EMBO/soda-nel huggingface dataset:
MIWI2 targets RNA Transcribed from piRNA-dependent Regions to drive DNA Methylation in Mouse Prospermatogonia (2018)
Figure 6

Figure 6. RMER4B sequence in the chr7 piRNA cluster is required for de novo DNA methylation of the Rasgrf1 DMR.

A. Depletion of piRNAs from chr7 piRNA cluster RMER4B sequence in chr7 piRNA clusterΔRMER4B/ΔRMER4B mice. Top panel shows overall view of chr7 piRNA cluster, and lower panel shows magnified view of RMER4B region in chr7 piRNA cluster. Only uniquely mapped piRNAs are shown.

B. Depletion of piRNAs that potentially target pit-RNA in chr7 piRNA clusterΔRMER4B/ΔRMER4B mice. piRNAs are mapped to RMER4B region of Rasgrf1 DMR (deleted sequence in Rasgrf1ΔRMER4B/ΔRMER4B mice) allowing two mismatches. Black bars represent piRNAs that are mapped to this region in antisense direction to pit-RNA. The numbers of genomic loci where each of these piRNAs is perfectly matched (loci), their read numbers (reads) and mismatches in the alignment between these piRNAs and Rasgrf1 RMER4B sequences (mm). All piRNAs shown here were perfectly matched to chr7 piRNA cluster. The numbers in parentheses show the total number of small RNA reads mapped to the genome.

C, D. DNA methylation status of the Rasgrf1 differentially methylated region (DMR) in spermatogonia (C), spermatocytes, spermatids, and sperms (D) from chr7 piRNA clusterΔRMER4B/ΔRMER4B mice. See Figures 4A and B for information about the region examined. Complete DNA methylation of H19 imprinted gene, which is methylated in the male germline, shows high purity of germ cells used for the analyses.

E. DNA methylation pattern in the progeny of male Rasgrf1ΔRMER4B/ΔRMER4B mice. DNA methylation status of the Rasgrf1 DMR in E12.5 whole embryos is shown. E12.5 whole embryos were obtained by the crosses shown in the panel. Two independent bisulfite-treated DNA sample were examined for Homo13 embryo.

EXTENDED VIEW FIGURE LEGEND
Fusobacterium nucleatum secretes amyloid-like FadA to enhance pathogenicity (2021)
Figure 2

Figure 2. Pre-FadA as a key component of amyloid-like FadA.

A. Single-letter amino acid (aa) sequence of FadA. The intact pre-FadA consists of 129 aa, with the first 18-aa (top row) constitute the signal peptide. The remaining 111 aa constitute mFadA. The residues that are replaced in the mutant proteins used in this study are shown in red.

B. Analysis of detergent resistant recombinant FadA polymers expressed in E. coli. An aliquot of 50 μg detergent-resistant pellets prepared from E. coli BL21(DE3) carrying the cloning vector pET21(b), pYWH417-6 (p_FadAc), or pYWH418 (p_mFadA) were analyzed by SDD-AGE, followed by Western blot analysis. FadA polymers were only detected in E. coli expressing FadAc, but not in mFadA or the vector control.

C. Reactivity of recombinant FadA and its variants with anti-amyloid fibril antibody OC. A total of 10 μg each of recombinant protein mFadA, FadAc, FadA-L-9A, FadA-L14A, FadA-S71A, and FadA-L76A purified from E.coli was slot-blotted onto nitrocellulose and incubated with polyclonal OC antibody at 1:5,000 dilution. Amyloid-like recombinant Rim4 (Berchowitz et al., 2015) was used as a positive control and histone H1 was used as a negative control. Ponceau stain (pink color) is shown as a loading control.

D. Thioflavin-T binding assay. FadA proteins at indicated concentrations were incubated with 10 μM Thioflavin-T at room temperature for 10 min. The fluorescent intensity was measured at excitation wavelength of 440 nm and emission wavelength of 500 nm. The experiment was performed in duplicate and repeated three times. The error bars indicate SD. *p<0.05, **p< 0.01, ***p<0.001 (compared to BSA, t-test).

