Journal: Journal of Cellular Physiology

Article Title: Statin regulated ERK5 stimulates tight junction formation and reduces permeability in human cardiac endothelial cells

doi: 10.1002/jcp.26064

Figure Lengend Snippet: siRNA mediated gene silencing of ERK5 induced HCMEC barrier perturbment. HCMEC were transfected with non‐silencing or ERK5 siRNA for 6 hr and allowed to reach confluence over 5 subsequent days. (a) Immunofluorescence imaging of tight junctions (ZO‐1, green), actin stress fibers (phalloidin, red), and nuclei (Hoechst, blue). Arrows indicate barrier perturbment. Scale bars: 10 μm. Results are from one experiment representative of three. (b) Western blot of ZO‐1, ERK5, ERK1/2, and GAPDH levels in HCMECs following siRNA induced ERK5 gene silencing. (c) HCMECs were plated on Thincerts™ containing 0.4 μm pores and permeability of 4 kDa FITC‐dextran across the HCMEC monolayer was assessed and compared to untransfected cells ( n = 4), mean ± s.d. ** p ≤ 0.01 compared to untransfected. (d) Assessment of TEER was conducted on HCMECs plated on Thincerts™ containing 0.4 μm pores ( n = 4), mean ± s.d. ** p ≤ 0.01 compared to untransfected

Article Snippet: For immunoprecipitation, lysates were incubated with ERK5 (2 μg/ml, #AF2848, R&D systems) or control IgG (2 μg/ml, goat) and protein G agarose.

Techniques: Transfection, Immunofluorescence, Imaging, Western Blot, Permeability

Journal: Journal of Cellular Physiology

Article Title: Statin regulated ERK5 stimulates tight junction formation and reduces permeability in human cardiac endothelial cells

doi: 10.1002/jcp.26064

Figure Lengend Snippet: Adenoviral mediated ERK5 expression stimulates HCMEC tight junction formation. HCMEC were transfected with Ad‐Control or Ad‐CA‐MEK5 and Ad‐ERK5 for 24 hr before treatment with doxorubicin 0.1 μM for 6 hr. (a) Immunofluorescence imaging of HCMEC tight junctions (ZO‐1, green), actin stress fibers (phalloidin, red), and nuclei (Hoechst, blue). Arrows indicate barrier perturbment. Scale bars: 10 μm. Results are from one experiment representative of three. (b) Western blot of ERK5, ERK1/2, phospho ERK1/2, and GAPDH levels in HCMECs. FLAG‐Tag was used to confirm transfection of Ad‐ERK5 and HA‐Tag was used to confirm transfection of Ad‐CA‐MEK5. (c) HCMECs were plated on Thincerts™ with 0.4 μm pores and permeability of 4 kDa FITC‐dextran across the HCMEC monolayer assessed and compared to Ad‐Control ( n = 4), mean ± s.d. * p ≤ 0.05 compared to Ad‐Control

Article Snippet: For immunoprecipitation, lysates were incubated with ERK5 (2 μg/ml, #AF2848, R&D systems) or control IgG (2 μg/ml, goat) and protein G agarose.

Techniques: Expressing, Transfection, Control, Immunofluorescence, Imaging, Western Blot, FLAG-tag, Permeability

Journal: Journal of Cellular Physiology

Article Title: Statin regulated ERK5 stimulates tight junction formation and reduces permeability in human cardiac endothelial cells

doi: 10.1002/jcp.26064

Figure Lengend Snippet: Simvastatin stimulates ERK5 phosphorylation in HCMECs. (a) HCMECs were incubated with simvastatin at a range of concentrations for 6 hr. Intracellular signaling responses were assessed by Western blotting for phosphorylation of ERK5 and ERK1/2 using phospho‐specific antibodies. Unprenylation of Rap1A was measured as a control for simvastatin activity. Total protein was measured for ERK5, ERK1/2, and Rap1. (b) HCMECs were incubated with 0.3 μM simvastatin at a range of time points. Intracellular signaling responses were assessed by Western blotting for phosphorylation of ERK5 and ERK1/2 using phospho‐specific antibodies. Unprenylation of Rap1 was measured as a control for simvastatin activity. Total protein was measured for ERK5, ERK1/2, and Rap1. (c) The level of ERK5 band shift following treatment with simvastatin, rosuvastatin, and pitavastatin was quantified relative to vehicle control. (d) Activation of ERK5 by band shift was determined (EC 50 concentration), mean ± s.d. ( n = 3). (e) HCMEC were transfected with siRNA to MEKK2, MEKK3, MEK5, and ERK5 for 6 hr and allowed to reach confluence over 5 subsequent days before treatment with 0.1% DMSO or 0.3 μM simvastatin for 6 hr. Western blot of ERK5, phosphorylated‐ERK5, MEKK2, MEKK3, MEK5, and GAPDH levels in HCMEC. (f) Quantification of ERK5 band shift ( n = 3) mean ± s.d. * p ≤ 0.05 compared to untransfected basal

