EMBO/soda-nel huggingface dataset:
Gut Inflammation Regulates Aβ and Tau Fibril Propagation to brain via a C/EBPβ/δ-secretase Pathway (2021)
Figure 4

Figure 4. Vagotomy attenuates DSS-elicited AD pathologies in 3xTg mice.

(A) Immunoblot showing p-C/EBP β, C/EBP β, AEP, APP, and Tau expression and processing in mouse colon of 3xTg mice with neither DSS treatment nor single side vagotomy, with both DSS treatment and single side vagotomy, or with DSS treatment but no single side vagotomy.

(B) Immunoblot showing p-C/EBP β, C/EBP β, AEP, APP, and Tau expression and proteolytic processing in mouse brain.

(C) AEP activity assay in colon lysates from pre-vagotomy DSS-treated, DSS-treated and vehicle-treated 3xTg mice. DSS treatment escalated AEP activity in colon of 3xTg mice, which was reversed by vagotomy. Data represent the mean ± SEM; representative data of three samples; ***P=0.0002 (***P< 0.001), **P=0.0013 (**P < 0.01) compared with control, one-way ANOVA.

(D) AEP activity assay with brain lysates from different brain hemispheres of single side pre-vagotomy DSS-treated 3xTg mice. Data represent the mean ± SEM; representative data of three samples; *P =0.0162 (*P < 0.05) compared with control, one-way ANOVA.

(E) Gastrointestinal permeability barrier defect as determined by FITC-dextran translocation in pre-vagotomy DSS-treated, DSS-treated and vehicle-treated 3xTg mice. Data represent the mean ± SEM; representative data of three samples; *P=0.0126 (*P < 0.001), ***P=0.0002 (***P < 0.001) compared with control, one-way ANOVA.

(F) Pro-inflammatory cytokines IL-6, IL1-β, and TNFα concentrations in colon lysates of vehicle-treated 3xTg mice, DSS-treated single side vagotomy 3xTg and DSS-treated 3xTg, respectively. Representative data of three samples; data are shown as mean ± SEM. *P=0.0372 (*P < 0.05), **P=0.0050 (**P < 0.01), ***P=0.0008 (***P <0.001), ****P<0.0001 compared with control, two-way ANOVA.

(G) Immunofluorescent staining of AEP (red) and C/EBP β (green), cleaved Tau N368 (red) and AT8 (green), cleaved APP C856 (red) and Aβ (green), T22 (red) and AT8 (green) and Iba-1(red) of the cerebral cortex in different brain hemispheres of pre-vagotomy DSS-treated 3xTg mice. Scale bar: 20 μm. White arrows indicate that C/EBP β and AEP, Tau N368 and AT8, as well as T22 and AT8 were co-localized in mouse brain, respectively.

(H) Representative electron microscopy of the synaptic structures in hippocampus. Red stars indicate the synapses. (Scale bar: 1 μm).

(I) The dendritic spines from the apical dendritic layer of the cerebral cortex region were analyzed by Golgi staining. (Scale bar: 5 μm).

(J) Quantitative analysis of the synaptic densities in DSS-treated mice. Representative data of five samples, data are shown as mean ± SEM. ****P < 0.0001, unpaired t tests.

(K) Quantitative analysis of the spine density. Representative data of five samples, data are shown as mean ± SEM. ***P =0.0001 (***P < 0.001), unpair t tests.
Gut Inflammation Regulates Aβ and Tau Fibril Propagation to brain via a C/EBPβ/δ-secretase Pathway (2021)
Figure 6

Figure 6. Colonic-injected Aβ PFFs, Tau N368 PFFs, AD patient brain extracts or 5xFAD brain extracts travel along the vagus nerve to brain stem in WT mice.

(A) Immunofluorescent staining showed ATTO 550-labeled Aβ PFFs (pre-formed fibrils) and Tau N368 PFFs traveling along the vagus nerve in WT mice at different time points. White arrows shows that ATTO 550 signals and Aβ PFFs (pre-formed fibrils) signals, ATTO 550 signals and Tau N368 PFFs were co-localized in the vagus nerve, indicating the positive signals were from the injected exogenous pre-form fibrils.

