EMBO/soda-nel huggingface dataset:
Therapeutic potential of targeting microRNA-10b in established intracranial glioblastoma: first steps toward the clinic. (2016)
Figure 8

Figure 8.

Continuous osmotic delivery of miR-10b inhibitor reduces growth of the established orthotopic GBM8 tumor xenografts.

A. Continuous osmotic delivery of miR-10b inhibitor markedly reduces tumor burden. The osmotic pumps, loaded with lipid nanoparticles formulated with 2’-O-MOE-PO miR-10b inhibitor or non-targeting control infused 2 μg of the oligonucleotides per day introtumorally, over 13 days. Tumors growth was monitored by the WBI, and the left panels illustrate tumor imaging of representative animals at the end of treatment. The bars represent average signal ratios for each group at day 13 (end of treatment), relative to day 2 (treatment initiation).

B. Representative immunostaining of tumors for PCNA proliferation marker.

C. Representative immunostaining of the tumors for KI67 proliferation marker.

D. Representative tumor immunostaining for cleaved Caspase 3 as a marker of apoptosis.

E. Tumor cell invasion was examined by fluorescence microscopy for mCherry-positive cells migrating through the tumor border.

F. Quantitative immunostainings analysis indicates that proliferation and apoptosis markers are affected by anti-miR-10b treatment. No significant effect on invasion of intracranial GBM8 was observed.

For each staining, the immunopositive area was quantified and normalized to DAPI-stained area using ImajeJ software. The 40-50 microscopic fields were quantified within four sections per each tumor, and average values of four tumors per group are presented. Statistical significance of the differences was determined by Student's t test, with p-values < 0.01 indicated by two asterisks, and p< 0.001 by three asterisks. Numbers of replicates and exact P-values are included in Appendix Table S4.
Therapeutic potential of targeting microRNA-10b in established intracranial glioblastoma: first steps toward the clinic. (2016)
Figure 9

Figure 9.

miR-10b inhibition reduces the growth of mouse GL261 glioma cells in vitro and GL261-derived intracranial tumors in immunocompetent mouse model.

A, B. miR-10b inhibition decreases GL261 cell viability. Cell viability was measured at days 3-7 after transfection with miR-10b inhibitor, non-targeting control, or Lipofectamine 2000 alone (mock).

A. Phase contrast photographs of GL261 cultures at day 6 post-transfection.

B. Growth curves of cultured GL261 cells, based on the viability assay.

C. InVivo-jet PEI – formulated 2’-O-MOE-PS/PO miR-10b inhibitor or non-targeting control were infused to orthotopic GL261 tumors by osmotic pumps, starting at day 6 after cell implantation. The uptake of ASOs was confirmed by IHC for PS-containing oligonucleotides (green). GL261 tumor cells expressing M-Cherry are red.

D. Osmotic delivery of miR-10b inhibitor markedly reduces GL261 tumor growth in immunocompetent Black 6 Albino mice. Mice photographs show tumor imaging in representative animals at day 3 after pump implantation, and average signals in photons per second are indicated. Tumor growth rates were calculated as ratios of the signals at day 3 of the treatment to day 1 prior to initiation of the treatment.

E. The efficacy of miR-10b inhibition in intracranial tumors was assessed by qRT-PCR analysis of the resected tumor tissues, with miR-10b expression levels normalized to miR-125b.

F. qRT-PCR analysis demonstrates that miR-10b target p21 was de-repressed in GL261 tumors upon miR-10b inhibition. mRNA expression levels were normalized to GAPDH.

Statistical significance of the differences was determined by Student's t test, with p-values < 0.05 indicated by asterisks and p< 0.001 by three asterisks. Numbers of replicates and exact P-values are included in Appendix Table S4.
Bacterial rhomboid proteases mediate quality control of orphan membrane proteins (2020)
Figure 3

Figure 3. HybA is a physiologic substrate of GlpG.

A Schematic of HybA and HybO fusions.

B Western blot analysis (probing with an anti-FLAG mAb) to detect cleavage of HybO with (+)/without (-) chromosomal (native) or pBAD33-encoded rhomboids (plasmid).

C Western blot analysis to detect cleavage of HybA with (+)/without (-) chromosomal (native) or pBAD33-encoded rhomboids (plasmid).

D Western blot analysis to detect cleavage of HybAG296F by endogenous or pBAD33-encoded rhomboid. Wild-type S. sonnei, Ss.