Article Snippet: For immunoprecipitation, lysates were incubated with ERK5 (2 μg/ml, #AF2848, R&D systems) or control IgG (2 μg/ml, goat) and protein G agarose.

Techniques: Phospho-proteomics, Incubation, Western Blot, Control, Activity Assay, Electrophoretic Mobility Shift Assay, Activation Assay, Concentration Assay, Transfection

Journal: Journal of Cellular Physiology

Article Title: Statin regulated ERK5 stimulates tight junction formation and reduces permeability in human cardiac endothelial cells

doi: 10.1002/jcp.26064

Figure Lengend Snippet: Statin induced ERK5 phosphorylation occurs via inhibition of protein geranylgeranylation. (a) Summary of the key substrates in the cholesterol biosynthesis pathway. HCMECs were treated with (b) mevalonolactone 50 μM, GGPP 10 μM, FPP 10 μM, squalene 10 μM, or cholesterol 10 μM for 24 hr in the presence and absence of simvastatin (0.3 μM for 6 hr). ERK5 expression was assessed by Western blotting. Unprenylation of Rap1A was measured as a control for simvastatin activity. The level of ERK5 activation assessed by mobility band shift was quantified and expressed as fold change relative to vehicle control ( n = 4) mean ± s.d. * p ≤ 0.05, ** p ≤ 0.01 compared to vehicle control basal. (c) HCMECs were treated with GGTI‐298 (10 μM) for a range of time points. Intracellular signaling responses were assessed by Western blotting for ERK5, unprenylated Rap1A and Rap1. (d) Immunofluorescence imaging of HCMEC tight junctions (ZO‐1, green), actin stress fibers (phalloidin, red), and nuclei (Hoechst, Blue) following treatment with GGTI‐298 (10 μM for 6 hr). Scale bars: 10 μm. (e) HCMEC were plated on Thincerts™ with 0.4 μm pores and permeability of 4 kDa FITC‐dextran across the HCMEC monolayer was assessed ( n = 4), mean ± s.d. * p ≤ 0.05 compared to vehicle control

Article Snippet: For immunoprecipitation, lysates were incubated with ERK5 (2 μg/ml, #AF2848, R&D systems) or control IgG (2 μg/ml, goat) and protein G agarose.

Techniques: Phospho-proteomics, Inhibition, Expressing, Western Blot, Control, Activity Assay, Activation Assay, Electrophoretic Mobility Shift Assay, Immunofluorescence, Imaging, Permeability

Journal: Journal of Cellular Physiology

Article Title: Statin regulated ERK5 stimulates tight junction formation and reduces permeability in human cardiac endothelial cells