(B) Immunofluorescent staining showed ATTO 550-labeled Aβ PFFs and Tau N368 PFFs spread into different brain regions in WT mice 3 months after colonic injection. Scale bar: 20 μm.

(C) Immunohistochemistry staining of human Aβ propagating along the vagus nerve from WT mice colonic-injected with PBS, AD brain extracts or 5xFAD brain extracts, using human specific Aβ antibody (BAN50). Black arrows indicate the human Aβ in mouse vagus nerve.

(D) Immunohistochemistry staining of human Tau propagating along the vagus nerve from WT mice colonic-injected with PBS or AD brain extracts, using human Tau specific antibody (HT7). Black arrows indicate the human Tau in mouse vagus nerve.
Gut Inflammation Regulates Aβ and Tau Fibril Propagation to brain via a C/EBPβ/δ-secretase Pathway (2021)
Figure 7

Figure 7. Colonic inoculation of recombinant Aβ PFFs or Tau N368 PFFs activates C/EBP β / δ-secretase pathway in WT mice brain.

(A) Immunofluorescent staining of AEP and p-C/EBP β in cerebral cortex of brains of WT mice 6 months after colonic inoculation with either Aβ PFFs or Tau N368 PFFs. Scale bar: 20 μm. White arrows indicate that AEP and p-C/EBP β signals (green) were colocalized with ATTO 550 signals (red), respectively.

(B-G) Morris water maze test. Data represent the mean ± SEM. Colonic-injected mice (n = 7 per group) were trained in the water maze over five days. Shown are the percentage of time spent in the target quadrant in the probe trail, *P=0.0362 (*P < 0.05),**P=0.0072 (**P < 0.01), unpaired t tests (B), Latency to platform, *P=0.0434, *P=0.0166 (*P < 0.05), two-way ANOVA (C), the area under curve of Latency (AUC Latency) (D), Swim Speed (E), Swim Path Distance (F), the area under curve of Swim Path Distance (AUC distance) (G).
Gut Inflammation Regulates Aβ and Tau Fibril Propagation to brain via a C/EBPβ/δ-secretase Pathway (2021)
Figure 8

Figure 8. Colonic-injection of AD brain extracts activates C/EBPβ/δ-secretase pathway in 3xTg mice brain.

(A) Immunofluorescent staining of AEP (red) and C/EBP β (green) in CA1 region from hippocampus of brains from colonic-injected PBS, colonic-injected AD brain extracts, and pre-Vagotomy colonic injected AD brain extracts 3xTg mice. Scale bar: 20μm.

(B) Quantitative analysis of AEP positive cells and C/EBP β positive cells, respectively. The density of both AEP and C/EBP β positive cells were significantly increased by colonic-injection of AD brain extracts and decreased through vagotomy before colonic-injection. Representative data of three samples, data are shown as mean ± SEM. **P =0.016 (**P < 0.01), ****P<0.0001, one-way ANOVA.

(C) Immunofluorescent staining of cleaved APPC586 (red) and Aβ (green) in cerebral cortex of brains from colonic-injected PBS, colonic-injected AD brain extracts, and pre-Vagotomy colonic injected AD brain extracts 3xTg mice.

(D) Quantitative analysis of cleaved APP C586 positive cells and Aβ positive cells, respectively. The density of both cleaved APP C586 positive cells and Aβ positive cells were significantly increased by colonic-injection of AD brain extracts and decreased through vagotomy before colonic-injection. Representative data of three samples, data are shown as mean ± SEM. ****P<0.0001, one-way ANOVA.

(E) Immunofluorescent staining of T22 (red) and HT7 (green) in cerebral cortex of brains from colonic-injected PBS, colonic-injected AD brain extracts, and pre-Vagotomy colonic injected AD brain extracts 3xTg mice.