E. Thioflavin-T binding assay in the presence of 0.1% SDS. FadA proteins (0.8 mg/ml) were incubated with 10 μM Thioflavin-T at room temperature for 10 min. The fluorescent intensity was measured at excitation wavelength of 440 nm and emission wavelength of 500 nm. The experiment was performed in duplicate and repeated three times. The error bars indicate SD. *p<0.05, **p< 0.01 (compared to BSA, t-test).

F. Western blot analysis of Fn12230 (Fn), fadA-deletion mutant US1 (∆fadA) and spontaneous mutant lam following SDS-PAGE. A total of 400 μl of each culture grown to OD600 of 1.0 was pelleted and loaded onto 12% SDS-PAGE. FadA protein was detected using anti-FadA mAb 7H7 at 1:4,000 dilution. Compared to the wild type, lam produced significantly less pre-FadA, but not mFadA.

G. Analysis of amyloid-like FadA produced by Fn 23726 and its ∆fap2 mutant. An aliquot of 50 μg detergent-resistant pellets was loaded onto each lane, followed by SDD-AGE and Western blot analysis as described above. The mutant produced significantly less amyloid-like polymers than the wild type.
Fusobacterium nucleatum secretes amyloid-like FadA to enhance pathogenicity (2021)
Figure 3

Figure 3. Detection of amyloid-like FadA in periodontal disease and CRC.

A. Detection of FadA in subgingival plaque samples by IHC. Three patients with periodontitis are shown here who provided plaque samples from their healthy sites (probing depth ≤ 3 mm, left panels) as well as periodontal diseased sites (probing depth ≥ 7 mm, middle panels). IHC was performed as described using mAb 7H7 at 1:800 dilution. Anti-mouse IgG isotype control of the diseased site is shown (right panels). The images were taken using 100X objective. Scale bar equals 50 μm.

B. Double immunofluorescence staining of paired normal and carcinoma tissues from two CRC patients. The frozen tissue sections were incubated with mAb 7H7 at 1:50 dilution and anti-amyloid antibody A11 at 1:25 dilution, or mouse and rabbit IgG control, followed by incubation with Alexa Fluor 555-conjugated goat anti-mouse and Alexa Fluor 680-conjugated donkey anti-rabbit, both at 1:1000 dilution. Co-staining of FadA (red) and amyloid oligomers (green) was observed in the carcinoma but not normal tissues. The images were taken under 60X objective. Scale bar equals 10 μm.
Fusobacterium nucleatum secretes amyloid-like FadA to enhance pathogenicity (2021)
Figure 5

Figure 5. Amyloid-like FadA mediates colonization in CRC and promotes tumor growth in vivo.

A. Analysis of Fn colonization in mouse xenograft tumors by IHC. An inoculum of 5×106 HCT116 cells was injected subcutaneously and bilaterally into the nude mice. Once the tumors became visible (after 3-4 days), approximately 5×106 CFU of Fn 12230 washed in PBS or 1 mg/ml Congo Red (CR) were injected into the tumors. The tumors were extracted one week later, and the formalin-fixed and embedded (FFPE) tissues were stained with mAb 7H7. Note the brown stains in tumor injected with PBS-washed tumor, but not in CR-washed tumor. Scale bar = 20 μM.

B. Effects of secreted amyloid-like FadA on tumor growth. HCT116 cells were injected into the nude mice as described above, followed by injection of 5×106 CFU of Fn 12230 (Fn, n=7), lam (n=3) or US1 (∆fadA, n=4). The tumor volumes were measured immediately before bacteria injection (Day 0, designated as 100%), and at 3 days after injection (Day 3). The horizontal lines represent averages of tumor volumes (left panel). The individual tumors are shown (right panel). *p < 0.05, **p < 0.01, ***p< 0.001 (t-test).

C. Effect of anti-amyloid antibody OC on FadAc-mediated tumor growth. HCT116 cells were injected into the nude mice (n=6) as described above, followed by injection of 4 μl of 2 mg/ml FadAc mixed with 6 μl OC antibody or rabbit IgG control (rIgG) into tumors on the opposite sides. The tumor volumes were measured on Day 0 and Day 5 as described above. The horizontal lines represent the averages (left panel). The individual tumor pairs are shown (right panel, n=6). *p < 0.05, **p < 0.01 (t-test).

