to decreased expression of p53 and p21. Therefore, the maintenance and function of intestine stem cell is improved.

Data information: Scale bars represent 150 μm for Fig. 6C; 20 μm and 10 μm for Fig. 6E up and lower panel. Data are presented as mean±s.e.m.

Expanded View Figure Legends
Identification of a novel mechanism of blood-brain communication during peripheral inflammation via choroid plexus derived extracellular vesicles (2016)
Figure 1

Figure 1. LPS injection induces changes in extracellular vesicles (EVs) and miRNAs in the cerebrospinal fluid (CSF).

(A) Representative transmission electron microscope (TEM) image showing the presence of EVs in the CSF in two independent experiments. (B) NanoSight quantification of the amount of particles in the CSF 0, 1, 2, 4 and 6 hours after i.p. LPS injection (n=3-5). (C) Size distribution of the EVs in vivo in the CSF before (black; n=5) and 6 h after (grey; n=3) LPS treatment determined by NanoSight analysis. (D-G) Quantitative real-time polymerase chain reaction analysis of miR-1a (D), miR-9 (E), miR-146a (F), and miR-155 (G) (n=4). RNA was isolated from pooled CSF (50 µl) from different mice (n=3). Data are displayed as mean ± SEM and analyzed by Student’s t-test. Significance levels are indicated on the graphs: *, 0.01 ≤ P < 0.05; **, 0.001 ≤ P < 0.01.
Identification of a novel mechanism of blood-brain communication during peripheral inflammation via choroid plexus derived extracellular vesicles (2016)
Figure 2

Figure 2. Primary choroid plexus epithelial (CPE) cells secrete miRNA-containing EVs upon LPS incubation in vitro.

(A-B) In vitro quantification (A) and size distribution (B) of EVs isolated from conditioned medium of primary CPE cells grown in transwell system after 12 h in the absence (black) or presence (grey) of LPS (n=5) determined by NanoSight analysis. (C-E) TaqMan qPCR assay for the quantification of miR-9 (C), miR-146a (D) and miR-155 (E) on the exosomal pellet isolated from conditioned medium of primary CPE cells grown in a transwell system and stimulated for 12 h with LPS (n=3). (F-I) Quantification of the miRNAs miR-1a (F), miR-9 (G), miR-146a (H) and miR-155 (I) by TaqMan qPCR assay from primary CPE cells grown in a transwell system without or with LPS stimulation (n=3). Significance levels are indicated on the graphs: *, 0.01 ≤ P < 0.05; **, 0.001 ≤ P < 0.01.
Identification of a novel mechanism of blood-brain communication during peripheral inflammation via choroid plexus derived extracellular vesicles (2016)
Figure 3

Figure 3. Effect of exosome inhibition on EV and miRNA secretion of primary CPE cells stimulated with LPS.

(A) In vitro quantification of EVs isolated from conditioned medium of LPS-stimulated primary CPE cells grown in a transwell system in the absence or presence of the exosome inhibitor GW4869 (n=3). (B-D) TaqMan assay quantification of the miRNAs miR-9 (B), miR-146a (C) and miR-155 (D) in supernatant of LPS-stimulated primary CPE cells grown in a transwell system and either left untreated or pretreated with GW4869 to inhibit exosome secretion (n=3). miR-1a levels were below detection limit. (E-H) TaqMan assay quantification of the miRNAs miR-1a (E), miR-9 (F), miR-146a (G) and miR-155 (H) in cell lysate of LPS-stimulated primary CPE cells grown in a transwell system left untreated or treated with GW4869 to inhibit exosome secretion (n=3). Data are displayed as mean ± SEM and analyzed by Student’s t-test. Significance levels are indicated on the graphs: *, 0.01 ≤ P < 0.05; **, 0.001 ≤ P < 0.01.
Identification of a novel mechanism of blood-brain communication during peripheral inflammation via choroid plexus derived extracellular vesicles (2016)
Figure 4

Figure 4. Systemic inflammation activates the exosomal machinery in the choroid plexus.

