EMBO/soda-nel huggingface dataset:
The high-energy transition state of the glutamate transporter homologue GltPh (2020)
Figure 3

Figure 3: Correlations between activity, dynamics and substrate affinity.

(A) Changes in the initial rates of substrate uptake (gray bars), transport domain dynamics (black bars), and l-Asp dissociation constant (white bars) of the mutant GltPh variants relative to the WT transporter. The transition frequencies were measured under non-equilibrium transport conditions, and frequencies obtained in the presence of d,l-TBOA were subtracted from the data. Error bars represent standard errors for at least three independent measurements.

(B) Cartoon representation of the crystal structure of Y204L/A345V/V366A GltPh in the presence of saturating concentrations of Na+ ions and l-Asp (colored as in Figure 1A; PDB accession number 6V8G) superimposed onto the structure of WT GltPh in the OFS (gray, PDB accession number 2NWX). l-Asp and Na+ are shown as spheres and colored by atom type.

(C) Close-up of the substrate-binding site. Green mesh represents an omit electron density map for l-Asp contoured at 4σ. Substrate-coordinating residues are shown as sticks.

(D) Superimposition of the mutant (colors) and the WT (gray) hairpins aligned on HP1. See also Appendix Figure S5 and Appendix Tables S3 and S5.
The high-energy transition state of the glutamate transporter homologue GltPh (2020)
Figure 4

Figure 4: Kinetic heterogeneity of the transport domain dynamics.

(A) Dwell-time distributions of the OFS (blue) and the IFS (red) for WT (top) and K290A (bottom) GltPh observed under equilibrium conditions in saturating Na+/l-Asp (left). Lines are fits to three exponentials. Data are averages and standard errors of at least three independent measurements.

(B) Representative EFRET trajectories of WT GltPh showing different transition frequencies. Raw data are in black, and idealizations are in brown. Scalebar is above the trajectories.

(C) Distribution of the WT (top) and the K290A GltPh (bottom) molecules with different mean transition frequencies (white bars). N is the number of trajectories longer than 90 s used in the analysis. The stacked black bars are fractions of trajectories without transitions. Pink bars show the expected binomial distribution if all trajectories shared the mean transition frequency of 0.04 s-1 for the WT and 0.34 s-1 for the K290A. Data are averages and standard errors of at least three independent measurements.

(D) 2D-histograms of the consecutive dwell lengths in the OFS (top) and the IFS (bottom). From left to right: calculated distribution of dwell times randomly selected from the distributions in panel A; measured distribution for WT GltPh and the K290A mutant.

(E) Representative trajectories of the WT (left) and the K290A (right) GltPh molecules showing switching between dynamic modes. Raw data are in black and idealizations are in brown (top). Black and grey bars under the trajectories indicate apparent slower and faster dynamic modes. Survival plots for the OFS and the IFS (middle). Solid lines are fits to a single (IFS) and double (OFS) exponentials. Dashed lines (OFS only) are rejected fits to single exponentials. Autocorrelation plots of the sequential dwell durations (bottom). Solid lines are fits to single exponentials to guide the eye. See also Appendix Figures S6-S9.
The high-energy transition state of the glutamate transporter homologue GltPh (2020)
Figure 5

Figure 5: Distributions of the OFS and the IFS lifetime pairs for individual molecules.

(A, B) 2D-histograms of lifetime pairs obtained for trajectories exhibiting single exponential behavior. N is the number of traces used. Scale bar shows relative density normalized by the number of molecules. Above and to the right of each panel are stacked histograms of, respectively, the OFS and the IFS lifetimes of the analyzed molecules (open bars) and the photobleaching time of molecules showing no transitions (black bars). Data from three independent measurements were combined for presentation.

(C) The mean lifetimes of the molecules falling within 30 % of the most populated bins of the 2D-histograms (yellow to red). The open blue bar represents the calculated mean OFS lifetime for WT GltPh (see main text for details). Shown are means and standard errors of the mean of at least three independent measurements.
A Cdc42-Borg4-Septin 7 axis regulates HSC polarity and function (2021)
Figure 1

Figure 1. Borg4 and septin 7 show a polar distribution in HSCs which is regulated by Cdc42 activity.