(F) Quantitative analysis of T22 positive cells. The density of T22 positive cells was significantly increased by colonic-injection of AD brain extracts and decreased through vagotomy before colonic-injection. Representative data of three samples, data are shown as mean ± SEM. ***P=0.0007 (***P < 0.001), ****P<0.0001, one-way ANOVA.
Dynamic expression of tRNA-derived small RNAs define cellular states (2019)
Figure 3

Figure 3. Effect of 5'-tsRNA knockdown on RA-induced differentiation of mESCs.

(A) Effect of ASO-mediated inhibition of 5'-tsRNAs on alkaline phosphatase activity in RA-treated mESCs. (n = 3), Error bars represent SEM, significance was calculated using unpaired t-test.

(B) Relative expression of stemness markers in RA-induced differentiation mESCs blocked for 5'-tsRNA function with ASOs. (n = 2) Error bars represent SEM, significance was calculated using unpaired t-test.

(C) Volcano plot of the transcriptome depicting the overexpressed transcripts (in green) and downregulated transcripts (in pink) in ASO treated-differentiating cells.

(D) KEGG pathway analysis of the transcripts upregulated in a RA-mediated differentiating mESCs blocked for tsRNA function.

(E) QPCR validation of transcripts involved in the stem cell signalling pathway that are upregulated upon 5'-tsRNA knockdowns using ASOs. (n =2) Error bars represent SEM, significance was calculated using unpaired t-test.

Data information: *, P value < 0.05; **, P value < 0.01; ***, P value < 0.001; ****, P value < 0.0001; n.s., not significant.
Dynamic expression of tRNA-derived small RNAs define cellular states (2019)
Figure 5

Figure 5. 5'-tsRNA-mediated regulation of cMyc through Igf2bp1 interaction.

(A) In vitro binding analysis of tsGlnCTG to Igf2bp1 in the presence and absence of antisense oligo (ASO) against tsGlnCTG. ASOs effectively disrupt the binding of tsGlnCTG to Igf2bp1. (n = 2), Error bars represent S.E.M.

(B) Quantification of the Igf2bp1 bound c-Myc mRNA between LIF- versus RA-treated mESCs (n = 2; see Fig. EV5F for duplicate data).

(C) Association of c-Myc transcript in different translating pools in RA treated mESCs as compared to LIF condition. (n = 2), Error bars represent SD, significance was calculated by one-tailed unpaired t-test.

(D) Relative enrichment of c-Myc mRNA in translating (80S and Polysome) and non-translating (mRNPs) pools (fractionated from polysome profiling) between ASO-treated and Mock-treated RA-induced differentiating mESCs. (n =2) Error bars represent SD, significance was calculated using one-tailed unpaired t-test.

(E) Relative levels of c-Myc mRNA in ASO-treated and Mock-treated RA-induced differentiating mESCs compared to LIF treated mESCs. (n = 3) Error bars represent SD, significance was calculated using one-tailed unpaired t-test.

(F) Epistatic analysis of 5'-tsRNAs and IGF2BP1 in regulating Myc transcriptional reporter activity. (n = 3), Error bars represent SD, significance was calculated using unpaired t-test.

(G) Schematic representing tsRNA based cMyc transcript regulation.

Data information: *, P value < 0.05, **, P value < 0.01; ***, P value < 0.001.
Rtt105 functions as a chaperone for Replication Protein A to preserve genome stability (2018)
Figure 1

Figure 1. Rtt105 binds RPA in vivo and in vitro.

(A) Identification of Rtt105-TAP associated proteins. Purified proteins were resolved on a 4-12% gel, revealed by silver staining, and identified by mass spectrometry (Table EV1). *Non-specific band also in control.

(B) Cells containing Rfa1-TAP were synchronized at G1, S, and G2/M phase and used for the TAP purification. Rfa1-TAP associated protein complexes were analyzed by Western blot using CBP, Rfa2, and Rtt105 antibodies (left panel). DNA content was monitored by flow cytometry (right panel).

(C) MBP-Rtt105 pulled down recombinant RPA, resolved on SDS-PAGE gels and visualized by Coomassie Brilliant Blue (CBB) staining. MBP was used as a negative control.

