EMBO/soda-nel huggingface dataset:
RAD54 promotes Alternative Lengthening of Telomeres by mediating branch migration (2020)
Figure 3

Figure 3: RAD54 oligomerization and ATPase activity regulate break-induced replication at ALT telomeres

A Representative images of combined EdU staining (Click-it) and DNA FISH for telomeres. HuO9 or SaOS2 cells were transfected with 20nM siRAD54#2 (representative images for siRAD54#1 not shown) for 48 hours followed by a 1.5 hr pulse of EdU. Pan-nuclear EdU stain represents S-phase cells. White arrows indicate non-S-phase cells containing EdU colocalizing with telomeres. Scale bars = 10 µm

B Quantification of data shown in A. HuO9 or SaOS2 cells were mock transfected or transfected with 20nM siRAD54#1 or 20nM siRAD54#2 for 48 hours followed by a 1.5 hr pulse of EdU. Cells with pan-nuclear EdU signal or greater than 8 EdU foci were excluded from analysis as S-phase cells. Non-S-phase cells were considered positive if they contained at least one EdU foci colocalizing with telomeres. Data were normalized to mock condition for each repeat. At least 100 non-S-phase cells were counted per condition per repeat, n=3. Values shown are mean±sd. Data were analyzed using two-way ANOVA followed by Dunnett's multiple comparison test.

C Representative DNA dot blot from C-circle assay on HuO9 cells, either mock transfected or transfected with 20nM siRAD54#2 for 48 hours.

D Quantification of C-circle assay in C. Signal was quantified with densitometry, background (- Φ29) was subtracted and signal was normalized to mock. N=5 biological replicates values shown are mean±sd. Conditions were analyzed using unpaired two-tailed student's t-test.

E Representative images of combined IF and DNA FISH for PML (IF) and telomeres (FISH). HuO9 or SaOS2 cells were mock transfected or transfected with 20nM siRAD54#2 for 48 hours. White arrows indicate large colocalization events. Blue arrows indicate small colocalization events. Scale bars = 10 µm

F Quantification HuO9 or SaOS2 cells were mock transfected or transfected with 20nM siRAD54#1 or 20nM siRAD54#2 for 48 hours. Cells were considered positive if they contained 1 large or 3 small colocalization events. At least 100 cells were counted per condition per repeat, n=3. Values shown are mean±sd. Data were analyzed using two-way ANOVA followed by Dunnett's multiple comparison test.

G Representative images of combined EdU staining (Click-it) and DNA FISH for telomeric DNA. HuO9 cells were forward transfected with 2µg empty vector (EV), RAD54-WT, RAD54-K189R, or S49E construct. After 24 hours, cells were transfected with 20 nM siRAD54#2 for 48 hours followed by a 1.5 hr pulse of EdU. White arrows indicate non-S-phase cells containing EdU colocalizing with telomeres. Scale bars = 10 µm

H Quantification of data shown in G. Non-S-phase cells were considered positive if they contained at least one EdU foci colocalizing with telomeres. Data were normalized to EV-Mock condition for each repeat. At least 100 non-S-phase cells were counted per condition per repeat, n=4 for EV, WT, S49E, n=3 for K189R. Values shown are mean±sd. Data were analyzed using two-way ANOVA followed by Sidak's multiple comparison test.
Foxp3 expression in induced regulatory T cells is stabilized by C/EBP in inflammatory environments (2018)
Figure 1

Figure 1 - C/EBP functions in the presence of inhibitory cytokines

A Real-time qRT-PCR analysis of C/EBPβ mRNA in CD4+ naïve T cells inactivated (naive) or cultured for 24 hr under conditions as indicated with DMSO or ATRA (all-trans retinoic acid). Data are representative of two independent experiments with consistent results and normalized with β-actin (mean and s.e.m. of quadruplicates).

B Immunoblot analysis of C/EBPβ in the nuclear fraction of naïve CD4+ T cells cultured for 2 d under conditions as indicated. The immunoblot was quantified using Image J.

