EMBO/soda-nel huggingface dataset:
Osteoclasts adapt to physioxia perturbation through DNA demethylation (2021)
Figure 1

Figure 1. Measurement of passive physiological range of oxygen tension (pO2) of osteoclasts.

(A) Schematic diagram of the two-photon intravital imaging and 2PLIM.

(B) Representative intravital image of calvarial bone marrow of Tcirg1EGFP/+ male mice treated with BTPDM1 showing osteoclasts (left, EGFP fluorescence) and 2PLIM image for pO2 changes (right, phosphorescence lifetime of BTPDM1). Scale bar, 20 μm.

(C) Phosphorescence lifetime in each osteoclast of calvarial bone marrow of mice upon exposure to ambient air (n = 91 from six mice). Data denote mean ± s.e.m.

(D, E) Change in pO2 of osteoclasts in the mice upon exposure to various concentration of oxygen from 21% to 14% pO2. Magnified PLIM images of osteoclasts under different peripheral oxygen saturation (SpO2, D). The pO2 of each osteoclast in these mice was plotted (E, n = 47 from three mice for each SpO2). Scale bar, 20 μm. Data denote mean ± s.e.m. **P < 0.01 (ANOVA).

(F) Effect of physioxia (5% pO2) or physiological hypoxia (2% pO2) on osteoclastogenesis. TRAP-stained cells (left panel) and the number of TRAP-positive cells with more than three nuclei (right). Scale bar, 100 μm. Data denote mean ± s.e.m. **P < 0.01 (n = 8 biological replicates; t-test).
Osteoclasts adapt to physioxia perturbation through DNA demethylation (2021)
Figure 2

Figure 2. Effect of oxygen deprivation on bone metabolism.

(A) μCT analysis of the femurs of 10-week-old male mice upon exposure to low oxygen concentration (14% pO2, n = 5) or ambient air (n = 7), and of 10-week-old Hif1aRank-/-; Hif2aRank-/- male mice upon exposure to low oxygen concentration (14% pO2, n = 6) or ambient air (n = 5) for 10 days (axial view of the metaphyseal region). Scale, 0.5 mm.

(B) Histological analysis of the proximal tibias of 10-week-old male mice upon exposure to low oxygen concentration (14% pO2) or ambient air for 10 days, and of 10-week-old Hif1aRank-/-; Hif2aRank-/- male mice upon exposure to low oxygen concentration (14% pO2) or ambient air (toluidine blue staining [arrow, osteoclasts]). Scale, 100 μm.

(C) Parameters for osteoclasts and osteoblasts during bone morphometric analysis of 10-week-old male mice under low oxygen concentration (n = 3) or ambient air (n = 5), and 10-week-old Hif1aRank-/-; Hif2aRank-/- male under low oxygen concentration (n = 3) or ambient air (n = 5) for 10 days. Data denote mean ± s.e.m. *P < 0.05; NS, not significant (t-test).
Osteoclasts adapt to physioxia perturbation through DNA demethylation (2021)
Figure 3

Figure 3. Effect of physiological hypoxia on hypoxia-inducible factor activity and energy metabolism.

(A) Protein expression of HIF-1α, HIF-2α, Blimp1, NFATc1, Ctsk and TRAP in control and Hif1aRank-/-; Hif2aRank-/- BMMs stimulated with RANKL and cultured under different conditions of oxygen concentration for 2 days.

(B) Effect of physioxia (5% pO2) or physiological hypoxia (2% pO2) on the formation of osteoclast from control and Hif1aRank-/-; Hif2aRank-/- BMMs. TRAP-stained cells (left panel) and the number of TRAP-positive cells with more than three nuclei (right). Scale bar, 100 μm. Data denote mean ± s.e.m. **P < 0.01; NS, not significant (n = 6 (control) and n = 6 (Hif1aRank-/-; Hif2aRank-/-) biological replicates; ANOVA).

(C) Expression of osteoclastogenic and canonical HIF-target genes in wild-type control and Hif1aRank-/-; Hif2aRank-/- BMMs under conditions of 5% or 2% oxygen. Data denote mean ± s.e.m. *P < 0.05; **P < 0.01; NS, not significant (n = 4 biological replicates; ANOVA).
Osteoclasts adapt to physioxia perturbation through DNA demethylation (2021)
Figure 4

Figure 4. Effect of oxygen deprivation on energy metabolism in vivo.