E Western blot analysis to detect cleavage of HybAP300A by endogenous or pBAD33-encoded rhomboid.

Data information: In (B-E), rhomboid substrates that are uncleaved, cleaved by GlpG, or by Rhom7, are marked by black, red, and blue arrows, respectively. Wild-type S. sonnei, Ss. Wild-type (WT)/inactive (SAHA: alanine substitution of the catalytic serine and histidine residues) enzymes were pBAD33-encoded (plasmid) in S. sonneiΔglpGΔrhom7.
Bacterial rhomboid proteases mediate quality control of orphan membrane proteins (2020)
Figure 4

Figure 4. GlpG specifically targets orphan HybA and does not influence hydrogenease-2 activity.

A Bacterial two-hybrid analysis with HybA and/or HybB fused chromosomally with the T25 and T18 domains of B. pertussis CyaA, respectively in the absence of endogenous CyaA. Bacteria were grown on LB agar containing 20 µg/ml X-gal at 37°C for 14 hours in the presence/absence of O2. Scale bar, 1 cm.

B Western blot analysis to detect HybA cleavage in S. sonneiΔrhom7 by wild-type (+) or inactive (S201A) GlpG expressed chromosomally (native) or from pUC19 (plasmid) with (+)/without (-) HybB. HybA was expressed from its native locus or a plasmid (pHybA).

C Quantification of the ratio of cleaved/uncleaved HybA in strains with (+) or without (-) HybB. mean ± S.D. of three experiments. *, p<0.05; ****, p<0.0001 (One-way ANOVA).

D Western blot analysis to detect GlpG-mediated cleavage of native tagged HybA at indicated times after blocking protein translation by the addition of chloramphenicol at T0 in the absence of HybB (-).

E Western blot analysis to detect GlpG-mediated cleavage of C-terminally sfCherry-3xFLAG tagged HybA at times after blocking protein translation by the addition of chloramphenicol at T0 in the presence of HybB (+).

F Growth of bacteria lacking hydrogenases (ΔhyaA-F ΔhycE +/- ΔhybO-G) or GlpG (ΔglpG), or expressing uncleavable HybB (hybAG296F) in 5% H2

G H2 consumption by S. sonnei. Bacteria were grown aerobically in LB overnight then diluted into M9 minimal media supplemented with 0.5% fumarate, 12.5 μg/ml nicotinic acid and 0.2% casamino acids in a sealed glass chromatography vial. The headspace was purged with 10% H2/90% argon and cultures were incubated at 37 °C with shaking at 180 rpm for 9 hours. H2 in the headspace was sampled and measured by gas chromatography.

H S. sonnei growth in 10% H2 with plasmid expressed wild-type or non-functional (pglpGSAHA) GlpG.

Data information: P-values were calculated by two-way ANOVA (F, H). Bars represent the mean ± S.D. n = 3 (F-H); *, p<0.05; ****, p<0.001. In (B, D, and E), HybA that is uncleaved or cleaved by GlpG is marked by black and red arrows, respectively.
Bacterial rhomboid proteases mediate quality control of orphan membrane proteins (2020)
Figure 5

Figure 5. GlpG and Rhom7 also target orphan components of formate dehydrogenases.

A Schematic of FdoH and FdnH fusions.

B Western blot analysis (probing with an anti-FLAG mAb) to detect cleavage of FdoH cleavage in S. sonneiΔrhom7 with (+)/without (-) FdoI.

C Western blot analysis to detect cleavage of FdnH in S. sonneiΔglpG with (+) or without (-) chromosomal Rhom7 (native), or wild-type (+)/inactive (SAHA) Rhom7 expressed from pBAD33 in S. sonneiΔglpGΔrhom7 with (+) or without (-) FdnI.

D Alignments of HybA, FdoH, and FdnH in S. sonnei with P. stuartii TatA and with their homologues from phylogenetically diverse organisms (S. sonnei, Salmonella enterica, Yersinia enterocolitica, Proteus mirabilis, Pseudomonas aeruginosa, and Burkholderia pseudomallei) highlighting conserved glycine (green) and proline (orange) residues.

E Western blot analysis to detect cleavage of wild-type (WT) or uncleavable (P300A) HybA by active (+) or inactive (S201A) chromosomal GlpG in the presence (+) or absence (-) of HybB.