C-F Flow cytometry of intracellular Foxp3 staining in CD4+ naïve T cells transduced with control retrovirus (MigRI) or retrovirus encoding A-C/EBP or C/EBPζ and cultured for 2 d under conditions as indicated. Representative experiments are shown in the left panel and pooled data with mean values from 8(C, E), 6(D) or 7(F) independent experiments are shown on the right. Dot plots are gated for CD4+GFP+ and numbers indicate percent Foxp3+ cells in the gate.

Data information: Statistical analysis was performed using unpaired two-tailed t-test (A), or (C-F) one-way ANOVA. (*p<0.05, ***p<0.001, ****p<0.0001; ns, not significant).
Foxp3 expression in induced regulatory T cells is stabilized by C/EBP in inflammatory environments (2018)
Figure 2

Figure 2 - C/EBPβ confers complete resistance to inhibitory effect of IFN-γ and IL-4 on iTreg generation

A, B Flow cytometry of intracellular Foxp3 staining in CD4+ naïve T cells transduced with control retrovirus (MigRI) or retrovirus encoding C/EBPβ and cultured for 2 d under conditions as indicated. Representative experiments are shown in the upper panel and pooled data from 4 (A), 3 (B) independent experiments are shown in the bottom. Dot plots are gated for CD4+GFP+ and numbers indicate percent Foxp3+ cells in the gate. Error bars represent mean ± s.e.m.

C Real-time qRT-PCR analysis of FACS-sorted GFP+ cells from CD4+ naïve T cells transduced with control retrovirus (MigRI) or retrovirus encoding C/EBPβ and cultured for 2 d in the conditions as indicated. Data are representative of two independent experiments with consistent results and normalized with β-actin (mean ± s.e.m. of triplicates).

D Body weight of RAG-2-/- mice after transfer with 5 x 105 CD4+CD45RBhigh cells alone (circles) or with 2 x 105 FACS-sorted CD4+GFP+ cells transduced with control retrovirus (MigRI) (squares) or retrovirus encoding C/EBPβ (triangles) and cultured for 2 d in the presence of TGF-β and culture supernatant. Each time point contains three (circles) or four (squares and triangles) mice in each group. Error bars represent mean ± s.d.

E Hematoxylin and eosin staining of the distal colons of mice (Scale bars, 200μm)

F Histological scores.

G Comparison of the spleen weight.

F, G Each symbol represents an individual mouse. Small horizontal lines indicate the mean (error bars, s.e.m).

Data information: Statistical analysis was performed using paired two-tailed t-test (A, B), one-way ANOVA (D,G), or Kruskal Wallis test (F). (*p<0.05, **p<0.01, ***p<0.001; ns, not significant)
Foxp3 expression in induced regulatory T cells is stabilized by C/EBP in inflammatory environments (2018)
Figure 3

Figure 3 - C/EBP is critical for the regulation of Foxp3 expression in vivo during iTreg generation

A, B Flow cytometry of intracellular Foxp3 staining of mesenteric lymph node cells of Balb/c mice that received RAG2-/-DO11.10 CD4+ naïve T cells transduced with control retrovirus (MigRI) or retrovirus encoding A-C/EBP or C/EBPβ. (A) The recipients were immunized via intravenous injection of OVA323-339 peptide (20 ug) 1 and 3 d after transfer of transduced cells and analyzed 5 d after the first immunization. (B) The recipients were fed 1% OVA solution in drinking water for 5 consecutive days. Representative experiments are shown in the left panel and pooled data from 4 (A) or 3 (B) independent experiments with mean values are shown on the right. Dot plots are gated for CD4+KJ.1.26+GFP+ (top) or CD4+KJ.1.26+GFP- (bottom) and numbers indicate percent Foxp3+ cells in the gate. Statistical analysis was performed using one-way ANOVA. (*p<0.05, **p<0.01; ns, not significant)
Foxp3 expression in induced regulatory T cells is stabilized by C/EBP in inflammatory environments (2018)
Figure 4

Figure 4 - C/EBPβ binds to methyl-CRE sequence in the Foxp3 TSDR and acts in a methylation-dependent manner

A EMSA of nuclear extracts of Jurkat cells transfected with indicated expression vectors and assessed with a probe covering the putative unmethyl- or methyl-CRE sequence in the TSDR.