(A) Representative intravital image of calvarial bone marrow of Rosa26GO-ATeam/+; Tg(Acp5-tdTomato) male mice showing osteoclasts (left, tdTomato fluorescence) and FLIM image for ATP changes (middle and right, fluorescence lifetime of EGFP). The FRET efficiencies of each osteoclast were plotted from the mice upon exposure to low oxygen atmosphere (14% pO2) or ambient air (right, n = 11 from three mice for each SpO2). Scale bar, 20 μm. Data denote mean ± s.e.m. not significant (ANOVA).

(B) Representative intravital image of calvarial bone marrow of Tg(Acp5-tdTomato) male mice showing osteoclasts (left, tdTomato fluorescence) and NAD(P)H autofluorescence lifetime images (middle and right). Average of autofluorescence lifetime of each osteoclast was plotted from the mice upon exposure to low oxygen concentration (14% pO2) or ambient air (right, n = 64 from three mice for each SpO2). Scale bar, 20 μm. Data denote mean ± s.e.m. NS, not significant (ANOVA).
Osteoclasts adapt to physioxia perturbation through DNA demethylation (2021)
Figure 5

Figure 5. Osteoclast-specific Tet2- and Tet3-deficient mice exhibit a high bone mass phenotype.

(A) Effect of Tet2 and Tet3 single deficiency on osteoclastogenesis. TRAP-stained cells (left panel) and the number of TRAP-positive cells with more than three nuclei (right). Scale bar, 100 μm. Data denote mean ± s.e.m. *P < 0.05; **P < 0.01 (n = 6 (control and Tet2Rank-/-) and n = 3 (Tet3aRank-/-) biological replicates; ANOVA).

(B) μCT analysis of the femurs of 10-week-old control (n = 6), Tet2Rank-/- (n = 5), Tet3Rank-/- (n = 5), Tet2Rank-/-; Tet3aRank+/- (n = 6), Tet2Rank+/-; Tet3aRank-/- (n = 6) and Tet2Rank-/-; Tet3aRank-/- (n = 4) male mice (axial view of the metaphyseal region). Scale, 0.5 mm.

(C) Histological analysis of the proximal tibias of 10-week-old control, Tet2Rank-/-, Tet3Rank-/-, Tet2Rank-/-; Tet3aRank+/-, Tet2Rank+/-; Tet3aRank-/- and Tet2Rank-/-; Tet3aRank-/- male mice (TRAP staining [red, osteoclasts]). Scale, 100 μm.

(D) Parameters for osteoclastic bone resorption during bone morphometric analysis of 10-week-old control (n = 10), Tet2Rank-/- (n = 5), Tet3Rank-/- (n = 6), Tet2Rank-/-; Tet3aRank+/- (n = 6), Tet2Rank+/-; Tet3aRank-/- (n = 6) and Tet2Rank-/-; Tet3aRank-/- (n = 4) male mice. Data denote mean ± s.e.m. **P < 0.01; NS, not significant (ANOVA).

(E) Effect of Tet2 and Tet3 double deficiency on osteoclastogenesis. TRAP-stained cells (left panel) and the number of TRAP-positive cells with more than three nuclei (right). Scale bar, 100 μm. Data denote mean ± s.e.m. **P < 0.01 (n = 6 (control), n = 10 (Tet2Rank-/-; Tet3aRank+/-), n = 3 (Tet2Rank+/-; Tet3aRank-/-) and n = 4 (Tet2Rank-/-; Tet3aRank-/-) biological replicates; ANOVA).
Osteoclasts adapt to physioxia perturbation through DNA demethylation (2021)
Figure 6

Figure 6. Tet2- and Tet3-mediated DNA demethylation is required for the activation of Blimp1-NFATc1 axis.

(A) Scatter plot of fold changes of RNA-seq data and MeDIP-seq data. The log2 fold changes of RNA-seq expression between wild-type control and Tet2Rank-/-; Tet3aRank+/- BMMs stimulated with RANKL for 2 days are shown on the x-axis, and log2 fold changes of MeDIP-seq expression (>0) are shown on the y-axis. The names of significant genes are labeled (FDR of differential expression analysis < 0.01, p-value of differential methylated region analysis < 0.01).