doi: 10.1002/jcp.26064

Figure Lengend Snippet: MEK5 inhibition prevents simvastatin induced tight junction formation and ERK5 phosphorylation. HCMECs were pre‐incubated with BIX02189 1 μM for 30 min priopr to addition of simvastatin 0.3 μM for 6 hr. (a) Immunofluorescence imaging of HCMEC tight junctions (ZO‐1, green), actin stress fibers (phalloidin, red), and nuclei (Hoechst, blue). Scale bars: 10 μm. Results are from one experiment representative of three. (b) Western blot of ZO‐1, ERK5, phospho ERK5, unprenylated Rap1A, and GAPDH levels in HCMECs. Level of ERK5 phosphorylation is quantified relative to vehicle control. Mean ± s.d. ( n = 3) * p ≤ 0.05 compared to vehicle control basal. (c) Permeability of 4 kDa FITC‐dextran through an endothelial monolayer on ThinCerts™, ( n = 4), mean ± s.d. * p ≤ 0.05, ** p ≤ 0.01 compared to vehicle control. (d) Assessment of TEER was conducted on HCMECs plated on Thincerts™ containing 0.4 μm pores ( n = 4), mean ± s.d. ** p ≤ 0.01 compared to vehicle control. (e) HCMECs were treated with simvastatin 0.3 μM for 6 hr before immunoprecipitation with IgG or ERK5 antibodies. Western blot of ZO‐1 following immunoprecipitation with ERK5 in HCMECs. (f) Level of ZO‐1 and ERK5 is quantified relative to vehicle control in IP lysates. Mean ± s.d. ( n = 3). ** p ≤ 0.01, compared to vehicle control

Article Snippet: For immunoprecipitation, lysates were incubated with ERK5 (2 μg/ml, #AF2848, R&D systems) or control IgG (2 μg/ml, goat) and protein G agarose.

Techniques: Inhibition, Phospho-proteomics, Incubation, Immunofluorescence, Imaging, Western Blot, Control, Permeability, Immunoprecipitation

Journal: Journal of Cellular Physiology

Article Title: Statin regulated ERK5 stimulates tight junction formation and reduces permeability in human cardiac endothelial cells

doi: 10.1002/jcp.26064

Figure Lengend Snippet: Simvastatin prevents doxorubicin induced barrier perturbment. HCMEC were pre‐incubated with simvastatin 0.3 μM for 6 hr before treatment with doxorubicin 0.1 μM for a further 6 hr. (a) Immunofluorescence imaging of HCMEC tight junctions (ZO‐1, green), actin stress fibers (phalloidin, red), and nuclei (Hoechst, blue). Scale bars: 10 μm. Results are from one experiment representative of three. (b) Western blot of ZO‐1, ERK5, ERK1/2, unprenylated Rap1A and Rap1 levels in HCMECs. Level of ERK5 activation is quantified relative to vehicle control. Mean ± s.d. ( n = 3) ** p ≤ 0.01 compared to vehicle control. (c) HCMEC were plated on Thincerts™ with 0.4 μm pores and permeability of 4 kDa FITC‐dextran across the HCMEC monolayer was assessed and compared to vehicle control ( n = 4), mean ± s.d. * p ≤ 0.05, ** p ≤ 0.01 compared to vehicle control. (d) Assessment of TEER was conducted on HCMECs plated on Thincerts™ with 0.4 μm pores ( n = 4), mean ± s.d. * p ≤ 0.05, ** p ≤ 0.01 compared to vehicle control

Article Snippet: For immunoprecipitation, lysates were incubated with ERK5 (2 μg/ml, #AF2848, R&D systems) or control IgG (2 μg/ml, goat) and protein G agarose.

Techniques: Incubation, Immunofluorescence, Imaging, Western Blot, Activation Assay, Control, Permeability

Journal: Journal of Cellular Physiology

Article Title: Statin regulated ERK5 stimulates tight junction formation and reduces permeability in human cardiac endothelial cells

doi: 10.1002/jcp.26064

Figure Lengend Snippet: ERK5 and ZO‐1 colocalize following simvastatin treatment in HCMECs. Immunofluorescence imaging of HCMEC tight junctions (ZO‐1, green), ERK5 (red), and nuclei (Hoechst, blue) following treatment with simvastatin 0.3 μM and/or doxorubicin 0.1 μM or BIX02189 1 μM for 6 hr. Scale bars: 10 μm. Arrows indicate co‐localization. Results are from one experiment representative of three

Article Snippet: For immunoprecipitation, lysates were incubated with ERK5 (2 μg/ml, #AF2848, R&D systems) or control IgG (2 μg/ml, goat) and protein G agarose.

