EMBO/soda-nel huggingface dataset:
MSCs Rescue Impaired Wound Healing in a Murine LAD1 Model by Adaptive Responses to Low TGF-β1 Levels (2020)
Figure 7

Figure 7. Release of TGF-β1 by MSCs is regulated by miR-21 / Smad7 signaling by sensing the environmental TGF-β1.

A. AT-MSCs were pretreated with a TGFβRI inhibitor SB431542 at 10 µM, or control DMSO for 1 h, and subsequently exposed to r.h. TGF-β1 at indicated concentrations. The cells were cultured for another 24 h and harvested for miR-21 assay. The expression of miR-21 was normalized on RNU6B. Data is given as mean ± SEM of three independent experiments.

B-D. AT-MSCs were transfected with 30 nM Smad7-siRNAs or control siRNA. Untransfected AT-MSCs served as control. RNA and protein were isolated 2 days after transfection for Smad7 expression by qPCR (B) and western blot (C). The remaining cells were treated with r.h. TGF-β1 at indicated concentrations for another 24 h. The expression of human TGF-β1 was analyzed by qPCR and normalized on human GAPDH (D). Data is given as mean ± SEM of three independent experiments.

E-G. AT-MSCs were transfected with 5 nM miR-21 mimic or control mimic, and were exposed to r.h. TGF-β1 2 days after transfection at indicated concentrations for another 24 h. The expression miR-21 was analyzed by qPCR-based miR-21 assay normalized on RNU6B (E), Samd7 protein level was shown by western blot (F), and human TGF-β1 expression was monitored by qPCR normalized on human GAPDH (G). Data is given as mean ± SEM of three independent experiments.

H-J. AT-MSCs were transfected with 50 nM miR-21 inhibitor or control inhibitor, and were exposed to r.h. TGF-β1 2 days after transfection at indicated concentrations for another 24 h, and subsequently subjected to the analysis of expression of miR-21 (H), Smad7 protein (I) and human TGF-β1 mRNA (J). Data is given as mean ± SEM of three independent experiments.

K-L. Full-thickness excisional wounds were produced on CD18-/- mice by 6 mm biopsy punches. One day after wounding, each wound received intradermal injection of 2.5x105 AT-MSCs that had been transfected with 50 nM miR-21 inhibitor or control inhibitor 2 days after transfection. CD18-/- wounds received mock injection with PBS served as control. Each wound region was digitally photographed at days 0, 3, and 6 post-wounding, and expressed as percentage of the initial wound size at day 0 (K). Wound tissues were harvested on day 6 post-wounding for quantification of total TGF-β1 protein by ELISA (L). Data is given as mean ± SEM; n = 5 wounds per group; *, p<0.05; **, p<0.01, by one-way ANOVA with Tukey's test. MSC_miR-21-IN, miR-21 inhibitor transfected AT-MSCs; MSC_ctrl-IN, control inhibitor transfected AT-MSCs.
Dual targeting of brain region-specific kinases potentiates neurological rescue in Spinocerebellar ataxia type 1 (2021)
Figure 1

Figure 1. Genetic screens reveal that ZBTB7A and ZBTB7B regulate ATXN1, and ZBTB7B shows more pronounced effect

(A) A diagram that outlines the steps of the genetic screen. ATXN1 reporter cell line produces RFP-conjugated ATXN1 and YFP internal control independently from a same transcript, and ATXN1 levels were monitored by measuring the ratio of red to yellow fluorescence intensity. 1) ATXN1 reporter cell line was transduced with lentivirus harboring shRNAs of 858 ubiquitination-related genes. 2)-3) The cells with the 5% lowest and 5% highest fluorescence ratio were sorted. 4) Illumina sequencing revealed enriched shRNAs in the two groups compared to the non-sorted group. 5) Identifying the genes targeted by these enriched shRNAs revealed 39 positive regulators including ZBTB7A and ZBTB7B, and 13 negative regulators of ATXN1 protein levels (See Table EV2 for the full list).