B ChIP-qPCR assay of C/EBPβ on the Foxp3 TSDR in naïve CD4+ T cells cultured for 2 d under conditions as indicated. Data are representative of two independent experiments with consistent results (mean ± s.e.m. of triplicates).

C, D Flow cytometry of intracellular Foxp3 staining in CD4+ naïve T cells transduced with control retrovirus (MigRI) or retrovirus encoding C/EBPβ and cultured for 2 d under conditions as indicated. Representative experiments are shown in the left panel and pooled data from 4 (C) or 5 (D) independent experiments with mean values are shown on the right. Dot plots are gated for CD4+GFP+ and numbers indicate percent Foxp3+ cells in the gate.

Data information: Statistical analysis was performed using one-way ANOVA (B-D). (*p<0.05, **p<0.01, ***p<0.001; ns, not significant).
Foxp3 expression in induced regulatory T cells is stabilized by C/EBP in inflammatory environments (2018)
Figure 6

Figure 6 - Human C/EBPβ also confers resistance to inhibitory effect of IFN-γ and IL-4 on human iTreg generation

A Flow cytometry of intracellular FOXP3 staining (right) or isotype control (left) in human CD4+CD25-CD45RA+ naïve T cells transduced with control retrovirus (Min) or retrovirus encoding hC/EBPβ and cultured for 2 d under conditions as indicated. Representative experiments are shown in the left panel and pooled data with mean±s.e.m are shown on the right. Histograms are gated for CD4+NGFR+. Statistical analysis was performed using unpaired two-tailed t test. (*p<0.05; ns, not significant).

B Flow cytometry of CFSE-labeled human CD4+CD25- T cells (responder) stimulated 4 d alone or together with NGFR+-sorted human CD4+CD25-CD45RA+ naïve T cells (suppressor) transduced with control retrovirus (Min) or retrovirus encoding hC/EBPβ and cultured for 5 d in the presence of hTGF-β, anti-hIFN-γ and anti-hIL-4 Abs. Histograms are gated for CFSE+. The ratio of responder to suppressor is 1:0.2 (top) or 1:0.4(bottom). Data are representative of two independent experiments with consistent results.

Expanded View Figure legends
Genetic and hypoxic alterations of the microRNA-210-ISCU1/2 axis promote iron-sulfur deficiency and pulmonary hypertension. (2015)
Figure 1

The miR-210-ISCU1/2 regulatory axis is activated in endothelium and remodeled vessels in PH

A RT–qPCR reveals that miR-210 was increased in lungs of mice with PH triggered by various conditions: VHL−/− as compared with VHL+/+ mice (N = 4/group), ***P < 0.0001 (first graph); hypoxia + SU5416 (Hyp + SU5416) (N = 6/group) as compared with normoxia + SU5416 (Norm + SU5416) (N = 7/group), **P = 0.0015 (second graph); Il6 transgenic versus littermate control mice (N = 4/group), **P = 0.0097 (third graph); and S. mansoni-infected mice (N = 4) compared with non-infected control mice (N = 5), ***P < 0.0001 (fourth graph).

B From animal subjects in (A), in situ hybridization (ISH, purple stain) revealed increased miR-210 in < 100-μm pulmonary vessels of mice suffering from PH, *P = 0.0493 for first graph, **P = 0.0015 for second graph, *P = 0.0391 for third graph.

C Representative ISH stain of miR-210 in < 100 μm pulmonary vessels of mice (bottom micrographs) exposed to Hyp + SU5416 compared with Norm + SU5416 (α-smooth muscle actin stain from serial sections, top row of micrographs).

D Increased miR-210 in < 200-μm remodeled pulmonary vessels of patients suffering from PAH (N = 19, Supplementary Table S1) as compared with non-PAH donor control lung (N = 10). Serial staining with hematoxylin and eosin is displayed in the top row of micrographs; quantification of miRNA ISH, right graph, *P = 0.0167.

E Increased levels of miR-210 in plasma drawn from the pulmonary circulation (pulmonary capillary wedge position, PCWP) of patients with elevated mean pulmonary arterial pressures (mean PAP ≥ 25 mmHg) compared with control subjects (mPAP < 25 mmHg, N = 5/group, demographics in Supplementary Table S2), *P = 0.0357.

