EMBO/soda-nel huggingface dataset:
TPC2 rescues lysosomal storage in mucolipidosis type IV, Niemann-Pick type C1 and Batten disease (2022)
Figure 2

Figure 2. TPC2 agonist effect on cholesterol accumulation and ultrastructural changes. (A-B) Confocal images (A) and statistical analysis (B) of cholesterol accumulation in human CTR, MLIV, JNCL, and NPC1 fibroblasts. Cholesterol accumulation was evident for NPC1 and MLIV fibroblasts, but not for JNCL fibroblasts. The images show filipin staining to visualize cholesterol accumulation and TO-PRO3 as nuclear staining. (C-D) TPC2-A1-P (30 µM, 48h) rescued NPC1 and MLIV cholesterol accumulation. (E-G) Confocal images (E-F) and statistical analysis (G) of MLIV patient fibroblasts mock-electroporated and treated with DMSO or electroporated with a gain-of-function hTPC2(M484L/G734E):mCherry TOPO 3.1 vector (white arrow heads) and treated with either DMSO or TPC2-A1-P (30 µM, 48h). (H-K) Confocal images (H-I) and statistical analysis (J-K) of human CTR and NPC1 patient fibroblasts treated with 50 nM mock siRNA (siSCR) or siRNA targeting TPCN2 (siTPC2) for 72 hours. Cells were then treated with DMSO or TPC2-A1-P (30 µM). Statistics (L) and electron microscopy images (M) of human CTR, MLIV, JNCL, and NPC1 fibroblasts. Effect of the treatment with TPC2 agonist (30 µM, 48h) was examined in NPC1 and MLIV cells. Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells); one-way ANOVA, post hoc Bonferroni's multiple comparisons test (B, D, E, L) or two-tailed Student's t-test (J and K). *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.
TPC2 rescues lysosomal storage in mucolipidosis type IV, Niemann-Pick type C1 and Batten disease (2022)
Figure 3

Figure 3. TPC2 agonist effect on lipofuscin and Gb3 accumulation. (A) Confocal images of CTR and JNCL fibroblasts. Images show LAMP1 staining and autofluorescence at 405 and 488 nm excitation wavelength, respectively, corresponding to the lipofuscin autofluorescence spectrum. Cells were treated with DMSO or TPC2-A1-P (30 µM) following cell cycle arrest (2h mitomycin C treatment). (B) Confocal images showing no autofluorescence signal at 594 nm excitation wavelength (used for LAMP1 staining). (C) Mean autofluorescence intensity in LAMP1+ area. (D-E) Confocal images of Gb3 accumulation stained with shigatoxin (STX) in CTR and JNCL fibroblasts. Cells were treated with DMSO or TPC2-A1-P (30 µM) after cell cycle arrest. Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells; one-way ANOVA, post hoc Bonferroni's multiple comparisons test. **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.
TPC2 rescues lysosomal storage in mucolipidosis type IV, Niemann-Pick type C1 and Batten disease (2022)
Figure 5

Figure 5. Effect of TPC2-A1-P on human neuronal LSD phenotypes. Cortical neurons were differentiated from iPSCs, generating lysosomal storage disease neurons and isogenic controls. (A-B) Lysosomal proteolysis was measured following pre-treatment with DMSO/TPC2-A1-P, using the magic red (MR) cathepsin B substrate, and performing FRAP measurements to assess the proteolysis rate. MCOLN1IVS3-2A>G (MLIV) neurons showed increased proteolysis, while CLN3D416G and CLN3ΔEx4-7 neurons (JNCL) exhibited either significantly lower or slightly reduced proteolysis rates, respectively. (C-D) Western blot analysis of cathepsin B (CtsB) in CTR and MCOLN1IVS3-2A>G neurons treated with TPC2-A1-P (30 µM) or DMSO. (E-G) Cortical neurons were treated with compounds and acidic compartments stained with LysoTracker (LyTr). Endolysosomal expansion was observed in MCOLN1IVS3-2A>G, CLN3D416G and CLN3ΔEx4-7 neurons, which was ameliorated by TPC2-A1-P (30 µM) treatment. (H-I) Electron microscopy analysis of neuronal rosettes (neuronal progenitor cells, NPC) treated with DMSO or TPC2-A1-P. TPC2-A1-P treatment significantly decreased the number of inclusion bodies (black arrow heads) in MCOLN1IVS3-2A>G. CLN3D416G and CLN3ΔEx4-7 lacked an appropriate assay window and showed no significant accumulation of inclusion bodies. However, CLN3ΔEx4-7 NPC showed significantly more mitochondria with aberrant cristae numbers (white arrow heads), a phenotype which was rescued by TPC2-A1-P (30 µM) treatment. Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells, obtained from at least three distinct neuronal differentiations); one-way ANOVA, post hoc Tukey's multiple comparisons test or two-tailed Student's t-test (C). **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.
TPC2 rescues lysosomal storage in mucolipidosis type IV, Niemann-Pick type C1 and Batten disease (2022)
Figure 6

