EMBO/soda-nel huggingface dataset:
Coordinated control of adiposity and growth by anti-anabolic kinase ERK7 (2020)
Figure 7

Figure 7: Sugarbabe is a downstream effector of ERK7 and PWP1

A, B. Expression of sugarbabe is downregulated in fat bodies upon knockdown of PWP1 (A) and ectopic expression of ERK7 (B) (N=3 replicates of 10 fat bodies/replicate for each genotype).

C. ERK71 mutants fail to maximally downregulate sugarbabe expression upon fasting (N=4 replicates of 10 larvae/replicate for each genotype).

D. Representative immunofluorescent images of fat bodies in a genetic epistasis experiment using ERK71 and sug mutants (sug17Δ/Df(2R)Exel7123) with LipidTOX and DAPI staining to visualize lipid droplets and nucleus, respectively. Scale bar: 50 µm.

E. sug mutant (sug17Δ/Df(2R)Exel7123) suppresses increased lipid droplet number observed in ERK71 mutants (N>20).

F. Ectopic sugarbabe expression in the fat body partially rescues TAG levels in the CG>ERK7 larvae (N=4 replicates of ≥10 larvae/replicate for each genotype).

G. sug mutant (sug17Δ/Df(2R)Exel7123) suppresses increased nuclear size phenotype of ERK71 mutant fat body cells (N ≥25 cells per genotype).

H, I. Ectopic Sugarbabe expression in the fat body partially restores the pupariation rate (H) (p<0.001 for the rescue, analyzed by Log-rank test, N=4 replicates of 30 larvae/replicate for each genotype) and pupal volume of CG>ERK7 larvae (I) (N=4 replicates of 10 pupae/replicate for each genotype).

J. Addition of 20% sucrose improves the growth rate and survival of CG>ERK7 larvae. Pupariation kinetics of CG>ERK7 larvae on 20% yeast or 20% yeast + 20% sucrose diet (p<0.0001 for the rescue, analyzed by Log-rank test, N=4 replicates of 30 larvae/replicate for each genotype and diet).

Data information: N stands for the number of biological replicates. Error bars display standard deviation (SD). * p<0.05, ** p<0.01, *** p<0.001 (Student's t test). Expression of RP49 was used for normalization of (A-C).

EXPANDED VIEW FIGURE LEGENDS
Triggering MSR1 promotes JNK-mediated inflammation in IL-4 activated macrophages (2019)
Figure 1

Figure 1: Alternative activation affects phagosomal proteolysis and lipolysis as well as phagocytosis of negatively charged particle.

(A) Internalization of apoptotic or necrotic GFP-expressing RAW264.7 cells. (B) Phagocytosis of fluorescent negatively charged carboxylated and positively charged amino microspheres (B) in primary M2 (IL-4) and M0 macrophages. Cytochalasin D (Cyto) (6 µM) was used as an inhibitor of phagocytosis, 1 h before phagocytosis. Data are shown as means of relative fluorescence units (RFU) ± standard error of the mean (SEM). (C-E) Real-time fluorescence assays for intraphagosomal proteolysis (C), acidification (D) and lipolysis (E) show substantially increased proteolysis, acidification and lipolysis in the phagosomes of M2(IL-4) macrophages. The kinetics of proteolysis, acidification and lipolysis of phagocytosed beads were plotted as a ratio of substrate fluorescence to calibration fluorescence. Beads were added to macrophages at 0 min. Shaded area in (C) & (D) represents SEM. (E) is a representative of three independent experiments. Leupeptin (100nM) and Bafilomycin (100nM) treatment serve as negative controls in (C) and (D), respectively.
Triggering MSR1 promotes JNK-mediated inflammation in IL-4 activated macrophages (2019)
Figure 2

Figure 2: Experimental workflow and the phagosome proteome of M2 (IL-4) macrophages.

(A) Workflow of the phagosome proteomics experiment. (B) Volcano plot of the phagosome proteome data. 1,766 proteins were quantified of which 121 proteins were significantly up-regulated and 62 proteins were down-regulated in M2(IL-4) macrophages. Selected proteins are indicated. (C) Heatmap of proteomics data shows high reproducibility between biological replicates. Selected proteins are highlighted. (D) Selected Gene Ontology (GO)-terms of biological processes significantly up-regulated and down-regulated on phagosomes of M2(IL-4) macrophages. Selected proteins of these GO-terms are highlighted. Error bars in (D) represent standard deviations from three biological replicates.
Triggering MSR1 promotes JNK-mediated inflammation in IL-4 activated macrophages (2019)
Figure 3

Figure 3: TAK1/MKK7/JNK complex is recruited to phagosomes of M2 (IL-4) macrophages in a K63 polyubiquitylation-dependent manner.