F, G From animals in (C) and humans in (D), immunohistochemistry (IHC) revealed that the miR-210 targets ISCU1/2 were reciprocally down-regulated in miR-210-enriched remodeled pulmonary vessels—namely in PH mice exposed to Hyp + SU5416 (F, ***P = 0.0002) and in human PAH patients (G, ***P = 0.0008).

H, I miR-210 expression (H) was increased (***P = 0.0003), and ISCU1/2 expression (I) was decreased (***P < 0.0001) in PECAM+ pulmonary vascular endothelial cells isolated from PH mice (Hyp + SU5416) as compared with control (Norm + SU5416) (N = 4/group, left bars).

Data information: In (A, B), mean expression of miR-210 in control groups was assigned a fold change of 1, to which all samples were compared. Error bars reflect SEM. Mouse tissue scale bar: 50 μm, human tissue scale bar: 100 μm.
Genetic and hypoxic alterations of the microRNA-210-ISCU1/2 axis promote iron-sulfur deficiency and pulmonary hypertension. (2015)
Figure 2

Impaired Fe-S cluster integrity in diseased pulmonary vasculature is driven by the miR-210-ISCU1/2 axis

After lentiviral delivery of GCN4 or GRX2 sensor genes to human PAECs, cellular fluorescence was measured by flow cytometry. Unlike control GCN4 sensors that homodimerized independent of Fe-S levels and induced consistent fluorescence, fluorescence derived from the GRX2 Fe-S-dependent sensors decreased in hypoxia, as displayed in representative flow cytometric plots (left) and by quantification of percentage of positive cells [(cell number in M2 gate)/(total cell number) × 100]. Immunoblotting revealed consistent expression of either GRX2 or GCN4 sensors (FLAG-tagged) in hypoxia (Hyp) compared with normoxia (Norm) (N = 3, ***P < 0.0001 for GRX2; N = 3, NS P = 0.1848 for GCN4).

In contrast to consistent fluorescence from control GCN4 sensors, Fe-S-dependent GRX2 sensor fluorescence was decreased by siRNA knockdown of ISCU1/2 (siISCU) as compared with control (siCont) (N = 3, ***P < 0.0001 for GRX2; N = 3, NS P = 0.1790 for GCN4).

GRX2, but not GCN4, sensor fluorescence was decreased after transfection of miR-210 oligonucleotide mimic (miR-210) as compared with control (miRC) (N = 3, ***P = 0.0002 for GRX2; N = 3, NS P = 0.0913 for GCN4). During hypoxic exposure, GRX2, but not GCN4, sensor fluorescence was increased after transfection of an antisense miR-210 inhibitor (AS210) as compared with control (ASC) (N = 3, **P = 0.003 for GRX2; N = 3, NS P = 0.1194 for GCN4).

After lentiviral delivery of sensor genes, Fe-S-dependent GRX2 sensor fluorescence was decreased in PECAM-positive cells from PH mouse lung (Hyp + SU5416) as compared with control non-PH mouse lung (Norm + SU5416, N = 3, ***P < 0.001).

By electron paramagnetic resonance (EPR) spectroscopy (representative VHL−/− versus control lung), Fe-S cluster signal was decreased (left graph) in VHL−/− mice lung tissue (VHL−/−, N = 3) as compared with WT control lung tissues (WT, N = 5), ***P = 0.0003.

By EPR, Fe-S-specific signal was decreased (right graph) in human PH-diseased lung tissue harvested at lung transplantation (PH, N = 3, Supplementary Table S3) compared with non-PH control donor lung tissues (control, N = 3), *P = 0.048.

Data information: In (A–C), sensor expression was confirmed by immunoblot for FLAG epitopes. Error bars reflect SEM. Source data are available online for this figure.
Genetic and hypoxic alterations of the microRNA-210-ISCU1/2 axis promote iron-sulfur deficiency and pulmonary hypertension. (2015)
Figure 3

miR-210 regulates Fe-S biogenesis, mitochondrial function, and downstream PH pathways in vivo and is necessary to induce hypoxic PH in mice

A Schema of comparing miR-210−/− and WT mice after hypoxia + SU5416 (PH) versus normoxia + SU5416 (control).