F Western blot analysis to detect cleavage of wild-type (WT) or modified (P259A) FdoH by active (+) or inactive (S201A) chromosomal GlpG in the presence (+) or absence (-) of FdoI.

G Western blot analysis to detect cleavage of wild-type (WT) or modified (P259A) FdnH with active (+) or inactive (SAHA) Rhom7 expressed from pBAD33 with (+) or without (-) FdnI.

Data information: In (B, C, E-G), rhomboid substrates that are uncleaved, cleaved by GlpG, or by Rhom7, are marked by black, red, and blue arrows, respectively.
Bacterial rhomboid proteases mediate quality control of orphan membrane proteins (2020)
Figure 6

Figure 6. Rhomboid cleavage licenses further degradation of substrates.

A Western blot analysis (probing with an anti-V5 mAb) to detect degradation of N-terminally V5-tagged wild-type (WT) or modified (P300A) HybA in S. sonneiΔrhom7 chromosomally expressing wild-type (+) or inactive (S201A) GlpG with (+) or without (-) HybB.

B Degradation of V5-tagged HybA at times after blocking protein translation at T0 in the presence (+) or absence (-) of HybB.

C Western blot analysis of N-terminally V5-tagged wild-type (WT) or modified (P259A) FdoH in S. sonneiΔrhom7 chromosomally expressing wild-type (+) or inactive (S201A) GlpG with (+)/without (-) FdoI.

D Western blot analysis of N-terminally V5-tagged wild-type (WT) or modified (P259A) FdnH in S. sonneiΔrhom7 with wild-type (+) or inactive (SAHA) Rhom7 expressed from pBAD33 with (+)/without (-) FdnI.

E and F Degradation of N-terminally V5-tagged wild-type (WT) or modified (P259A) FdoH (E) or FdnH (F) in S. sonneiΔrhom7 with wild-type (+) or inactive (S201A, SAHA) GlpG expressed chromosomally (native) or from pUC19 (plasmid) without FdoI or FdnI (-), respectively, +/- exposure to 400 µM CuCl2 for 30 min.

Data information: Rhomboid substrates that are uncleaved, cleaved by GlpG, or Rhom7, are marked by black, red, and blue arrows, respectively. Degradation products post rhomboid cleavage are marked by green arrows.
Bacterial rhomboid proteases mediate quality control of orphan membrane proteins (2020)
Figure 7

Figure 7. Rhomboids prevent aggregation of orphan substrates in the inner membrane.

A Western blot analysis probing the localisation and status of plasmid-encoded N-terminally V5-tagged wild-type (WT) or modified (P300A) HybA in S. sonneiΔrhom7ΔhybB with wild-type (+) or inactive (SAHA) GlpG expressed from pUC19. Whole cell lysate (Whole cell.), Soluble (Sol.), Membrane (detergent-solubilised, Mem.), and the Aggregate (Agg.) fractions are shown. HybA that is uncleaved, cleaved only by GlpG, and further degraded post GlpG cleavage is marked by black, red, and green arrows, respectively.

B Model of rhomboid-mediated quality control by selectively targeting orphan components of multi-protein respiratory complexes.

EXPANDED VIEW FIGURES AND TABLES
Critical Regulation of a NDIME/MEF2C Axis in Embryonic Stem Cell Neural Differentiation and Autism (2020)
Figure 1

Figure 1. LncRNA NDIME is required for proper neural differentiation.

A. The production of Sox1-GFP+ neural-lineage cells during 7 days of neural differentiation, as shown by microscopy. Scale bars, 100 µm.

B. qRT-PCR analysis of NDIME expression during 7 days of neural differentiation (D3, P = 0.068; D5, P = 0.0049; D7, P = 0.0003).

C. Relative expression level of NDIME mRNA in the Sox1-GFP- population and Sox1-GFP+ population at day 7 of neural differentiation (P = 0.0118).

D. mESCs were cultured in differentiation medium alone, or with 10% FBS, at day 5 of neural differentiation as shown by microscopy. Scale bars, 100 µm.

E. Temporal expression profile of NDIME by qRT-PCR. mESCs were cultured in differentiation medium alone or with 10% FBS (D3, P = 0.074; D5, P = 0.0048; D7, P = 0.0034).

