EMBO/soda-nel huggingface dataset:
Fine Tuning of Histone Demethylase KDM6A/B Improves the Development of Nuclear Transfer Embryo (2018)
Figure 6

Figure. 6 KDM6B knockdown improved the SCNT embryo birth rate and DMD-ntES derivation.

A. Experimental scheme for generating dual reporter mice and SCNT.

B. Representative fluorescence images of siRNA-control or siRNA-6B injected SCNT embryos. Scale bar, 50 μm.

C. Quantification of embryos that expression tdTomato after injection with siRNA-control or siRNA-6B. Numbers of observed embryos are indicated. n ≥ 3, Error bars, s.e.m., ***P < 0.001 according to two-tailed Student's t-test.

D. Representative images of green fluorescence in Oct4::EGFP reconstructed blastocysts derived from injected siRNA-control or siRNA-6B SCNT embryos. Representative images from ≥ 25 embryos analyzed in three independent micromanipulations are shown. Green fluorescence indicates that the Oct4::EGFP transgene has been expressed. Scale bar, 50 μm.

E. Oct4::EGFP was up-regulated significantly in ICSI embryos compared with SCNT embryos at blastocyst stages, as determined by RT-qPCR. Data shown are mean expression values relative to Gapdh. The value in ICSI embryos was set as 1. Error bars, s.e.m., n ≥ 3. *P < 0.05, ***P < 0.001 by two-tailed Student's  t-test.

F. Representative image of cloned mice derived by siRNA-6B injected SCNT embryos.

G. Bar graph showing the efficiency of attachment to the feeder cells (left) and si6B-mdx-ntES derivation (right). The mdx sick mice tail-tip fibroblasts as nuclear donors. N, total number of embryos analyzed for each condition. Results are from four replicate experiments.

H. Immunostaining images of si6B-mdx-ntES expressed pluripotency markers. Scale bar, 50 μm.

I. The si6B-mdx-ntES possessed multiple-differentiation potential, as shown in embryoid body.  Scale bar, 100 μm.

J. An image of a chimeric mouse derived from si6B-mdx-ntES.
Fine Tuning of Histone Demethylase KDM6A/B Improves the Development of Nuclear Transfer Embryo (2018)
Figure 7

Figure. 7 Analyses of Molecular Features of knockdown KDM6B assisted SCNT.

A. Schematic illustration of the experimental procedures.  We combined the live-cell imaging, blastomere biopsy and single-cell RNA sequencing to accurate analysis.

B. Heatmap comparing ZGA genes expression between WT-2 and NT-2 and si6B-NT embryos (FC > 5, FPKM > 5 in each replicate; left). A total of 1,813 DEGs are classified into two groups by unsupervised hierarchical clustering. KEGG and GO analysis of the two groups by unsupervised hierarchical clustering (right).

C. Scatter plots comparing the si6B-NT and NT-2 genes expression. The higher expression genes in siRNA-6B and NT-2 are colored with red and blue, respectively (FC > 5, FPKM >5).

D. MA plot comparing gene expression between si6B-NT and NT-2. The data analyzed derive from two independent biological replicates. Arrows represent data points outside of the plotting area. DEG, differentially expressed genes.

E. Venn diagram showing the overlap between the genes that failed to be activated in SCNT 2-cell embryos and derepressed in knock-down KDM6B (left). Heat-map, KEGG and GO enrichment showing the expression pattern of 319 overlap genes (FC > 5, FPKM >5; right).

F. Schematic illustration of the experimental approach.

G. Representative localization of Xist expression in the nuclei of SCNT embryos injected with siRNA-6A, siRNA-6B or siRNA-Xist (left). Arrows indicate the blastomeres enlarged in the bottom panels. The ratios of blastomeres classified according to the positive or negative expression of Xist analyzed by RNA-FISH (right). Each bar represents a single embryo.

H. Large-scale qPCR analysis of Xist and eight X-linked genes in 4-cell stage male embryo. Each rectangle bar represents the expression value detected by single embryo RT-qPCR. The number of embryos in each group is indicated. Coloured bars indicate expression levels.

