EMBO/soda-nel huggingface dataset:
MYDGF protects bone mass by inhibiting osteoclastogenesis and promoting osteoblast differentiation (2021)
Figure 2

Fig 2

BMT alleviates bone loss and improves bone metabolism in mice.

As described in Fig EV3A, BMT was conducted on 12-month-old CKO and WT mice with normal diet, and then the recipients from each group were performed for analysis 3 months after interventions (n=10，there were 10 mice in each group, and 6 mice in each group were selected for detection at the end of the experiment).

A Representative images of micro-CT reconstruction, scale bar, 2mm.

B-E Quantitative micro-CT analysis of BMD (B), Tb.BV/TV (C), Tb. N (D) and Tb. Sp (E) (n=6). F, G Three-point bending measurement of tibia maximum load (F) and stiffness (G) (n=6).

H Representative images of H&E staining (upper panel), TRAP staining (middle panel) and calcein double-labeling (lower panel) in distal femora, scale bar, 100 µm for H&E staining. 50 µm for TRAP staining, 50μm for calcein double-labeling.

I, J Quantification of number for osteoblasts (I) and number of osteoclasts (J) in distal femora (n=6).

K Quantification of bone formation rate per bone surface (BFR/BS) (n=6).

Data information: Results are shown as mean ± SEM. *P＜0.05, **P＜0.01 and ***P＜0.001 (Student's t test).
MYDGF protects bone mass by inhibiting osteoclastogenesis and promoting osteoblast differentiation (2021)
Figure 3

Fig 3

Bone marrow specific overexpression of MYDGF ameliorates bone loss and improves bone metabolism in mice.

As shown in Fig EV3J, in situ myeloid specific MYDGF overexpression in bone marrow was performed on CKO mice aged 9 months. Micro-CT analysis and histomorphometric analysis of distal femora were performed after 3 months intervention.

A Representative images of micro-CT reconstruction of distal femora, scale bar, 2mm.

B-E Quantitative micro-CT analysis of BMD (B), Tb.BV/TV (C), Tb. N (D) and Tb. Sp (E) (n = 6).

F, G Three-point bending measurement of tibia maximum load (F) and stiffness (G) (n=6).

H Representative images of H&E in distal femora, scale bar, 100 µm.

I Quantification of number for osteoblasts in distal femora (n=6).

J Representative images of TRAP staining in distal femora, scale bar, 50µm

K Quantification of number for osteoclasts in distal femora (n=6).

L Representative images of calcein double-labeling, scale bar, 50μm.

M Quantification of bone formation rate per bone surface (BFR/BS) (n=6).

Data information: Results are shown as mean ±SEM. *P<0.05, **P<0.01, ***P<0.001 (Student's t test).
MYDGF protects bone mass by inhibiting osteoclastogenesis and promoting osteoblast differentiation (2021)
Figure 4

Fig 4

MYDGF inhibits RANKL-induced osteoclastogenesis.

A-C Primary BMMs from WT and CKO mice were induced with M-CSF (100 ng/ml) and RANKL (50 ng/ml) for osteoclast differentiation. Representative TRAP staining images (A), scale bar, 200μm. Quantification of osteoclast number (B) percentage of osteoclast area (C). Results are shown as mean ± SEM, ***P<0.001 (Student's t test).

D-F BMMs with treatment of conditioned medium of BMCs from WT or CKO mice for 7 days of osteoclast differentiation with or without RANKL (50 ng/ml). M-CSF (100 ng/ml) was present in all settings. Representative TRAP staining images, upper panel represent cultured without RANKL, lower panel represent cultured with RANKL (50 ng/ml; D), scale bar, 200μm. Quantification of osteoclast number of TRAP-positive cells (E) and percentage of osteoclast area (F). Results are shown as mean ± SEM, *P<0.05 (Student's t test).

G-M BMMs from WT mice were cultured in the presence of M-CSF (100 ng/ml) and RANKL (50 ng/ml) with indicated amount of rMYDGF for 7 days. Representative TRAP staining images, scale bar, 200 μm (G). Quantification of osteoclast number (H) and percentage of osteoclast area (I). Representative images of resorption pits on Osseo Assay surface, scale bar, 100μm (J). Quantification of resorption pits number (K) and percentage of resorption area (L). Representative images of fluorescence of cell nuclei and F-actin rings (M) scale bar, 100μm. Results are shown as mean ± SEM, *P < 0.05 (one way ANOVA).