Techniques: Immunofluorescence, Imaging

Journal: Journal of Cellular Physiology

Article Title: Statin regulated ERK5 stimulates tight junction formation and reduces permeability in human cardiac endothelial cells

doi: 10.1002/jcp.26064

Figure Lengend Snippet: ERK5 cellular translocation following simvastatin treatment in HCMECs. HCMECs were treated with simvastatin 0.3 μM for 6 hr before subcellular fractionation performed. (a) Western blot of ZO‐1 and ERK5. ATP1A1, actin, and lamin B were used as positive controls to show cellular fraction specificity in membrane, cytoplasm, and nuclear fractions respectively. Whole cell lysate (WCL). (b) Quantification of the total ERK5 level in each compartment ( n = 3), mean ± s.d. * p ≤ 0.05 compared to vehicle control for each fraction. (c) Quantification of ERK5 band shift relative to each fraction's vehicle control ( n = 3), mean ± s.d. * p ≤ 0.05 compared to vehicle control for each fraction

Article Snippet: For immunoprecipitation, lysates were incubated with ERK5 (2 μg/ml, #AF2848, R&D systems) or control IgG (2 μg/ml, goat) and protein G agarose.

Techniques: Translocation Assay, Fractionation, Western Blot, Membrane, Control, Electrophoretic Mobility Shift Assay

Journal: Molecular cell

Article Title: Stabilization of ERK-Phosphorylated METTL3 by USP5 Increases m 6 A Methylation

doi: 10.1016/j.molcel.2020.10.026

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ERK5 (D315V) Rabbit mAb , Cell signaling , 12950.

Techniques: Control, Recombinant, Luciferase, Mutagenesis, Modification, Multiplex Assay, CRISPR, Generated, Ubiquitin Proteomics, Software

Journal: Molecular Endocrinology

Article Title: ERα-XPO1 Cross Talk Controls Tamoxifen Sensitivity in Tumors by Altering ERK5 Cellular Localization

doi: 10.1210/me.2016-1101

Figure Lengend Snippet: A, ERK5 (upper panel) and pERK5 (lower panel) immunostaining after 45 minutes of 4-OH-TAM treatment in tamoxifen-sensitive MCF-7 cells and tamoxifen-resistant MCF-7 TAM R cells that were kept in 4-OH-TAM for the indicated times. MCF-7 cells or MCF-7 TAM R cells at different stages of resistance progression were treated with 1 μM 4-OH-TAM for 45 minutes, and immunofluorescence microscopy was performed with an antibody specific to ERK5 or pERK5. Nuclei were stained with DAPI. Three fields per slide were quantified (n = 3). A one-way ANOVA model was fitted to assess the contribution of tamoxifen resistance progression on 4-OH-TAM treatment-induced ERK5 or pERK5 nuclear localization. When the main effects were statistically significant at α = .05, pairwise t tests with a Newman-Keuls correction were used to identify the time that ERK5 or pERK5 localization was significantly different from parental MCF-7 cells. *, P < .05; **, P < .01; ***, P < .001; ****, P < .0001. B, Nuclear localization of pERK5 decreases as tamoxifen resistance progresses. MCF-7 parental cells or MCF-7 TAM R cells that were kept in media containing 4-OH-TAM for 50 or 100 weeks were fractionated, and whole-cell lysate and cytosolic or nuclear fractions were subjected to Western blot analysis using pERK5, Lamin1b (as nuclear fraction marker), and β-actin (as cytosolic fraction marker) antibodies. C, ERK5 activity regulated migratory potential in MCF-7 TAM R cells. MCF-7 TAM R cells were infected with AdCMV or dominant-negative AdERK5 for 24 hours and then seeded on the upper chamber of a transwell system for migration assays (B) or invasion assays (C). The number of cells that migrated/invaded to the bottom side of the chambers was counted. Values are presented as mean ± SEM from two independent experiments. AdERK5, adenovirus construct for ERK5.

Article Snippet: The following antibodies were used: pERK5 Thr218/Tyr 220 (number 3371; Cell Signaling), Lamin B1 (ab16048; Abcam), NUP153 (A301–789A), KPNA3 (A301–626A), NUP205 (A303–935A), RANGAP1 (A302–026A), KPNA2 (A300–483A), XPO1 (A300–469A), ERK5 (A302–656A), all from Bethyl Laboratories, and ERK2 antibody (D-2 sc-1647; Santa Cruz Biotechnology) were used in 1:1000 dilution. β-Actin (Sigma; SAB1305546) was used in 1:10 000 dilution.