(D) GST-tagged full length (FL) and Rtt105 deletion mutants (Schematic of the Rtt105 truncations is shown in the upper panel) were purified and used to pull down Rfa1 protein. GST proteins were used as a negative control. Isolated protein complexes were resolved on 15% SDS-PAGE gels and visualized by CBB staining.

(E) Rfa1-TAP purification was performed using Rfa1-TAP rtt105∆ strains expressing WT and indicated Rtt105 mutant forms. pRS313 serves as vector control (Vc).
Rtt105 functions as a chaperone for Replication Protein A to preserve genome stability (2018)
Figure 2

Figure 2. RTT105 deletion leads to a reduction of RPA binding at replicating regions genome wide.

(A) Schematic for Rfa1 and Rfa2 ChIP-Seq (this Figure) and BrdU IP-Seq (Fig 6). Yeast cells were arrested with α factor at G1 phase, then released into fresh YPD medium containing BrdU to label newly synthesized DNA as well as to allow cell enter S phase at two conditions: (i) in medium with 0.2 M HU for 45 min at standard growth temperature (25 oC), (ii) in medium without HU for 72 min at low temperature (16oC) to slow down S phase progression. Cells were then collected for Rfa1/Rfa2-ChIP, or BrdU-IP. The resulted DNAs were detected by sequencing.

(B) Snapshots of Rfa1 ChIP-Seq peak at ARS305 from WT and rtt105∆ cells released into HU-medium.

(C) The average Rfa1 ChIP-Seq read density from cells released into HU-medium around ACS sites. ACS: ARS consensus sequence.

(D) Snapshots of Rfa2 ChIP-Seq peaks at ARS305 from cells released at 16°C for 72 min.

(E) The average Rfa2 ChIP-Seq read density surrounding ACS sites from cells released at 16°C for 72 min.

(F) Rfa1 occupancy analysis of cells progression through S phase. G1-arrested cells were released into fresh YPD medium to allow cell progression through S phase at standard growth temperature (25°C). Equal amounts of cells were collected at each time point. Rfa1 ChIP were performed using IgG beads for TAP-tag. ARS305 and ARS607, early fired replication origins; ARS607+14kb, a corresponding 14kb away region of ARS607. The mean and standard deviation (SD) of three biological replicates were shown. The single-tailed non-parametric Wilcoxon test was performed as described in methods (**: 0.001≤P value<0.01).
Rtt105 functions as a chaperone for Replication Protein A to preserve genome stability (2018)
Figure 3

Figure 3. Rtt105 has roles in both RPA nuclear import and loading RPA to replicating DNA.

(A) The nuclear localization of Rfa1 proteins is altered in rtt105∆ mutant cells. Rfa1 tagged with GFP at its C terminus was used to analyze the localization of Rfa1-GFP fusion protein (Green) and the nuclear envelop is visualized with the Nup49-mCherry fusion protein (Red) using fluorescence microscopy. DIC: differential interference contrast. Scale bar: 5μm.

(B) NLS-RFA1-GFP rescues the nuclear localization of Rfa1 in rtt105∆ mutant cells. The wild type RFA1 gene was fused with an SV40 large T Antigen nuclear localization sequence (NLS) at its 5'-end and a GFP gene at its 3' end to obtain a construct, driven by the RFA1 promoter to express NLS-RFA1-GFP fusion protein. The engineered construct was then transformed into WT or rtt105∆ mutant yeast cells to replace its endogenous RFA1 expression. The resulting yeast cells were then visualized under microscope, and GFP signals enriched in the nuclei were scored. RFA1-GFP lacking the NLS sequence was transformed into rtt105∆ rfa1∆ mutant cells as a control. DAPI staining indicates nuclear DNA. Statistical significance was evaluated based on Student's t-tests (***: P value<0.001).

(C) The experimental scheme for the Rfa2 ChIP in indicated yeast strains.