I. Image of full-term cloned pups derived from NT Sertoli cells injected with siRNA-control, siRNA-Xist, or siRNA-Xist-6B.
Functional divergence of the two Elongator subcomplexes during neurodevelopment (2022)
Figure 3

Figure 3. Patient-derived ELP4/6 mutations inhibit Elongator activity despite the proper complex assembly

A. Model of the murine Elongator complex (Elp123456) prepared using cryo-EM structure of yeast Elp123456 (yELP123456; PDB:). The [4Fe4S] cluster of Elp3 subunit is indicated by the orange cube. Murine Elp123 (mElp123) domain organization scheme: WD40 - beta-transducin repeat domain; TPR - tetratricopeptide repeat domain; Elp1* - C-teminal domain of second Elp1 subunit; DD- dimerization domain of two Elp1 subunits (top); CP1/CP2 - predicted contact points between Elp123 and Elp456. Murine Elp123456 model with depicted ELP4/6 mutations (bottom). Color code for molecules: mElp1 - orange, mElp2 - yellow, mElp3 - pink, mElp4 - green, mElp5 - blue, mElp6 - brown, Elp4 mutations (L296/L294 - magenta and Y91/Y90 - violet), Elp6 mutations (L118 - red and L126 - brown).

B. Gel filtration profiles from Superose 6 Increase 10/300 GL column for all reconstituted Elp123456 variants.

C. Pull-down experiments of the assembled mElp123456 complexes carrying patients-derived mutations. The mElp123WT subcomplexes were immobilized on the Dynabeads (strep) via Elp3 protein and incubated with the Elp456 variants. The inputs and pull downs were loaded on the SDS-PAGE gels and visualized by Coomassie staining. The molecular mass marker is indicated on the left (M).

D. Co-immunoprecipitation analyses of Elongator complexes in patient-derived fibroblast cell. Whole cell lysates (left) and eluates after immune-precipitation (right) are shown for control fibroblasts (ctrl) and cultured patient fibroblasts (Elp4Y91C/L296I (patient 1); Elp6L118W/L118W (patient 2), Elp6L118W/L118W (patient 3). Used antibodies for immobilization (ELP1) and detection (ELP1, ELP3, ELP4 , ELP5 and α-tubulin) are indicated. n = 4 independent experiments.

E. Normalized acetyl-CoA hydrolysis activity assay for mElp123456 variants incubated either without tRNA (left panel) or with human bulk tRNA (right panel). Statistical analysis: one-way ANOVA (α = 0.05). Statistically significant differences are indicated (*p ≤ 0.05; ****p ≤ 0.0001; ns - not significant), n = 3 independent experiments. Data represent mean ± SD.
Noncoding RNA circBtnl1 suppresses self-renewal of intestinal stem cells via disruption of Atf4 mRNA stability (2023)
Figure 1

Figure 1．CircBtnl1 is highly expressed in intestinal stem cells.

(A) A heatmap of circRNAs from small intestine crypts of C57BL/6 mice. The top eight highly expressed circRNAs (circOgdh, circRasa2a, circBtnl1, circMed13I, circVapa, circSlc43a2, circCnot6I, circAlg12) are listed.

(B) Small intestine crypts were isolated from C57BL/6 mice for organoid culture. The top eight upregulated circRNAs were depleted in organoid with a shRNA strategy, followed by organoid formation. Organoid formation ratios are shown as mean ± s. d.， n = 3 mice. *P < 0.05, **P < 0.01, NS, not significant, by one-tailed Student's t-test.

(C) Organoid images of small intestine (SI) (up) and colon (down) grown under circBtnl1 deletion and control. Scale bars，200 μm.

(D) One-week-old mice were euthanized for longitudinal sections. A global view of the section is shown in the left panel and the indicated tissues are shown to the right. 1, Brain; 2, Heart; 3, Liver; 4, Lung; 5, Stomach 6, SI (Small Intestine). Scale bars, 500 μm (left panel); 100 μm (magnified panels).