F. Distribution of NDIME in the cytoplasm and nucleus at day 7 of neural differentiation. The XIST RNA and GAPDH mRNA were considered as the positive controls for the nuclear and cytosolic fractions, respectively. Data are represented as mean, and error bars represent SEM in all panels. n= 3.

G. Verification of the efficiency of knockout of exon 3 of NDIME in mESCs via qRT-PCR.

H, I. Knockout of NDIME strongly inhibited neural differentiation, as shown by microscopy (H) and FACS (I) analyses during 7 days of neural differentiation. Scale bars, 100 µm.

J. Knockout of NDIME inhibits the expression of neural-lineage genes at day 5 of neural differentiation.

K. NESTIN and GFP immunostaining of the cells in (H) at day 5 of neural differentiation. Nuclei were stained with Hoechst 33342 (blue). Scale bars, 100 µm.

L. NESTIN and GFP immunostaining of Ctrl, NDIME-/-, and NDIME-/-+Rs26-NDIME cells at day 5 of neural differentiation. Scale bars, 100 µm.

M. Expression of neural-lineage genes of Ctrl, NDIME-/-, and NDIME-/-+Rs26-NDIME cells at day 5 of neural differentiation.

N. Relative expression of Emx1, Brn2, Sox5, and Zeb2 mRNA in different differentiation stages of Ctrl and NDIME-knockout cells (compared with control cells).

Data information: Data in (B, C, E, G, I, J, and N) are represented as mean ± SEM, n= 3. *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired two-tailed Student's t-test). Data in (M) are represented as mean ± SEM, n = 4. **P < 0.01, ***P < 0.001 (one-way ANOVA with Bonferroni post-hoc test). NS-non‐significant.
Critical Regulation of a NDIME/MEF2C Axis in Embryonic Stem Cell Neural Differentiation and Autism (2020)
Figure 2

Figure 2. NDIME functions upstream of its neighbor gene Mef2c.

A, B. Expression levels of MEF2C (A) (D0, P = 0.0020; D3, P = 0.0025; D5, P = 0.0003; D7, P = 0.0142) of Ctrl and NDIME-/- cells during 7 days of neural differentiation, and protein level of MEF2C (B) following knockout of NDIME at day 5 of neural differentiation.

C. Western blot analysis of the effects of MEF2C overexpression in NDIME-knockout cells at day 5 of neural differentiation.

D, E. Overexpression of MEF2C in the NDIME-knockout cells rescued neural differentiation defects at day 5 of neural differentiation, as shown by microscopy (D) and FACS (E). Scale bars, 100 µm.

F. NESTIN and GFP immunostaining of the cells in (D) at day 5 of neural differentiation. Scale bars, 100 µm.

G. Expression of neural-lineage genes in Ctrl, NDIME-/-, and NDIME-/-+Mef2c cells during 7 days of neural differentiation.

Data information: Data in (A) are represented as mean ± SEM, n= 3. *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired two-tailed Student's t-test). Data in (E and G) are represented as mean ± SEM, n = 4. Asterisks indicate a difference between NDIME-/- and Ctrl, whereas hash tags indicate a difference between NDIME-/-+Mef2c and NDIME-/-. */#P < 0.05, **/##P < 0.01, ***/###P < 0.001 (one-way ANOVA with Bonferroni post-hoc test). NS-non‐significant.
Critical Regulation of a NDIME/MEF2C Axis in Embryonic Stem Cell Neural Differentiation and Autism (2020)
Figure 3

Figure 3. NDIME blocks PRC2 binding at the Mef2c promoter.

A, B. Enrichment of H3K27me3 (A) and RNA polymerase II (B) at the Mef2c promoter in the NDIME-knockout cells at day 5 of neural differentiation compared with control cells.

C. NDIME interacts with EZH2 (P = 0.0043) and SUZ12 (P = 0.0003), but not EED (P = 0.6720) and EZH1 (P = 0.0951) compared with IgG, as determined by RIP using EZH2, SUZ12, EED, and EZH1 antibodies at day 5 of neural differentiation. Nuclear RNA U1 as a negative control. The presence of NDIME and U1 was measured by qRT-PCR with or without reverse transcriptase (RT).

D, E. NDIME levels following EZH2-RIP (P = 0.0028) (D) or SUZ12-RIP (P = 0.0015) (E) in HEK293T cells compared with pcDNA3.1. Nuclear RNA U1 was used as a negative control. The presence of NDIME and U1 was measured by qRT-PCR with or without reverse transcriptase (RT).