(B) Western blot analysis of Atxn1 levels in cerebellar granule neurons (CGNs) after knockdown of Zbtb7a and/or Zbtb7b (n = 3).

(C) Western blot analysis of Atxn1 levels in the cerebella of wild-type (n = 3), Zbtb7a+/- (n = 4), Zbtb7b+/- (n = 3), Zbtb7a+/-; Zbtb7b+/- (n = 4) mice.

(D) Western blot analysis of Atxn1 levels in CGNs after overexpressing Zbtb7a and/or Zbtb7b (n = 3).

(E) Genetic interaction of wild-type human ATXN1(30Q) with either ZBTB7A or ZBTB7B expressed in the Drosophila eyes. Co-expression of ATXN1(30Q) with ZBTB7A or ZBTB7B severely disrupted the external Drosophila eye structure and increased ATXN1 levels. Scale bar: 100 μm

Data information: (B-D) Mean ± SD, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, one-way ANOVA.
Dual targeting of brain region-specific kinases potentiates neurological rescue in Spinocerebellar ataxia type 1 (2021)
Figure 2

Figure 2. ZBTB7B regulates ATXN1 indirectly through activating RSK3 expression

(A) ZBTB7B's domain structure marked with the mutations in BTB and ZF domain used in this study.

(B) Two representative biological functions of ZBTB7B: 1) changing target gene expression; 2) recruiting target proteins for ubiquitination. CUL3: Cullin3, E2: ubiquitin-conjugating enzyme, SUB: substrate.

(C) Western blot analysis of ATXN1 levels after overexpression of ZBTB7B wild-type (WT), zinc-finger domain mutant (ZFmut) or BTB domain mutants (BTBmut1 and BTBmut2) (n = 3).

(D) qRT-PCR results of Atxn1 mRNA levels in the cerebella of wild-type (n = 3), Zbtb7b+/- (n = 4), and Zbtb7b-/- mice (n = 4).

(E) MA plot (M: log ratio; A: mean average) of the differentially expressed genes (DEG) analysis from RNA-seq samples of the cells overexpressing either WT or ZFmut ZBTB7B. Four filters and the number of genes kept after applying each filter are described. Two genes that passed the 4th filter are labeled.

(F) Expression heatmap of the 16 genes that passed the 3rd filter. Color scale: row z-score.

(G) qRT-PCR results of RSK3 mRNA levels across different CRISPR knockout cell lines (n = 2).

(H) Epistatic relationship between ZBTB7B and RSK3 in terms of ATXN1 regulation. ATXN1 levels were measured after overexpression of ZBTB7B in the RSK3 knockout cell line (sgRSK3) and control cell lines (WT and sgControl) (n = 3). ZBTB7B+RSK3: co-overexpression of both ZBTB7B and RSK3.

(I) qRT-PCR results of RSK3 mRNA levels after knockdown of ZBTB7B in Daoy cells (n = 2).

(J) qRT-PCR results of Rsk3 mRNA levels in the cerebella of Zbtb7b conditional knockout mice (n = 6).

(K) Position frequency matrix of ZBTB7B binding DNA sequence and a table of the predicted ZBTB7B binding sites on human RSK3 promoter. Start and End position numbers are described based on TSS. The seventy base pairs upstream RSK3 promotor includes top two potential binding sites and was used for luciferase assays. C: mutated into A in Fig EV2D.

(L) Luciferase assay result of RSK3 promoter in HEK 293T and HeLa cells transfected with either an empty or ZBTB7B expressing vector (n = 3).

Data information: (C, D) Mean ± SD, ***P<0.001 ****P<0.0001, one-way ANOVA. (G-I) Mean ± SD, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA. (J, L) Mean ± SD, *P<0.05, ***P<0.001, ****P<0.0001, two-tailed t-test.
Dual targeting of brain region-specific kinases potentiates neurological rescue in Spinocerebellar ataxia type 1 (2021)
Figure 3

Figure 3. RSK3 regulates ATXN1 by phosphorylating S776.