Techniques: Immunostaining, Immunofluorescence, Microscopy, Staining, Western Blot, Marker, Activity Assay, Infection, Dominant Negative Mutation, Migration, Construct

Journal: Molecular Endocrinology

Article Title: ERα-XPO1 Cross Talk Controls Tamoxifen Sensitivity in Tumors by Altering ERK5 Cellular Localization

doi: 10.1210/me.2016-1101

Figure Lengend Snippet: A, Verification of XPO-1 levels in patient tumor samples. mRNA from tumor FFPE samples were isolated. After the quality of RNA was verified, qPCR was run for XPO1 and 36B4 as control. A one-way ANOVA model was fitted to assess the contribution of subtype to expression of XPO1 mRNA. When the main effects were statistically significant at α = .05, pairwise t tests with a Newman-Keuls correction were used to identify the subtype that had the highest level of XPO1 expression. ***, P < .01. B, Impact of XPO1 inhibitor on cell cycle progression. Cells were treated with Veh or 1 μM 4-OH-TAM in the presence or absence of 10 −7 M SXR. Cell numbers were examined using the FACS analysis. C, Anchorage-independent growth of tamoxifen-resistant BT474 cells treated or nontreated with XPO1 inhibitor (SXR). Colony formation was visualized with Giemsa staining. On the right are representative pictures of spheroid, single colonies formed by BT474 cells. A two-way ANOVA model was fitted to assess the contribution of ligand (Veh, E2, or tamoxifen) and inhibitor (Ctrl and SXR) treatment on anchorage-dependent growth of BT474 cells. When the main effects were statistically significant at α = .05, pairwise t tests with a Bonferroni correction were used to identify whether treatment were statistically different from each other. **, P < .01; ****, P < .0001. D, XPO1 overexpression in MCF-7 and BT474 cell increase ERK5 activation. MCF7 cells (left panel) or BT474 cells (right panel) were infected with AdCMV as control or AdXPO1. XPO1 mRNA overexpression was detected by qPCR. XPO1 protein overexpression was assessed by Western blot of immunofluorescence analysis. A t test was applied to assess whether AdXPO1 virus infection resulted in statistically significant overexpression of XPO1 in each cell line. *, P < .05 E, XPO1 overexpression in MCF-7 and BT474 cells increase cell proliferation. MCF-7 cells (left panel) and BT474 cells (right panel) were infected with AdCMV as control or AdXPO1, and then cells were treated with increasing doses of 4-OH-TAM in the presence or absence of 100 nM SXR. Ctrl, control; FACS, fluorescence-activated cell sorter.

Article Snippet: The following antibodies were used: pERK5 Thr218/Tyr 220 (number 3371; Cell Signaling), Lamin B1 (ab16048; Abcam), NUP153 (A301–789A), KPNA3 (A301–626A), NUP205 (A303–935A), RANGAP1 (A302–026A), KPNA2 (A300–483A), XPO1 (A300–469A), ERK5 (A302–656A), all from Bethyl Laboratories, and ERK2 antibody (D-2 sc-1647; Santa Cruz Biotechnology) were used in 1:1000 dilution. β-Actin (Sigma; SAB1305546) was used in 1:10 000 dilution.

Techniques: Isolation, Control, Expressing, Staining, Over Expression, Activation Assay, Infection, Western Blot, Immunofluorescence, Virus, Fluorescence

Journal: Molecular Endocrinology

Article Title: ERα-XPO1 Cross Talk Controls Tamoxifen Sensitivity in Tumors by Altering ERK5 Cellular Localization

doi: 10.1210/me.2016-1101

Figure Lengend Snippet: As ERα (+) breast tumors acquire resistance to tamoxifen, a group of nuclear transport proteins including XPO1 will be up-regulated, increasing ERK5 export from nucleus. Thus, 1) ERα, which is in the nucleus, will not have the partners to elicit proper transcriptional responses to tamoxifen and 2) ERK5, which partners with other cytoplasmic proteins, will now contribute to increased tumorigenicity and tamoxifen resistance.