(A) Representative confocal images of CD63, RAB5 and ANXA2 (red) in the choroid plexus (CP) 0, 4 and 8 h after LPS treatment. Hoechst (blue) was used to stain the nucleus. The dotted line indicates the ependymal cells that line the ventricle and the square boxes indicate the zoomed insert images displayed at the right corner of each image. Scalebar = 100 µm. (B-C) Representative TEM images showing the presence of MVBs in the CPE cells before (B) and 6 hours after (C) LPS administration in vivo. White arrow heads point to exosomes present in MVBs (n=3). Scale bar = 9 µm. (d-f) Quantification of amount of MVBs per cell section (D), amount of exosomes per MVB (E), and amount of exosomes per cell section (F), based on TEM analysis. (G-J) Quantitative real-time polymerase chain reaction (qPCR) analysis of miR-1a (G), miR-9 (H), miR-146a (I), and miR-155 (J). Data are presented as relative expression normalized with housekeeping miRs by TaqMan qPCR assay (0 h, n=4; 1 h, n=5; 6 h, n=5; 24 h, n=3). (K) NanoSight analysis of CSF isolated from LPS-injected mice followed by icv injection of vehicle or GW4869, a neutral sphingomyelinase inhibitor that inhibits exosome secretion (n = 8). (L) qPCR analysis of the expression of miR-1a, miR-9, miR-146a and miR-155 in the choroid plexus of mice injected with LPS and then icv injected with vehicle (black) or GW4869 (grey), a neutral sphingomyelinase inhibitor that inhibits exosome secretion (n = 4). (M) NanoSight analysis of supernatant of choroid plexus explants from PBS- or LPS-injected mice (n = 6). Data are displayed as mean ± SEM and analyzed by Student’s t-test. Significance levels are indicated on the graphs: *, 0.01 ≤ P < 0.05; **, 0.001 ≤ P < 0.01; ***, 0.0001 ≤ P < 0.001.
Identification of a novel mechanism of blood-brain communication during peripheral inflammation via choroid plexus derived extracellular vesicles (2016)
Figure 5

Figure 5. The choroid plexus reponds to different systemic inflammatory triggers.

(A-B) Representative confocal images of choroid plexus (CP) on brain sections from naive mice and four hours after LPS injection (n=3). Brain sections were stained for TLR4 (A) and TNFR1 (B) (red), pan-cytokeratine (panCK; green) and the nuclei were stained with Hoechst (blue). The ependymal cells aligning the ventricles are marked with a dotted line. Scalebar = 100 µm. (C) NanoSight analysis of EVs in the CSF in sham-operated (black) relative to CLP-treated (grey) mice 10 hours after surgery. (D) qPCR analysis of the expression of miR-1a, miR-9, miR-146a and miR-155 in the choroid plexus of sham-operated mice (black) and mice subjected to CLP (grey) 10 hours after surgery. (E) qPCR analysis of the expression of miR-1a, miR-9, miR-146a and miR-155 in the CSF of sham-operated mice (black) and mice subjected to CLP (grey) 10 hours after surgery. (F) NanoSight analysis of EVs in CSF of control (black) and TNF-injected (25 µg/20 g; grey) mice 6 hours after TNF challenge. (G) qPCR analysis of the expression of miR-1a, miR-9, miR-146a and miR-155 in the choroid plexus of control mice (black) and in mice injected with TNF (grey) 6 hours after injection. (H) qPCR analysis of the expression of miR-1a, miR-9, miR-146a and miR-155 in the CSF of control mice (black) and on mice 6 hours after TNF injection (grey). Data are displayed as mean ± SEM and analyzed by Student’s t-test. Scalebar 100 µm. Significance levels are indicated on the graphs: *, 0.01 ≤ P < 0.05; **, 0.001 ≤ P < 0.01; ***, 0.0001 ≤ P < 0.001.
Identification of a novel mechanism of blood-brain communication during peripheral inflammation via choroid plexus derived extracellular vesicles (2016)
Figure 7

Figure 7. EVs can cross the ependymal layer and deliver the miRNA cargo to the brain.