F, G. Enrichment of EZH2 (F) and SUZ12 (G) at the Mef2c promoter in NDIME-knockout cells at day 5 of neural differentiation compared with control cells.

H. Expression of neural-lineage genes at day 5 of neural differentiation in the NDIME-knockout cells after adding the EZH2 inhibitor GSK126 (7.5 µM) partially rescued neural differentiation defects, as shown by qRT-PCR; the wild-type and NDIME-knockout cells treated with DMSO.

Data information: Data in (A-G) are represented as mean ± SEM, n= 3. *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired two-tailed Student's t-test). Data in (H) are represented as mean ± SEM, n = 4. **P < 0.01, ***P < 0.001 (one-way ANOVA with Bonferroni post-hoc test). NS-non‐significant.
Critical Regulation of a NDIME/MEF2C Axis in Embryonic Stem Cell Neural Differentiation and Autism (2020)
Figure 4

Figure 4. Exons 1-3 of NDIME antagonize EZH2-mediated epigenetic silencing of Mef2c.

A. RNA secondary structure was predicted based on MFE and partition function through RNAfold WebServer (left panel, and the colour scale represents positional entropy). The thermodynamic ensemble of RNA structures (right panel).

B. RIP analysis using EZH2 antibody for the co-transfection of Flag-EZH2 and NDIME exons 1-3 (P = 0.0177) or exon 4 (P = 0.4335) in HEK293T extracts compared with IgG.

C. Verification of the efficiency of overexpression of exons 1-3 or exon 4 of NDIME in NDIME-knockout cells at day 5 of neural differentiation via qRT-PCR. Three different primers P1, P2, and P3 were used to analyze NDIME or exons of NDIME expression in Ctrl, NDIME-/-, NDIME-/-+Rs26-Exon123, and NDIME-/-+Rs26-Exon4 cells (compared with control cells). The position of primers P1, P2, and P3 are shown in the schematic diagram.

D, E. FACS (D), expression of neural-lineage genes (E), in Ctrl, NDIME-/-, NDIME-/-+Rs26-Exon123, NDIME-/-+Rs26-Exon4 cells at day 5 of neural differentiation.

F. Microscopy of the control, NDIME-/-, NDIME-/-+Rs26-Exon123, and NDIME-/-+Rs26-Exon4 cells at day 5 of neural differentiation. Scale bars, 100 µm.

G. NESTIN and GFP immunostaining of the cells in (F) at day 5 of neural differentiation. Scale bars, 100 µm.

H. Enrichment of H3K27me3 at the Mef2c promoter of the cells in (F) at day 5 of neural differentiation.

I. Western blot analysis of expression of MEF2C in the cells in (F) at day 5 of neural differentiation.

Data information: Data in (B and C) are represented as mean ± SEM, n= 3. *P < 0.05, ***P < 0.001 (unpaired two-tailed Student's t-test). Data in (D, E and H) are represented as mean ± SEM, n = 4. ***P < 0.001 (one-way ANOVA with Bonferroni post-hoc test). NS-non‐significant.
Critical Regulation of a NDIME/MEF2C Axis in Embryonic Stem Cell Neural Differentiation and Autism (2020)
Figure 5

Figure 5. The NDIME/MEF2C axis promotes the neural gene expression programs and regulates numerous ASD risk genes expression.

A. Summary of the DEGs in the NDIME-knockout cells compared with control cells at day 5 of neural differentiation.

B. Analysis of significant GO terms for genes that were downregulated by at least two-fold in the NDIME-knockout cells at day 5 of neural differentiation (hypergeometric test, P < 0.05).

C. Summary of the DEGs in the MEF2C-knockdown cells compared with control cells at day 5 of neural differentiation.

D. Analysis of significant GO terms for genes that were downregulated by at least 2-fold in the MEF2C-knockdown cells compared with control cells at day 5 of neural differentiation (hypergeometric test, P < 0.05).

E. Pathway enrichment analysis for the DEGs in the NDIME-knockout cells compared with control cells at day 5 of neural differentiation.

F. Pathway enrichment analysis for the DEGs in the Mef2c-knockdown cells compared with control cells at day 5 of neural differentiation.