(A) Western blot analysis of ATXN1 after knockdown of RSK3 in Daoy cells. Bottom left panel: qRT-PCR results of RSK3 mRNA. Bottom right panel: densitometry of the western blot image in the top panel (n = 3 except for shATXN1 where n = 1).

(B) Western blot analysis of ATXN1 after overexpression of RSK3, ZBTB7B, or eGFP in Daoy cells (n = 3).

(C) Western blot analysis of ATXN1(82Q) after overexpression of either wild-type or inactive RSK3 in Daoy cells expressing ATXN1(82Q) (n = 3).

(D) In vitro kinase assay of RSK3 with ATXN1. MSK1 was used as a positive control. Asterisk: auto-phosphorylated RSK3.

(E) In vitro kinase assay of four different RSK members with ATXN1.

(F) In vitro kinase assay of RSK3 with N-terminal (F1), middle (F2), and C-terminal fragment (F3) of ATXN1.

(G) Mass spectrometry analysis of phosphorylated residues in ATXN1 F3 after in vitro kinase assay with RSK3.

(H) Western blot analysis of ATXN1 after a treatment with RSK3 inhibitor (BI-D1870) on SCA1 patient iPSC-derived neurons (n = 3).

(I) Western blot analysis of total and phospho-S776 ATXN1 after a co-treatment with MSK1 (SB-747651A) and RSK3 inhibitor (BI-D1870) on SCA1 patient iPSC-derived neurons (n = 3).

Data information: RSK3 antibody used in (B, C) is only suitable for detecting overexpressed RSK3 but not for endogenous RSK3. (A-C) Mean ± SD, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA. (H, I) Mean ± SD, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA.
Dual targeting of brain region-specific kinases potentiates neurological rescue in Spinocerebellar ataxia type 1 (2021)
Figure 4

Figure 4. Reducing RSK3 rescues behavioral phenotypes in SCA1 animal models by decreasing ATXN1

(A) Images of Drosophila eyes expressing human ATXN1(82Q) with either knockdown or overexpression of the Drosophila RSK3 homolog Sk6II. Scale bar: 100 μm (top) and 20 μm (bottom).

(B) Improvement of motor performance by the knockdown of Sk6II in Drosophila SCA1 model expressing ATXN1(82Q) in the central nervous system. Mean ± SEM, *P < 0.05, Linear mixed-effect model ANOVA, n = 10/genotype, four times of measurements.

(C) A representative image of a mouse brain 4 weeks after AAV injection of shRsk3 into 12 weeks old SCA1 mice (Atxn1154Q/2Q) cerebellum. Scale bar: 2 mm.

(D) Western blot analysis of wild-type (2Q) and mutant Atxn1 (154Q) in the cerebella of 16 weeks old SCA1 mice dissected 4 weeks after the stereotaxic injection of AAV9 harboring shControl (n = 3), shRsk3 (n = 3), or shAtxn1 (n = 2). Mean ± SD, *P<0.05, ***P<0.001 ****P<0.0001, one-way ANOVA.

(E) Western blot analysis of wild-type (2Q) and mutant Atxn1 (154Q) in the brainstem (BS) and cerebella (CBM) of 8 weeks old Atxn1154Q/2Q (n = 6) and Atxn1154Q/2Q; Rsk3+/- mice (n = 7). Mean ± SD, *P<0.05, **P<0.01, two-tailed t-test.

(F) Rotarod motor performance test result of Atxn1154Q/2Q; Rsk3+/- mice at 11-12 weeks old. Mean ± SEM, *P<0.05, two-way ANOVA, n = 35, 31, 34, 38 for WT, Rsk3+/-, Atxn1154Q/2Q, Atxn1154Q/2Q; Rsk3+/- genotype, respectively.
Dual targeting of brain region-specific kinases potentiates neurological rescue in Spinocerebellar ataxia type 1 (2021)
Figure 5

Figure 5. Differential expression of RSK3 and MSK1 in mouse and human brains

(A) Fluorescent western blot analysis of Atxn1, Msk1, and Rsk3 in seven different brain regions of wild-type mice at 9 weeks old (n = 3). Each protein levels were represented relative to the protein levels in brainstem. Mean ± SD, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA.