(E) Northern blot analysis of circBtnl1 in different tissues of two-month-old mice, 18S RNA served as a loading control.

(F， G) RNA fluorescence in situ hybridization of circBtnl1 in small intestine tissues (F) and organoid (G). Green region shows the distribution of circBtnl1 using antisense probe, gray region shows the nuclei staining by DAPI. For (F)，scale bars，5um; for (G)，scale bars，20 um.

(H) Relative distribution of circBtnl1 in mice ISCs determined by RT-qPCR in different cell fractions. U1 was served as the nuclear RNA marker and GAPDH was served as the cytoplasmic RNA marker. Data are shown as means ± SD. Data are representative of at least three independent experiments.
Noncoding RNA circBtnl1 suppresses self-renewal of intestinal stem cells via disruption of Atf4 mRNA stability (2023)
Figure 2

Figure 2．Deletion of circBtnl1 promotes the self-renewal of ISCs.

(A) Small intestine images of circBtnl1+/+ and circBtnl1-/-mice. n = 3 mice were used for SI detection. Small intestine length are shown as means ± SD. **P < 0.01 by two-tailed Student's t-test.

(B) H&E staining of global view small intestine (left) and all of three enlarged small intestine regions: duodenum, jejunum and ileum from circBtnl1+/+ and circBtnl1-/- mice (right). Scale bars, 50 μm. Number of crypts were calculated as means ± SD. **P < 0.01 by two-tailed Student's t-test in right panel. 3 fields were calculated for each group.

(C) Representative images of Ki67 staining of in small intestinal tissues from circBtnl1+/+ and circBtnl1-/- mice. For all representative images, at least three independent experiments were performed with similar results. For every microscope field, average Ki67+ ISC numbers per crypt are shown as means ± SD. **P < 0.01 by two-tailed Student's t-test in right panel. Scale bar, 50 μm.

(D) EdU staining of organoids from the circBtnl1+/+ and circBtnl1-/- mice under circBtnl1 overexpression. circBtnl1+/+ and circBtnl1-/- mice crypts were collected for organoid formation, and 10 μM EdU solution was added to the cultured organoid four hours before collected. Scale bars, 50 μm.

(E) H&E staining of small intestine tissues from indicated mice at different time points (D0 - D7) after 8 Gy radiation damage. Number of crypts were calculated as means ± SD. **P < 0.01 by two-tailed Student's t-test in right panel. n = 5 fields were calculated for each group. Scale bar, 50 μm.

(F) Quantification of organoid formation from intestinal crypts isolated from circBtnl1+/+ and circBtnl1-/- mice induced by radiation (8 Gy) on D5 and D7. (n = 3 circBtnl1+/+ and circBtnl1-/- mice were used and for each mouse, three separate organoid assays were performed). Crypt area were calculated as means ± SD. **P < 0.01 by two-tailed Student's t-test in right panel. Scale bar，200 μm.

(G) Confocal microscopy of ISCs makers Olmf4 in organoids from circBtnl1+/+ and circBtnl1-/- mice. Scale bars, 30 μm. Three independent experiments were performed with similar results, and representative experiments are shown. For every microscope field, average Olmf4+ ISC numbers per crypt are shown as means ± SD. **P < 0.01 by two-tailed Student's t-test in right panel in right panel.
Noncoding RNA circBtnl1 suppresses self-renewal of intestinal stem cells via disruption of Atf4 mRNA stability (2023)
Figure 3

Figure 3．CircBtnl1 binds Ddx3y in ISCs.

(A) Small intestine crypts from WT mice were lysed and incubated with biotin-labeled and linearized sense (circBtnl1 transcripts), anti-sense, or Sepharose 4B beads control. Eluted fractions were resolved by SDS-PAGE followed by silver staining and mass spectrometry. Representative protein Ddx3y was shown on the right.

(B, C) CircBtnl1 was co-localized with Ddx3y protein in small intestine tissue (B) and organoid (C) by RNA FISH, followed by immunofluorescence staining. Scale bars, 5 μm.