D. Effect of detergent-resistant pellets prepared from Fn 12230 (Fn) and US1 (∆fadA) on tumor growth. HCT116 cells were inoculated into the nude mice as described above. In one group of mice (n=3), sarkosyl-resistant pellets prepared from Fn 12230 and US1 (∆fadA) were injected into the tumors on opposite sides. In another group (n=5), sarkosyl-resistant pellets prepared from Fn 12230 were mixed with OC or rabbit IgG control (rIgG) before injecting into the tumors on opposite sides. The tumor volumes were measured described above. The vertical lines represent the standard deviations. The individual tumor pairs are shown (right panel). *p < 0.05, **p < 0.01, ***p< 0.001 [compared to US1 (∆fadA), t-test].
Fusobacterium nucleatum secretes amyloid-like FadA to enhance pathogenicity (2021)
Figure 6

Figure 6. Amyloid-like FadA induces periodontal bone loss in mice.

A. Approximately 1x109 CFU of Fn 12230, US1 (∆fadA), and lam suspended in carboxymethylcellulose (CMC) were orally administered to C57BL/6 mice three times a week for 10 weeks. CMC alone was administered as a control. Maxillae from mice inoculated with CMC alone (n=5), Fn 12230 (n=5), US1 (∆fadA; n=5), and lam (n=4) were harvested and fixed in 4% paraformaldehyde and stored in 70% ethanol, followed by microCT (μCT) scanning using a Scanco vivaCT 80 system at 55 kVp, 145 µA, and 250 ms integration time. Shown in the figure are reconstructed grayscale images. ImageJ was used to measure difference in bone height from the cementoenamel junction (CEJ) to the alveolar crest between palatal roots of first and second molars (see arrows).

B. The average bone loss of each group shown in (A), with the lines above each bar representing standard deviations. *p< 0.05. **p< 0.01 (t-test).
Generation of a self-cleaved inducible Cre for efficient temporal genetic manipulation (2019)
Figure 1

Figure 1. Generation and characterization of sCreER.

A Schematic diagram showing the working principle for sCreER.

B Schematic diagram showing steps of switch of sCreER into Cre.

C Generation of Npr3-sCreER or Npr3-CreER knock-in alleles by homologous recombination.

D Schematic diagram showing the experimental strategy.

E Whole-mount fluorescence view of E14.5-15.5 hearts from Npr3-sCreER;R26-tdTomato and Npr3-CreER;R26-tdTomato mice.

F Immunostaining for tdTomato, ESR and CDH5 on heart sections. C.M., compact myocardium; T.M., trabecular myocardium. Arrowheads, tdTomato+ESR- endocardial cells.

G Quantification of the percentage of tdTomato+ or ESR+ endothelial cells. Data are mean ± s.e.m.; n = 5.

H Isolation of tdTomato- or tdTomato+ cells from E14.5 Npr3-sCreER;R26-tdTomato and Npr3-CreER;R26-tdTomato mice by FACS.

I qRT-PCR of Cre, ESR, Npr3 and Cdh5 from tdTomato+ cells. Data are mean ± s.e.m.; n = 5; *P < 0.05.

J Western blotting of ESR and GAPDH in tdTomato- and tdTomato+ cells. Data are mean ± s.e.m.; n = 5; *P < 0.05; n.s., non-significant. Scale bars, 500 µm. Each figure is representative of 5 individual biological samples.
Generation of a self-cleaved inducible Cre for efficient temporal genetic manipulation (2019)
Figure 2

Figure 2. sCreER has significantly higher recombination efficiency than CreER by R26-Confetti reporter.

A Schematic diagram showing recombination readouts by crossing of Npr3-CreER or Npr3-sCreER with R26-Confetti reporter.

B Schematic figure showing experimental strategy.

C Whole-mount fluorescence images of E14.5 Npr3-CreER;R26-Confetti and Npr3-sCreER;R26-Confetti mouse hearts. No Tam is used as control for leakiness of Npr3-sCreER.

D, E Immunostaining for CDH5 on heart sections shows a significantly increase of endothelial cell labeling in Npr3-sCreER;R26-Confetti heart compared with Npr3-CreER;R26-Confetti heart (Tam). *P < 0.05; Data are mean ± s.e.m.; n = 5. Scale bars, 500 µm in c; 100 µm in d. Each image is representative of 5 individual biological samples.
Generation of a self-cleaved inducible Cre for efficient temporal genetic manipulation (2019)
Figure 3

Figure 3. Recombination efficiency by sCreER or CreER on R26-LZLT reporter.