(B) Representative BaseScope assay images of Rsk3 in the whole brain sagittal sections of wild-type (top) and Rsk3-/- mice (bottom) at 5-6 weeks old. The areas within the black squares are magnified in the right panels in each genotype. Scale bar = 2 mm in whole brain images; 50 μm in the magnified images.

(C) Representative IF images of Msk1 in the whole brain sagittal sections of wild-type (top) and Msk1-/- mice (bottom) at 6 months old. The areas within the white squares are magnified in the right panels in each genotype. Scale bar = 2 mm in whole brain images; 50 μm in the magnified images.

(D) Western blot analysis of total and active form of Rsk3 and Msk1 in the cerebella and brainstem of 12 weeks old WT and SCA1 mice (n = 4). Relative Rsk3 and Msk1 levels were represented into log2 scale. The protein levels of the two kinases are significantly different between the two brain regions. Mean ± SD, *P<0.05, **P<0.01, ****P <0.0001, two-way ANOVA.

(E) Western blot analysis of ATXN1, RSK3, pRSK3, MSK1, and pMSK1 in the postmortem cerebella, pons, and medulla oblongata of human controls and SCA1 patients (n = 3 per genotype). Total protein was used for normalization, and protein levels are represented relative to cerebellar protein levels in control group. C: Cerebellum, P: Pons, M: medulla oblongata. Values from P and M are combined and presented in BS. Mean ± SEM, *P<0.05, **P<0.01, two-way ANOVA, n = 3 (CBM) and 6 (BS).

Data information: BS: brainstem, CBM: cerebellum, MB: midbrain, ST: striatum, HC: hippocampus, CTX: cortex, OB: olfactory bulbs.
Dual targeting of brain region-specific kinases potentiates neurological rescue in Spinocerebellar ataxia type 1 (2021)
Figure 6

Figure 6. Differential effect of Rsk3 and Msk1 on Atxn1 regulation and disease phenotypes in SCA1 mice brainstem

(A) Western blot analysis of total and pS776 wild-type and mutant Atxn1 in the brainstem of 8 weeks old SCA1 mice with heterozygous knockout of either one or both kinases. Mean ± SD, *P<0.05, **P<0.01, ****P<0.0001, one-way ANOVA, n = 7, 6, 4, 7 following the order of genotypes in the western blot, respectively.

(B) Plethysmography data measuring breathing ability (total ventilated gas volume per minute) of 34 weeks old mice with different genotypes. Mean ± SD, *P<0.05, two-tailed t-test, n = 12, 13, 15, 13, 15, 9, 15, 13 following the order of genotypes in the graph, respectively.

(C) Representative immunohistochemistry images of Iba1+ reactive microglia in the coronally sectioned hypoglossal nucleus of 36 weeks old mice with different genotypes. Yellow arrows indicates Iba1+ microglia, and larger pyramidal shaped cells are motoneurons. scale bar = 50 μm.

(D) Quantification of the number of Iba1+ cells shown in (C). Mean ± SD, *P<0.05, ***P<0.001, one-way ANOVA, n = 3 mice per genotype, each dot is an averaged measurement from six sections per mouse.

(E) Representative Hematoxylin and Eosin stained motoneurons in the coronally sectioned hypoglossal nucleus (white dashed line) of 36 weeks old mice with different genotypes. The motoneurons are dark purple with pyramidal shape within the nucleus. C: central canal. Scale bar = 100 μm.

(F) Quantification of the number of motoneurons shown in (E). Mean ± SD, *P<0.05, one-way ANOVA, n = 3 mice per genotype, each dot is an averaged measurement from six sections per mouse.