Figure 6. Effect of TPC2-A1-P on lysosomal exocytosis and autophagy. (A) Confocal images of plasma membrane (PM) LAMP1 immunofluorescence in CTR fibroblasts. LAMP1 on the PM is expressed as fold-change relative to DMSO-treated cells. (B) Statistical analysis of lysosomal exocytosis data as shown in A. (C) Lysosomal exocytosis in CTR, MLIV, NPC1 and JNCL human fibroblasts. PM-localized LAMP1 was measured by flow cytometry, expressed as % of CTR DMSO-treated cells. Ionomycin in A-C (4 µM; 10 min treatment) was used as positive control. TPC2-A1-P and ML-SA1 (30 µM, each; 90 min treatment in A-C). (D) Cartoon showing lysosomal exocytosis. Statistics (B-C): Shown are mean values ± SEM. n > 3 for each tested condition (in B each dot represents an imaged frame containing several cells, in C each dot is the mean FITC intensity value expressed as percentage obtained from at least 1*104 events); two-way ANOVA, post hoc Dunnett's (B) or Tukey's (C) multiple comparisons test, *p-value < 0.05; ***p-value < 0.001; ****p-value < 0.0001. (E) Cartoon showing the roles of LC3 and P62 in the autophagic pathway. (F-G) Immunoblot analysis of endogenous LC3 (LC3I-II) following TPC2-A1-P or ML-SA1 (30 µM, each) treatment, alone or with BafA1, under fed (complete media) or starvation (HBSS) conditions in CTR, MLIV and NPC1 patientfibroblasts. Graphs show densitometry of LC3II bands normalized to actin. (H) Immunoblot analysis of endogenous LC3 (LC3I-II) following TPC2-A1-P (30 µM) or DMSO treatment, under fed (complete Neurobasal/B27) or starvation (DMEM/F12 free) conditions in CTR and MLIV iPSC derived cortical neurons. Graphs show densitometry of LC3II bands normalized to actin. (L-M) Immunoblot analysis of endogenous SQSTM1 (P62) upon TPC2-A1-P or ML-SA1 (30 µM, each) treatment, under fed (complete media) or starvation (HBSS) conditions in CTR, MLIV and NPC1 patient fibroblasts. Statistics (G, H, J): Shown are mean values ± SD. n = 3 lysates per condition pooled from 3 independent experiments; two-tailed Student's t-test. *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001.
TPC2 rescues lysosomal storage in mucolipidosis type IV, Niemann-Pick type C1 and Batten disease (2022)
Figure 7

Figure 7. Expression of TPC2 in human and mouse brain. The TPC2 reporter mouse Tpcn2IRES-Cre/eR26-τGFP was generated as previously described (Wen et al, 2011; Wyatt et al, 2017). (A-B, E) Both neurons and glia were found to express Tpc2 in the corpus callosum, the hippocampus, and the cerebellum (so = stratum oriens, sp = stratum pyramidale, sr = stratum radiatum, mo = molecular layer, gr = granular layer). Subpopulations of astrocytes (Gfap) and microglia (Iba1) express Tpc2 (E). (C) 1 week (1w)- or 8 week (8w)-old mouse brains were dissected, and brain Tpc2 transcript mapped. (D) A cDNA array was used to map TPC2 transcripts in the human brain. (E) Tpc2 expression in the Tpc2 reporter mouse, mouse brain, and human brain is summarized as cartoons, finding highest expression in cerebellum and hippocampus (top panels). Affected brain regions in the lysosomal storage diseases MLIV, JNCL, and NPC1 based on patient and mouse data are colour-coded.
Pharmacological reversal of synaptic and network pathology in human MECP2-KO neurons and cortical organoids (2020)
Figure 1

Figure 1. Human MECP2-KO neurons exhibit alterations in synapse-relevant genes and pathways.