N-P As shown in Fig EV4E, BMMs were co-cultured with osteoclasts in presence of 1,25(OH)2D3 and PGE2 with conditioned medium of BMCs from WT or CKO mice for 5 days. Representative images of osteoclasts co-cultured with osteoblasts, scale bar, 200μm (N). Quantification of osteoclast number (O) and percentage of osteoclast area (P). Results are shown as mean ± SEM, *P < 0.05 (Student's t test).

Data information: Each experiment was repeated 5 times.
MYDGF protects bone mass by inhibiting osteoclastogenesis and promoting osteoblast differentiation (2021)
Figure 5

Fig 5

MYDGF promotes osteoblast differentiation.

A-C BMSCs cultured in osteogenesis induction medium with treatment of conditioned medium of BMCs from WT or CKO mice for ALP staining assay at 7 days or Alizarin Red S (ARS) staining assay at 14 days. Representative images of ALP staining (upper panel) and ARS staining (lower panel) of BMSCs, scale bar, 100μm (A). Quantitative analysis of ALP activity (B) and calcium mineralization (C).

D-F BMSCs cultured in osteogenesis induction medium with treatment of 0ng/ml (vehicle) or 100ng/ml rMYDGF. Representative images of ALP staining (upper panel) and ARS staining (lower panel) of BMSCs, scale bar, 100 mm (D). Quantitative analysis of ALP activity (E) and calcium mineralization (F).

G-I Primary calvarial osteoblasts cultured in osteogenesis induction medium with treatment of conditioned medium of BMCs from WT or CKO mice for ALP staining assay at 7 days or ARS staining assay at 14 days. Representative images of ALP staining and ARS staining of osteoblasts, scale bar, 100μm (G). Quantitative analysis of ALP activity (H) and calcium mineralization (I).

J-L Primary calvarial osteoblasts cultured in osteogenesis induction medium with treatment of 0ng/ml (vehicle) or 100ng/ml rMYDGF. Representative images of ALP staining and ARS staining of osteoblasts, scale bar, 100μm (J). Quantitative analysis of ALP activity (K) and calcium mineralization (L).

Data information: Results are shown as mean ± SEM, *P<0.05, **P<0.01, ***P<0.001(Student's t test). Each experiment was repeated 5 times.
MYDGF protects bone mass by inhibiting osteoclastogenesis and promoting osteoblast differentiation (2021)
Figure 6

Fig 6

MYDGF facilitates the progress of bone defects healing.

As shown in Fig EV4J, Bone defects models were conducted on young (3-month-old) and aged (18-month-old) mice, AAV-GFP or AAV-MYDGF intra-marrow injection once 2 weeks before bone defects surgery on young and aged mice, and the defects area was analyzed after 2 weeks (n=10, there were 10 mice in each group, and 5 mice in each group were selected for detection at the end of the experiment).

A Micro-CT reconstruction of the calvarial defects. The red dotted lines indicate the position of the original defect margin, scale bar, 1mm.

B H&E staining of the calvarial defects. The dotted lines indicate the position of the original defect margin, scale bar, 200μm.

C, D Quantitative micro-CT analysis of BMD (C) and BV/TV (D) of the regenerated bone in calvarial defects (n=5).

E, F Histomorphometric analysis of osteoblast surfaces (Ob.s/BS) (E) and osteoclast surfaces (Oc.s/BS) (F) of the regenerated bone in calvarial defects (n=5).

G Micro CT reconstruction of the femoral cortical bone defects. The red dotted lines indicate the position of the original defect margin, scale bar, 1mm.

H H&E staining of the femoral cortical bone defects. The dotted lines indicate the position of the original defect margin, scale bar, 200μm.

I, J Quantitative micro-CT analysis of BMD (I) and BV/TV (J) of the regenerated bone in calvarial defects (n=5).

K, L Histomorphometric analysis of osteoblast surfaces (Ob.s/BS) (K) and osteoclast surfaces (Oc.s/BS) (L) of the regenerated bone in calvarial defects (n=5).

Data information: Results are shown as mean ± SEM, *P<0.05, **P<0.01, ***P<0.001 (Student's t test).
MYDGF protects bone mass by inhibiting osteoclastogenesis and promoting osteoblast differentiation (2021)
Figure 7

Fig 7

MYDGF prevents OVX-induced bone loss and ameliorates age-related osteoporosis.

As shown in Fig EV4L, OVX mice and 18-month-old male mice serve as the model of osteoporosis, AAV-MYDGF or AAV-GFP were injected into bone marrow cavity every 3 weeks for 12 weeks (n=10, there were 10 mice in each group, and 6 mice in each group were selected for detection at the end of the experiment).