(G) Kaplan-Meier survival graphs of SCA1 mice with heterozygous knockout of either one or both kinases. The data of Msk1+/-, Rsk3+/-, and Msk1+/-; Rsk3+/- mice overlap those of wild-type mice. *P<0.05, Gehan-Breslow-Wilcoxon test, n = 15, 15, 12, 13, 22, 11, 20, 19 following the order of genotypes in the legend on top of the figure, respectively.
Dual targeting of brain region-specific kinases potentiates neurological rescue in Spinocerebellar ataxia type 1 (2021)
Figure 7

Figure 7. Differential effect of Rsk3 and Msk1 on Atxn1 regulation and disease phenotypes in SCA1 mice cerebella

(A) Western blot analysis of total and pS776 wild-type and mutant Atxn1 in the cerebella of 8 weeks old SCA1 mice with heterozygous knockout of either one or both kinases. Mean ± SD, **P<0.01, ***P<0.001, one-way ANOVA, n = 7, 6, 4, 7 following the order of genotypes in the western blot, respectively.

(B) Rotarod motor performance test result of 11-12 weeks old SCA1 mice with heterozygous knockout of either one or both kinases. Mean ± SEM, *P<0.05, two-way ANOVA, n = 18, 12, 13, 11, 19, 13, 15, 13 following the order of genotypes in the legend on top of the figure, respectively.

(C) Representative immunofluorescent (IF) images of cerebellar molecular layer between the cerebellum lobule V and VI across different genotypes at 36 weeks old, stained with anti-Calbindin 1 antibody. Scale bar = 100 μm.

(D) Quantification of the thickness of top and bottom molecular layers shown in (C). Mean ± SD, *P<0.05, **P<0.01, ***P<0.001, one-way ANOVA, n = 3 mice per genotype, each dot is an averaged measurement from three sections per mouse.
The time-of-day of myocardial infarction onset affects healing through oscillations in cardiac neutrophil recruitment (2016)
Figure 1

Figure 1. Circadian oscillations of neutrophils, adhesion molecules and chemokines in the mouse heart at steady state.

A Baseline blood counts of granulocytes. One-way ANOVA; n = 8 mice per ZT; *P = 0.0001 ZT5 vs. ZT13.

B Flow cytometry quantification of neutrophils in digested hearts. The representative dot plots show the gating strategy for cardiac neutrophils (CD45+Ly6G+CD11b+) at ZT5. One-way ANOVA; n = 5 mice per ZT; *P = 0.0001 ZT5 vs. ZT13.

C Representative immunostainings for neutrophils in the myocardium (left ventricle), identified as Ly6G-positive (20x magnification).

D Cardiac mRNA expression levels normalized to HPRT. One-way ANOVA; n = 5 mice per ZT; ZT5 vs. ZT17: *P = 0.0064 (CXCL1), *P = 0.0007 (CXCL2), *P = 0.0007 (CXCL5), *P = 0.0001 (ICAM-1), *P = 0.0009 (VCAM-1), *P = 0.0181 (CCL3), *P = 0.0360 (CCL5).

E Mean fluorescence intensity (MFI) of CXCX2 expression by neutrophils in blood and heart. One-way ANOVA; n = 3 mice for ZT1, ZT17, ZT21 and n = 5 for ZT5, ZT9, ZT13; ZT5 vs. ZT13: *P = 0.0425 (blood), *P = 0.0078 (heart).

Data information: All data are expressed as mean ± SEM.
The time-of-day of myocardial infarction onset affects healing through oscillations in cardiac neutrophil recruitment (2016)
Figure 2

Figure 2. Inflammatory response after MI during active (ZT13) or resting phase (ZT5).