(A) Representative confocal image of brain parenchyma, four hours after intracerebroventricular injection of PKH26 labelled EVs (red). Astrocytes are stained with GFAP (white), nuclei with Hoechst (blue), and pan-cytokeratin (panCK; green) was used to label the choroid plexus. The dotted line shows the ventricular border and the white arrow heads point to EVs that crossed the ependymal cell layer. (B-C) Representative confocal images showing the uptake of double-labelled EVs (Ribogreen; green, PKH26, red) by astrocytes stained with GFAP (white) (B) and microglia cells stained with IBA1 (white) (C) incubated on mixed cortical cultures. Boxed areas are shown as zoomed images on right. Cell nuclei are stained with Hoechst (blue). The in vivo and in vitro uptake experiments were performed 3 and 2 times (n=3), respectively. Scalebar 100 = µm.
Identification of a novel mechanism of blood-brain communication during peripheral inflammation via choroid plexus derived extracellular vesicles (2016)
Figure 8

Figure 8. EVs repress mRNAs in target cells and transfer a pro-inflammatory message that is exosome dependent.

(A-B) qPCR gene expression analysis of miRNA target genes Mapk3, Notch1, Dicer, Tab2, Sox2, Calm2, Smad2, Smad5, Dnmt3 and Irak1 in mixed cortical cultures incubated with EVs isolated from CSF from untreated (black) or LPS treated (grey) mice (A) and in brain tissue (B) before (black) and 8 h after LPS injection (grey) (n=3). (C) qPCR gene expression analysis of inflammatory genes Il-1β, Tnf, Il-6, iNos and iκbα by qPCR in mixed cortical cultures (left; in vitro) incubated with EVs isolated from CSF from untreated (black) or LPS treated (grey) mice and in brain tissue (right; in vivo) before (black) and 8 h after LPS injection (grey) (n=3). (D) Cytokine analysis (IL-6, IL-1β and TNF) of supernatant of mixed cortical cultures (left; in vitro) incubated with EVs isolated from untreated (black; n=3) or LPS treated (grey; n=3) mice and of CSF (right; in vivo) from untreated (black; n=3) and LPS treated mice (grey; n=6). (E-F) qPCR gene expression analysis of inflammatory genes Il-1β, Tnf, Il-6, iNos and iκbα (E) and miRNA target genes Mapk3, Notch1, Dicer, Tab2, Sox2, Calm2, Smad2, Smad5, Dnmt3 and Irak1 (F) in mixed cortical cultures incubated with CD63-depleted EVs isolated from untreated mice (black) and mice treated with LPS for 6 h (grey) (n=3). (G-H) qPCR gene expression analysis of miRNA target genes Mapk3, Notch1, Dicer, Tab2, Sox2, Calm2, Smad2, Smad5, Dnmt3 and Irak1 (G) and inflammatory genes Il-1β, Tnf, Il-6, iNos and iκbα (H) in brain tissue from LPS-injected mice icv injected with vehicle (black) or GW4869 (grey; n=7). Data are displayed as mean ± SEM and analyzed by Student’s t-test. Significance levels are indicated on the graphs: *, 0.01 ≤ P < 0.05.
Male infertility associated Ccdc108 Regulates Multiciliogenesis via the Intraflagellar Transport Machinery (2022)
Figure 1

Figure 1. Ccdc108 is essential for ciliogenesis in multiciliated epidermis of the Xenopus embryo.

A, B. Depletion of Ccdc108 markedly affects bead movement by decreasing velocity (Movies EV 1-3). Fluorescent beads were applied to the dorsal surface of Xenopus embryos. Bead tracks every 500 ms over 2 s are shown (A). Plot of average bead velocity shows significantly reduced bead flow in ccdc108 morphants, which can be fully rescued by co-injecting with Xenopus ccdc108 mRNA (B). 150 beads from 15 embryos for each condition. Arrowheads indicate beads that showed no movement on the embryo surface through the recording.

C, D. Defective cilia formation in ccdc108 morphants. Embryos at stage 27 were fixed and stained with the acetylated tubulin (Ac-tub) antibody and/or Cep164 antibody. mRNA of a membrane-bound form of GFP (mGFP) was co-injected with each morpholino to indicate targeted cells. Representative confocal images (C) and 3D-SIM images (D) of the epidermis for each condition and plots show significantly reduced cilia number in Ccdc108 depleted embryos. (C) 20 images of 20 embryos and (D) > 80 MCCs from 6 embryos for each condition.

E. Scanning electron microscope images of Xenopus embryos display defective cilia formation in Ccdc108 depleted MCCs. White arrowheads mark MCCs with reduced ciliation. Boxed areas are magnified to show the details.

