EMBO/soda-nel huggingface dataset:
Metabolic response to point mutations reveals principles of modulation of in vivo enzyme activity and phenotype (2021)
Figure 4

Figure 4: Filamentation in mutant DHFR strains is completely reversible and does not represent TLD. (A) Mutant W133V was grown in amino acid supplemented M9 medium (M9+AA) for 4 hours at 42°C, and subsequently placed on M9 agar pads and their growth was monitored at room temperature. Shown are phase contrast images taken from different time points throughout the time-lapse experiment. Unlike cells experiencing TLD, an irreversible phenomenon, W133V DHFR cells recover and resume growth at low temperature. (B and C) WT cells were treated with different concentrations of Tmp at 42°C for varying amounts of time in amino acid supplemented M9 medium (panel B) or in M9 media supplemented with both amino acids and GMP (panel C), following which they were spotted on M9+AA plates and allowed to grow at 30°C. Colonies were counted next day. In the presence of only amino acids, there was no loss in viability for any concentration of Tmp (panel B), despite extensive filamentation (Figure EV1E). In contrast, in the presence of amino acids and GMP, the cells showed sharp loss in viability when grown at high Tmp concentrations. In both panels, error bars represent SD of two biological replicates. The dashed line represents cfu at one-hour timepoint. (D) WT (with and without 5μg/ml Tmp) and mutants were grown for 6 hours at 42°C in M9+AA medium without or with GMP, and subsequently diluted serially and spotted on M9+AA agar plates and allowed to grow at 30°C till visible colonies were formed. While WT treated with high Tmp shows loss in cfu, indicating TLD, no loss in viability was observed for WT or mutants. (E) Expression of SOS response genes recA, recN and sulA when mutant strains and Tmp treated WT cells are grown in M9 medium supplemented with amino acids (black bars) or in the presence of both amino acids and GMP (gray bars) at 42°C (I91L+W133V was grown at 40°C). Error bars represent SD of 2-3 biological replicates.
Metabolic response to point mutations reveals principles of modulation of in vivo enzyme activity and phenotype (2021)
Figure 5

Figure 5: Supplementation of dTMP alleviates dTDP/dTTP levels and rescues filamentation. (A) Distribution of cell length of WT and mutant strains when grown in M9 medium supplemented with amino acids (gray) or with both amino acids and 1mM dTMP (pink). WT, W133V and V75H+I155A strains were grown at 42°C while I91L+W133V and V75H+I91L+I155A mutants were grown at 40°C. 1mM dTMP largely rescues filamentation of mutant strains (* indicates the median cell lengths were significantly different, Mann-Whitney test, p-value <0.001). The central band in the box plots represents the median of the distribution, the box ends represent the 25th and 75th percentile, the whiskers represent the 10th and 90th percentile, while the dots represent the 5th and 95th percentile. Data was usually obtained from 2-3 biological replicates. The number of cells used to derive the boxplot distributions in the different panels range usually between 150 to 300 (please refer to Figure 5 source data for exact number of cells for each dataset). (B) Abundances of selected nucleotides in I91L+W133V mutant when grown for 4 hours at 40°C in M9 medium supplemented with both amino acids and 1mM dTMP. Metabolite levels were normalized to those of WT grown under similar conditions. dTDP and dTTP levels recover and are now comparable to dTMP levels. Error bars represent SEM of 3 biological replicates. The dashed line in represents value of 1 for WT. (C) Mutant V75H+ I91L+I155A grows very poorly (in terms of colony forming units, cfu) on a minimal media agar plate supplemented with amino acids (M9+AA) at 40°C. Supplementation of additional dTMP increases the cfu by several orders at the same temperature, while supplementation with both pyrimidine (dTMP) and purine (GMP) allows it to grow as good as WT. In comparison, WT was grown at 42°C under different supplementation conditions. In all cases, cultures were 7-fold serially diluted for the next spot. The three rows (two rows for WT) in each condition represent biological replicates. (D) Comparison of growth rates of WT and mutant DHFR strains at 42°C (40°C for I91L+W133V and V75H+I91L+I155A) in minimal medium that is supplemented with amino acids and/or 1mM dTMP. Except for W133V and to a lesser extent for V75H+I155A, the effect of dTMP on growth rates is only modest. Error bars represent SEM of 3 biological replicates.
Metabolic response to point mutations reveals principles of modulation of in vivo enzyme activity and phenotype (2021)
Figure 6