A Flow cytometry analysis of neutrophils in hearts and representative immunostainings for neutrophils in the infarct area, identified as Ly6G-positive (5x and 20x magnifications). Two-way ANOVA followed by Bonferroni post-hoc test; n=5 mice for no MI, n = 5 for 12h post MI, n = 3 for 24h post MI and n = 3 for 72h post MI in both ZT groups; ZT5 vs. ZT13: *P = 0.0161 (12h MI), *P = 0.003 (24h MI).

B Flow cytometry analysis of neutrophils in bone marrow. Two-way ANOVA followed by Bonferroni post-hoc test; n = 5 mice for no MI, n = 5 for 12h post MI, n = 4 for 24h post MI and n = 4 for 72h post MI in both ZT groups; ZT5 vs. ZT13: *P = 0.0471 (12h MI), *P = 0.0035 (24h MI).

C Blood counts of granulocytes. Two-way ANOVA followed by Bonferroni post-hoc test; n = 9 mice for no MI, n = 10 for 12h post MI, n = 13 for 24h post MI and n = 10 for 72h post MI in both ZT groups; ZT5 vs. ZT13: *P = 0.0364 (24h MI).

D Flow cytometry analysis of neutrophils in hearts and bone marrow under inverted light cycle conditions. Two-way ANOVA followed by Bonferroni post-hoc test; n = 3 mice for no MI in both ZT groups, n = 5 for ZT5 and n = 6 for ZT13 24h post MI; ZT5 vs. ZT13: *P = 0.0147 (24h MI).

E Representative gating strategy for GMPs in the bone marrow, identified as identified as lineage negative (CD11b-, Gr1-, B220-, CD3- and Ter119-) and Sca-1-, c-kit+, CD16/32+, CD34+. Flow cytometry quantification of GMP in the bone marrow. Two-way ANOVA followed by Bonferroni post-hoc test; n = 4 mice for no MI and 24h post MI in both groups; ZT5 vs. ZT13: *P = 0.0077 (no MI), *P = 0.0013 (24h MI).

F CXCL12 levels in bone marrow lavage. Two-way ANOVA followed by Bonferroni post-hoc test; n = 7 mice for no MI and n = 6 for 24h post MI in both groups; ZT5 vs. ZT13: *P = 0.0077 (no MI), *P = 0.0013 (24h MI).

G Plasma levels of pro-inflammatory cytokines and chemokines. Two-way ANOVA followed by Bonferroni post-hoc test; n = 11 mice for no MI in both ZT groups, n = 7 for ZT5 and n = 8 for ZT13 24h post MI; ZT5 vs. ZT13: *P = 0.0271 (CXCL12, no MI), *P = 0.0108 (TNFα, 24h MI), *P = 0.001 (G-CSF, 24h MI), *P = 0.005 (CXCL1, 24h MI), *P = 0.0005 (CXCL2, 24h MI), *P = 0.0016 (CCL3 24h MI), *P = 0.0144 (CCL5, 24h MI).

Data information: All data are expressed as mean ± SEM.
The time-of-day of myocardial infarction onset affects healing through oscillations in cardiac neutrophil recruitment (2016)
Figure 3

Figure 3. MI during active phase (ZT13) leads to infarct expansion and reduced cardiac function.

A Permanent LAD occlusion was perfomed at ZT5 or ZT13. TTC staining (white, infarct; red, vital myocardium) and quantification of infarct size normalized to the left ventricle (LV). Student`s t-test; n = 4 mice for ZT5 and n = 5 for ZT13 MI; *P = 0.0041.

B Plasma troponin I levels 24 hours after MI. Student`s t-test; n = 4 mice for ZT5 and n = 5 for ZT13 MI; *P = 0.0007.

C Flow cytometry analysis of dead cardiomyocytes (CD45-, zombie+) 24 hours after MI. Student`s t-test; n = 3 mice in both groups; *P = 0.0072.

D Survival rates after MI and cause of death. Log-rank test; n = 87 mice in both groups; *P = 0.0006.

E Myofibroblasts within infarcts were quantified by alpha smooth muscle actin (αSMA) staining as ratio between stained and total area of random fields. Student`s t-test; n = 4 mice in both groups; *P = 0.0134.