(B) MeDIP-qPCR analysis to validate regions that are hypermethylated in Tet2Rank-/-; Tet3aRank+/- BMMs stimulated with RANKL for 2 days. Data denote mean ± s.e.m. *P < 0.05; **P < 0.01 (n = 3-5 biological replicates; t-test).

(C) Gene expression of TET target candidates in BMMs stimulated with RANKL for 2 days. Data denote mean ± s.e.m. **P < 0.01 (n = 4-7 biological replicates; t-test).

(D) Genome browser view of the expression and the methylation site distribution in the Prdm1 gene locus of control and Tet2Rank-/-; Tet3aRank+/- BMMs stimulated with RANKL for 2 days.

(E) Bisulfate sequencing analysis of selected region of the second intron of the Prdm1 gene in control and Tet2Rank-/-; Tet3aRank+/- BMMs stimulated with RANKL for 2 days.

(F) Effect of retroviral NFATc1 expression on osteoclastogenesis of control and Tet2Rank-/-; Tet3aRank+/- BMMs stimulated with RANKL for 3 days. TRAP-stained cells (left panel) and the number of TRAP-positive cells with more than three nuclei (right). Scale bars, 100 μm. Data denote mean ± s.e.m. **P < 0.01 (n = 3 biological replicates; ANOVA).

(G) Bisulfate sequencing analysis of the selected region of the second intron of the Prdm1 gene in BMMs stimulated with RANKL and culture under 5% and 2% oxygen for 3 days. (H) Effect of retroviral NFATc1 expression on osteoclastogenesis of BMMs stimulated with RANKL and culture under 5% and 2% oxygen for 5 days. Control and Tet2Rank-/-; Tet3aRank+/- BMMs stimulated with RANKL for 3 days. TRAP-stained cells (left panel) and the number of TRAP-positive cells with more than three nuclei (right). Scale bars, 100 μm. Data denote mean ± s.e.m. **P < 0.01 (n = 3 biological replicates; ANOVA).
SARS-CoV-2 targets neurons of 3D human brain organoids (2020)
Figure 2

Figure 2. SARS-CoV-2 deregulates of Tau in cortical neurons

A. Tau immunoreactivity (magenta) specifies the cortical plate (CP) surrounding the luman (L) (i). Selected optic sections at high magnification (ii and iii) and highresolution imaging (iv) show Tau localization only in axons of cortical neurons. Note the somas of neurons are free from Tau protein. At least eight organoids from four different batches (n=4) are tested. Figures display scale bars.

B. Tau localization in SARS-CoV-2-positive neurons (AB4, green) in selected optic sections (i). Note, in contrast, to control groups, SARS-CoV-2-exposed organoids display mislocalized Tau (magenta) majorly into the somas of neurons (arrowheads). Selected confocal slices are shown to distinguish Tau mislocalization into neuronal soma (arrowheads). At high magnification, neuronal soma is further specified by the perinuclear localization of SARS-CoV-2 (green) (ii and iii). Bar diagrams at right quantifies the percentage of neurons (Mock and SARS-CoV-2 exposed) exhibiting mislocalized Tau (iv) and the fraction of SARS-CoV-2-positive neurons exhibiting Tau-positive axons spanning different cortical areas (v). For statistics, at least 300 cells from six organoids from four different batches (n=4) were tested. Figures display scale bars. Unpaired t test with Welch's correction, ***P<0.001. Data presented as mean + SD.

C. Schematic cartoon of differential Tau distribution in mock compared to SARS-CoV-2-positive neurons. In mock, Tau is sorted mainly to axons. In SARS-CoV-2 positive neurons, Tau is missorted to the soma (determined by Pan-Tau antibody). Furthermore, phosphorylated Tau (at T231) majorly localizes in the soma (bottom panel, determined by Tau AT-180 antibody, see below).

D. In contrast to controls (i), Tau AT180 antibody (magenta) that specifically recognizes the phosphorylated Threonine 231 of Tau protein distinctly localizes at the somas of SARS-CoV-2-positive neurons (AB4, green) (ii). At least four organoids from two different batches (n=2) are tested. Figures display scale bars.

E. Co-localization of SARS-CoV-2 (AB4, green) and phosphorylated Tau protein (magenta) at somas of cortical neurons revealed by high-resolution imaging and de-convolution. Representative images from at least 300 cells examined. Figures display scale bars.