(A,B) Level of expression of borgs and septins in young and aged LT-HSCs. mRNA levels of borgs and septins were normalized to the level of expression of GAPDH and borgs are shown relative to the level of Borg 2 expression in young LT-HSCs. Septins are shown relative to the level of septin 7 expression in young LT-HSCs. At least three biological replicates (n=3) are shown. Error bars indicate mean±SEM, unpaired t-test, **P<0.01, ***P<0.001, ****P<0.0001.

(C) Percentage of cells polarized for septin 7 among young, aged and aged LT-HSCs treated with CASIN (5µM). At least 3 biological repeats, at least 50 cells were scored per sample. Error bars indicate mean±SEM, unpaired t-test, **P<0.01, ***P<0.001.

(D,E) Representative immunofluorescence microscopy images of the distribution of septin 7 (red) or borg3 or borg4 (green) in young, aged and aged LT-HSCs treated with CASIN (5µM). Nuclei were stained with DAPI (blue), scale bar= 5µm.
A Cdc42-Borg4-Septin 7 axis regulates HSC polarity and function (2021)
Figure 2

Figure 2. The physical interaction between Cdc42-Borg4-Septin 7 is regulated by the activity of Cdc42.

(A) Representative immunofluorescence confocal microscopy images of young, aged and aged + CASIN (5µM) treated LT-HSCs for the proximity ligation assay (red signal), testing the extent of close physical interaction of Cdc42 and borg4. A red fluorescent signal indicates close proximity of the two proteins tested. Nuclei were stained with DAPI (blue), scale bar= 5µm.

(B) Quantification of the level of fluorescent signal in young, aged and aged + CASIN (5µM) treated LT-HSCs to quantify the Cdc42-borg4 proximity interaction. Data are normalized to the mean fluorescence signal of aged LT-HSCs. 3 biological repeats, n=at least 17 cells per condition, error bars indicate mean±SEM, unpaired t-test, ****P<0.0001.

(C) Representative immunofluorescence confocal microscopy images of young, aged and aged + CASIN (5µM) treated LT-HSCs for the proximity ligation assay (red signal), testing the extent of close physical interaction of borg4 and septin 7. A red fluorescent signal indicates close proximity of the two proteins tested. Nuclei were stained with DAPI (blue), scale bar= 5µm.

(D) Quantification of the level of fluorescent signal in young, aged and aged + CASIN (5µM) treated LT-HSCs to quantify the borg4-septin 7 proximity interaction. Data are normalized to the mean fluorescence signal of aged LT-HSCs. 3 biological repeats, n=at least 17 cells per condition, error bars indicate mean±SEM, unpaired t-test, ***P<0.001, ****P<0.0001.
A Cdc42-Borg4-Septin 7 axis regulates HSC polarity and function (2021)
Figure 3

Figure 3. Borg4 or septin 7 regulate the distribution of polarity proteins in HSCs.

(A,B) Representative immunofluorescence microscopy images of the distribution of septin 7 (red), Cdc42 (red) and tubulin (green) in LT-HSCs from borg4fl/fl or borg4∆/∆ mice. Nuclei were stained with DAPI (blue), scale bar= 5 µm.

(C) Percentage of cells with a polar distribution of septin 7, Cdc42 or tubulin in borg4fl/fl or borg4∆/∆ LT-HSCs. At least 3 biological repeats, at least 50 cells were scored per sample. Error bars indicate mean±SEM, Two-way ANOVA analysis, ***P<0.001, **P<0.01, *P<0.05.

(D,E) Representative immunofluorescence microscopy images of the distribution of borg4 (red), Cdc42 (red) or tubulin (green) in LT-HSCs from septin 7fl/fl or septin 7∆/∆ mice. Nuclei were stained with DAPI (blue). scale bar= 5 µm.

(F) Percentage of cells with a polar distribution of borg4, Cdc42 or tubulin in septin 7fl/fl or septin 7∆/∆ LT-HSCs. At least 3 biological repeats, at least 30 to 50 cells were scored per sample. Error bars indicate mean±SEM, Two-way ANOVA analysis, **P<0.01, *P<0.05.
A Cdc42-Borg4-Septin 7 axis regulates HSC polarity and function (2021)
Figure 4

Figure 4. HSCs devoid of borg4 or septin 7 show impaired function upon transplantation.