Figure 6: In vivo enzyme kinetics of Tmk is highly cooperative and different from that in vitro. (A) in vitro activity of purified Thymidylate Kinase (Tmk) as a function of dTMP concentration shows Michaelis-Menten (MM) like kinetics. ATP concentration is saturating at 1mM. The KM of dTMP is 13μM. The error bars represent SD of three technical replicates. (B) For a 10-fold drop in intracellular dTMP concentration in mutant relative to WT (as seen in I91L+W133V mutant), we calculate dTDP levels in mutant (relative to WT) as a function of various assumed intracellular concentrations of dTMP in WT (shown along x-axis), as absolute value of this is not known experimentally, assuming MM kinetics (black line) as shown in panel (A) or Hill kinetics with coefficient of 2.5 (red line) as shown in panel (C). The dotted line corresponds to the experimentally observed dTDP ratio of 0.01 for I91L+W133V mutant. This ratio is realized only for the Hill-like curve. (C) Apparent in vivo activity kinetics of Tmk enzyme using steady state dTMP and dTDP levels obtained from metabolomics. The plot includes data from WT, mutants W133V and I91L+W133V, as well as WT treated with 0.5μg/ml Trimethoprim, obtained at different time points during growth. Data points represent metabolite levels for all individual biological replicates without averaging. The black data points were acquired during growth in the presence of different concentrations of external dTMP (0.25, 0.5, 1, 2 and 5mM), while red points were from conditions with no external dTMP. Both black and red solid lines represent fit to Hill equation. (D) The green points were acquired during growth of WT, W133V and I91L+W133V mutants in the presence of different concentrations of thymidine, which follow the red curve. The inset plot shows the same graph with selected datapoints for I91L+W133V mutant. The cyan circle shows I91L+W133V mutant in the absence of any metabolite supplementation, while red and black points indicate metabolite levels following thymidine and dTMP supplementation respectively. (E) A flow chart summary of the actual chain of events triggered by destabilizing mutations in DHFR that eventually lead to bacterial filamentation, as revealed in this study.
Pr-set7/SETD8 Promotes the Reactivation of Neural Stem Cells (2021)
Figure 1

Figure 1. Pr-set7 is required for NSC reactivation.

(A) Larval NSCs of control (ctrl; yw) and pr-set7716 (716) at 24h ALH were stained for Deadpan (Dpn) and Miranda (Mira).

(B, C) Quantifications of cellular extensions (B) and NSC diameters (C) for genotypes in (A).

In (B), n=1055, 12 brain lobes [BL] for control; n=1000, 15 BL for pr-set7716. In (C), n=101 for control; n=112 for pr-set7716.

(D) A schematic representation of the pr-set7716 mutation. pr-set7716 contains a G to A point mutations at 831 nucleotides (nt). Drosophila Pr-set7 contains a SET domain (in yellow), which is required for its methyl transferase activity.

(E) Larval NSCs of control (ctrl; yw) and pr-set7716/Df(3R)ED5644 (716/Df) at 96h ALH were labelled with Dpn and Mira.

(F, G) Quantifications of cellular extensions (F) and NSC diameters (G) for genotypes in (E). In (F), n=1488, 15 BL for control; n=2014, 20 BL for pr-set7716/Df. In (G), n=92 for control; n=93 for pr-set7716/Df.

(H) Quantifications of EdU incorporation for genotypes in (I). Control, n=1365, 16 BL; pr-set7716/Df, n=1006, 16 BL.

(I) Larval brains of control (ctrl; yw) and pr-set7716/Df at 96h ALH were labelled with Dpn and EdU.

(J, K) Western blot and quantification analysis of larval brain extracts of control (ctrl; yw), pr-set7716/Df and UAS-pr-set7 induced with insc­-Gal4 at 72h ALH (n=2 as biological replicates). Blots were probed with anti-Pr-set7 antibody or anti-Actin antibody (loading control). Pr-set7 levels were normalized against actin intensity.

(L) Larval NSCs of control (ctrl; yw), pr-set7716/Df, and UAS-pr-set7 induced with insc­-Gal4 at 72h ALH were stained for Pr-set7, Mira and DNA.

