EMBO/soda-nel huggingface dataset:
MicroRNA-483 amelioration of experimental pulmonary hypertension (2020)
Figure 4

Figure 4. Decreased level of PAH-induced genes in EC-miR-483 Tg rats.

(A) Lung ECs were isolated from EC- miR-483 transgenic rats (Tg) and their wild-type littermates (WT), n = 3 in each group. MiR-483-3p/-5p levels were measured by qPCR.

(B) Serum from WT and Tg rats (n = 6) were collected. MiR-483-3p and -5p associated with CD144 were enriched by immunoprecipitation with anti-CD144. Levels of miR-483-3p/-5p were measured by qPCR.

(C, D) Lung ECs isolated from miR-483-Tg rat and WT littermates were exposed to normoxia or hypoxia (0.2% O2) for 24 hr. Proliferation and migration of ECs were measured by flow cytometry and wound healing assay, respectively.

(E-H) Tg and WT rats were injected with saline or MCT. Expression levels of TGF-β, TGFBR2, β-catenin, CTGF, IL-1β, and ET-1 mRNA and protein in lung tissues were measured by qPCR and Western blot, respectively (E). Levels of miR-483-3p/-5p in serum and lung tissues were measured by qPCR (F). Ago1- or Ago2-associated miRNAs (G) and mRNAs (H) were enriched from the isolated lung tissues by immunoprecipitation with anti-Ago1 or anti-Ago2 and quantified by qPCR (n = 3×3, samples were pooled from three animals for each assay, and 3 independent experiments [a total of 9 animals] were performed).

Data information: Values are expressed as mean ± SEM. Data are representative of three independent experiments (C-D). Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups, exact P values were shown in Appendix Table S3).
MicroRNA-483 amelioration of experimental pulmonary hypertension (2020)
Figure 5

Figure 5. EC-miR-483 Tg rats show ameliorated PH.

(A-B) EC-miR-483-Tg and WT rats were injected with saline or MCT. Analysis of mPAP and RV hypertrophy [RV/(LV+S)] for the indicated groups (7 rats per group) (A, B). Vascularization evaluated by H&E staining of pulmonary arteries (C). Vascular integrity evaluated by intravenous injection of Evans blue dye (D).

(E, F) EC-miR-483-Tg rats and WT littermates were injected with SU5416 and then exposed to hypoxia for 3 weeks and had reoxygenation for 2 weeks or injected with DMSO and exposed to normoxia for 5 weeks. Analysis of mPAP (E) and RV hypertrophy [RV/(LV+S)] (F) for the indicated groups.

Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups). Scale bar: 20 µm (C) or 0.5 cm (D).
MicroRNA-483 amelioration of experimental pulmonary hypertension (2020)
Figure 6

Figure 6. Lenti-miR-483 inhalation ameliorates MCT-induced PAH.

(A) Ctrl- or MCT-treated rats were intratracheally delivered Lenti-pre-miR-483-GFP or Lenti-GFP at Day 21 and Day 28 after the start of MCT treatment (S+GFP, M+GFP, and M+483).

(B, C) qPCR analysis of miR-483-3p/-5p in serum and lung tissue from PH (MCT-treated) rats.

(D) Protein and mRNA levels of TGF-β, TGFBR2, β-catenin, CTGF, IL-1β, and ET-1 measured by Western blot and qPCR, respectively (n = 3×3, samples were pooled from three animals for each assay, and 3 independent experiments [a total of 9 animals] were performed).

E-I Analysis of mPAP (E), RV hypertrophy [RV/(LV+S)] (F), and systemic blood pressure (SBP) (G) for the indicated groups (7 rats in each group).

(H) RV hypertrophy shown by the H&E staining and RV/(LV+S) quantification.

(I) Vascularization revealed by H&E staining (n = 7 rats in each group).

(J) Survival rate of 3 groups (n = 15 rats in each group).

Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups, exact P values were shown in Appendix Table S3). Scale bar: 0.5 cm (H) or 20 µm (I).
Identification of a candidate therapeutic autophagy-inducing peptide (2013)
f1

a, Immunoprecipitation of Flag–beclin 1 constructs with Nef–HA in HeLa cells 24 h post-transfection. b, GFP–LC3-positive dots (autophagosomes) in MCF7 cells expressing GFP–LC3 and Flag–beclin 1 constructs grown in either normal medium or starved in EBSS for 2 h. c, Sequences of beclin 1 amino acids 267–284, Tat–beclin 1 (T-B) and Tat-scrambled (T-S) control peptide. Red letters indicate amino acid substitutions to enhance hydrophilicity. d, Biochemical assessment of autophagy (p62 and LC3 immunoblots) in peptide-treated HeLa cells (3 h). e, f, Representative images (e) and quantification of GFP–LC3-positive dots (f) in peptide-treated HeLa/GFP–LC3 cells (30 &mgr;M, 3 h). Scale bars, 20 &mgr;m. g, GFP–LC3-positive dots in siRNA-transfected peptide-treated HeLa/GFP–LC3 cells (30 &mgr;M, 3 h). h, Model of Tat–beclin 1 peptide (left) based on corresponding elements of the beclin 1 evolutionarily conserved domain (ECD) structure (centre). Essential phenylalanine side chains, magenta; positions of solubility mutations, pink; lipid interaction site, yellow. ECD surface representation (right) illustrates exposure of corresponding peptide (cyan). i, p62 and LC3 immunoblots in peptide-treated HeLa cells (3 h). In b, f, g, bars represent mean ± s.e.m. of triplicate samples (50–100 cells per sample). Similar results were observed in three independent experiments. *P  0.05, **P  0.01; t-test.
Identification of a candidate therapeutic autophagy-inducing peptide (2013)
f2

a, HeLa cells were treated with biotin-conjugated peptides (30 &mgr;M, 3 h) and proteins bound to peptides were analysed by immunoblot with anti-GAPR-1. b-T-B, biotin–Tat–beclin 1; b-T-S, biotin–Tat-scrambled. b, Immunoprecipitation of Flag–beclin 1 with GAPR-1–Myc in HeLa cells 24 h post-transfection. c, GFP–LC3-positive dots in GAPR-1 siRNA-transfected peptide-treated HeLa/GFP–LC3 cells (20 &mgr;M, 3 h) with or without 100 nM bafilomycin A1. Bars represent mean ± s.e.m. of triplicate samples (50–100 cells per sample). Similar results were observed in three independent experiments. NC, non-silencing control. d, Localization of beclin 1 and GM130 (a Golgi marker) in HeLa cells stably transduced with empty vector (left panel) or GAPR-1–Myc (middle panel) or transfected with GAPR-1 siRNA (right panel) and treated with peptide (20 &mgr;M, 1 h). Scale bar, 20 &mgr;m. e, Immunoblot of beclin 1 in post-nuclear supernatant (PNS) and Golgi-enriched fractions in HeLa cells stably transduced with empty vector or GAPR-1–Myc after peptide treatment (20 &mgr;M, 2 h). f, WIPI2 dots in cells in the experimental conditions shown in d. Bars represent mean ± s.e.m. for 100–150 cells. *P  0.05, ***P  0.001; t-test. †††P  0.001; two-way ANOVA for comparison of magnitude of changes between groups.
Identification of a candidate therapeutic autophagy-inducing peptide (2013)
f3

a, Percentage of cells with small htt103Q aggregates (left) and number of aggregates per cell (right) in HeLa cells expressing doxycycline (Dox)-repressible CFP–htt103Q after daily treatment with doxycycline or peptide (20 &mgr;M, 4 h per day) for 2 days. Bars represent mean ± s.e.m. of triplicate samples (60–120 cells per sample). Similar results were observed in three independent experiments. b, Filter trap assays for htt103Q large and small aggregates in HeLa/htt103Q cells. c, Viral titres in HeLa cells infected with 0.1 plaque-forming units (p.f.u.) per cell of SINV, CHIKV or WNV (strain TX02) and treated with peptide (10 &mgr;M, 4–8 h post-infection). Values represent geometric mean ± s.e.m. for triplicate samples of supernatants collected 18 h post-infection (SINV) or 24 h post-infection (CHIKV and WNV). Similar results were observed in three independent experiments. d, Bacteria colony-forming units (c.f.u.) in primary BMDMs infected with L. monocytogenes Dgr;actA mutant strain DPL-4029 for 30 min and treated with peptide (10 &mgr;M from 0 to 2 h post-infection). Bars represent mean ± s.e.m. of triplicate samples. Similar results were observed in three independent experiments. e, HIV-1 p24 antigen release in primary human MDMs infected with HIV-1 24 h after initiation of daily peptide treatment. Values represent mean ± s.e.m. of triplicate samples. Similar results were observed in MDMs from three independent donors. f, HIV-1 p24 antigen release in MDMs transduced with nonspecific scrambled shRNA (shNS) or ATG5 shRNA (shATG5) and treated daily with peptide (5 &mgr;M). Values represent mean ± s.e.m. of triplicate wells. Similar results were observed in MDMs from two independent donors. g, LC3 immunoblot of MDMs transduced with the indicated shRNA at day 0 and day 10 after HIV-1 infection. *P  0.05; **P  0.01; ***P  0.001; t-test.
Identification of a candidate therapeutic autophagy-inducing peptide (2013)
f4