F Masson´s trichome staining of fibrosis (blue, collagen; red, vital myocardium) and quantification relative to total LV (*P = 0.0095) as well as LV anterior wall thickness (*P = 0.0068) 7 days after MI. Student`s t-test; n = 6 mice for ZT5 and n = 7 for ZT13 MI.

G Analysis of relative collagen type I content identified by Sirius Red staining 7 days after MI. Student`s t-test; n = 4 mice for ZT5 and n = 5 for ZT13 MI; *P = 0.0175.

H Echocardiographic assessment of ejection fraction (EF), end-systolic-volume (ESV) and end-diastolic-volume (EDV). Two-way ANOVA; n = 6 mice for no MI for both groups, n = 6 for ZT5 and n = 9 for ZT13 at 72h post MI, n = 8 for both groups at 7d post MI and n = 7 for ZT5 and n = 5 for ZT13 14d post MI; ZT5 vs. ZT13: *P = 0.0001 (EF, 3d), *P = 0.0042 (EF, 7d), *P = 0.0121 (EF, 14d); *P = 0.0253 (ESV, 3d), *P = 0.0421 (ESV, 7d), *P = 0.0005 (ESV, 14d); *P = 0.0053 (EDV, 14d).

Data information: All data are expressed as mean ± SEM.
The time-of-day of myocardial infarction onset affects healing through oscillations in cardiac neutrophil recruitment (2016)
Figure 4

Figure 4. Limiting neutrophilic inflammation during active phase (ZT13) reduces MI damage.

A Permanent LAD occlusion was performed at ZT13 followed by injection of isotype or Ly6G antibody 45min after surgery and then every 24 hours.

B Flow cytometry analysis of cardiac neutrophils 24 hours after ZT13 MI. The dotted line indicates cardiac neutrophil counts 24h after ZT5 MI, shown in Fig. 2A. Student`s t-test; n = 3 mice for isotype and n = 5 for Ly6G injected mice; *P = 0.0004.

C Plasma TNFα levels 24 hours after ZT13 MI. Student`s t-test. n=8 independent samples for isotype and n=6 for Ly6G injected mice; *P = 0.0019.

D Infarct size relative to left ventricular area (LV) and plasma troponin I levels 24 hours after ZT5 or ZT13 MI. Student`s t-test; For infarct size n = 3 mice in both groups for ZT5 and n = 4 mice for isotype and n = 3 mice for Ly6G at ZT13; Isotype vs. Ly6G: *P = 0.0158 (ZT13). For troponin levels n = 8 mice for isotype and n = 6 mice for Ly6G with ZT13 MI; *P = 0.0012.

E Masson's-trichrome staining of fibrosis (blue, collagen; red, vital myocardium) and quantification relative to total area of the LV (*P = 0.0084); morphometric quantification of the LV anterior wall thickness (*P = 0.0027) 7 days after ZT13 MI. Student`s t-test; n = 5 mice for isotype and n = 4 mice for Ly6G.

F Analysis of collagen type I fibers within infarcts identified by Sirius Red staining 7 days after ZT13 MI. Student`s t-test; n = 5 mice for isotype and n = 4 mice for Ly6G; *P = 0.0375.

G Echocardiographic measurement of ejection fraction (EF), end-systolic-volume (ESV) and end-diastolic-volume (EDV) before and after ZT13 MI. Two-way ANOVA; n = 6 mice for no MI in both groups, n = 9 for isotype and n = 5 for Ly6G at 72h post MI, n = 8 for isotype and n = 4 for Ly6G at 7d post MI. Isotype vs. Ly6G: *P = 0.0001 (EF, 3d), *P = 0.0227 (EF, 7d); MI *P = 0.0133 (ESV, 3d), *P = 0.0004 (ESV, 7d); *P = 0.0017 (EDV, 7d).

Data information: All data are expressed as mean ± SEM.