(M, N) Quantifications of NSC diameters (M) at 24h ALH and EdU incorporation (N) for genotypes in (O). In (M), n=152 for control; n=129 for pr-set7716/Df. In (N), for control, n=601, 13 BL at 24h ALH; n=988, 14 BL at 48h ALH; n=813, 9 BL at 72h ALH and n=829, 10 BL at 96h ALH. For pr-set7716/Df, n=566, 12 BL at 24h ALH; n=808, 13 BL at 48h ALH; n=1032, 10 BL at 72h ALH and n=825, 9 BL at 96h ALH.

(O) Larval brains of control (ctrl; yw) and pr-set7716/Df at 24h, 48h, 72h and 96h ALH were labelled with Dpn and EdU.

Data information: Quantification data are presented in as mean ± SD (B, C, F, G, H, M, N). n numbers in (B, F, H, N) were total NSCs counted with total brain lobes measured, while each brain lobe considered as one biological replicate. Yellow dotted boxes indicate the region of zoomed-in images (O). White arrows point to NSCs and yellow arrowheads point to cellular extensions of NSCs. The central brain is to the left of white dotted lines (I, O). Statistical analyses were done comparing two different genotypes using Mann-Whitney test (B, C, F, G, H, M, N). ***P < 0.001. Scale bars, 5 μm (A, E, L), 15 μm (O), 30 μm (I).
Pr-set7/SETD8 Promotes the Reactivation of Neural Stem Cells (2021)
Figure 2

Figure 2. Pr-set7 levels increase with NSC reactivation and is required for H4K20me1 in larval brains.

(A) Larval NSCs of control (ctrl; yw) and pr-set7716/Df(3R)ED5644 (716/Df) at 0h, 24h and 48h ALH were stained for Pr-set7, Dpn and Mira. Yellow dotted boxes, zoomed-in area.

(B) Quantifications of the corrected total cell fluorescence (CCF) for genotypes in (A). CCF = Integrated Density - (area of selected cell × mean fluorescence of background readings). At 0h ALH, CCFs were 735.7±220.7 (n=168) for control, 302.1±150.0 (n=160) for 716/Df. At 24h ALH, CCFs were 2238.3±628.4 (n=158) for control, 310.4±207.1 (n=113) for 716/Df. At 48h ALH, CCFs were 3834.0±924.2 (n=124) for control, 465.9±295.0 (n=126) for 716/Df. Data are presented as mean ± SD. Statistical analyses were done comparing between the CCFs at later time points with the CCFs for control at 0h ALH using Mann-Whitney test . ***P < 0.001.

(C) Larval NSCs of control (ctrl; yw), pr-set7716/Df, pr-set7716/Df with UAS-pr-set7 induced with insc-Gal4, and pr-set7716/Df with pr-set7-BAC at 24h ALH were stained for H4K20me1, Dpn, Mira and DAPI. Control: n=108; pr-set7716/Df: n=87; pr-set7716/Df with UAS-pr-set7 induced with insc-Gal4: n=167 and pr-set7716/Df with pr-set7-BAC: n=104.

(D) A schematic representation of the FUCCI.

(E) Larval NSCs with FUCCI constructs induced with insc-Gal4 at 24h were stained for GFP, RFP, Dpn, and Prospero (Pros).

(F) Quantifications of quiescent NSCs in different cell-cycle stages from FUCCI flies (induced with insc-Gal4) in control (and insc-Gal4>FUCCI) and pr-set7716/Df. Quiescent NSCs of insc-Gal4>FUCCI: 76.1%±1.5% in G2, 1.7%±0.2% in S and 22.2%±1.3% in G0 (n=343). Quiescent NSCs of insc-Gal4>FUCCI; pr-set7716/Df: 74.4%±1.0% in G2, 0.9%±0.2% in S and 24.7%±0.8% in G0 (n=344).

Data information: Quantification data are presented in as mean ± SD (B, F). Statistical analyses were done comparing two different genotypes using Mann-Whitney test (B, F). ns: non-significant. White arrows point to NSCs. Scale bars, 5 μm (C, E), 15 μm (A).
Pr-set7/SETD8 Promotes the Reactivation of Neural Stem Cells (2021)
Figure 3

Figure 3. cdk1 and ebd1 are identified as potential targets of Pr-set7 in NSC-specific TaDa in vivo profiling.