a, GFP–LC3-positive dots in tissues of 6-week-old GFP–LC3 mice treated with the indicated peptide (20 mg kg−1 i.p., 6 h). A minimum of ten fields was counted per tissue section. Bars represent mean ± s.e.m. for three mice. Similar results were observed in three independent experiments. b, p62 immunoblot of brains of 5-day-old GFP–LC3 mice treated with the indicated peptide (20 mg kg−1 i.p., 6 h). c, Survival curves of 5-day-old C57BL/6J mice infected with CHIKV (106 p.f.u. s.c.) and treated daily with peptide (15 mg kg−1 i.p. beginning 1 day post-infection). d–f, Representative images of WNV envelope antigen and TdT-mediated dUTP nick end labelling (TUNEL) staining (d) (T, Tat alone), quantification of cell death in brain (e), and survival curves (f) for 5-day-old C57BL/6J mice infected with WNV (Egypt strain 101, 1 p.f.u. intracerebral (i.c.)) and treated daily with peptide (d-amino acid forms, 20 mg kg−1 i.p. beginning 1 day post-infection). Images in d are from cerebral cortex day 6 post-infection. Similar results were observed in all regions of the brain for three mice per group. Scale bar, 20 &mgr;m. Bars in e represent mean ± s.e.m. TUNEL-positive cells per unit area of brain for three mice. g, Geometric mean + s.e.m. viral titres of WNV-infected mouse brains day 6 post-infection. Values represent combined data for 12–20 mice per treatment group from 10 to 12 litters. Data in c and f represent combined survival probabilities for three and four independent litters, respectively, in each group. Similar results were observed in each independent experiment. *P  0.05; **P  0.01; ***P  0.001; NS, not significant; t-test.
WRN helicase and mismatch repair complexes independently and synergistically disrupt cruciform DNA structures (2022)
Figure 1

Figure 1 - WRN helicase unfolds cruciform DNA

A A schematic representation of the cruciform detection assay.

B Representative cruciform detection assay. Supercoiled DNA (cruciform) and linear DNA (no cruciform) were resolved on a 1% agarose gel, stained with GelRed. The % of cruciform-containing molecules are expressed as averages; n = 3.

C Representative polyacrylamide gel showing purification of recombinant WRN. The gel was stained with Coomassie Brilliant blue.

D Cruciform unfolding assay with increasing WRN concentrations and cruciform consisting of random inverted repeats (random-IR). Top, quantitation of cruciform unfolding. The amount of linear DNA from the "No protein" lane was subtracted from all other samples. Averages shown; n = 3 technical replicates; error bars, SEM.

E Cruciform unfolding assays as in (D) with WRN, WRN K577M, and WRN E84A (all used at 10 nM), carried out either in the presence or absence of ATP, or with ATP-ƔS. Averages shown; n ≥ 3 technical replicates; error bars, SEM.

F Electrophoretic mobility shift assays with WRN and WRN K577M, using pUC19 with the random-IR cruciform structure as a substrate.

G Representative cruciform unfolding assays with increasing concentrations of WRN, together with human RPA (hRPA, 30 nM), yeast S. cerevisiae RPA (yRPA, 30 nM), or human mitochondrial SSB (hmtSSB, 50 nM). pUC19 with the random-IR cruciform structure was used as a substrate.