A Pluripotent stem cells (PSCs) with one of two distinct exonic loss-of-function mutations (Q83X nonsense or K82 frameshift (K82fs)) at the MECP2 locus were generated and differentiated into neurons via a neural progenitor cell (NPC) intermediate.

B Immunofluorescent staining confirmed the absence of MeCP2 in MECP2-KO PSCs, NPCs, and neurons. Scale bar = 10 µm.

C RT-qPCR array revealed differential gene expression between control and MECP2-KO 28-day neurons. The P values are calculated based on a Student's t-test of the replicate 2-ΔΔCt values for each gene in the control group and KO groups (WT83/Q83X cell lines were used; N = 3 clones from each genotype).

D Overtime quantitative gene expression profile of many of the involved genes relevant to synaptic function (days 14 and 28 post differentiation; normalized by control NPC expression).

E Single-cell RT-qPCR analysis distinguished populations of control and KO 28-day neurons. Principal Component Analysis (PCA) of 441 cells projected onto the first two components. Overlaid populations of WT83/Q83X and WT82/K82fs neurons are shown.

F Violin plots of selected genes showing the comparison between control and MECP2-KO neurons from the single-cell analyses (log2(expression) values) that overlapped with the results obtained via RT-PCR array.

G Volcano plot illustrates differences in expression patterns of target genes of neurons from the qPCR single-cell analyses. The dotted lines represent differentially expressed genes between the groups at P < 0.05 (unpaired Student's t-test).

H Left: Schematic of laminar cortical markers. Right: Top: Quantitation of laminar cell markers based on the single-cell RT-qPCR analysis (two-way analysis of variance (ANOVA), N = 3 replicates per genotype, F3,24 = 3.31, *P = 0.04, with difference in the proportion of cortical layers V and VI, 95% confidence interval (CI) [8.31, 42.41]); Bottom: altered proportions of neurotransmitter markers (two-way ANOVA, N = 3 replicates per genotype, F5,24 = 27.33, ***P < 0.0001, with differences in the proportions of glutamatergic, 95% CI [22.33, 33.60], and cholinergic, 95% CI [0.166, 11.43], neurons).

I Gene ontology (GO) analysis of targeted genes relevant to synaptic function displaying the differences between MECP2-KO and control neurons. For the full list of markers used see Dataset EV1.

Data information: Note that asterisks signify statistically significant difference from MECP2-KO. Data are presented as mean ± standard error of the mean (s.e.m.).
Pharmacological reversal of synaptic and network pathology in human MECP2-KO neurons and cortical organoids (2020)
Figure 3

Figure 3. Treatment of MECP2-mosaic neurospheres with Nefiracetam and PHA 543613.

A Schematic showing the formation of control, MECP2-KO, and mosaic MECP2-KO/control neurospheres (WT83/Q83X cell lines were used).

B, C Immunofluorescent staining (B) and quantitation (C) confirmed the graded decrease in MeCP2 expression from control to 50% MECP2 mosaic to full MECP2-KO neurospheres. Scale bar = 50 µm.

D 8-week MECP2-KO neurospheres (N = 44) exhibited decreased size (diameter; **P < 0.01), but 50% MECP2-mosaic neurospheres (CTR/KO; N = 44) were unchanged from controls (one-way ANOVA, F2,150 = 6.03, P = 0.003; Dunnett's multiple comparisons test; N = 65). Left: representative brightfield images of neurospheres; Right: neurosphere size quantification. Scale bar = 200 µm.

E Calcium imaging analysis showing calcium transients normalized by control (one-way ANOVA, F2,72 = 15.62, P < 0.0001; Dunnett's multiple comparisons test vs control: MECP2-mosaic, ***P < 0.001; MECP2-KO, ***P < 0.001). WT83/Q83X cell lines were used; N = 44 Q83X neurospheres and 65 WT83 neurospheres.