F. Defective cilia formation occurs in ccdc108 CRISPR mutants. Embryos at one cell stage were injected with Cas9 protein with or without the sgRNA against ccdc108 (108sg) and fixed at stage 27. Representative 3D-SIM images and plot show significantly reduced cilia number in ccdc108 CRISPR mutants. > 70 MCCs from 6 embryos for each condition. Cell membranes (mGFP, purple), cilia (Ac-tub, green) and basal bodies (Cep164, yellow) were labeled with indicated antibodies.

G. In situ hybridization chain reaction (HCR) reveals that ccdc108 mRNA is specially detected in MCCs. Embryos at stage 35 were fixed and subjected to in situ HCR (red). Embryos were then incubated with the Ac-tub antibody (cyan) and DAPI (gray).

Data information: Quantification data were collected from three independent experiments. Unpaired two-tailed t-test was performed (*** p<0.001). Mean ± s.d. values are presented.
Male infertility associated Ccdc108 Regulates Multiciliogenesis via the Intraflagellar Transport Machinery (2022)
Figure 2

Figure 2. The function of Ccdc108 in regulating multiciliogenesis is evolutionarily conserved across species.

A. In situ HCR reveals that ccdc108 mRNA is specially detected in ciliated organs in zebrafish. Zebrafish embryos at the long-pec stage (48 hours post fertilization) were fixed and subjected to in situ HCR (purple). Embryos were then incubated with the Ac-tub antibody (green).

B. Representative images and plots show that ccdc108 morphants display severe hydrocephalus (white arrowhead) in zebrafish, which was rescued by expression of Xenopus Ccdc108.

C, D. Representative confocal images and plots show that ccdc108 morphants displayed ciliogenesis defects in neuromasts (C) and olfactory placodes (D). Zebrafish embryos were fixed and stained with the Ac-tub antibody (green), phalloidin (purple) and Hoechst (blue).

E. Scanning electron microscope images of zebrafish embryos display a failure in cilia formation in Ccdc108 depleted olfactory placodes.

F. CCDC108 shows similar expression pattern to that of indicated cilia related proteins in mEPCs. Cells were collected at the indicated day after serum starvation were used for immunoblotting. IFT81 is an IFT complex protein, and RSPH1 is a component of the ciliary radial spoke structure. GAPDH was used as loading control.

G. Four independent shRNAs (108-i1, -i2, -i3 and -i5) effectively depleted CCDC108 without affecting the indicated cilia related proteins. GAPDH served as loading control. Ctrl-i, a control shRNA.

H. Representative 3D-SIM images and plot reveal that depletion of CCDC108 leads to a significant reduction in multiciliogenesis in mEPCs. mEPCs infected with lentivirus expressing indicated shRNA were serum starved for five days and subjected to immunostaining. GFP-CETN1 (blue) marked mEPCs infected with lentivirus. Basal bodies (CEP164, purple) and cilia (Ac-tub, green) were labeled with indicated antibodies. > 60 MCCs from three independent repeats for each condition were counted.

Data information: Quantitative data were from three independent repeats. Unpaired two-tailed t-test was performed (*** p<0.001; * p<0.05). Mean ± s.e.m (B, C, and D) and mean ± s.d. (H) values are presented.
Male infertility associated Ccdc108 Regulates Multiciliogenesis via the Intraflagellar Transport Machinery (2022)
Figure 3

Figure 3. Ccdc108 is required for apical trafficking of basal bodies and F-actin enrichment in MCCs of the Xenopus epidermis.

A. Depletion of Ccdc108 causes disorganized basal body distribution in MCCs. Representative 3D-SIM images (x-y) and 3D-reconstructions (x-z) of MCCs of embryos show a failure of apical trafficking of basal bodies in Ccdc108 depleted cells. Cell membranes (mGFP, blue), basal bodies (Cetn1, green) and distal appendages (Cep164, purple) were labeled with indicated antibodies. Select area of Cep164 channel was zoomed to show centriolar Cep164 accumulations. Orange arrowheads and numbers mark individual centriole. > 70 MCCs from 6 embryos for each condition were counted.