(A) Immunoblot (IB) analysis showing the recruitment of TAK1, MKK7, JNK and TAB1/TAB2 to the phagosome in M2(IL-4) macrophages, while MKK4 is not recruited. Rab7a, a phagosomal marker, was used as a loading control for phagosomes and vimentin was used as a loading control for total cell lysates. The white line in the JNK blot shows other bands were cut out. Full blot can be seen in Figure EV4C. (B) Quantitation of three independent IB experiments by imageJ of non-saturated blots for TAK1, MKK7, TAB1 and TAB2 expression on phagosomes of resting M0 macrophages and M2(IL-4) macrophages. Error bars represent SEM. *p<0.01, **p<0.001 (Student's T-test) (C) IB showing enrichment of K63 polyubiquitylated proteins on the phagosome of M2(IL-4) macrophages compared to M0 macrophages. (F) Treatment with the UBC13 inhibitor NSC697923 reduces recruitment of TAB1, TAB2, TAK1 and MKK7 to the phagosome of M2(IL-4) macrophages, indicating a K63-polyubiquitylation dependent translocation for these proteins. (C) and (D) are representatives of three and two independent experiments, respectively.
Triggering MSR1 promotes JNK-mediated inflammation in IL-4 activated macrophages (2019)
Figure 4

Figure 4: Phagosomal MSR1 is K63 polyubiquitylated and interacts with Tab1/Tab2/Tak1/MKK7.

(A) Workflow for TUBE pulldown of K63-polyubiquitylated proteins from phagosomal extracts. (B) Selected ubiquitylated phagosomal proteins identified by TUBE-MS approach. (C) Sequences alignment of N-terminal region of murine and human MSR1 shows high sequence identify and conserved ubiquitylated lysine. (D) Tab2-TUBE pull-downs from phagosome extracts of M2(IL-4) macrophages treated with the K63 specific deubiquitylase (DUB) AMSH-LP or the unspecific DUB USP2. (F) MSR1 immunoprecipitation from M2(IL-4) macrophage phagosomes shows that TAB1, TAB2, TAK1 and MKK7 bind to polyubiquitylated MSR1. TAK1 shows a specific pattern of post-translational modifications indicative of its activation. (D-F) are representative of two independent experiments.
Triggering MSR1 promotes JNK-mediated inflammation in IL-4 activated macrophages (2019)
Figure 5

Figure 5: MSR1 triggering activates JNK, which induces a proinflammatory stimulus.

(A) Immunoblot against MSR1 of TUBE pulldowns of MSR1 WT and KO BMDMs shows increasing amounts of ubiquitylated MSR1 upon alternative activation and further increases upon MSR1 ligation with fucoidan or oxLDL. (B) Msr1+/+ (WT) and Msr1-/- (KO) M0 macrophages and M2(IL-4) macrophages untreated or stimulated with the MSR1 ligands fucoidan or oxLDL (50 μg/ml, 30 min) were analysed for the phosphorylated and the total forms of JNK1/2 and MSR1. ITGAM (CD11b) serves as a loading control. Both fucoidan and oxLDL activate JNK in a MSR1-dependent manner. (C) IL4-activated MSR1 knock-out BMDMs were transfected with WT or K27R MSR1 and treated with 50 μg/ml Fucoidan for 1 hr. Mutation of the ubiquitylation site K27 abolishes MSR1 signalling. (D) qPCR data of Tnfa, Il1b and Ccl2 mRNA levels in WT and MSR1 KO M0 and M2(IL-4) BMDMs shows an MSR1-dependent increase of pro-inflammatory cytokines in response to MSR1 ligation by fucoidan. (E) Inhibition of JNK by JNK-IN8 reduces expression of Tnfa and Ccl2 upon MSR1 ligation, showing that it is JNK-dependent. (F) Flow cytometry analysis of cell surface markers in WT and MSR1-/- M0 macrophages and M2(IL-4) macrophages untreated or stimulated with the MSR1 ligands fucoidan (50 μg/ml, 24 h). Data shows MSR1-dependent increase of the early activation markers CD54, CD69 and CD86 and a decrease of the M2 marker CD301b/Mgl2. CD11b serves as a control. Error bars represent SEM. ***p<0.0001; ****p<10-5 (Student's T-test) (A-B) Data are representative of three independent experiments.
Triggering MSR1 promotes JNK-mediated inflammation in IL-4 activated macrophages (2019)
Figure 6