(D and E) An increase in nuclear localization of Rfa1 could not rescue the RPA binding defects at the replication regions in rtt105∆ cells. The percentage of Rfa2 ChIP-DNA over the total input DNA was calculated. The mean and standard error (SE) of three biological replicates were shown. Statistical significance was evaluated based on Student's t-tests (*: 0.01≤P value<0.05).
Rtt105 functions as a chaperone for Replication Protein A to preserve genome stability (2018)
Figure 4

Figure 4. Rtt105 enhances the binding efficiency of RPA to ssDNA in vitro.

(A) Purified RPA and Rtt105-RPA complex were resolved on 15% SDS-PAGE gels and analyzed by silver staining.

(B and C) EMSA assay was performed to analyze ssDNA binding ability of RPA or Rtt105-RPA complex. Cy3-labeled 30nt ssDNA was used as the substrate, and reaction mixtures were resolved on native PAGE gels. Quantification of the RPA-bound ssDNA is shown in (C). The mean values ± SD from three independent experiments were plotted, with P values derived from two-way analysis of variance (ANOVA) (***: P value < 0.001).

(D) Recombinant Rtt105-RPA complex was mixed with a 30nt-ssDNA in a 2:1 ratio, and the reaction product were subjected to gel filtration chromatography. Equal volume of fraction 5-10 were resolved on SDS-PAGE for detecting proteins by Ruby staining (upper panel) or were resolved by native agarose gel for detection of Cy3-ssDNA signal (lower panel).

(E) GST-RPA pull down Rtt105 with increased amount of 30nt ssDNA, and the bound proteins were resolved on SDS-PAGE and visualized by silver staining.
Rtt105 functions as a chaperone for Replication Protein A to preserve genome stability (2018)
Figure 5

Figure 5. Rtt105 affects the binding mode of RPA to ssDNA.

(A) Schematic for ssDNA curtain assay. Briefly, ssDNA curtains were formed by flowing 1 nM RPA-eGFP for 3 minutes. Then 5 nM RPA-mCherry pre-mixed with or without 5 nM Rtt105 wild-type (WT) or E171L172AA (EL) mutant proteins were injected into the flow cell. Data were acquired with a TIRF microscope (Nikon, Inverted Microscope Eclipse Ti-E). Lasers were shuttered at 2-second intervals, and the exposure time was 100 ms. Each kymogram with two channels, in which green and red represent eGFP and mCherry signal, respectively.

(B and C) Kymograms showing most ssDNA molecules two distinct stretching patterns of RPA-ssDNA molecules. Pattern 1, slow stretching; Pattern 2, fast stretching. The counts of the two patterns in each reaction were plotted in (C). Please note a fraction of individual ssDNA shows both patterns.

(D) Rtt105 increases the rate of ssDNA stretching upon RPA binding. Stretching rate was defined as the length change of ssDNA upon binding to RPA-mCherry in unit time as shown in (B, white arrow). All error bars represent SD for each data set. Statistical significance was evaluated based on Student's t-tests (*: 0.01≤P value<0.05; **: 0.001≤P value<0.01).

(E) Rtt105 increases the extent of ssDNA stretching upon RPA binding. Length increment = DNA length at 8 minutes / DNA length at 3 minutes. All error bars represent SD for each data set. Statistical significance was evaluated based on Student's t-tests (**: 0.001≤P value<0.01; ****: P value<0.0001).
Rtt105 functions as a chaperone for Replication Protein A to preserve genome stability (2018)
Figure 6

Figure 6. Rtt105 is important for DNA replication and genome stability maintenance. The procedure for BrdU IP-Seq was described in Fig 2A.

(A) Snapshots of BrdU IP-Seq peaks at ARS305 from cells released into YPD medium containing HU for 45 min.

(B) The average BrdU IP-Seq read density around fired ACS from cells released into YPD medium containing HU for 45 min sites.

(C) Snapshots of BrdU IP-Seq peaks at ARS305 from cells released into YPD medium at 16°C for 72 min.

(D) The average BrdU IP-Seq read density around ACS sites from cells released into 16°C for 72 min.

(E) Rad52-GFP foci were counted in WT and rtt105∆ cells, the percentage of cells with Rad52-foci is reported. Error bars indicate one standard deviation from three independent experiments with at least 150 cells counted in each replicate. Statistical significance was evaluated based on Student's t-tests (****: P value<0.0001).