(A-H) Micro-CT reconstruction, H&E staining and histomorphometric analysis of trabecular bone from distal femurs in Sham +GFP, OVX +GFP and OVX + MYDGF groups (n=6). Representative images of micro-CT reconstruction, scale bar, 2mm (A). Representative images of H&E staining in distal femora, scale bar, 500μm for upper panel,100μm for lower panel (D). Quantitative micro-CT analysis of BMD (B), Tb.BV/TV (C), Tb. N (E) and Tb. Sp (F). Three-point bending measurement of tibia maximum load (G) and stiffness (H).

(I-P) Micro CT reconstruction, H&E staining and histomorphometric analysis of trabecular bone from distal femurs in aged mice with AAV-GFP or AAV-MYDGF (n=6). Representative images of micro-CT reconstruction, scale bar, 2mm (I). Representative images of H&E staining in distal femora, scale bar, 500μm for upper panel,100μm for lower panel (J). Quantitative micro-CT analysis of BMD (K), Tb.BV/TV (L), Tb. N (M) and Tb. Sp (N). Three-point bending measurement of tibia maximum load (O) and stiffness (P).

Data information: Results are shown as mean ± SEM, *P＜0.05, **P＜0.01, ***P＜0.001 (Student's t test).
MYDGF protects bone mass by inhibiting osteoclastogenesis and promoting osteoblast differentiation (2021)
Figure 8

Fig 8

MYDGF inhibits RANKL-induced NF-κB signaling during osteoclastogenesis.

A qRT-PCR analysis for Nfatc1, Ctsk, c-Fos, Mmp9 and Acp5 mRNA expression in BMMs cultured in osteoclastogenesis induction medium for 48 hours.

B Nfatc1 protein expression in BMMs cultured in osteoclastogenesis induction medium for different times.

C Representative immunostaining (left panel) and relative expression level (right panel) of Nfatc1 on the trabecular bone surface, scale bars for left panel are 50μm (n=5).

D, E KEGG pathway analysis indicated the altered function of NF-κB signaling pathway (D). Heatmap of the NF-κB pathway associated genes (E). Experiment was repeated 3 times.

F-I Representative WB images (F) and the protein levels of IKBα, P65, PKCβ treated with or without rMYDGF for RANKL -induced osteoclastogenesis (G-I)

J, K Luciferase activities of NF-κB (J) and Nfatc1 (K) as quantified from the BMMs treated with indicated intervention.

L The p65 nuclear translocation in BMMs, scale bar, 25μm.

M BMMs were treated with control, RANKL or MG132 for osteoclastogenesis, then with or without rMYDGF for 24 hours. IgG, P65 and histone antibodies were used to ChIP and RT-PCR was performed to determine Nfatc1 promoters.

N Representative TRAP staining images of BMMs treated with rMYDGF or indicated inhibitors, scale bars, 200μm.

O, P Quantification of osteoclast number (O) and percentage of osteoclast area (P).

Data information: Results are shown as mean ± SEM. *P＜0.05, **P＜0.01, ***P＜0.001 (Student's t test). Each in vitro experiment was repeated at least 3 times.
MYDGF protects bone mass by inhibiting osteoclastogenesis and promoting osteoblast differentiation (2021)
Figure 9

Fig 9

MYDGF promotes osteoblastogenesis via MAPK1/3-STAT3 pathway.

A qRT-PCR analysis for Runx2, Alp, Osx, Ocn mRNA expression in mice BMSCs cultured in osteogenesis medium with or without 100 ng/ml rMYDGF induction for 48 hours.

B Runx2 protein expression in BMSCs cultured in osteogenesis induction medium for different times.

C-G Representative WB images (C) and the protein levels of Smad2/3, mTOR, STAT3(S), STAT3(Y), MAPK1/3 and JAK1/2 proteins expression (D-G).

H The mRNA levels of Runx2 in BMSCs cultured in osteogenesis medium induction for 48 hours with indicated interventions.

I, J Quantitative analysis of ALP activity (I) and calcium mineralization (J) in BMSCs cultured in osteogenesis medium induction for 48 hours with indicated intervention.

K Representative immunostaining (left panel) and relative expression level (right panel) of Runx2 on the trabecular bone surface, scale bars for left panel are 20μm (n=5).

L BMSCs were treated with control and stattic for osteoclastogenesis, then with or without rMYDGF for 24 hours. IgG, STAT3 and histone antibodies were used to ChIP and RT-PCR was performed to determine Runx2 promoter.