Figure 6: MSR1 ubiquitylation and JNK activation correlates in human tumour associated macrophages

(A) Immunohistochemistry analysis of MSR1 in patient ovarian, uterus and sarcoma cancers showing tumour-associated macrophages. (B) Pull-downs of K63 polyubiquitin chains and IB analysis of five human primary cancers shows a correlation between the amount of polyubiquitylated MSR1 and JNK activation in an ovarian tumour. (C) Working model: MSR1 is activated by ligation through many different substrates, including apoptotic cells, fucoidan or oxidised LDL (Signal 1). However, only when the macrophage is IL-4-activated (Signal 2) becomes MSR1 ubiquitylated by an unknown E2/E3 ligase. This ubiquitylation recruits Tab2/3, Tak1, Mkk7 and finally JNK, thereby allowing MSR1 to signal directly through the JNK signalling pathway which induces pro-inflammatory gene transcription.
Controlled X-chromosome activation dynamics defines meiotic potential of female mouse in vitro germ cells (2022)
Figure 1

Figure 1. A tailor-made system to trace X-chromosome inactivation and reactivation dynamics during PGCLC induction.

(A) Schematic representation of the features implemented in the XRep cell line. A hybrid background in which cells carry one X chromosome from M.m musculus (Xmus) and one from M.m castaneus (Xcas). The cell line carries an rtTA under the control of the Rosa26 locus and piggyBac transposon-based vectors with doxycycline (Dox)-responsive promoters driving the expression of Prdm14, Blimp1 and Tfap2c. The Xmus carries a GFP reporter and a truncation of the Tsix transcript while the Xcas carries a tdTomato reporter.

(B) Overview of the adapted PGCLC differentiation timeline. Stages of the culture system are shown.

(C) Representative FACS data of primordial germ cells specific surface markers CD61 and SSEA1 in ESCs, EpiLCs d4 and PGCLCs d5. Numbers indicate the percentages of SSEA1+/CD61+ gated cells over time. Shown are contour plots gated on live cells.

(D) Immunostaining of PGCLCs d5 cryosections for SOX2 (magenta) and TFAP2C (cyan). Barplot indicates the quantification of SOX2+ cells, TFAP2C+ cells, and SOX2+/TFAP2C+. n=1150 cells, from n=3 separate inductions, using two biological clones. The white squares represent the position of the magnified region at the bottom. Scale bar, 50 µm and 10 µm for the magnified region.

(E) Representative culture showing the X-activity reporter during PGCLC induction. Images for bright field (BF), XGFP, and XTomato were taken for ESCs, EpiLC d4 and PGCLC d5. Scale bar, 50 μm.

(F) Representative FACS data showing XGFP and XTomato distribution during PGCLC induction. Numbers indicate the percentage of cells gated according to the XGFP and XTomato status (grey = X-inactive, green/red = X-active). Dashed line indicates the transition from X-active to X-inactive according to XGFP levels. XGFP and XTomato percentages in ESCs, EpiLCs and d1 full bodies are calculated from the entire cell population, while in PGCLC d2 to PGCLC d5 are calculated from SSEA1+/CD61+ PGCLCs as indicated. Shown are histograms gated on live cells.
Controlled X-chromosome activation dynamics defines meiotic potential of female mouse in vitro germ cells (2022)
Figure 2

Figure 2. Gene expression analysis reveals two PGCLC subpopulations.

(A) PCA of gene expression dynamics during PGCLC differentiation. 4 biological replicates are shown. n = top 500 most variable genes. PGCLCs were sorted for SSEA1 and CD61 expression and further divided into XGFP+ and XGFP-. Axes indicate the variance. Arrows indicate hypothetical trajectory. Shapes indicate the biological clone (clone A11 = square, clone E9 = rhombus).