B By immunohistochemistry (IHC), ISCU1/2 was unchanged in < 100 μm pulmonary vessels of miR-210−/− mice exposed to Hyp + SU5416 (N = 6) versus Norm + SU5416 (N = 7), NS P = 0.2833.

C Fe-S-dependent Complex I-specific activity was decreased in WT PH mice as compared with control (N = 7/group, left bars, ***P < 0.0001), but activity was preserved in the lungs of miR-210−/− mice in either condition (N = 8/group, right bars), NS P = 0.3693.

D IHC demonstrated that pulmonary vascular GLUT1 was increased in WT PH mice (left bars, ***P = 0.0006), but GLUT1 was unchanged in miR-210−/− mice in either condition (right bars, NS P = 0.9967).

E 3-nitrotyrosine (3-NT) was increased in pulmonary vessels of WT PH mice but was reduced in miR-210−/− mice in either condition (N = 6/group) (***P < 0.0001, NS P = 0.4087).

F In PH conditions, endothelin-1 was decreased in miR-210−/− lung tissue compared with WT tissue (N = 5/group), **P = 0.007.

G PCNA was increased in WT PH pulmonary vessels but was decreased in miR-210−/− tissue exposed to either condition (N = 5/group) (**P = 0.0096, *P = 0.0124, *P = 0.0263 for miR-210−/−).

H PCNA was increased in PECAM+ endothelial cells from PH versus non-PH mice (N = 3/group, **P = 0.0052).

I Unlike WT mice (black bars) demonstrating increased right ventricular systolic pressure (RVSP) after Hyp + SU5416 (N = 10) versus Norm + SU5416 (N = 11), hemodynamic dysregulation was significantly alleviated in miR-210−/− mice (white bars, N = 11) (***P < 0.0001, *P = 0.0258, **P = 0.0059).

J Compared with WT controls (N = 9), miR-210−/− mice (N = 8) displayed a blunted increase of the Fulton index (RV/LV + S) under PH versus baseline conditions (expressed as a ratio of RV/LV + S under Hyp + SU5416 versus Norm + SU5416, *P = 0.031).

K–M Under PH (black bars, N = 8/group) versus baseline conditions (white bars, N = 6/group), pulmonary vascular remodeling was alleviated in miR-210−/− mice, as visualized via histology (L), and confirmed by decreased % arteriolar muscularization (K, **P = 0.001, **P = 0.0045 for miR-210−/−, *P = 0.0158) and decreased vessel wall thickness (M, ***P < 0.0001, **P = 0.0086).

Data information: Error bars reflect SEM. Mouse tissue scale bar: 50 μm.
Genetic and hypoxic alterations of the microRNA-210-ISCU1/2 axis promote iron-sulfur deficiency and pulmonary hypertension. (2015)
Figure 4

MiR-210 is sufficient to induce pulmonary vascular dysfunction in mice

A Schema for forced miR-210 expression in vivo (N = 8/group).

B, C Intrapharyngeal delivery of miR-210 mimic increased miR-210 in whole lung (***P = 0.0002) (B) and in < 100-μm pulmonary vessels (**P = 0.007) (C).

D miR-210 mimic also repressed ISCU1/2 levels in those same caliber vessels (N = 6/group), **P = 0.0022.

E Endothelin-1 was increased in mouse lung tissue after delivery of miR-210 mimic (N = 6/group), *P = 0.0368.

F–H miR-210 mimic delivery increased RVSP (N = 8/group) (F) and vascular remodeling (α-smooth muscle actin stain, α-SMA), as evidenced by increased percent of muscularized (< 100 μm) pulmonary vessels (G) and increased medial thickening relative to vessel diameter when compared with miR-Control (H) (N = 6/group). **P = 0.0021 for (F), **P = 0.0072 for (G), *P = 0.0335 for (H).

Data information: Error bars reflect SEM. Mouse tissue scale bar: 50 μm.
Genetic and hypoxic alterations of the microRNA-210-ISCU1/2 axis promote iron-sulfur deficiency and pulmonary hypertension. (2015)
Figure 5

Antisense inhibition of miR-210 protects against and improves existing manifestations of PH in vivo

A Schema of the strategy to determine whether antisense inhibition of miR-210 (anti-miR-210) prevents PH secondary to Hyp + SU5416 exposure (“prevention protocol”).