M, N The protein levels of MAPK1/3 and JAK1/2 expression.

O, P Representative WB diagram (O) and the protein levels of STAT3(S) and MAPK1/3 expression (P).

Data information: Results are shown as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001(Student's t test). Each in vitro experiment was repeated 5 times.
HRAS-driven cancer cells are vulnerable to TRPML1 inhibition (2019)
Figure 3

Fig 3. MCOLN1 expression is necessary and sufficient for proliferation of cells expressing oncogenic HRAS.

(A-C) Bar graphs showing the relative cell numbers in the indicated cell types following the indicated treatments. Cell numbers were assessed after a 48-hour period of growth in cultures seeded at equal densities. All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was pairwise Student's t-tests with Bonferroni post hoc correction to account for multiple pairwise testing in (A) and (B).

(D) Representative images showing Ki67-labeling in xenografts generated from implantation of HN31 cells stably expressing control shRNA (left) or MCOLN1 shRNA (right). Pseudocolored panels show the representative distribution Ki67 positive or negative nuclei. Scale bars shown (0.5 mm) apply to all panels.

(E) Bar graphs showing fraction (left) and total number (right) of Ki67 labeled nuclei in the indicated cell types. All values represent mean±SEM. Values in the bar graph on the right were normalized to control mean. Data points represent values from independent eggs. Statistical test employed was Student's t-test.

(F) Bar graphs showing the relative cell numbers in UMSCC-22A-HRASWT cells following the indicated perturbations. All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.

(G) Kaplan-Meier curves showing the survival of patients stratified on the basis of MCOLN1 expression in BLCA and HNSC patients whose tumors were wild-type for HRAS (right) or carried oncogenic HRAS mutations (left). Statistical test employed was the Mantel-Cox log-rank test.
HRAS-driven cancer cells are vulnerable to TRPML1 inhibition (2019)
Figure 4

Fig 4. TRPML1 inhibition or MCOLN1 knockdown attenuated ERK phosphorylation in mammalian cells expressing activated HRAS

(A) Representative Western blots generated using extracts from cells of expressing oncogenic HRAS and controls treated with ML-SI1 as indicated. The primary antibodies used are indicated on the right.

(B) Bar graph showing quantification of the Western blots shown in (A). All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.

(C-D) Representative Western blots generated using extracts from the indicated cell types after the indicated perturbations. Primary antibodies used are indicated on the right.

(E) Bar graph showing quantification of the Western blots in (C). All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.

(F) Bar graph showing quantification of the Western blots in (D). All values represent mean±SEM. Data points represent values from biological replicates. Statistical tests employed were Student's t-test and ANOVA.
HRAS-driven cancer cells are vulnerable to TRPML1 inhibition (2019)
Figure 5

Fig 5. Effects of oncogenic dRas on cell proliferation and ERK phosphorylation is attenuated in trpml-deficient Drosophila

(A) Bar graph showing relative levels of trpml mRNA in Drosophila larval brain extracts from control animals and animals expressing dRasG12V in glia. All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.

(B) Representative images of Drosophila 3rd instar larvae expressing the indicated transgenes in glial cells. repo-GAL4 was used to drive the expression of UAS-dRasG12V in glia. Images shown are volumetric reconstructions of confocal stacks. The various anatomical structures of the larval brain are annotated. VNC refers to the 'ventral nerve chord', which is distinct from dorsal hemispheres of the brain. GFP staining represents the total glial membranes in the larval brains. Scale bar, 100μm.

(C) Bar graph showing relative glial cell volume in larvae of the indicated genotypes. All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.

(D) Representative Western blots generated using macrophage extracts isolated from 3rd instar larvae of the indicated genotypes. He-GAL4 was used to drive the expression of UAS-dRasG12V in macrophages. The primary antibodies used are indicated on the right.

(E) Bar graph showing quantification of the Western blots shown in (D). All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.

(F) Representative Western blots generated using fat-body extracts isolated from 3rd instar larvae of the indicated genotypes. cg-GAL4 was used to drive the expression of UAS-HRASG12V in larval fat-bodies. The primary antibodies used are indicated on the right.

(G) Bar graph showing quantification of the Western blots shown in (F). All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.
HRAS-driven cancer cells are vulnerable to TRPML1 inhibition (2019)
Figure 6

Fig 6. TRPML1 is required for nanoclustering and levels of HRASG12V at the plasma membrane

(A) Schematic showing the methodology utilized for preparation of plasma membrane sheets from MDCK cells expressing GFP-HRASWT or GFP-HRASG12V. After preparation of plasma membrane sheets, gold conjugated antibodies are used to visualize clusters of GFP-tagged proteins in electron micrographs.