H Quantitation of assays such as in (G). The amount of linear DNA from the "No protein" lane was subtracted from all other samples. Averages shown; n ≥ 3 technical replicates; error bars, SEM.
WRN helicase and mismatch repair complexes independently and synergistically disrupt cruciform DNA structures (2022)
Figure 2

Figure 2 - WRN unfolding of cruciforms prevents DNA cleavage by the SLX1-SLX4CCD nuclease.

A Representative assays with cruciform DNA (consisting of random-IR) either without or with WRN incubation prior to cruciform detection by SLX1-SLX4CCD. The ScaI restriction site is 931 bp away from the cruciform site cleaved by SLX1-SLX4CCD. Combined activity of ScaI and SMX results in 931 and 1795 bp bands indicated by the green arrows. The intensity of these bands decreases upon WRN incubation, resulting predominantly in linear DNA cut by ScaI, suggesting that WRN removes the substrate for SLX1-SLX4CCD. The catalytic dead version of SLX1-SLX4CCD (SLX1CD-SLX4CCD), where SLX1 carries an R41A mutations in its nuclease domain, has no activity on the cruciform DNA.

B Quantitation of assays such as in (A). Averages shown; n = 3 technical replicates; error bars, SEM.
WRN helicase and mismatch repair complexes independently and synergistically disrupt cruciform DNA structures (2022)
Figure 3

Figure 3 - TA repeats enhance cruciform DNA formation

A Comparison of cruciform substrates based on random inverted repeats (left) and TA repeats (right). Top, a schematic cartoon; Bottom, DNA sequence.

B Mapping of TA repeats cruciform structure. T7 Endonuclease I activity, followed by SspI, cleaves the cruciform within negative supercoiled DNA, producing two bands of the expected size (607 bp and 2119 bp, indicated by the green arrows, lane 2). Simultaneous incubation of the substrate with SspI and EcoRI allows EcoRI cutting at the TA repeats sequence (lane 5), while the extruded cruciform is refractory to EcoRI digestion (lane 4, the * indicates scDNA).

C Representative cruciform detection assays. Plasmid DNA was subjected to Cruciform extrusion procedure, where indicated. Supercoiled DNA (cruciform, refractory to EcoRI) and linear DNA (no cruciform, cleaved by EcoRI) were resolved on a 1% agarose gel, stained with GelRed. Top, quantitation of extruded cruciform in % with respect to the amount of total DNA that is present in the lane. Averages shown; n = 4 technical replicates; error bars, SEM.

D Representative cruciform unfolding assays with increasing WRN concentrations, using pUC19 with either random inverted repeats or TA repeats as a substrate. Top, quantitation of cruciform unfolding. The amount of linear DNA from the "No protein" lane was subtracted from all other samples. Averages shown; n = 3 technical replicates; error bars, SEM.
WRN helicase and mismatch repair complexes independently and synergistically disrupt cruciform DNA structures (2022)
Figure 4

Figure 4. Cruciform DNA unfolding is not a shared activity of RecQ family helicases

A Recombinant BLM and S. cerevisiae Sgs1 used in this study. The gel was stained with Coomassie Brilliant blue.

B Representative cruciform unfolding assay with increasing concentrations of WRN, BLM, and Sgs1. Cruciform DNA consisting of random inverted repeats was used.

C Quantitation of assays such as (B). Averages shown; n ≥ 3 technical replicates; error bars, SEM.

D Quantitation of DNA unwinding from experiments such shown in (EV4D). Averages and individual data points shown; n = 2 technical replicates.

E Recombinant WRN-3A (S991A T1152A S1256A) used in this study. The gel was stained with Coomassie Brilliant blue.

F Representative cruciform unfolding assays with increasing concentrations of WRN and WRN-3A. Cruciform DNA consisting of random inverted repeats was used.

G Quantitation of assays such as in (F). Averages shown; n = 3 technical replicates; error bars, SEM.
WRN helicase and mismatch repair complexes independently and synergistically disrupt cruciform DNA structures (2022)
Figure 5

Figure 5 - WRN helicase core and RQC domains are responsible for cruciform DNA unfolding

A Primary structure of WRN. Full-length and truncation WRN variants are shown.