(F) Rad53 phosphorylation was analyzed in WT and rtt105∆ cells by immunoblotting of protein extracts with anti-Rad53 antibodies. Ponceau S (Pon S) staining was applied as a loading control.

(G) Deletion of RTT105 leads to an increased rate of gross chromosomal rearrangements (GCRs). Wild-type (WT) and rtt105∆ mutant strains integrated with the yWSS439-5ori∆ yeast artificial chromosomes (YACs). GCRs rates in the evaluation of telomere marker loss were assayed and calculated as previously described (Huang & Koshland, 2003). Data represent the mean (±SD) of three independent experiments. Statistical significance was evaluated based on Student's t-tests (**: 0.001≤P value<0.01).

(H) Ten-fold serial dilutions WT or rtt105∆ cells in two different backgrounds were assayed on normal growth media (YPD) and on media containing the indicated DNA damaging agents, methyl-methane sulfonate (MMS), camptothecin (CPT), bleomycin (Bleo), and hydroxyurea (HU).
The nucleoporin ELYS regulates nuclear size by controlling NPC number and nuclear import capacity (2019)
Figure 1

Figure 1: An imaging-based siRNA screen for gene knockdowns that reduce nuclear size. (A) The screen approach is depicted, and details are available in the Methods. Throughout the study, a premalignant breast cancer cell line was used (MCF-10AT1k.cl2), unless otherwise indicated. Images and data shown in this figure were obtained directly from the screen. Scale bar, 20 µm. (B) Montages of representative DAPI-stained nuclei are shown for gene knockdowns that led to reduced nuclear size. Cell-to-cell variability in the amount of ELYS knockdown likely accounts for apparent nuclear size variability. Ten nuclei per column, organized by maximum nuclear size z-score. Scale bar, 25 µm. (C) Median nuclear size z-scores are plotted for gene knockdowns that led to reduced nuclear size. Data are based on three different siRNA oligo sequences for each gene and two biological replicates. Error bars represent the SEM for biological replicates.
The nucleoporin ELYS regulates nuclear size by controlling NPC number and nuclear import capacity (2019)
Figure 2

Figure 2: ELYS knockdown reduces NPC density and nuclear levels of lamin B2, Ran, and NTF2. MCF-10AT1k.cl2 cells were transfected with control or ELYS siRNA. (A) Representative images of cells stained for lamin B2 and B1 are shown. (B) Representative images of cells stained for lamin A/C and B1 are shown. (C) For each experiment, nuclear lamin B1, B2, and A/C staining intensities were quantified for 44-159 nuclei per condition (96 nuclei on average) and normalized to the negative control. Three biological replicates, data from one representative experiment shown. (D) Representative images of cells stained for FG-Nups (mAb414) and lamin B1 are shown. (E) Representative images of cells stained for Nup133 and lamin B1 are shown. (F) For each experiment, nuclear mAb414 and Nup133 staining intensities were quantified for 31-83 nuclei per condition (60 nuclei on average) and normalized to the negative control. To measure NPC densities, confocal NE surface images were acquired for mAb414 and Nup133 stained nuclei. NPC numbers were counted per unit area for 18-50 nuclei per condition (30 nuclei on average) and normalized to the negative control. Two biological replicates, data from one representative experiment shown. (G) Representative images of cells stained for Ran and lamin B1 are shown. (H) Representative images of cells stained for NTF2 and lamin A/C are shown. (I) Nuclear Ran and NTF2 staining intensities were quantified and normalized to the negative control. To measure the N/C staining intensity ratio of a given cell, the average fluorescence intensity of a nuclear region was divided by the average fluorescence intensity of a cytoplasmic region. For each experiment, 54-216 nuclei were quantified per condition (112 nuclei on average). Two biological replicates, data from one representative experiment shown.

Two-tailed Student's t-tests assuming equal variances: ***, p<0.005; NS, not significant. Scale bars are 10 µm. Error bars represent SD.