(A) A schematic representation of the TaDa analysis performed in Drosophila NSCs.

(B, C) The binding profile of Pr-set7 on cdk1 gene region (B) and ebd1 gene region (C) in NSCs. The green peaks represent positive binding and red means no binding, which are based on the log2 ratio between Dam-Pr-set7 and Dam control (Y-axis).

(D) qPCR analysis of pr-set7, cdk1, ebd1 and arm in larval brains of control (ctrl; yw) and pr-set7716/Df(3R)ED5644 (716/Df) at 24h ALH. n=7 as biological replicates. Statistical analyses were done comparing between control (GAPDH1, GAPDH2, Tbp1 and Rpl32) and pr-set7716/Df, using Mann-Whitney test. The mRNA levels of control were normalized to 1. Data are presented as mean ± SD calculated from all replicates.

(E) Western blot analysis of larval brain extracts of control (ctrl; yw) and pr-set7716/Df at 24h ALH. Blots were probed with anti-Cdk1 antibody and anti-GAPDH1 antibody (loading control).

(F) Quantification of normalized Cdk1 intensity against GAPDH in (E). n=7 as biological replicates.

(G) Larval NSCs of control (ctrl; yw) and pr-set7716/Df at 24h ALH were stained for Ebd1, Dpn and Mira. Yellow dotted boxes, zoomed-in area. White arrows point to NSCs.

(H, I) Quantifications of nuclear Ebd1 (H) and the corrected total cell fluorescence (CCF) (I) in genotypes in (G). In (H), n=756, 9 BL for control; n=765, 9 BL for pr-set7716/Df. In (I), CCF = Integrated Density - (Area of selected cell × Mean fluorescence of background readings). CCFs were 1102.2±493.2 (n=99) for control (ctrl; yw) and 144.0±88.0 (n=98) for 716/Df.

(J) Western blot analysis of larval brain extracts of control (ctrl; yw) and pr-set7716/Df at 24h ALH. Blots were probed with anti-Arm antibody or anti-GAPDH1 antibody (loading control).

(K) Quantification of normalized Arm levels against GAPDH in (J). n=5 as biological replicates.

Data information: Data are presented in (D, F, H, I, K) as mean ± SD. n numbers in (H) were total NSCs counted with total brain lobes measured, while each brain lobe was considered as one biological replicate. Statistical analyses were done comparing two different genotypes using Mann-Whitney test (D, F, H, I, K). *P < 0.05, **P < 0.01, and ***P < 0.001. Scale bars, 15 μm (G).
Pr-set7/SETD8 Promotes the Reactivation of Neural Stem Cells (2021)
Figure 4

Figure 4. Cdk1 functions downstream of Pr-set7 and is both necessary and sufficient for NSC reactivation on fed condition.

(A) Larval brains of control and cdk1 RNAi (BDSC#35350, #36117, and #40950) under insc­-Gal4 at 24h ALH were stained for Deadpan (Dpn) and Miranda (Mira).

(B) Quantifications of cellular extensions for genotypes in (A). Control: n=942, 11 BL; BDSC#35350: n=1056, 13 BL; BDSC#36117: 16.0%±3.5% (n=1059, 12 BL); BDSC#40950: 16.7%±3.1% (n=933, 11 BL).

(C) Larval brains of control and cdk1 RNAi (BDSC#35350, #36117 and #40950) under insc­-Gal4 at 24h ALH were labelled with Dpn and EdU.

(D) Quantifications of NSCs without EdU incorporation for genotypes in (C). Control: n=849, 10 BL; BDSC#35350: n=741, 10 BL; BDSC#36117: 87.4%±8.0% (n=697, 10 BL); BDSC#40950: 92.6%±2.4% (n=621, 8 BL).

(E) Larval brains of control, UAS-cdk1-Myc, and UASp- cdk1.T14A.Y15F.VFP (cdk1.CA) under insc­-Gal4 at 6h ALH were labelled with Dpn and EdU.

(F, G) Quantifications of EdU incorporation (F) and NSC diameters (G) for genotypes in (E). In (F), n=345, 6 BL for control; n=524, 8 BL for cdk1-Myc and n=280, 6 BL for cdk1.CA. In (G), n=123 for control; n=135 for cdk1-Myc and n=137 for cdk1.CA.