(B) PCA of gene expression dynamics compared to in vivo samples from (Nagaoka et al, 2020). n = top 500 most variable genes, calculated including in vivo samples. Grey arrow indicates putative developmental trajectory. Shapes indicate the replicates (clone A11 = square, clone E9 = rhombus, in vivo samples = circle).

(C) MA plot of differential gene expression changes between XGFP- and XGFP+ PGCLCs as determined by RNA-seq. Log2-mean expression (log2-normalised counts from DESeq2) on the x-axis and the log2-fold change on the y-axis are shown. Significantly upregulated and downregulated genes are highlighted in red and green respectively. False Discovery Rate (FDR) < 0.001. Non-significant genes with log2-mean expression between 0 and 0.2 were removed for easier plot visualisation.

(D) Expression levels (normalised DEseq2 counts) of selected differentially expressed genes between XGFP- and XGFP+ PGCLCs during the differentiation time course. Genes with FDR < 0.001 were considered significantly differentially expressed. Points indicate expression of individual biological replicates.

(E) Heatmap of RNA-seq normalised counts showing the Zscore across PGCLC induction timepoints of 31 manually selected and manually ordered marker genes belonging to the categories reported on the side.

(F) Selected GO terms enriched in XGFP- PGCLCs and XGFP+ PGCLCs.

(G) FACS analysis of cell cycle using DAPI. Numbers indicate the percentage of cells in G1, S and G2/M respectively.

(H) Alkaline phosphatase staining for ESCs, XGFP+ PGCLCs and XGFP- PGCLCs grown for 7 days in 2i/LIF medium on immortalised mouse embryonic fibroblasts.

(I) Barplot indicating the absolute numbers of Alkaline Phosphatase (AP) positive colonies in each cell type after 7 days of culture in 2i/LIF medium on immortalised mouse embryonic fibroblasts. Y-axis is in square root scale (sqrt) for better plot visualisation. Each white dot represents one technical replicate.
Controlled X-chromosome activation dynamics defines meiotic potential of female mouse in vitro germ cells (2022)
Figure 4

Figure 4. Single-cell RNA-seq of maturing germ cells using the rOvary system.

(A) Schematic illustration of the single-cell RNA-seq experiment and the isolated populations during germ cell maturation in rOvaries. The first 24h of culture are indicated as d0. rOvary = reconstituted Ovary, d = day of rOvary culture; XGFPint. = XGFPintermediate.

(B) (i) Imaging of XGFP and XTomato reporters in rOvaries d5 aggregated with E13.5 gonadal and mesonephric cells. Scale bars = 50μm. BF = bright field. (ii) FACS gating strategy for single-cells sorted XTomato+ cells against XGFP intensities. Numbers indicate the percentage of gated live cells over the total population. Numbers in brackets indicate the percentage of gated cells over the XTomato+ population.

(C) UMAP projection labelled with FACS sorted populations. XGFPint. = XGFPintermediate.

(D) UMAP embedding based on Shared Nearest Neighbor (SNN) modularity clustering identified 5 clusters, termed Mitotic 1 (n = 77), Mitotic 2 (n = 97), Pre-meiotic 1 (n = 62), Pre-meiotic 2 (n = 90) and Meiotic (n = 53) labelled with different colours. Arrows indicating cell trajectories, inferred by RNA velocity analysis.

(E) Marker gene expression projected onto the UMAP plot.

(F) Heatmap of gene expression dynamics throughout germ cell maturation clusters. Selected genes belong to the category "early germ cell" and "meiotic". Zscore is shown.

(G) Schematic representation of the IVDi (In Vitro Differentiation) maturation system. The stages of oogenesis in culture for 11 days are indicated. The condition of culture is indicated above. rOvary = reconstituted Ovary. Agg = aggregation day. AA = Ascorbic Acid.

(H) Immunofluorescence images of SYCP3 (red), DAZL (yellow) and DAPI at agg11 of IVDi tissue maturation from XGFP- PGCLCs. IVDi = in vitro differentiation. White squares indicate the positions of the magnified section shown below. Top panel scale bar = 100 μm. Middle panel scale bar = 10 μm. Bottom panel scale bar = 50 μm.