F 8-week neurosphere size (diameter) remained unchanged by treatment with either drug (one-way ANOVA, F6,344 = 0.52, P = 0.79; N = 44 Q83X neurospheres and 65 WT83 neurospheres).

G Cell viability was improved or unharmed in treated 8-week neurospheres (one-way ANOVA, F9,104 = 6.81; Dunnett's multiple comparisons test vs CTR/KO untreated: Nefi + PHA 1 µM, **P < 0.01 and Z = 2.974; Nefi + PHA 10 µM, ***P < 0.001 and Z = 3.733; N = 8-24 neurospheres per condition).

H, I Calcium transient frequency (number of peaks in 10 mins recording) normalized by CTR/KO untreated (H; one-way ANOVA, F9,239 = 6.18; Dunnett's multiple comparisons test vs CTR/KO untreated: Nefi 1 µM, *P < 0.05 and Z = 0.780; Nefi + PHA 10 µM, ***P < 0.001 and Z = 1.972; N = 15-61 neurospheres per condition) and calcium transient amplitude normalized by CTR/KO untreated (I; one-way ANOVA, F9,241 = 1.27, P = 0.255; N = 16-62 neurospheres per condition).

J, K MEA spike frequency heatmap (J) and quantification (K) normalized by CTR/KO untreated (one-way ANOVA, F5,38 = 1.21, P = 0.321; Dunnett's multiple comparisons test vs CTR/KO untreated: PHA, P = 0.65 and Z = 1.134; Nefi, P = 0.98 and Z = 0.06; PHA + Nefi, P = 0.86 and Z = 0.94; N = 7-8 wells per condition). 'X' on activity heatmap signifies absence of a neurosphere in that position.

Data information: Nefi: Nefiracetam; PHA: PHA 543613. Note that asterisks in F-K signify a statistically significant difference in treated MECP2-mosaic (CTR/KO) neurospheres compared to untreated CTR/KO neurospheres. Data are presented as mean ± s.e.m.
Aberrant upregulation of CaSR promotes pathological new bone formation in ankylosing spondylitis (2020)
Figure 1

Figure 1. CaSR+ osteoblasts accumulated in the spinal entheseal tissues from AS patients.

(A) An illustration of spinal ligament tissues collection.

(B) SOFG staining of spinal entheseal tissue and Immunohistochemical analysis of CaSR+ and Runx2+ cells in human spinal tissue. The bottom panels show higher magnification of the boxed area in the top panels.

(C) Quantitative analysis of the number of CaSR+ and Runx2+ cells per area (mm2). n=5, Student's t-test.

(D) RT-qPCR analysis of expression of CaSR in human spinal tissue. n=3, Student's t-test.

(E) Western blot analysis of expression of CaSR in human spinal tissue.

(F) Immunofluorescence analysis of CaSR+ (Green) and Runx2+ (Red) cells in human spinal tissue.

(G) Quantitative analysis of CaSR+, Runx2+ and CaSR+ Runx2+ cells number per area (mm2) and CaSR+ cell percentage. n=5, Student's t-test.

(H) Immunofluorescence analysis of CaSR+ (Green) and OCN+ (Red) cells in human spinal tissue.

(I) Quantitative analysis of CaSR+, OCN+ and CaSR+ OCN+ cells number per area (mm2). n=5, Student's t-test.

Data information: SP: spinous process; IL: interspinous ligament; SL: supraspinous ligament; UIL: uncalcified interspinous ligament; TZ: transitional zone. Data shown as mean ± SD.. ** P < 0.01 compared between groups. Scale bar: 100μm.
Aberrant upregulation of CaSR promotes pathological new bone formation in ankylosing spondylitis (2020)
Figure 2

Figure 2. CaSR+ osteoblasts mediate new bone formation in PGIS model

(A) μCT images of the spine in PGIS model (2D and 3D reconstruction). Arrow head shows spinal ankylosis. n=5 per group. μCT images of new bone (red area) in hind paws (2D and 3D reconstruction) in DBA/1 model compared to baseline.

(B) Incidence of spinal ankylosis in PGIS model. n=5 per cage of total 3 cages per group, Fisher's exact test.