A Schematic diagram showing Npr3-sCreER or Npr3-CreER mouse crossed with a newly generated Rosa26 reporter R26-LZLT.

B Schematic diagram showing experimental strategy.

C Whole mount fluorescent images of E14.5 or E15.5 Npr3-sCreER;R26-LZLT and Npr3-CreER;R26-LZLT mouse hearts. No tam is as control for detecting leakiness of sCreER or CreER.

D Representative Immunostaining images for tdTomato, ESR and CDH5 on heart sections.

E Quantification of the percentage of endocardial cells expressing tdTomato in Npr3-sCreER;R26-LZLT (sCreER) or Npr3-CreER;R26-LZLT (CreER) hearts. Data are mean ± s.e.m.; n = 5; *P < 0.05. Scale bars, 500 µm. Each figure is a representative of 5 individual biological samples.
Generation of a self-cleaved inducible Cre for efficient temporal genetic manipulation (2019)
Figure 4

Figure 4. Increased efficiency of gene deletion by sCreER, compared with CreER. A Schematic diagram showing Kdr gene deletion by sCreER.

B Schematic figure showing experimental design.

C Immunostaining for VEGFR2 on Npr3-sCreER;Kdrflox/flox heart section from mice without tamoxifen treatment (No Tam).

D, E Immunostaining for VEGFR2 on mouse heart sections after tamoxifen treatment (Tam). A, atrium; V, ventricle; TM, trabecular myocardium.

F, G Immunostaining for tdTomato, VEGFR2 and CDH5 on heart sections from mice of four different genotypes. Quantification data showed the percentage of VEGFR2+ endocardial cells (ECs) in tdTomato+ (tdT+) cells in the TM regions. Cartoon images (bottom) indicate tdTomato+ cells (red) and VEGFR2+ cells (green) in the TM or compact myocardium (CM). Data are mean ± s.e.m.; n = 5. Scale bars, 100 µm. Each image is a representative of 5 individual biological samples.
Generation of a self-cleaved inducible Cre for efficient temporal genetic manipulation (2019)
Figure 5

Figure 5. Generation and characterization of Col1a2-sCreER and Col1a2-CreER alleles.

A Schematic diagram showing knock-in of sCreER or CreER into Col1a2 locus by homologous recombination.

B Schematic figure showing experimental strategy.

C Whole-mount fluorescence images of Col1a2-sCreER;R26-tdTomato or Col1a2-CreER;R26-tdTomato hearts. Inserts indicate bright-field images.

D Immunostaining for tdTomato and PDGFRa on heart sections shows similar patterning of tdTomato and PDGFRa signals.

E Immunostaining for tdTomato and ESR on heart sections. Arrowheads indicate valve mesenchyme.

F Quantification of the percentage of PDGFRa+ cells expressing tdTomato in Col1a2-sCreER;R26-tdTomato (sCreER) or Col1a2-CreER;R26-tdTomato (CreER) hearts. n.s., non-significant; Data are mean ± s.e.m.; n = 6; n.s., non-significant.

G Quantification of the percentage of tdTomato+ cells expressing ESR. *P < 0.05; Data are mean ± s.e.m.; n = 6; *P < 0.05.

H Immunostaining for PDGFRa in heart sections shows a significant increase of fibroblast labeling in valves of Col1a2-sCreER;R26-Confetti heart compared with Col1a2-CreER;R26-Confetti heart (Tam).

I Quantification of the percentage of fibroblasts expressing fluorescent reporters in cardiac valves. Data are mean ± s.e.m.; n = 5; *P < 0.05. Scale bars, 500 µm in c-e; 100 µm in h. Each figure is a representative of 5-6 individual biological samples.
Budding yeast Rif1 binds to replication origins and protects DNA at blocked replication forks (2018)
Figure 1

Figure 1. The C-terminus of yeast Rif1 is dispensable for control of DNA replication

A. Structure of budding yeast Rif1 and C-terminally truncated mutant Rif1-∆C594. RBM: Rap1-binding motif; CTD: C-terminal domain.