F. The bar diagram quantifies the fraction of Tau AT180-positive neurons that co-localize with SARS-CoV-2-positive neurons. For statistics, at least 250 cells from four organoids and two independent batches (n=2) were examined. Unpaired t test with Welch's correction. Data presented as mean + SEM.
SARS-CoV-2 targets neurons of 3D human brain organoids (2020)
Figure 3

Figure 3. SARS-CoV-2 induces of neuronal death

A. Cells from mock-organoids display a healthy nucleus labeled by DAPI (blue) (i). SARS-CoV-2-positive cells (green) display condensed (middle panel, ii) and fragmented DNA (bottom panel, iii, arrows). At least 75 cells from two (n=2) independent batches of organoids were examined. Figures display scale bars.

B. Compare to mock organoids (i) SARS-CoV-2-exposed organoids (ii) display increased TUNEL-positive cells (displayed as inverted LUT) at the cortical plate that is specified by TUJ-1 (magenta). At least four organoids from two (n=2) independent batches of organoids were examined. Figures display scale bars. The bar diagram below quantifies the frequencies of TUNEL-positive cells between mock and SARS-CoV-2-exposed organoids. Four organoids from two (n=2) independent batches were examined. Unpaired t test, *P<0.05. Data presented as mean + SD.

C. Most of the SARS-CoV-2-positive cells (AB4, green) are TUNEL-positive (i) and some of the SARS-CoV-2-positive cells are caspase-positive (ii). Caspase-positive cells in SARS-CoV-2-exposed organoids display pT231Tau localization at the cell soma specified by AT-180 (ii), which are not observed in in mock organoids (iii). Arrowheads point SARS-CoV-2-positive cells (AB4, green) that are also positive for TUNNEL (red), caspase 3 (yellow) and Tau AT-180 (magenta). Figures display scale bars. Bar diagrams at right quantifies proportions of TUNEL and caspase-positive cells among SARS-CoV-2-positive cells. The second graph below quantifies the proportions of pT231Tau-positive cells among caspase positive cells between control and virus exposed groups. At least 400 cells from 4 organoids and two independent (n=2) batches were examined. Unpaired t test, **P<0.01. Data presented as mean + SEM. TUNEL-positive cells in control un-exposed organoids do not contain pT231Tau (iii). Figures display scale bars. Bar diagrams at right quantifies proportions of pT231Tau-positive cells among TUNNEL-positive cells between control and virus exposed groups. At least 350 cells from 4 organoids and two independent (n=2) batches were examined. Unpaired t test, ***P<0.001. Data presented as mean + SEM.
In vivo generation of human CD19-CAR T cells results in B cell depletion and signs of cytokine release syndrome (2018)
Figure 1

Fig. 1: CAR T cell generation in human PBMC transplanted mice

(A) Ex vivo generation of CAR T cells. Activated human PBMC were left untransduced or incubated with CD8-LVCD19CAR at an MOI of 2. Five days later, expression of CD19-CAR and CD8 were determined on CD3+ cells. Numbers indicate the percentage of cells in the respective gate. (B) Experimental outline for in vivo CAR generation. 1x107 human PBMC were engrafted into naïve NSG mice or NSG mice that had been intraperitoneally (i.p.) injected with 5x105 Raji cells (Raji +) six days before. One day later, 2x106 t.u. of CD8-LVCD19CAR (filled circles) or CD8-LVRFP (grey triangles) were i.p. injected, respectively. As further control, another group of mice received PBS (open circles). Seven days later, mice were sacrificed and organs and cells were removed for further analysis. (C) Detection of CAR T cells by vector copy numbers (VCN). Genomic DNA was isolated from peritoneal cavity, spleen and blood cells. VCN were determined in technical duplicates by qPCR for two individual mice of each group. Presence of B cells in the transplanted PBMC is indicated below. (D-F) Cells isolated from the peritoneal cavity (peritoneum), spleen, or blood were evaluated by flow cytometry for the percentages of human CD8+ in CD3+ cells (D), of CAR+ or RFP+ cells in the CD8+ and CD8- fractions, respectively (E), and of human CD19+ cells (F) within the fraction of human CD45+ cells. Representative density plots are shown for the peritoneal cells. The gating strategy is represented in Appendix Fig. S1A. (G) Mice were transplanted with B-cell depleted human PBMC and then received CD8-LVCD19CAR (filled circle) or PBS (open circle). As control, CD8-LVCD19CAR or PBS were injected into mice transplanted with non-depleted PBMC. Data represent mean ± SD for all groups (CD8-LVCD19CAR: n=6 in D, n=4 in G; CD8-LVRFP: n=4; PBS: n=4 in G, all others n=3). Statistical significance was determined using MANN-Whitney test; ns: not significant.
In vivo generation of human CD19-CAR T cells results in B cell depletion and signs of cytokine release syndrome (2018)
Figure 2