(H) Larval brains of control (ctrl; yw), cdk1-Myc overexpression under insc­-Gal4, pr-set7716/Df(3R)ED5644 (716/Df), and pr-set7716/Df with cdk1-Myc induced with insc-Gal4 at 24h ALH were labelled with Dpn and EdU.

(I, J) Quantifications of EdU incorporation (I) and NSC diameters (J) for genotypes in (H). In (I), n=1334, 16 BL for control; n=1299, 15 BL for insc>cdk1; n=1221, 18 BL for pr-set7716/Df and n=1372, 16 BL for pr-set7716/Df with cdk1-Myc induced with insc-Gal4. In (J), n=153 for control; n=152 for insc>cdk1; n=156 for pr-set7716/Df and n=159 for pr-set7716/Df with cdk1-Myc induced with insc-Gal4.

Data information: Data were presented in quantification graphs as mean ± SD (B, D, F, G, I, J). n numbers in (B, D, F, I) were total NSCs counted with total brain lobes measured, while each brain lobe was considered as one biological replicate. White arrows point to NSCs and yellow arrowheads point to cellular extensions of NSCs (A). Statistical analyses were done comparing among different genotypes using ordinary one-way ANOVA (B, D, F, G, I, J). ***P < 0.001. Scale bars, 5 μm (A), 30 μm (C, E, H). Control: insc­-Gal4/+ UAS-dicer2 in (A-G).
Pr-set7/SETD8 Promotes the Reactivation of Neural Stem Cells (2021)
Figure 5

Figure 5. Ebd1 promotes NSC reactivation and its overexpression suppresses NSC reactivation defects in pr-set7 mutants.

(A) Larval NSCs of control (ctrl; yw) and ebd1240 at 24h ALH were stained for Deadpan (Dpn) and Miranda (Mira).

(B) Quantifications of cellular extensions for genotypes in (A). Control: n=886, 11 BL; ebd1240: n=864, 12 BL.

(C) Larval brains of control (ctrl; yw) and ebd1240 at 24h ALH were labelled with Dpn and EdU.

(D) Quantifications of NSC without EdU incorporation for genotypes in (C). Control: n=1034, 12 BL; ebd1240: n=1139, 16 BL.

(E) Larval NSCs of grainyhead (grh)>CD8-GFP at 0h, 24h and 48h ALH were stained for CD8, Dpn, and Ebd1. Yellow dotted boxes indicate the region of zoomed-in images.

(F, G) Quantifications of nuclear/cytoplasmic Ebd1 (F) and the corrected total cell fluorescence (CCF) of Ebd1 in NSCs (G) for genotypes in (E). In (F), n=1044, 14 BL at 0h ALH; n=712, 10 BL at 24h ALH and n=1122, 11 BL. In (G), CCF = Integrated Density - (area of selected cell × mean fluorescence of background readings). For CCFs of Ebd1, 128.5±140.7 (n=162) at 0h ALH, 938.7±1176.8 (n=150) at 24h ALH and 1868.2±1025.1 (n=161) at 48h ALH.

(H) Larval brains of control (ctrl; yw), ebd1-Flag overexpression under insc­-Gal4, pr-set7716/Df(3R)ED5644 (716/Df) and pr-set7716/Df with ebd1 induced with insc-Gal4 at 24h ALH were labelled with Dpn and EdU.

(I) Quantifications of NSCs without EdU incorporation for larval brains in (H). Control: n=1162, 13 BL; insc>ebd1: n=997, 11 BL; pr-set7716/Df: n=928, 13 BL and pr-set7716/Df with ebd1 induced with insc-Gal4: n=1284, 16 BL.

Data information: Data are presented in (B, D, F, G, I) as mean ± SD. n numbers in (B, D, F, I) were total NSCs counted with total brain lobes measured, while each brain lobe was considered as one biological replicate. White arrows point to NSCs and yellow arrowheads point to cellular extensions of NSCs (A, E). Statistical analyses were done comparing two different genotypes using Mann-Whitney test (B, D, F). For F, statistical analyses were done comparing the percentage at later time points with the ones at 0h ALH. Statistical analyses were done comparing among different genotypes using ordinary one-way ANOVA (G, I). ns: non-significant; *P < 0.05; ***P < 0.001. Scale bars, 5 μm (A), 15 μm (E), 30 μm (C, H).
Pr-set7/SETD8 Promotes the Reactivation of Neural Stem Cells (2021)
Figure 6

Figure 6. A reciprocal regulation between Cdk1 and Ebd1 during NSC reactivation.