(C) Quantitative analysis of structural parameters of spinal ankylosis by μCT analysis. n=5 per group, one‐way ANOVA, Bonferroni post hoc.

(D) RT-qPCR analysis of expression of CaSR in spinal ligament tissuse. n=3, Student's t-test.

(E) H&E, SOFG and Immunofluorescence analyses of the spine specimen of PGIS model compare to baseline. n=5 per group.

(F) Quantitative analysis of CaSR+, Runx2+ and CaSR+ Runx2+ cells number and CaSR+ cell percentage in the spine specimen of PGIS model. n=5 per group, one‐way ANOVA, Bonferroni post hoc.

(G) H&E, SOFG and Immunofluorescence analyses of the spine specimen of PGIS model at 30 Weeks compare to baseline. GP: Growth plate. n=5 per group.

(H) Quantitative analysis of CaSR+, OCN+ and CaSR+ OCN+ cells number in the spine specimen of PGIS model. n=5 per group, one‐way ANOVA, Bonferroni post hoc.

Data information: AF: AF: Annulus fibrosus; GP: Growth plate; NB: New bone. Data shown as mean ± SD. ** P < 0.01 compared between groups. Scale bar: 100μm.
Aberrant upregulation of CaSR promotes pathological new bone formation in ankylosing spondylitis (2020)
Figure 3

Figure 3. CaSR+ osteoblasts mediate new bone formation in DBA/1 model.

(A) μCT images of pathological new bone formation in DBA/1 model. n=9 per group.

(B) Incidence of ankle pathological new bone formation in DBA/1 model. n=9 per cage of total 3 cages per group, Fisher's exact test.

(C) Quantitative analysis of structural parameters of pathological new bone by μCT analysis. n=9 per group, one‐way ANOVA, Bonferroni post hoc.

(D) RT-qPCR analysis of expression of CaSR in hind paw tissue. n=3, Student's t-test.

(E) H&E, SOFG and Immunofluorescence analyses of pathological new bone formation in hind paws. n=9 per group.

(F) Quantitative analysis of CaSR+, Runx2+ and CaSR+ Runx2+ cells number and CaSR+ cell percentage in pathological new bone formation sites. n=9 per group, one‐way ANOVA, Bonferroni post hoc.

(G) H&E, SOFG and Immunofluorescence analyses of pathological new bone in hind paws. n=9 per group.

(H) Quantitative analysis of CaSR+, OCN+ and CaSR+ OCN+ cells number in pathological new bone formation sites. n=9 per group, one‐way ANOVA, Bonferroni post hoc.

Data information: CB: Cortical bone; NB: New bone. Data shown as mean ± SD. ** P < 0.01 compared between groups. Scale bar: 100μm.
Aberrant upregulation of CaSR promotes pathological new bone formation in ankylosing spondylitis (2020)
Figure 5

Figure 5. CaSR promotes osteogenic differentiation and new bone formation through PLCγ signalling pathway.

(A) Alizarin red staining of hBMSC cells for 11 days.

(B) Quantification of Alizarin Red staining at 570 nm (A570). n=3, one‐way ANOVA, Bonferroni post hoc.

(C) RT-qPCR analysis of osteogenesis markers in hBMSC cells. n=3, one‐way ANOVA, Bonferroni post hoc.

(D) Alizarin red staining of hBMSC cells.

(E) Quantification of Alizarin Red staining at 570 nm (A570). n=3, one‐way ANOVA, Bonferroni post hoc.

(F) RT-qPCR analysis of osteogenesis markers in hBMSC cells. n=3, one‐way ANOVA, Bonferroni post hoc.

(G) Western blot analysis of phosphorylation of PLCγ protein levels in hBMSC cells.

(H) Western blot analysis of phosphorylation of PLCγ protein levels in hBMSC cells treated with SR and NPS-2143.

(I) Immunofluorescence analysis of spine in PGIS model. Quantitative analysis of CaSR+, p-PLCγ+ and CaSR+ p-PLCγ+ cells number in spine of PGIS model. n=5, one‐way ANOVA, Bonferroni post hoc.