(B) Weighted mean univariate K-function curves of immunogold labeled GFP-HRASG12V under indicated conditions, n ≥ 15 for each condition. The gold point patterns were analyzed with univariate K-functions expressed as L(r)-r. The bar graph on the right shows the peak value Lmax of the L(r)-r curve, which represents the extent of GFP-HRASG12V nanoclustering. Values are normalized to DMSO average and represent mean±SEM (n ≥ 15 for each condition). Statistical significances of differences were evaluated in bootstrap tests.

(C) Bar graph showing GFP-HRASG12V content in the inner leaflet of the plasma membrane is quantified as the number of gold particles per 1μm2 region from electron micrographs. Values are normalized to DMSO average and represent mean±SEM, n ≥ 15 for each condition. Statistical test employed was Student's t-test with Bonferroni post hoc correction to account for multiple pairwise comparisons.

(D) The extent of GFP-HRASG12V nanoclustering quantified by Lmax values in cells exposed to the indicated perturbations. Values are normalized to DMSO average and represent mean±SEM (n ≥ 15 for each condition). Statistical significances of differences were evaluated in bootstrap tests.

(E) Same as (C) but in cells exposed to the indicated perturbations. Values shown are normalized to DMSO average.

(F) Representative Western blots generated using extracts from HN31 cells stably expressing the MCOLN1 shRNA. Samples treated with exogenous cholesterol for 3 hours are indicated at the bottom. Primary antibodies used are indicated on the right.

(G) Bar graph showing quantification of the Western blots in (F). All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was ANOVA.

(H) Schematic summarizing the effects of decreased TRPML1 activity on HRASWT and HRASG12V. Our data indicate that TRPML1 inhibition diminished HRASG12V nanoclustering and plasma membrane levels. Addition of cholesterol restored these parameters. Only the nanoclustering of HRASWT was affected by ML-SI1.
HRAS-driven cancer cells are vulnerable to TRPML1 inhibition (2019)
Figure 7

Fig 7. TRPML1 inhibition results in diminished de-esterification and redistribution of cellular cholesterol

(A) Schematic showing the role of TRPML1 in vesicular trafficking. TRPML1 is active in the membrane of late-endosomes and facilitates fusion of these vesicles with lysosomes or the plasma membrane. Channels delivered to the plasma membrane are redelivered to the late-endosomes via endocytosis. Whereas delivery of vesicles to the lysosomes is required for cholesterol de-esterification in that compartment, vesicular fusion with the plasma membrane is required for cholesterol recycling.

(B-D) Bar graph showing the relative levels of total or free/total cholesterol in HN31 cell extracts exposed to the indicated perturbagens (B-C), or larval brain extracts from animals of the indicated genotypes (D). Values represent mean±SEM. Data points represent values from biological replicates. Statistical tests employed were Student's t-test and ANOVA.

(E) Confocal images of MDCK cells expressing mCherry-D4H. Colocalization with the cortical actin (phalloidin) indicates that mCherry-D4H is predominantly localized to the plasma membrane in DMSO treated cells. Within 24 hours of ML-SI1 treatment, the mCherry-D4H redistributes to intracellular punctae. Although minimal mCherry-D4H signal remained at the plasma membrane in ML-SI1-treated cells, the phalloidin signal (cortical actin) remained unchanged. Scale bar (10μm) shown in the panel on the top right applies to all panels.

(F) Bar graph showing quantification of mCherry-D4H punctae in DMSO or ML-SI1-treated cells. All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.

(G) Confocal images of MDCK cells expressing mCherry-D4H and LAMP1-GFP treated with ML-SI1. Scale bar (10μm) shown in the panel on the top right applies to all panels.

(H) Confocal images of control and MCOLN1 shRNA-expressing HN31 cells treated with the cholesterol sensor, filipin. Scale bar (10μm) shown in the panel on the top applies to both panels.

(I) Bar graph showing the quantification of the internalized filipin staining in HN31 cells after MCOLN1 knockdown. All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.

(J) Fraction of phase-separated GPMVs at the indicated temperatures. Colors of the lines indicate the treatment conditions of MDCK cells prior to generation of GPMVs. Dashed vertical lines indicate the miscibility temperature of the indicated populations of GPMVs. Values shown represent mean±SEM. Lines were generated by fitting the data to sigmoidal functions.

(K) Bar graph showing miscibility temperatures of GPMVs isolated from MDCK cells exposed to the indicated conditions. All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.

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