(D) Immunoblotting analysis of Ddx3y in RNA pull-down samples by circBtnl1 sense, antisense and control probes in mice ISCs.

(E) Immunoblotting analysis of Ddx3y in RNA pull-down eluate by circBtnl1 probes in circBtnl1+/+ and circBtnl1-/- ISCs.

(F) Domain mapping analysis of circBtnl1-binding domains of Ddx3y protein. Different domains of Ddx3y protein were incubated with circBtnl1, followed by RNA pulldown assay and Western blot.

(G) Electrophoretic mobility shift assay (EMSA) using biotin-labeled circBtnl1 and recombinant Ddx3y.

(H) Truncated fragments of biotinylated circBtnl1 were incubated with small intestine crypts lysates, followed by RNA pulldown assay and Western blot. Schematic diagram of circBtnl1 truncated fragments was shown in Figure S5C.

(I) Organoid formation was conducted with Ddx3y knockdown (Ddx3y KD), Ddx3y overexpression (oeDdx3y) and Ddx3y binding domain deletion overexpression (oeDdx3y△160-260) in circBtnl1+/+ and circBtnl1-/- ISCs. Typical images and organoid formation ratios are shown as means ± SD. **P < 0.01 by two-tailed Student's t-test. Scale bars, 200 μm.

(J) Confocal microscopy of cell proliferation makers Ki67 in Ddx3y KO and control organoids from circBtnl1+/+ and circBtnl1-/- mice. Scale bars，20 μm. Three independent experiments were performed with similar results, and representative experiments are shown.
Noncoding RNA circBtnl1 suppresses self-renewal of intestinal stem cells via disruption of Atf4 mRNA stability (2023)
Figure 4

Figure 4．CircBtnl1 inhibits Atf4 expression through facilitation of its mRNA stability.

(A) Volcano plot analysis of differentially expressed stem factors from circBtnl1+/+ and circBtnl1-/- mice ISCs. Representative genes were shown.

(B) Small intestine crypt cells were isolated from circBtnl1+/+ and circBtnl1-/- two-month-old mice, followed by RNA extraction and q-PCR analysis for top ten upregulated transcription factors. n = 3 independent samples. Results of relative fold changes are shown as means ± SD. **P < 0.01 by two-tailed Student's t-test.

(C) Small intestine crypts were isolated from C57BL/6 mice for organoid culture. The top ten upregulated transcription factors were depleted in organoid with a shRNA strategy, followed by organoid formation. Organoid-formation ratios are shown as mean ± s. d., n = 3 mice. *P < 0.05, **P < 0.01, ***P < 0.001, NS, not significant, by two-tailed Student's t-test.

(D) Immunoblotting analysis of Ddx3y in RNA pull-down samples by Atf4 sense, antisense and control probes in mice ISCs.

(E) Immunoblotting analysis of Ddx3y in RNA pull-down samples by Atf4 and control probes in circBtnl1+/+ and circBtnl1-/- mice ISCs.

(F) Immunoblotting analysis of Ddx3y in RNA pull-down samples by Atf4 sense probe and gradient circBtnl1 in mice ISCs.

(G) Domain mapping analysis of circBtnl1-binding domains of Ddx3y protein. Different domains of Ddx3y protein were incubated with circBtnl1, followed by Atf4 pulldown assay and Western blot.

(H, I) RNA blot analysis of Atf4 mRNA in circBtnl1+/+ and circBtnl1-/- ISCs treated with 2 μg ml-1 actinomycin D for the indicated times by Northern blot analysis (H) and q-PCR (I). n = 3 independent experiments. Results of relative fold changes are shown as means ± SD. *P < 0.05, ** P < 0.01, *** P < 0.001, NS, not significant, by two-tailed Student's t-test.

(J) Confocal microscopy of ATF4 in Ddx3y knockdown and overexpression organoids from circBtnl1+/+ and circBtnl1-/- mice. Scale bars, 20 μm.

(K, L) Expression levels of Ddx3y and Atf4 in circBtnl1+/+ and circBtnl1-/- ISCs were examined by real-time PCR (K) and Western blot (L), three independent experiments were performed. Results of relative fold changes are shown as means ± SD. ** P < 0.01, NS, not significant, by two-tailed Student's t-test.