(J) Immunofluorescence analysis of Achilles tendon enthesis compartment in SMTS model. Quantitative analysis of CaSR+, p-PLCγ+ and CaSR+ p-PLCγ+ cells number in Achilles tendon enthesis compartment. n=5, one‐way ANOVA, Bonferroni post hoc.

(K) Immunofluorescence analysis of pathological new bone on dorsal surface of hind paws. Quantitative analysis of CaSR+, p-PLCγ+ and CaSR+ p-PLCγ+ cells number in pathological new bone formation sites. n=9, one‐way ANOVA, Bonferroni post hoc.

(L) μCT images of the PCT in SMTS model (2D). Arrow heads shows bony projection.

(M) SOFG staining of Achilles tendon enthesis compartment in SMTS model.

(N) Quantitative analysis of area of UF and CF. n=5, Student's t-test

Data information: AF: Annulus fibrosus; CB: Cortical bone; NB: New bone; UF: Uncalcified fibrocartilage; CF: Calcified fibrocartilage; PCT: posterior calcaneal tuberosity. Data shown as mean ± SD. n = 5 for PGIS (I) and SMTS models (J); n = 9 for DBA/1 (K). * P < 0.05，** P < 0.01 compared between groups. Scale bar: 100μm.
Aberrant upregulation of CaSR promotes pathological new bone formation in ankylosing spondylitis (2020)
Figure 6

Figure 6. Multiple inflammatory cytokines induce CaSR up-regulation in osteogenic precursor cells.

(A) RT-qPCR and Western Blot analyses of CaSR expressions in MC3T3-E1 cells treated with IL-1β. n=3, one‐way ANOVA, Bonferroni post hoc.

(B) RT-qPCR and Western Blot analyss of CaSR expressions in MC3T3-E1 cells treated with TNFα. n=3, one‐way ANOVA, Bonferroni post hoc.

(C) RT-qPCR and Western Blot analyses of CaSR expressions in MC3T3-E1 cells treated with IL-17A. n=3, one‐way ANOVA, Bonferroni post hoc.

(D) RT-qPCR and Western Blot analyses of CaSR expressions in MC3T3-E1 cells treated with IL-22. n=3, one‐way ANOVA, Bonferroni post hoc.

(E) RT-qPCR and Western Blot analyses of CaSR expressions in MC3T3-E1 cells treated with IL-23. n=3, one‐way ANOVA, Bonferroni post hoc.

Data information: The data are presented as the means ± SD from one representative experiment of three independent experiments performed in triplicate. # P <0.05 ** P < 0.01 compared between groups.
Aberrant upregulation of CaSR promotes pathological new bone formation in ankylosing spondylitis (2020)
Figure 7

Figure 7. Inflammatory signalling pathways are activated in osteogenic precursor cells in AS animal models.

(A) Immunofluorescence analysis of spine in PGIS mice. Quantitative analysis of CaSR+ p-p65+ cells and CaSR+ p-Stat3+ cells number in pathological new bone formation of PGIS model. n = 5, one‐way ANOVA, Bonferroni post hoc.

(B) Immunofluorescence analysis of pathological new bone of hind paws in DBA/1 mice. Quantitative analysis of CaSR+ p-p65+ cells and CaSR+ p-Stat3+ cells number in pathological new bone formation sites. n = 9, one‐way ANOVA, Bonferroni post hoc.

(C) Immunofluorescence analysis of Achilles tendon enthesis compartment in SMTS model. Quantitative analysis of CaSR+ p-p65+ cells and CaSR+ p-stat3+ cells number in PCT. n=5, Student's t-test.

(D) Schematic presentation of multiple inflammatory cytokines from inflammatory cell up-regulate hyperstimulation of CaSR through NF-κB/p65 and JAK/Stat3 pathways. Hyperstimulation of CaSR induce osteogenic differentiation through PLCγ signalling pathway in osteogenic precursor cell to promote pathological new bone formation in AS.

Data information: AF: Annulus fibrosus; GP: Growth plate; CB: Cortical bone; NB: New bone; UF: Uncalcified fibrocartilage; CF: Calcified fibrocartilage; PCT: posterior calcaneal tuberosity. Data shown as mean ± SD. ** P < 0.01 compared between groups. Scale bar: 100μm.

Expended view legend