Fig. 2: CAR T cell generation in HSC-transplanted mice

(A) Experimental outline. IL-7 was injected intravenously into HSC-humanized NSG mice before 2x106 t.u. of CD8-LVCD19CAR (CAR), or PBS as control (PBS), were administered. Distinct symbols for each individual animal are used throughout B-F (M16: lilac triangle; M19: blue triangle). Open symbols indicate animals from the vector-injected group devoid of PCR-detectable CAR T cells. (B) CAR T cell levels in the CD8+ and CD8- T cells harvested from blood, spleen and bone marrow of PBS (PBS) or vector-injected (CAR) mice determined by flow cytometry. (C) Detection of CAR T cells by determining vector copy numbers (VCN) in genomic DNA isolated from CD8+ enriched cells harvested from bone marrow. (D) Detection of CAR T cells in bone marrow quantified by flow cytometry. (E) CD19+ B cell levels in blood, spleen and bone marrow determined by flow cytometry. (F) Relative human CD19+ B cell level in blood calculated by normalizing the levels at the day the animals were sacrificed to those before vector administration. Data represent mean ± SD for all groups. N=6 in PBS group; n=10 in CAR group. Statistical significance was determined using One-way ANOVA test with Bonferroni correction.
In vivo generation of human CD19-CAR T cells results in B cell depletion and signs of cytokine release syndrome (2018)
Figure 3

Fig. 3: Cytokines and histopathology

(A) Cytokine levels in plasma of individual mice obtained from blood at 7 weeks after vector injection. The distinct symbols used for each individual mouse are identical to the ones used in Fig. 2. Mean values ± SD of n=2 technical replicates. (B) Immunohistochemistry of paraffin embedded sections from the lungs, brain meninges, brain striatum, spleen and liver of the vector-injected mouse M16 (CAR) and a control mouse injected with PBS (PBS) stained against CD8 (αCD8), the CAR (αCAR), CD4 (αCD4), or CD19 (αCD19). Black arrowheads point at infiltrated lymphocytes, red arrowheads at histiocytes, and the yellow line indicates B-lymphocyte rich zones.

Expanded View Figure Legends
D-Aspartate treatment attenuates myelin damage and stimulates myelin repair (2018)
Figure 1

Figure 1. Effects of D-Asp exposure on OPC proliferation and differentiation.

A. RT-PCR of CNPase (left) and MBP (right) mRNAs expression in MO3.13 precursors under control conditions and following 10-200 μM D-Asp exposure for 3 days. Graphs show quantification of ratio of CNPase, and MBP to L19.

B. Western blotting (left) and densitometric analysis (right) of MBP expression in absence or in presence of 10-200 μΜ D-Asp exposure for 5 days.

C. Cell growth analysis of human MO3.13 oligodendrocytes in absence or in presence of 200 μM D-Asp for 1-5 days. The density of M03.13 oligodendrocytes was daily recorded through trypan blue dye exclusion. Mean of daily measurements were recorded. The data of each experimental group were normalized to the density of cells plated at day 0 and expressed as percentage of ctrlday0.

D. FACS-based cell cycle distribution analysis after PI incorporation of MO3.13 oligodendrocytes in absence or in presence of 200 μM D-Asp for 3 days. Representative FACS plots of biological replicates are shown (n=5 independent experimental sessions).

E. FACS-based cell cycle distribution analysis after PI incorporation of rat primary OPC in absence or in presence of 100 μM D-Asp for 1 day. Representative FACS plots of biological replicates are shown (n=3 independent experimental sessions).