(I) Quantification of SYCP3+ cells (oocytes in cyst and primary follicles) in IVDi tissues at agg11. Each dot represents one IVDi tissue performed in 3 biological replicates.
Controlled X-chromosome activation dynamics defines meiotic potential of female mouse in vitro germ cells (2022)
Figure 6

Figure 6. Comparison of prophase I progression and germ cell maturation capacity of XGFP+ and XGFP- PGCLCs by m220 feeder expansion culture.

(A) Schematic representation of the m220 stromal feeders expansion culture to compare the meiotic capacity of XGFP- and XGFP+ PGCLCs. Meiosis is induced via addition of Retinoic Acid (RA) and Bone Morphogenetic Protein 2 (BMP2). c0 = starting day of culture, c9 = culture day 9 and last day of culture.

(B) Representative images for the expression of XGFP (green) and SYCP3 (red) in germ cells at c5, c7 and c9 from XGFP+ and XGFP- PGCLCs. All cells with SYCP3 signal irrespective of localization pattern or intensity were scored as SYCP3+ cells. Cells were counterstained with DAPI (grey). Scale bars = 10 μm.

(C) Number of SYCP3+ cells per m220 culture day originating from XGFP+ and XGFP- PGCLCs. Each white dot represents a biological replicate (n = 3). P-values shown are from a two-sided Mann-Whitney U test. n.s. = not significant.

(D) Percentage of XGFP+ cells among SYCP3+ cells at the indicated m220 culture day, originating from XGFP+ or XGFP- PGCLCs. Green bars show constitutive GFP expression in XGFP+ PGCLC-derived cells, while striped bars signify XGFP reactivation in XGFP- cells. Each white dot represents a biological replicate (n = 3).

(E) Representative images showing stages of meiotic prophase I from culture day 9 (c9) germ cells from XGFP+ and XGFP- PGCLCs. c9 germ cells were spread and immunostained for SYCP3 (red), and γH2AX (grey). Scale bars = 10 µm.

(F) Quantification of meiotic progression in culture day (c9) expanded germ cells derived from XGFP+ and XGFP- PGCLCs. The graphs show the percentages of the meiotic stage for SYCP3+ cells. Lepto, leptotene; Zygo, zygotene; Pachy, pachytene; Diplo, diplotene. Numbers indicate detected cells per meiotic stage and original XGFP status.

(G) Representative tile scan merged images of XGFP (green) and Xtomato (red) in germ cells at c7 from XGFP+ and XGFP- PGCLCs. Cells were counterstained with DAPI (grey). Scale Bar = 1 mm. The white squares contain a magnified image of the region depicted by the white dotted squares (Scale Bar = 1 µm).

(H) Density ridge plot showing size of cellular aggregates in (G) measured using the XTomato signal. X-Axis shows area in µm2 scaled as log2. Dashed line represents the mean. XFGP- mean = 1320 µm2, XGFP+ mean = 5514 µm2. XGFP- n = 297 aggregates, XGFP+ n = 373 aggregates. P-value from two-sample unpaired Wilcoxon-Mann-Whitney test with R defaults.
The miRNA-212/132 family regulates both cardiac hypertrophy and cardiomyocyte autophagy (2012)
f1

(a) Overexpression of miRNA precursors from a library identified miRNAs enhancing cardiomyocyte growth and brain natriuretic peptide (BNP) secretion. The miRNA family, miR-212/132, is highlighted by red circles. (b,c) Effects of miR-212 and miR-132 precursors and inhibitors (anti) on cardiomyocyte cell size as compared with the effects of scrambled (Scr) controls (n=5–13). Representative images used for quantification of cardiomyocyte cell size are shown in c. (d) Effects of various pro-hypertrophic stimuli on miR-212 and miR-132 expression in neonatal cardiomyocytes (n=6–10). (e) miR-212 and miR-132 expression levels during pressure-induced left ventricular hypertrophy 3 and 21 days after transaortic constriction (TAC) surgery of mice (n=4). (f) Cardiomyocyte diameters after Sham operation or 3, 14 and 35 days after TAC (n=4 per group). (g,h) miR-212 and miR-132 expression (normalized to sno-202 levels) in fractionated cardiomyocytes and cardiac fibroblasts derived from adult mice after 3 and 35 days of TAC. All values represent mean±s.e.m. *P0.05; **P0.01; ***P0.005. Scale bar in c represents 50 μm. ACTN2, α-cardiac actinin; Ang II, angiotensin-2; a.u., arbitrary unit; d, day; DAPI, 4′,6-diamidino-2-phenylindole; FC, fold change; NS, no significant difference; P/I, phenylephrine/isoprenaline.
The miRNA-212/132 family regulates both cardiac hypertrophy and cardiomyocyte autophagy (2012)
f2