(A-F) Percentage of B-cells (B220 positive cells), T-cells (CD3 positive cells) and myeloid cells (Mac-1+ or Gr+ positive cells) among BM cells, percentage of LT-HSCs, ST-HSCs and LMPPs in LSK cells and percentage of CLPs in lin-Sca1C-kit low cells in borg4fl/fl or borg4∆/∆ or septin 7fl/fl or septin 7∆/∆ animals at steady state (n=at least 3). Error bars indicate mean±SD, *P<0.05, Two-way ANOVA analysis, **P<0.01, unpaired t-Test.

(G) Experimental setup for competitive bone marrow transplantation experiments.

(H) Frequency of donor derived cells (borg4fl/fl or borg4∆/∆) in peripheral blood 4, 8,16 and 20 weeks post transplantation (n=12 per group, error bars indicate mean±SD, ****P<0.0001, Two-way ANOVA analysis).

(I) Frequency of donor derived cells (septin 7fl/fl or septin 7∆/∆) in peripheral blood 8,16 and 21 weeks post transplantation (n=12 per group, error bars indicate mean±SD, ****P<0.0001, Two-way ANOVA analysis).

(J-O) Percentage of donor derived (borg4fl/fl or borg4∆/∆ or septin 7fl/fl or septin 7∆/∆) B-cells (B220 positive cells), T-cells (CD3 positive cells) and myeloid cells (Mac-1+ or Gr+ positive cells) among BM cells, percentage of donor derived LT-HSCs, ST-HSCs and LMPPs in LSK cells and percentage of donor derived CLPs in lin-Sca1C-kit low cells in animals at post- transplantation (n=12). Error bars indicate mean±SD, ****P<0.0001, **P<0.01 and *P<0.05, Two-way ANOVA analysis and 2 tailed unpaired t-test.
A Cdc42-Borg4-Septin 7 axis regulates HSC polarity and function (2021)
Figure 5

Figure 5. Lack of septin 7 in LT-HSCs impairs HSC proliferation.

(A) Percentage of single LT-HSCs from septin 7fl/fl of septin 7∆/∆ mice that completed first and second division up to 48 hours post initiation of culture. (192 sorted HSCs cells were analysed, error bars indicate mean±SD).

(B) Percentage of single LT-HSCs septin 7fl/fl of septin 7∆/∆ dying up to 48 hours (192 sorted HSCs were analysed, error bars indicate mean±SD).

(C). Frequency of colonies with distinct cell size 5 days after initiation of culture of individual LT-HSCs from septin 7fl/fl of septin 7∆/∆ mice (192 sorted HSCs were analysed, error bars indicate mean±SD).
Antagonizing miRNA-19a/b augments PTH anabolic action and restores bone mass in osteoporosis in mice (2022)
Figure 1

Fig. 1 Inhibition of miR-19a/b increases bone mass in vivo.

A Venn diagram illustrating the number of miRNAs whose expression was reduced in mouse bones in vivo upon injection of PTH (n=4) or an antibody against Sclerostin (Scl-Ab; n=4) compared to vehicle (veh; n=4) control.

B Quantification of the expression of miR-19a and miR-19b in bones of mice treated with PTH (n=15) or Scl-Ab (n=14) compared to veh (n=13) control.

C Quantification of the expression of miR-19a and miR-19b in calvarial osteoblasts upon in vitro stimulation with PTH or Wnt3a compared to veh control (n=3).

D Alkaline phosphatase staining of differentiating calvarial osteoblasts transfected with a scrambled (scr) control miRNA, anti-sense microRNA (anti-miR) inhibiting miR-19a or miR-19b or a combination thereof (anti-miR-19a/b) (representative image of 4 independent experiments).

E Quantification of alkaline phosphatase staining of calvarial osteoblasts transfected with scr, anti-miR-19a, anti-miR-19b or anti-miR-19a/b (n=4).

F, G Expression analysis of osteoblast marker genes (F) Runx2 and (G) Type I Collagen (Col1a1) in calvarial osteoblasts transfected with scr, anti-miR-19a, anti-miR-19b or anti-miR-19a/b (n=4).

H Images of von Kossa-stained histological sections of the proximal tibiae of 12-week-old male C57Bl/6J mice and fluorescence double labeling to visualize bone formation (insets) after 4-weeks of treatment with anti-miR-19a/b (n=12), scr control oligonucleotides (n=11) or veh (n=8). Scale bars indicate 1 mm (black) and 50 μm (white).