(M) Organoid formation was conducted with Atf4 knockdown (Atf4 KD) from circBtnl1+/+ and circBtnl1-/- organoid. Typical images and organoid formation ratios (n = 3) are shown. Scale bars, 300 μm. **P < 0.05, NS, not significant, by one-tailed Student's t-test.
Noncoding RNA circBtnl1 suppresses self-renewal of intestinal stem cells via disruption of Atf4 mRNA stability (2023)
Figure 5

Figure 5．CircBtnl1 deficiency promotes ATF4 onto Sox9 promoter to trigger its expression.

(A) Putative ATF4-binding motifs are predicted in the mouse Sox9 promoter (up) by Homer assay. ATF4-binding motif CACAATGCCCC were shown in mouse and human Sox9 promoter.

(B) ChIP was performed to identify binding regions of ATF4 on Sox9 promoter in ISCs, followed by qPCR. IgG enrichment served as a control. Results are shown as means ± SD. **P < 0.01 by two-tailed Student's t-test. Data are representative of at least three independent experiments.

(C) Luciferase reporter assays were performed in Sox9 promoter-depletion (-160~-120) and control cells. Data are shown as means ± SD. **P < 0.01 by two-tailed Student's t-test. Data are representative of at least three independent experiments.

(D, E) ChIP assay was performed using anti-H3K4me3 (D) or anti-H3K27me3 (E) with circBtnl1+/+ and circBtnl1-/- ISCs lysates. **P < 0.01 by two-tailed Student's t-test. Data are representative of at least three independent experiments.

(F) DNase I accessibility assay was conducted using circBtnl1+/+ and circBtnl1-/- ISCs. n = 3 independent samples. **P < 0.01 by two-tailed Student's t-test.

(G) circBtnl1+/+ or circBtnl1-/- ISCs were subjected to nuclear run-on assay, followed by detection of Sox9 transcription through qPCR analysis. Results of relative fold changes are shown as means ± SD. ** P < 0.01 by two-tailed Student's t-test.

(H) Confocal microscopy of Sox9 in Atf4 knockdown (Atf4 KD) organoids from circBtnl1+/+ and circBtnl1-/- mice. Scale bars, 30 μm.

(I) Crypts were isolated from circBtnl1+/+ and circBtnl1-/- mice for organoid culture, and the circBtnl1 overexpression organoid was conducted from circBtnl1-/- organoid. RNA was extracted from the organoid and the expression of Sox9 was analyzed by RT-PCR. Results of relative fold changes are shown as means ± SD. **P < 0.01 by two-tailed Student's t-test. Data are representative of at least three independent experiments.

(J) Immunoblotting analysis of ATF4 and Sox9 in organoids from Ddx3y overexpression (oe Ddx3y) and Atf4 knockdown (Atf4 KD) as indicated in circBtnl1+/+ and circBtnl1-/-. Results are representative of two independent experiments.
Noncoding RNA circBtnl1 suppresses self-renewal of intestinal stem cells via disruption of Atf4 mRNA stability (2023)
Figure 6

Figure 6．Sox9-mediated signaling is required for ISC maintenance.

(A) Immunoblotting analysis of Sox9 and Olfm4 (stem cell maker) in small intestine crypts from circBtnl1+/+; sgSox9 and circBtnl1-/-; sgSox9 mice, vector served as control. Results are representative of two independent experiments.

(B, C) Representative images of Ki67 staining of in small intestine (B) and colon (C) from circBtnl1+/+ mice. For all representative images, at least three independent experiments were performed with similar results. Scale bar, 30 μm.

(D) H&E staining of small intestinal tissues from indicated mice. Two-month-old mice were used and n = 3 fields were taken for each group. Scale bar, 100 μm. Number of crypts were calculated as means ± SD. *P < 0.05, **P < 0.01 by two-tailed Student's t-test in right panel.