(a) Overexpression construct of miR-212/132 under the control of the α-MHC promoter. (b) Expression levels of miR-212 and miR-132 in heart samples of individual wild-type (WT) and miR-212/132 transgenic (TG) mice as assayed by RT–PCR analysis against the mature forms of corresponding microRNAs. Rnu6b was used as housekeeping control. (c) Survival rate of two different miR-212/132 TG mouse families (TG-Fam23, TG-Fam43) versus WT controls was analysed by Kaplan–Meier survival assay (n=87, 65 and 53 for WT, TG-Fam23 and TG-Fam43, respectively). (d) Morphology of explanted hearts from TG and WT mice at 10 weeks after birth. Scale bar, 1 cm. (e,f) Heart-to-body-weight ratios (e) and cardiomyocyte diameter (f) in 8-week-old α-MHC-miR-212/132 transgenic mice compared with their wild-type littermates (n=5–7). Scale bar, 50 μm. (g–i) Echocardiographic analysis of cardiac dimensions and function for α-MHC-miR-212/132 TG mice and WT controls (n=16–18) (g) end-systolic area, (h) end-diastolic area, (i) fractional shortening. All values in e–i represent mean±s.e.m. *P0.05; **P0.01; ***P0.005. DAPI, 4′,6-diamidino-2-phenylindole; WGA, wheat germ agglutinin (membrane stain).
The miRNA-212/132 family regulates both cardiac hypertrophy and cardiomyocyte autophagy (2012)
f3

(a) Heart-to-body-weight ratios for 12-week-old miR-212/132 null (KO) and wild-type (WT) mice (n=5–6). (b) Cardiomyocyte cell size of neonatal miR-212/132 null and WT mice (n=5–6 isolations). (c–f) Heart-to-body-weight ratios (c), cardiomyocyte diameter (d), cardiac fibrosis (e) and lung wet weight (f) in Sham-operated WT mice and miR-212/132 null and WT mice 3 weeks after TAC surgery (n=4–7). Scale bar, 50 μm. (g–i) Echocardiographic analysis of cardiac dimensions and function in Sham-operated WT mice and miR-212/132 null and WT mice 3 weeks after TAC (n=4–11). (g) End-diastolic area, (h) end-systolic area and (i) fractional shortening. All values represent mean±s.e.m. *P0.05; **P0.01; ***P0.005; #P0.05 compared with WT TAC; ###P0.005 compared with WT TAC; PSR, picrosirius red (collagen stain). DAPI, 4′,6-diamidino-2-phenylindole; WGA, wheat germ agglutinin.
The miRNA-212/132 family regulates both cardiac hypertrophy and cardiomyocyte autophagy (2012)
f4

(a) Luciferase activity levels upon cotransfection of a luciferase construct containing wild-type (WT) or mutated (Mut.) 3′UTR of FoxO3 with scrambled (Scr) control (Ctrl), pre-miR-132 or pre-miR-212 (n=9). (b,c) Expression levels of FoxO3 on mRNA (b) and protein levels (c) in hearts of WT and α-MHC-miR-212/-132 transgenic (TG) mice. M: Size marker (n=9–13). (d) FoxO3 mRNA levels in neonatal rat cardiomyocytes transfected with Scr Ctrl, anti-miR-212 and anti-miR-132 after phenylephrine (PE, 10 μM) treatment (n=6–9; P-values against Scr+PE). (e–g) Expression levels of atrogin-1 (e) and Mcip1 (f) and calcineurin phosphatase activity levels (g) in hearts of WT and α-MHC-miR-212/132 TG mice (n=5–9). (h) Luciferase activity levels showing the NFAT transcriptional activity in cardiomyocytes transfected with Scr Ctrl, pre-miR-132 or pre-miR-212 (n=5). (i,j) Mcip1.4 (i) and atrogin-1 (j) mRNA levels in WT and miR-212/132 null (KO) mice 3 weeks after transaortic constriction (TAC) or Sham operation (n=5–7 per group). All values represent mean±s.e.m. *P0.05; **P0.01; ***P0.005. #P0.05 compared with WT TAC. FC, fold change.