F. Confocal microscopic images displaying the coexpression of MAG or MBP with Phalloidin-594 (a-b and c-d, respectively) in rat primary OPC cultured in absence or in presence of 100 μM D-Asp for 4 days. (e), Single representative MBP++ cell. Scale bars: 50 μm in a-d; 10 μm in e. (f-g); Quantitative analysis of Alexa-594-phalloidin-positive oligodendrocytes (f) or MAG+ and MBP++ oligodendrocytes (g) scored in control and D-Asp exposed cells. Alexa-594-phalloidin-positive oligodendrocytes were scored in 4 categories, according to their morphological complexity. In each category, data were normalized on the total number of oligodendrocytes.

Data information: The values represent the means ± S.E.M. Level of significance was determined by using: in A, left panel, one-way ANOVA P<0.0001 followed by Tukey's post hoc test, *P< 0.05 versus control (n=3); A, right panel, one-way ANOVA P<0.0001 followed by Tukey's post hoc test, *P<0.05 versus control (n=3); B, one-way ANOVA P=0.0001 followed by Bonferroni post hoc test, *P<0.05 versus control (n=3); C, for each day, two-tailed Student's t test, *P<0.05 versus control at 3 days (n=5); F, for each category two-tailed Student's t test, *P<0.05 versus control (n=3). See the exact P‐values from comparisons tests in Appendix Table S1.

Source data are available online for this figure
D-Aspartate treatment attenuates myelin damage and stimulates myelin repair (2018)
Figure 3

Figure 3. Effect of the NMDA receptors antagonist and NCX3 blockers on D-Asp-induced myelin marker expression.

A. RT-PCR of CNPase mRNA expression in oligodendrocyte MO3.13 progenitors under control conditions and following 200 μM D-Asp exposure for 3 days, in absence or in presence of 10 μM MK-801 (left panel), or 30 nM YM-244769 or 100 nM BED (right panel). Graphs show quantification of ratio of CNPase to L19.

B. RT-PCR of MBP mRNA expression in MO3.13 cells under control conditions and following D-Asp exposure for 3 days, in absence or in presence of 10 μΜ MK-801 (left panel), or 30 nM YM-244769 or 100 nM BED (right panel) Graphs show quantification of ratio of MBP to L19.

C. (a-j); Confocal images displaying the expression of NG2 and MAG proteins in rat primary OPC cultured in presence of D-Asp for 3 days, in absence (a-c) or in presence of 10 μM MK-801 (d-f), or 30 nM YM-244769 (g-i) or 100 nM BED (j-l).

C. (m-n); Quantitative analysis of NG2+ and MAG+ cells in rat primary OPC cultured in presence of D-Asp for 3 days, in absence or in presence of 10 μM MK-801, 30 nM YM-244769 or 100 nM BED.

D. RT-PCR of NCX3 mRNA expression under control conditions and following 10-200 μM D-Asp exposure or 100 nM PMA for 3 days;

E. RT-PCR of NCX3 mRNA expression following 200 μM D-Asp exposure, in absence or in presence of 10 μM MK-801.

F. RT-PCR of NCX1 mRNA expression following 200 μM D-Asp exposure, in absence or in presence of 10 μM MK-801. Graphs show quantification of ratio of NCX1 and NCX3 to L19.

Data information: The values represent the means ± S.E.M from 3 independent experimental sessions. Level of significance was determined by using: in A, left, one-way ANOVA P=0.009 followed by Tukey's post hoc test, *P< 0.05 versus control, ˄P< 0.05 versus D-Asp (n=3); A, right, one-way ANOVA P=0.0001 followed by Tukey's post hoc test, *P< 0.05 versus control, ˄P< 0.05 versus D-Asp (n=3); B, left, one-way ANOVA P=0.0001 followed by Tukey's post hoc test, *P< 0.05 versus control, ˄P< 0.05 versus D-Asp (n=3); B, right, one-way ANOVA P=0.0004 followed by Tukey's post hoc test, *P< 0.05 versus control, ˄P< 0.05 versus D-Asp (n=3); C, m and C, n, one-way ANOVA P<0.0001 followed by Bonferroni post hoc test, *P< 0.05 versus control, ˄P< 0.05 versus D-Asp (n=3); D, one-way ANOVA P=0.0001 followed by Tukey's post hoc test, *P< 0.05 versus control, ˄P< 0.05 versus D-Asp (n=3); E, one-way ANOVA P=0.0083 followed by Tukey's post hoc test, *P<0.05 versus control, ˄P< 0.05 versus D-Asp (n=3), n.s, not significant. See the exact P‐values from comparisons tests in Appendix Table S3.