B. Rif1-∆C594 retains function to control DNA replication. Growth of a cdc7-1 rif1-∆C594 mutant was compared with growth of cdc7-1 RIF1 and cdc7-1 rif1∆ strains at 23ºC (permissive temperature for cdc7-1 allele), 26ºC (mild restrictive temperature) and 30ºC (strict restrictive temperature).
Budding yeast Rif1 binds to replication origins and protects DNA at blocked replication forks (2018)
Figure 2

Figure 2. Full-length and C-terminally truncated Rif1 proteins bind distinct chromosomal loci.

A. Specimen overview of Chromosome VI-left region showing results of ChIP-Seq analysis of Rif1 and Rif1-∆C594 proteins. ChIP sequence reads were normalised against sequence reads from corresponding input samples, and relative enrichment is plotted for Chromosome VI coordinates 1-80,000. Y-axis shows enrichment values (linear scale, range is 0 to 3.5). Values below 1 are shown in grey, and values above 1 (i.e. sequences enriched in ChIP samples) are coloured blue (Rif1) and red (Rif1-∆C594). Plots show ChIP analysis results from cells arrested by α-factor (G1), released from α-factor at 16ºC for 60 min and 90 min, or released from α-factor into 0.2 M HU for 60 min at 23ºC.

B. Rap1-dependent association of Rif1 with the promoter regions of Rap1-controlled genes. ChIP enrichment around PAU3 (left) and MAM3 (right), both genes whose transcription is controlled by Rap1. Values above 1 (i.e. enriched) shown by overlaid blue and red histograms for Rif1 and Rif1-∆C594, respectively. Values below 1 shown in grey.
Budding yeast Rif1 binds to replication origins and protects DNA at blocked replication forks (2018)
Figure 3

Figure 3. Rif1 and Rif1-∆C bind replication origins, centromeres, and tRNA genes

A. ChIP-Seq analysis of Rif1 and Rif1-∆C594 proteins shows tRNA gene and origin binding, with widened peaks at early origins ARS606 and ARS607 in HU block. Plots show Chromosome VI genome coordinates 160,000- 210,000. Plot colours here and in following Figures are as in Fig 2B. Widened peaks are not observed in unperturbed S phase samples, or for Rif1-∆C594.

B. Association of Rif1-∆C594 at replication origins is enhanced in HU block. ChIP enrichment of Rif1 and Rif1-∆C594 around early origin ARS1426 (left) and late origin ARS1412 (right).

C. Differential association of Rif1 and Rif1-∆C594 to centromeres. ChIP enrichment of Rif1 and Rif1-∆C594 around the CEN4 locus.
Budding yeast Rif1 binds to replication origins and protects DNA at blocked replication forks (2018)
Figure 4

Figure 4. Analysis of origin binding reveals full-length Rif1 binds broad regions near early origins in HU.

A. ChIP-qPCR confirmation of Rif1 and Rif1-∆C594 association with replication origins. ChIP was performed using cells arrested in α-factor (open bars) or HU (grey bars), and qPCR analysis performed for early origin ARS1426 (left) and late origin ARS1412 (right). Values shown are 'normalised ChIP efficiencies' obtained by subtracting the value obtained at a control locus (see Materials and Methods). Bars indicate the averages of two biological replicates, with values from each replicate shown by open circles.

B. Numbers of early and late origins associated with Rif1 and Rif1-∆C594 peaks. Plot showing numbers of early and late origins bound by Rif1 and Rif1-∆C594 in G1 and HU-blocked cells, based on peak-calling results.

C. Replication timing programme is intact in rif1-∆C594 cells. Replication of selected origins at an HU block analysed by BrdU incorporation. Cells were synchronised by α-factor and released into the medium containing 0.2 M HU and 1.13 mM BrdU. Plots show the percentage of total ARS607, ARS422.5, or ARS1412 DNA pulled down by IP with anti-BrdU, calculated from two biological replicate samples. Bars indicate the average of two biological replicates and open circles the values from each replicate. Insets in ARS422,5 and ARS1412 panels show the same data with Y-axis scales adjusted to 0 - 0.03%.