(A) Larval NSCs of control (ctrl; yw) and cdk1B47 at 24h ALH were labelled for Earthbound1 (Ebd1), Deadpan (Dpn), and Miranda (Mira).

(B) Quantifications of the corrected total cell fluorescence (CCF) of Ebd1 in genotypes in (A). CCF = Integrated Density - (Area of selected cell × Mean fluorescence of background readings). CCFs were 846.9±394.2 for control (ctrl; yw; n=167, 14 BL) and 354.3±245.3 for cdk1B47 (n=211, 13 BL).

(C, D) Larval NSCs of control (UAS-dicer2) and UAS-cdk1-Myc induced with insc-Gal4 at 6h ALH were labelled for Ebd1, Dpn, and Mira. (D) Quantifications of the CCF of Ebd1 in genotypes in (C). CCFs were 111.3±80.2 for control (n=157, 12 BL) and 346.6±221.1 for cdk1-Myc (n=181, 12 BL).

(E) qPCR analysis of cdk1 in larval brains of control (ctrl; yw) and ebd1240 at 24h ALH. n=3 as biological replicates. Statistical analyses were done comparing control (GAPDH1, GAPDH2, Tbp1 and Rpl32) and ebd1240, using two-tailed Student's t-test. The mRNA levels of control were normalized to 1.

(F, G) Larval NSCs of control (ctrl; yw), ebd1240 and pr-set7716/Df(3R)ED5644 (716/Df) at 24h ALH were stained for Senseless (Sens), Dpn and Mira. (F) Quantifications of the CCF in genotypes in (G). CCFs were 1349.7±792.5 for control (n=166, 13 BL), 505.8±395.9 for ebd1240 (n=206, 14 BL) and 859.7±619.0 for 716/Df (n=174, 13 BL).

(H) A working model. Pr-Set7 promotes NSC reactivation by positively regulating Cdk1 and Wingless pathway transcriptional co-activator Ebd1 in NSCs. Cdk1 upregulates Ebd1 in NSCs, while Ebd1 may maintain an equilibrium of Cdk1 level during reactivation.

Data information: Data are presented in (B, D, E, G) as mean ± SD. n numbers in (B, D, G) were total NSCs counted with total brain lobes measured, while each NSC was considered as one biological replicate. White arrows point to NSCs (A, C, F). Yellow dotted boxes indicate the region of zoomed-in images (A, C, F). Statistical analyses were done comparing between two different genotypes using Mann-Whitney test (B, D) and two-tailed Student's t-test (E). Statistical analyses were done comparing among different genotypes using ordinary one-way ANOVA (G). *P < 0.05; ***P < 0.001. Scale bars, 15 μm (A, C, F).

Expanded View Figure legends
MYDGF protects bone mass by inhibiting osteoclastogenesis and promoting osteoblast differentiation (2021)
Figure 1

Fig 1

Myeloid specific MYDGF deletion increased bone loss and decreased bone strength in young and aged mice.

As described in Fig EV1H, WT and CKO mice aged 4 weeks were selected and divided into 4 groups, and fed a normal diet until to 3 months or 18 months, respectively (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 bone mineral density (BMD) (B), trabecular bone volume (Tb.BV/TV) (C), trabecular bone number (Tb. N) (D) and trabecular separation (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 in distal femora, scale bar, 50µm.

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

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

L, M Representative images of Ocn+ (red) and DAPI (blue) immunostaining (L) and quantification of Ocn+ cells on trabecular (M), scale bar, 50µm (n=6).

N, O Representative images of TRAP (red) and DAPI (blue) immunostaining (N) and quantification of TRAP cells on trabecular (O), scale bar, 50µm (n=6).

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

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

R, S The serum levels of CTX (R) and PINP (S) (n=6).

Data information: Results are shown as mean ± SEM. *P＜0.05, **P＜0.01 and ***P＜0.001 (Student's t test).