I Bone histomorphometric analysis of the proximal tibiae of the same animals as in (H) Abbreviations: BV/TV, bone volume/tissue volume; MS/BS, mineralizing surface/bone surface; BFR/BS, bone formation rate/bone surface; MAR, mineral apposition rate; OS/BS, osteoid surface/bone surface; Ob.S/BS, osteoblast surface/bone surface; N.Ob/BS, number of osteoblasts/bone surface; ES/BS, eroded surface/bone surface; Oc.S/BS, osteoclast surface/bone surface; N.Oc/BS, number of osteoclasts/bone surface.

J Images of von Kossa-stained histological sections of the fourth lumbar vertebral body of 12-week-old mice after 4-weeks of treatment with anti-miR-19a/b, scr or veh. Scale bar indicates 1 mm.

K Quantification of bone mass (BV/TV) of the fourth lumbar vertebral body of 12-week-old mice after 4-weeks of treatment with anti-miR-19a/b (n=12), scr (n=12) or veh (n=8).

Data information: Mean values ± SEM. Three or four groups were compared using one-way ANOVA followed by Tukey's post-hoc analysis. *p<0.05, **p<0.01, ***p<0.001 vs. veh, #p<0.05, ###p<0.001 vs. scr.
Antagonizing miRNA-19a/b augments PTH anabolic action and restores bone mass in osteoporosis in mice (2022)
Figure 2

Fig. 2 Anti-miR-19a/b treatment does not cause cytotoxic effects.

A Images of von Kossa-stained histological sections of the bone and bone marrow (upper panel) and of hematoxylin-eosin-stained liver sections (lower panel) of 12-week-old mice after treatment with anti-miR-19a/b (n=12), scrambled (scr) control oligonucleotides (n=11) or vehicle (veh) (n=8). Scale bars indicate 100 μm (upper panel) and 50 μm (lower panel).

B, C Quantification of the femur length (B) and width (C) after treatment with anti-miR-19a/b (n=8), scr (n=7) or veh (n=8).

D, E Quantification of the expression of miR-19a (D) and miR-19b (E) in the bone, bone marrow and liver after treatment with anti-miR-19a/b (n=4), scr (n=4) or veh (n=3).

F-H Quantification of the expression of the housekeeping genes glyceraldehyde-3-phosphate dehydrogenase (Gapdh), beta-2 microglobulin (b2m), beta-actin 1 (β-actin 1) and beta-actin 2 (β-actin 2) in the bone (F), bone marrow (G) and liver (H) after treatment with anti-miR-19a/b (n=4), scr (n=4) or veh (n=3).

I, J CTG assay-based quantification of luminescence signal intensity as surrogate parameter of metabolically active osteoblasts of the MC3T3-E1 cell line (I) and of hepatocytes of the HepG2 cell line (J) treated with anti-miR-19a/b, scr or veh and after stimulation with staurosporine to induce cell death as positive control. n= 6.

Data information: Mean values ± SEM. More than three groups were compared using one-way ANOVA followed by Tukey's post-hoc analysis. **p<0.01, ***p<0.001 vs. veh, ###p<0.001 vs. scr.
Antagonizing miRNA-19a/b augments PTH anabolic action and restores bone mass in osteoporosis in mice (2022)
Figure 3

Fig. 3 Anti-miR-19a/b increases bone mass in a Tgif1-dependent manner.

A Immunoblot of Tgif1 protein expression in calvarial osteoblasts transfected with scrambled (scr), or with anti-miR-19a, anti-miR-19b and anti-miR-19a/b (left), or with miR-19a, mir-19b and mir-19a/b mimics (right) as indicated. Immunoblot for Actin was used as loading control. Normalized fold expression and molecular weight in kilo Dalton (kD) are indicated (representative image of 3 independent experiments).

B Luciferase activity in MC3T3-E1 cells transfected with a reporter plasmid containing the 3' untranslated region (UTR) of the wild type (WT) Tgif1 mRNA (Tgif1-3'UTR-WT) or an alternative version bearing mutations (mut, marked in red) of the putative miR-19a/b binding site (yellow box) to disable the binding of miR-19a/b (Tgif1-3'UTR-mut). In addition to the respective reporter plasmid, cells were co-transfected with scr, or with anti-miR-19a, anti-miR-19b and anti-miR-19a/b (left), or with miR-19a, mir-19b and mir-19a/b mimics (right) as indicated. n=6.