D. Peaks of Rif1 become broader in HU-arrested cells. Box and whisker plot shows peak widths of full length Rif1 at early and late origins in G1 and HU-blocked cells. Analysis was performed on those origins detected by peak calling as associated with a Rif1 peak. Boxes show the range of 25th to 75th percentiles, with horizontal lines within the boxes representing 50th percentiles. Whiskers represent 95% confidence intervals. Outliers are presented as open circles. Numbers of origins analysed are: 46 Early origins in G1, 56 Late origins in G1, 85 Early origins in HU, and 24 Late origins in HU.
CSF progranulin increases in the course of Alzheimer's disease and is associated with sTREM2 (2018)
Figure 1

Figure 1. Association of CSF PGRN with mutation status, age and gender

(A) CSF PGRN is increased in mutation carriers (MC) compared to non-carriers (NC). (B) CSF PGRN is not associated with age in either NC or MC. (C) CSF PGRN is increased in males compare to females. (D) CSF PGRN levels do not differ among MC participants carrying a PSEN1, PSEN2 or APP mutation.

The blue or red bars in (A), (C) and (D) represent the mean and the standard deviation (SD). Group comparisons were assessed by a linear model adjusting by age, gender and APOE ε4 status. The solid lines in (B) indicate the regression line for each of the groups and the 95% confidence interval (CI) calculated by a linear model adjusting by gender and APOE ε4 status. The standardized regression coefficients (β) and the P-values are also shown. In graph (B), the individual values are not shown in order to protect participants' confidentiality. All analysis and graphs are performed excluding 3 PGRN values outliers. Including the outliers in the analysis rendered similar results (Appendix Table S1).

Abbreviations: APP, amyloid precursor protein; CSF, cerebrospinal fluid; ns, non-significant; PSEN1, presenilin 1; PSEN2, presenilin 2.
CSF progranulin increases in the course of Alzheimer's disease and is associated with sTREM2 (2018)
Figure 2

Figure 2. Changes of CSF PGRN as a function of EYO

(A) CSF PGRN as a function of EYO in mutation carriers (MC, red) and non-carriers (NC, blue). The solid lines indicate the regression line for each of the groups and the 95% confidence interval (CI) calculated by a linear model adjusting by gender. The interaction term of mutation status and EYO is significant (P = 0.041), also when including PGRN outliers and participants with EYO > +20 (P = 0.030). Individual data points are not displayed to prevent disclosure of mutation status.

(B) The graph depicts the standardised differences of CSF PGRN between MCs and NCs as a function of EYO, in the context of other biomarker and cognitive changes. The curves were generated by the linear model that best fit each marker (see statistics section and Appendix Table S2). CSF PGRN is significantly increased in MC compared to NC 10 years before the expected symptom onset (shadowed area) after brain amyloidosis and brain injury (as measured by CSF T-tau) have started, and shortly before CSF sTREM2 starts to increase.

Abbreviations: Aβ1-42: amyloid-β 42; CSF, cerebrospinal fluid; MC, mutation carriers; MMSE, Mini-Mental State Examination; NC, non-carriers; T-tau, total tau.
CSF progranulin increases in the course of Alzheimer's disease and is associated with sTREM2 (2018)
Figure 3

Figure 3. CSF PGRN levels across the Alzheimer's continuum

Scatter plot representing the levels of CSF PGRN in healthy controls (depicted in blue) and the different stages of the Alzheimer's continuum (depicted in red). The analysis and graphs were performed excluding CSF PGRN outliers (1 'healthy control', 1 'Preclinical AD A+/TN-', 4 'AD CDR = 0.5' and 1 'AD CDR = 1'). Including them yielded a similar result and CSF PGRN was still significantly higher in the 'AD CDR = 1' group compared to the 'healthy controls' (P = 0.001) and 'Preclinical AD A+/TN-' (P = 0.0001) groups.

P-values were assessed by a one-way analysis of covariance adjusted by age, gender and APOE ε4, followed by Bonferroni corrected pair-wise post hoc comparisons.

Abbreviations: A: amyloid-β biomarker status; AD: Alzheimer's disease; CDR: clinical dementia rating; CSF, cerebrospinal fluid; N: neurodegeneration biomarker status; T: tau pathology biomarker status.