B. Transmission electron microscopy images of Xenopus embryos displaying aberrant apical trafficking and docking of centrioles in Ccdc108 depleted MCCs. Orange arrowheads mark centrioles. Percentage of centrioles docked to plasma membrane in each field were scored. At least 7 cells for each sample were counted.

C. Representative 3D-SIM images reveal that depletion of Ccdc108 has no effect on apical expansion in MCCs. Embryos treated as in Figure 1C were fixed and stained with phalloidin (purple). Cilia (Ac-tub, gray) were also presented. Apical area of MCCs in each condition were measured as described in Methods and plotted. > 80 MCCs from 6 embryos for each condition.

D. Representative confocal images show a significant reduction in apical actin following depletion of Ccdc108. Cell boundaries and apical actin network were marked with mGFP (green) and phalloidin (purple) and phalloidin intensity levels measured and plotted. > 80 MCCs from 6 embryos for each condition.

Data information: Quantitative data in (A, C, and D) were from three independent experiments. Unpaired two-tailed t-test was performed (*** p<0.001 ; ** p<0.01; * p<0.05). Mean ± s.d. values are presented.
Male infertility associated Ccdc108 Regulates Multiciliogenesis via the Intraflagellar Transport Machinery (2022)
Figure 4

Figure 4. Ccdc108 localizes to the ciliary axoneme and basal body in ciliated MCCs, and its ciliary accumulation is dependent on the Ccdc108-IFT interaction.

A. Live imaging of MCCs shows the distribution of Ccdc108 in MCCs cilia and basal bodies. Embryos expressing GFP-Ccdc108 (green) and Cetn4-RFP (purple) were imaged at stage 27. To better visualize ciliary structures, maximum intensity projections of images stacks for regions containing cilia and basal bodies are shown separately.

B, C. Ccdc108 interacts with IFT proteins. IFT candidate proteins were identified by Shotgun mass spectrometric analysis (B). Interactions were further determined by coimmunoprecipitation of endogenous IFT proteins (C) from HEK293T cells with GFP-Trap agarose beads. The table lists unique peptide numbers of indicated IFT proteins in the GFP and GFP-Ccdc108 samples.

D. Live imaging of MCCs shows both Ccdc108 and Ift74 proteins localize to the ciliary axoneme and the basal bodies in MCCs. Embryos expressing mCherry-Ccdc108 (green) and GFP-Ift74 (purple) were imaged at stage 27.

E. Identification of an evolutionarily conserved seven amino acid motif (dashed box) is essential for the Ccdc108 interaction with IFT-B proteins. Multiple protein sequences were analyzed with Clustal Omega program (* indicates fully conserved residue positions; : and . indicate strongly and weakly conserved residue positions respectively). Interactions were determined by coimmunoprecipitation of endogenous IFT74 and IFT81 from HEK 293T cells expressing GFP-luciferase, GFP-Ccdc108 and mutant proteins.

F Live imaging of MCCs shows the inability of Ccdc108 mutant proteins lacking the seven amino acid IFT interaction domain to localize to cilia. Embryos expressing GFP tagged Ccdc108 mutant proteins (green) and Cetn4-RFP (purple) were imaged at stage 27.
Male infertility associated Ccdc108 Regulates Multiciliogenesis via the Intraflagellar Transport Machinery (2022)
Figure 5

Figure 5. The Ccdc108-IFT interaction is essential for proper multiciliogenesis in MCCs.

A-C. The IFT-interaction domain is required for Ccdc108 multiciliogenesis function in Xenopus embryos: (A) impaired multiciliogenesis, (B) defective apical migration/docking of centrioles, and (C) reduced enrichment of apical F-actin. Embryos at stage 27 were fixed and stained with the acetylated tubulin (A; Ac-tub; green), Cep164 (B; purple) and Cetn1 (B; green) antibodies or phalloidin (purple). mRNA of a membrane-bound form of GFP was co-injected with each morpholino to indicate targeted cells (A,B). Typical D-SIM images (A,B) and confocal images (C) are present.

Embryos were treated as described in Figure 1C and expressed mGFP or GFP-Ccdc108 mutants. Plots show results from three independent experiments. (A) > 70 MCCs from 6 embryos, and (C) > 70 MCCs from 6 embryos for each condition. Unpaired two-tailed t-test was performed (*** p<0.001; **p<0.01). Mean ± s.d. values are presented.