C Images of von Kossa-stained histological sections of the proximal tibiae of 12-week-old male mice of the genotypes Dmp1-Cre-;Tgif1fl/fl treated weekly with scr (n=8) or anti-miR-19a/b (n=10) and Dmp1-CreTg;Tgif1fl/fl treated weekly with scr (n=7) or anti-miR-19a/b (n=14) for 4 weeks. Scale bar indicates 1 mm.

D Bone histomorphometric analysis of the proximal tibiae of the same animals as in (C). For abbreviations see legend to figure 1.

E Images of von Kossa-stained histological sections of the fourth lumbar vertebral bodies of the same animals as in (C) and (F) quantification of the BV/TV. Scale bar represents 1 mm.

Data information: Mean values ± SEM. Statistical tests were performed with one-way ANOVA followed by Tukey's post-hoc analysis (B), or the non-parametric Kruskal-Wallis test to compare more than two groups (D, F), in which normal distribution could not be assumed. *p<0.05, **p<0.01, ***p<0.001 vs. scr.
Antagonizing miRNA-19a/b augments PTH anabolic action and restores bone mass in osteoporosis in mice (2022)
Figure 4

Fig. 4 Inhibition of miR-19a/b enhances the bone anabolic effect of PTH treatment.

A Immunoblot of Tgif1 protein expression in calvarial osteoblasts after stimulation with PTH (+) or vehicle (-) and after transfection with scrambled (scr), anti-miR-19a, anti-miR-19b or anti-miR-19a/b as indicated. Immunoblot for Actin was used as loading control. Normalized fold expression and molecular weight in kilo Dalton (kD) are indicated (representative image of 3 independent experiments).

B Images of von Kossa-stained histological sections of the proximal tibiae of 12-week-old male wild type mice after treatment with scr oligonucleotides and vehicle (veh) (n=8), anti-miR-19a/b and veh (n=10), scr and intermittent PTH (n=12) or a co-treatment with anti-miR-19a/b and PTH (n=10) for 4 weeks. Scale bar indicates 1 mm.

C Histomorphometric analysis of the proximal tibiae of the same animals as in (B) after treatment with intermittent PTH or veh and/or weekly injections of anti-miR-19a/b or scr control for 4 weeks. For abbreviations see legend to figure 1.

D Images of von Kossa-stained histological sections of the fourth lumbar vertebral bodies of the same animals as in (B). Scale bars indicate 1 mm.

E Histomorphometric analysis of the fourth lumbar vertebral body. For abbreviations see legend to figure 1, scr, veh n=8; anti-miR-19a/b, veh n= 10; scr, PTH n=12; anti-miR-19a/b, veh n=10.

F Analysis of serum carboxy-terminal collagen crosslinks (CTX) in the same animals as in (B).

Data information: Mean values ± SEM. One-way ANOVA followed by Tukey's post-hoc analysis was used to compare four groups. *p<0.05, **p<0.01 ***p<0.001 vs. scr, veh; #p<0.05, ##p<0.01, ###p<0.001 vs. anti-miR-19a/b, veh; §p<0.001, §§p<0.001, §§§p<0.001 vs scr, PTH.
Antagonizing miRNA-19a/b augments PTH anabolic action and restores bone mass in osteoporosis in mice (2022)
Figure 5

Fig. 5 Inhibition of miR-19a/b restores bone mass in a mouse model of osteoporosis.

A Time course of the relative change (compared to sham-operated and scr-treated control, n=10) of bone mass (BV/TV, bone volume/tissue volume) in the tibiae of female mice in which osteoporosis was induced by ovariectomy (OVX) 21 days before start of weekly intravenous (i.v.) treatment with scrambled (scr, n=6) or anti-miR-19a/b (n=8).

B-D Micro-Computed Tomography (μCT) of the distal femora (B, C) and midshaft femoral cross sections (D) of the same animals as in A, after termination of the experiment.