B Recombinant full-length and truncation WRN variants used in this study are shown. The gel was stained with Coomassie Brilliant blue.

C Quantitation of DNA unwinding from assays such as shown in (EV5). Averages shown; n = 3 technical replicates; error bars, SEM.

D Cruciform unfolding assays with increasing concentrations of WRN variants.

E Quantitation of assays such as shown in (D). Averages shown; n = 3 technical replicates; error bars, SEM.
WRN helicase and mismatch repair complexes independently and synergistically disrupt cruciform DNA structures (2022)
Figure 6

Figure 6 - MutSα, MutSβ and to a lesser degree MutLα MMR complexes unfold cruciform structures

A MutL homologue heterodimers used in this study: MutLα (MLH1-PMS2), MutLβ (MLH1-PMS1) and MutLγ (MLH1-MLH3).

B MutS homologue heterodimers used in this study: MutSα (MSH2-MSH6), MutSβ (MSH2-MSH3) and MutSγ (MSH4-MSH5).

C Cruciform unfolding assays with MutSβ, MutS⍺, MutL⍺, and MutLβ. Cruciform DNA based on random inverted repeats was used.

D Quantitation of assays such as in (C) and (EV6A). Averages shown; n = 3 technical replicates; error bars, SEM.

E Representative electrophoretic mobility shift assays with the MutS and MutL homologue heterodimers, using an oligonucleotide-based Holliday junction as a substrate. Red asterisk indicates the position of the radioactive label.

F Representative cruciform unfolding assays with MutSβ and MutS⍺, carried out without or with ATP, using the random-IR cruciform as a substrate. Top, quantitation of cruciform unfolding. Averages shown; n = 3 technical replicates; error bars, SEM.

G Representative cruciform unfolding assays with MutSβ and MutS⍺, carried out without or with hRPA, using the random-IR cruciform as a substrate. Top, quantitation of cruciform unfolding. Averages shown; n = 3 technical replicates; error bars, SEM.
WRN helicase and mismatch repair complexes independently and synergistically disrupt cruciform DNA structures (2022)
Figure 7

Figure 7 - The activities of WRN and MMR proteins synergize in the unfolding of a TA cruciform

A Representative polyacrylamide gel showing recombinant MutSβ, MutSβ-3K255A (MSH3 with mutation K255A, deficient in mismatch recognition) and MutSβ-3K902A (MSH3 with mutation K902A, ATPase-deficient). The gel was stained with Coomassie Brilliant blue.

B Electrophoretic mobility shift assay with MutSβ, MutSβ-3K255A and MutSβ-3K902A, using either dsDNA (50 bp) or dsDNA bearing one extrahelical T, as a substrate. 6% native acrylamide gel was used.

C Electrophoretic mobility shift assays with MutSβ and MutSβ-3K255A, using circular pUC19 with the AT repeat cruciform structure, as a substrate. 0.8% native agarose gel was used.

D Cruciform unfolding assays with MutSβ, MutSβ-3K255A and MutSβ-3K902A. Bottom, representative experiments; top, quantitation, averages shown; n = 3 technical replicates; error bars, SEM.

E Representative polyacrylamide gel showing recombinant MutLα, MutL⍺-1E34A (MLH1 with mutation E34A, ATPase-deficient) and MutL⍺-2E41A (PMS2 with mutation E41A, ATPase-deficient). The gel was stained with Coomassie Brilliant blue.

F Unfolding of TA cruciform by WRN, MutLα and its ATPase-deficient variants. Bottom, representative experiments; top, quantitation; averages shown; n = 3 technical replicates; error bars, SEM.

G Unfolding of TA cruciform by WRN and MMR proteins. Bottom, representative experiments; top, quantitation; averages shown; n = 4 technical replicates; error bars, SEM.

H Unfolding of TA cruciform by MutLα and wild-type WRN or helicase dead WRN-K577M. Bottom, representative experiments; top, quantitation, averages shown; n = 4 technical replicates; error bars, SEM.
A stress-induced tyrosine-tRNA depletion response mediates codon-based translational repression and growth suppression (2020)
Figure 3

Figure 3. Cell growth repression upon oxidative stress and tRNATyrGUA depletion.