G. Overlap of DEGs between the NDIME-knockout cells and the MEF2C-knockdown cells.

H. Analysis of significant GO terms for genes that were dysregulated in (G) (hypergeometric test, P < 0.05).

I. Heatmap illustrating the expression of selected neurectoderm genes and mESCs-specific genes that were shown as log2 FPKM in Ctrl, NDIME-/- , shCtrl and shMef2c mESCs at day 5 of neural differentiation. Each lane corresponds to an independent biological sample.

J. qRT-PCR analysis of representative co-upregulated and co-downregulated genes in (G) at day 5 of neural differentiation.

K. Overlapping between co-regulated genes in (G) and gene sets of interest (hypergeometric test, P = 0.0174).

L. Relative expression of selectively down-regulated ASD risk genes from the DEGs of the NDIME-knockout cells compared with control cells at day 5 and from the DEGs of the MEF2C-knockdown cells compared with control cells at day 5.

Data information: Data in (J and L) are represented as mean ± SEM, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired two-tailed Student's t-test).
Critical Regulation of a NDIME/MEF2C Axis in Embryonic Stem Cell Neural Differentiation and Autism (2020)
Figure 6

Figure 6. Overexpression of NDIME in the hippocampus rescues autism-like social deficits in mice.

A. qRT-PCR analysis of NDIME (P = 0.0051) and MEF2C (P = 0.0062) in VPA-treated mice and saline-treated mice; n = 9 per group.

B. Enrichment of EZH2 at the Mef2c promoter in VPA-treated mice and saline-treated mice (compared with saline-treated mice); n = 9 per group.

C. Western blot analysis of the protein levels of MEF2C and GAPDH in the hippocampus of VPA-treated mice (n = 9) and saline-treated mice (n = 10).

D. Immunofluorescence images for MEF2C in the hippocampus of VPA-treated mice and saline-treated mice. Nuclei were stained with Hoechst 33342 (blue); n = 9 per group. Scale bars, 50 µm.

E. Statistical analysis of the grayscale levels of MEF2C in (C) by ImageJ (P = 0.0074); saline-treated mice (n = 10), VPA-treated mice (n = 9).

F. Statistical analysis of the Mean MEF2C staining intensity/unit area in (D) by ImageJ (P = 0.0096); n = 9 per group.

G. Immunofluorescence images for GFP in the hippocampus of saline-treated mice, VPA-treated mice, and VPA-treated mice with viral-mediated expression of NDIME; n = 9 per group. Scale bars, 100 µm.

H. Immunofluorescence images for MEF2C of mice in (G); n = 9 per group. Scale bars, 50 µm.

I-K. Adeno-associated virus overexpression of NDIME in the dentate gyrus (DG) of the dorsal hippocampus of VPA-treated mice rescued autism-like social deficits. Open field test (I), three-chamber test (J), and elevated-plus maze (EPM) test (K).

Data information: Data in (A, B, E, and F) are represented as mean ± SEM. *P < 0.05, **P < 0.01 (unpaired two-tailed Student's t-test). Data in (I-K) are represented as mean ± SEM; n = 9 per group. Asterisks indicate a difference between VPA+AAV-GFP and Saline+AAV-GFP, whereas hash tags indicate a difference between VPA+AAV-NDIME-GFP and VPA+AAV-GFP. #P < 0.05, **/##P < 0.01, ***P < 0.001 (one-way ANOVA with Bonferroni post-hoc test). NS-non‐significant.
USP34 controls osteogenic differentiation and bone formation by regulating BMP2 signaling (2018)
Figure 1

Figure 1. USP34 is required for osteogenic differentiation of human MSCs

A Representative images of alkaline phosphatase (ALP), Alizarin Red S (ARS) and Van Gieson's staining of human MSCs.

B, C Quantitative analyses of the ALP activity and calcium mineralization. Results are shown as mean ± SEM; n=6; **: p<0.01 and ***: p<0.001 by t test.

D, E Quantitative RT-PCR and western blot analyses of the expression of osteogenic markers. Cells were cultured in osteogenic medium for 5 days. Results are shown as mean ± SEM; n=3; *: p<0.05, **: p<0.01 and ***: p<0.001 by t test.

F H&E staining of the MSCs-mediated ectopic bone formation. Scale bar, 50 μm.

G Quantitative analysis of bone volume versus total tissue volume (BV/TV). Results are shown as mean ± SEM; n=6. *: p<0.05 by t test.