E-G Quantification of trabecular BV/TV by μCT in femora (E), tibiae (F), and the fourth lumbar vertebral bodies (G) of the same animals as in (A-D) 70 days after OVX.

H Histomorphometric analysis of the proximal tibiae of the same mice as in (A-D) after termination of the experiment. For abbreviations see legend to figure 1.

I Analysis of the number TRAP-positive multinucleated cells (No of MNCs), number of nuclei per osteoclast (No of nuclei/OC) and osteoclast size (OC size) after 4 days of ex vivo osteoclast differentiation of bone marrow macrophages isolated from sham-operated (n=11) or ovariectomized mice treated with scr (n=12) or anti-miR-19a/b (n=12).

J Expression analysis of the osteoclast marker genes Trap and Cathepsin K (CatK) in bone marrow macrophages isolated from sham-operated (n=2) or ovariectomized mice treated with scr (n=3) or anti-miR-19a/b (n=3) after 4 days of differentiation.

Data information: Mean values ± SEM. One-way ANOVA followed by Tukey's post-hoc analysis was used for statistical analysis. *p<0.05, **p<0.01, ***p<0.001 vs. sham; scr, #p<0.05, ##p<0.01, ###p<0.001 vs. OVX; scr.
Antagonizing miRNA-19a/b augments PTH anabolic action and restores bone mass in osteoporosis in mice (2022)
Figure 6

Fig. 6. Anti-miR-19a/b suppresses osteoblast-mediated osteoclast differentiation through inhibition of Rankl expression.

A, B Expression of miR-19a (A) and miR-19b (B) during osteoclast differentiation of bone marrow macrophages (n=3).

C Representative images of TRAP-stained osteoclast cultures of bone marrow cells transfected with scrambled (scr) control oligonucleotide or anti-miR-19a/b (representative images of 3 independent experiments). Scale bar indicates 200 μm.

D Quantification of multinucleated TRAP-positive multi-nucleated cells (MNCs) after 4 days of osteoclast differentiation of bone marrow cells transfected with scr (n=4) or anti-miR-19a/b (n=8).

E, F Representative images (E) and quantification of MNCs (F) after transfection with scr or anti-miR-19a/b and stimulation with conditioned medium (CM) collected from Ocy454 cells that were stimulated with vehicle (veh) or PTH (n=6). Scale bar indicates 200 μm.

G Rankl mRNA expression in Ocy454 cells transfected with scr or anti-miR-19a/b and stimulated with veh or PTH (n=12).

H Quantification of RANKL protein abundance in CM collected from Ocy454 cells transfected with scr or anti-miR-19a/b and stimulated with veh or PTH (n=14).

I, J Expression of Rankl (I) and PP2A-Cβ (J) mRNA in Ocy454 cells transfected with control GapmeR or GapmeR against Tgif1 and treated with veh or PTH (n=3).

K Rankl mRNA expression in Ocy454 cells transfected with control GapmeR or GapmeR against PP2A-Cβ and treated with veh or PTH (n=3).

L Immunoblot of Crtc2 protein expression in the cytoplasm and nucleus of Ocy454 cells transfected with control GapmeR or GapmeR against PP2A-Cβ and stimulated with veh or PTH. Immunoblot for Lamin A/C was used as loading control and purity control of the nuclear fraction. Normalized fold expression and molecular weight in kilo Dalton (kD) are indicated (representative image of 3 independent experiments).

M Immunoblot of Crtc2 protein expression in the cytoplasm and nucleus of Ocy454 cells transfected with scr or anti-miR-19a/b and stimulated with veh or PTH. Immunoblot for Lamin A/C was used as loading control and purity control of the nuclear fraction. Normalized fold expression and molecular weight in kilo Dalton (kD) are indicated (representative image of 3 independent experiments).

Data information: Mean values ± SEM. Student's t-test was used for statistical analysis of two groups (D). One-way ANOVA followed by Tukey's post-hoc analysis was used for statistical analysis in all other experiments. *p<0.05, **p<0.01, ***p<0.001 vs. Day 1 (B), vs. scr; veh (F-H), vs. scrambled control Gapmer (C); veh (K-L), ##p<0.01, ###p<0.001 vs. scr; veh (F-H), vs. C; veh (K-L), §p<0.05, §§p<0.01, §§§p<0.001 vs. scr; veh (F-H), vs. C; veh (K-L).