(E) H&E staining of intestinal tissues from indicated mice at different time points after 8 Gy of radiation damage. Two-month-old mice were used and n = 3 fields were taken for each group. Number of crypts were calculated as means ± SD. *P < 0.05, **P < 0.01 by two-tailed Student's t-test in right panel. Scale bar, 50 μm.

(F) Representative organoid images of circBtnl1+/+; sgSox9 and circBtnl1-/-; sgSox9 mice, sgCtrl served as control. Organoid formation ratios (n = 3) are shown. Organoid formation ratios are shown as mean ± s. d. *P < 0.05 by two-tailed Student's t-test. Scale bars, 200 μm.
Enhancing protective microglia activities with a dual function TREM2 antibody to the stalk region (2020)
Figure 1

Figure 1. Screening and molecular characterization of anti-mouse TREM2 antibodies

(A) Schematic representation of TREM2 processing by ADAM10/17. Cleavage occurs C-terminal of residue His157. The entire ectodomain (residues 19-171) was used for immunization of rats to generate TREM2 antibodies. sTREM2 (soluble TREM2), CTF (C-terminal fragment).

(B) Immunoblot analysis of membrane fractions of HEK293 Flp-In cells stably overexpressing both mouse TREM2 and mouse DAP12 upon treatment with 4D9 antibody reveals increased levels of membrane-bound TREM2 similar to what can be achieved by ADAM protease inhibition using the GM6001 inhibitor. An isotype antibody was used as a negative control. Calnexin served as a loading control. Levels of membrane-bound TREM2 were quantified by MSD ELISA. Data represent the mean ± SEM (n=3). One-way ANOVA, Tukey's post hoc test; p (DMSO vs GM) = 0.0011; p (DMSO vs isotype) = 0.992; p (isotype vs 4D9) = 0.0005; n.s., not significant.

(C) Immunoblot analysis of conditioned media from HEK293 Flp-In cells stably overexpressing both mouse TREM2 and mouse DAP12 upon treatment with 4D9 antibody reveals decreased levels of sTREM2 similar to what can be achieved by ADAM protease inhibition using the GM6001 inhibitor. An isotype antibody was used as a negative control. sAPPα served as a loading control. Note that heavy and light chains of the antibodies used for treatment are also detected and annotated. Levels of sTREM2 were quantified by MSD ELISA. Data represent the mean ± SEM (n=3). One-way ANOVA, Tukey's post hoc test; p (DMSO vs GM) < 0.0001; p (DMSO vs isotype) = 0.6372; p (isotype vs 4D9) < 0.0001; n.s., not significant.

(D) 4D9 antibody selectively detects TREM2 on the cell surface of HEK293 Flp-In cells stably overexpressing mouse TREM2 and mouse DAP12. An anti-HA antibody was used as a positive control while empty vector transfected HEK293 Flp-In cells were used as a negative control. Scale bar= 10µm.

(E) Peptide ELISAs detect anti-mouse TREM2 antibody binding to tiled stalk region peptides, full length stalk peptide or a truncated ADAM cleavage site peptide. The binding epitope of 4D9 antibody is located 12 amino acids N-terminal of the ADAM cleavage site at His 157.

(F) Sequence comparison of mouse TREM2 and human TREM2 shows substantial sequence conservation around the 4D9 epitope (upper panel). Immunoblot analysis demonstrates that antibody 4D9 is highly specific for mouse TREM2 and does not detect human TREM2 or mouse TREM1 (lower panel).

(G) 4D9 binding to mouse TREM2 ECD is competed off by a stalk region peptide. A competition ELISA demonstrates that a dose titration of stalk peptide reduces binding of 4D9 to TREM2 ECD with an EC50 of 1.3uM. Data represent the mean ± SEM (n=3).

(H) Surface plasmon resonance binding kinetics of increasing concentrations of 4D9 antibody to mouse TREM2 ectodomain evaluated by BIAcore, Kon = 5.9 x 105 M-1s-1, Koff = 4.0 x 10-5 s-1, KD = 68 pM. 4D9 binding to human TREM2 or mouse TREM1 was undetectable.