B–D Intravenous delivery of anti-miR-210 down-regulated miR-210 expression in whole lung tissue (N = 7/group), ***P < 0.0001 (B), the pulmonary vasculature as assessed by in situ staining (N = 7/group), ***P = 0.0003 (C), and PECAM+ vascular endothelial cells derived from whole lung tissue (N = 4/group), **P = 0.0013 (D).

E Correspondingly, anti-miR-210 delivery resulted in preservation of ISCU1/2 expression in < 100-μm pulmonary vessels as compared to anti-miR-Control (N = 5/group), **P = 0.0011.

F Similarly, ISCU1/2 expression was preserved in PECAM+ endothelial cells derived from mice treated with anti-miR-210 (N = 4/group), **P = 0.0016.

G Endothelin-1 was decreased in lung tissue from mice treated with anti-miR-210 during Hyp + SU5416 exposure (N = 6/group), *P = 0.0181.

H As assessed by in situ immunofluorescence, PCNA was decreased in pulmonary vessels (< 100 μm) after anti-miR-210 delivery, **P = 0.001.

I–K Anti-miR-210 delivery (N = 10/group) ameliorated the elevation of RVSP, **P = 0.0031 (i) and vascular remodeling, as reflected by increased percent of muscularized arterioles (N = 8/group, **P = 0.0091) (J) and increased medial thickness relative to vessel diameter in < 100-μm pulmonary vessels (N = 6/group, **P = 0.0021) (K).

L Schema of the strategy to determine whether anti-miR-210 improves existing PH (“reversal protocol”).

M, N Pharmacologic inhibition of miR-210 (N = 7/group) down-regulated pulmonary expression of miR-210, ***P < 0.0001 (M), and preserved ISCU1/2 expression in < 100-μm pulmonary vessels, **P = 0.0015 (N).

O–Q In contrast to anti-miR-Control (N = 8), anti-miR-210 ameliorated the elevation of RVSP (N = 10/group, ***P < 0.0001) (O) and pulmonary vascular remodeling (N = 7/group, **P = 0.0022, *P = 0.0167) (P–Q).

Data information: Error bars reflect SEM. Mouse tissue scale bar: 50 μm.
Genetic and hypoxic alterations of the microRNA-210-ISCU1/2 axis promote iron-sulfur deficiency and pulmonary hypertension. (2015)
Figure 6

Antisense inhibition of miR-210 specifically in vascular endothelium improves existing manifestations of PH in vivo

A Following the schema of Fig5A in the presence of hypoxia but the absence of SU5416, intravenous delivery of anti-miR-210 ameliorated the elevation of RVSP compared with anti-miR-Control (N = 6/group, ***P = 0.001).

B Schema of the strategy to determine whether delivery of anti-miR-210 specifically to the vascular endothelium via packaging with 7C1 nanoparticles improves hypoxia-induced PH (“7C1 reversal protocol”).

C, D Consistent with specific delivery to the vascular endothelium after intravenous administration (Dahlman et al, 2014), 7C1-mediated delivery of anti-miR-210 (N = 6/group) decreased miR-210 expression in PECAM+ pulmonary vascular endothelial cells, **P = 0.0013, (D) but not PECAM-negative pulmonary cells, NS P = 0.3775 (C).

E Correspondingly, endothelial-specific anti-miR-210 (N = 6/group) increased ISCU1/2 expression in PECAM+ cells even in the presence of chronic hypoxia, ***P = 0.0003.

F–I Delivery of anti-miR-210 (N = 5/group) decreased endothelin-1 in hypoxic mouse lung tissue, *P = 0.0198 (F). As a result, in contrast to anti-miR-Control (N = 8), endothelial-specific anti-miR-210 ameliorated the elevation of RVSP (N = 6), **P = 0.0024 (G), and pulmonary vascular remodeling (N = 6/group), **P = 0.0021, *P = 0.0117 (H–I).

Data information: Error bars reflect SEM. Mouse tissue scale bar: 50 μm.