(A) Growth curves of MCF10A cells exposed to oxidative stress (200μM H2O2) relative to control cells (n=3). Two-way ANOVA was used to test for significance.

(B) Northern blot of MCF10A cells expressing a control short-hairpin RNA or a hairpin targeting tRNATyrGUA.

(C) Western blot of MCF10A expressing a control short-hairpin RNA or a hairpin targeting the tyrosine-tRNA synthetase, YARS (red arrow). HSC70 was used as a loading control.

(D) Growth curves of MCF10A cells expressing RNAi targeting mature tRNATyrGUA or YARS relative to cells expressing a control hairpin (n=3). Two-way ANOVA was used to test for significance.

(E) Cell growth of MCF10A cells transiently transfected with a tRNATyrGUA overexpression vector relative to an empty control vector (n=3). A one-tailed Mann-Whitney test was used to test for significance at day 3.

Data information: Data represent mean ± s.e.m. *p < 0.05 and ***p < 0.001
A stress-induced tyrosine-tRNA depletion response mediates codon-based translational repression and growth suppression (2020)
Figure 4

Figure 4. A set of growth promoting genes are sensitive to tRNATyrGUA depletion.

(A) Cells depleted of tRNATyrGUA or YARS were processed for label free quantitation by mass spectrometry to identify proteins that were reduced by a log2-fold change of 0.5 or more. This set was overlapped with proteins containing a higher than median abundance of Tyr codon content to identify candidate mediators of the pleiotropic effects of tRNATyrGUA depletion.

(B) GO functional analysis of the 109 candidate gene-set from (A).

(C) Quantitative western blot validation depicting abundances of protein targets (EPCAM, SCD, and USP3) identified from (A). HSC70 was used as a loading control and is not modulated upon molecular perturbation of tRNATyrGUA.

(D) Quantification of western blot analysis in (C) (n=4). A one-tailed Mann-Whitney test was used to test for statistical significance.

(E) Growth curves for MCF10A cells were transfected with either control siRNA or two independent siRNA targeting EPCAM, SCD, or USP3. Note that the control cell growth curve is the same in all graphs and were plotted separately for clarity and does not represent independent experiments. Two-way ANOVA was used to test for significance (n=3).

Data information: Data represent mean ± s.e.m. *p < 0.05, **p < 0.01
A stress-induced tyrosine-tRNA depletion response mediates codon-based translational repression and growth suppression (2020)
Figure 5

Figure 5. Global ribosome occupancy analysis from tRNATyrGUA-depleted cells reveals reduced translation efficiency for Tyr-enriched genes.

(A) Quantitative western blot EPCAM, SCD, and USP3 in MCF10A cells 24 hours after treatment with H2O2 (200μM). HSC70 was used as a loading control.

(B) Quantification of western results in (A) (n=9). A one-tailed Mann-Whitney test was used to test for statistical significance.

(C) A schematic of the codon-based USP3 reporter. A Myc-tagged WT or mutant reporter with 5 Tyr codons mutated to Ala codons were cloned upstream of a Luciferase used for transfection normalization.

(D) Quantitative western blot for the Myc-tag and Luciferase (top) with normalized fluorescent intensities (bottom) are shown (n=3).

(E) Ribosome occupancy of 20-22nt RPFs in tRNATyrGUA-depleted cells compared to control cells reveal greater occupancy at both tyrosine codons in tRNATyrGUA-depleted cells using a Wilcoxon test.

(F) Genes were sorted based on their changes in GC-corrected translation efficiency (TE) values, with reduced TE in tRNATyrGUA-depleted cells shown in left and enhanced TE shown on right. The red bars over each column depict the range of values in that bin. Distribution of genes with high tyrosine codon content across these three bins were assessed using mutual information calculation and testing (see methods for details). For visualization, we used the hypergeometric distribution to assign p-values to the overlap between tyrosine-rich genes and each bin. We then defined an enrichment score as -log of p-value, if there was a significant enrichment. If the overlap is significantly fewer than expected by chance, log of p-value is used instead (depletion). The resulting enrichment score is then shown as a heatmap with gold depicting positive enrichment.

Data information: Data represent mean ± s.e.m. *p < 0.05 and ***p < 0.001