Article Snippet: The following antibodies were used: pERK5 Thr218/Tyr 220 (number 3371; Cell Signaling), Lamin B1 (ab16048; Abcam), NUP153 (A301–789A), KPNA3 (A301–626A), NUP205 (A303–935A), RANGAP1 (A302–026A), KPNA2 (A300–483A), XPO1 (A300–469A), ERK5 (A302–656A), all from Bethyl Laboratories, and ERK2 antibody (D-2 sc-1647; Santa Cruz Biotechnology) were used in 1:1000 dilution. β-Actin (Sigma; SAB1305546) was used in 1:10 000 dilution.

Techniques:

Journal: eLife

Article Title: Targeting mir128-3p alleviates myocardial insulin resistance and prevents ischemia-induced heart failure

doi: 10.7554/eLife.54298

Figure Lengend Snippet: ( A ) Pri-mir128-1 promotor luciferase activity in H9C2 was increased by long-term hypoxia (N = 4 experiments). ( B ) Schematic figure showing CEBP consensus binding sites in the intronic promoter region between exon1 and exon2 of R3hdm1 gene conserved in human, rat, and mouse. ( C–D ) Cebpβ was downregulated in myocardium after 4w-MI (N = 6–8 mice) and ARCMs (N = 4 experiments) stressed by short and long-lasting hypoxia. ( E–F ) Measurements of Pri-mir128-1 , mir128-3p and Irs1 were performed in ARCMs with CEBPβ overexpression ( Cebpb o/e) under long-term hypoxia or Cebpb knockdown in response to short-term hypoxia (N = 4–6 experiments). ( G ) Pri-mir128-1 promotor luciferase activity was augmented by CEBPβ deficiency (N = 3 experiments). ( H ) MAPK7 (also known as ERK5) phosphorylation and expression were decreased in the myocardium four weeks after MI (N = 7 mice). ( I ) MAPK7 was activated by acute hypoxic stress, while it was declined by prolonged stimulation in ARCMs (N = 3 experiments). ( J–K ) qPCR analyses of Cebpb and mir128-3p in Mapk7 -deficient or Mapk7 -overexpressing ( Mapk7 o/e) ARCMs (N = 6 experiments). ( L ) Chromatin Immunoprecipitation (ChIP) was performed on ARCMs DNA using anti-CEBPβ antibody (anti-IgG antibody as negative control) (N = 3–5 experiments). Hypoxia-induced CEBPβ binding to the proximal Pri-mir128-1 promoter was inhibited by MAPK7 deficiency. Data were normalized to the input chromatin. ( M ) Immunofluorescent staining of ARCMs demonstrated that hypoxia-triggered nuclear translocation of phosphorylated CREB1 or CEBPβ (red) was blocked by Mapk7 knockdown. DAPI was used for nuclear staining (blue) (N = 5 experiments). Scale bar: 30 µm. ( N ) Immunoprecipitation showed that hypoxia promoted association of phosphorylated CREB1 and CEBPβ in ARCMs; however, the affinity was reduced in the absence of MAPK7. IgG was used as negative control. ( O ) Decreased CEBPβ and reduced IRS1 were detected in the absence of MAPK7 or CREB1, while the reduction was rescued by CREB1 overexpression in Mapk7 -knockdown ARCMs (N = 6 experiments). Data presented as mean ± SEM. Comparisons between two groups were performed using two-tailed Student’s t-test. One-way or two-way ANOVA followed by Bonferroni post hoc tests were employed as appropriate. Figure 3—source data 1. Role of MAPK7 and CEBPβ in mir128-3p transcriptional regulation.

Article Snippet: Cells were transfected with 100 nmol/L of control siRNA (AGGUAGUGUAAUCGCCUUG, Sigma), rat Mapk7 siRNA (AAAGGGUGCGAGCCUAUAU, Sigma), rat Creb1 siRNA (Silencer Select s135439, Ambion), rat Cebpb siRNA (Silencer Select s127566, Ambion), rat Irs1 siRNA (Silencer Select s129870, Ambion), human Mapk7 siRNA (SignalSilencer 7301, Cell Signaling), human Mapk7 cDNA (a gift from Cathy Tournier’s lab), human Creb1 cDNA (ID #82203, Addgene), or human Cebpb cDNA (isoform of LAP, ID #15738, Addgene) following the manufacturer’s instructions.

Techniques: Luciferase, Activity Assay, Binding Assay, Over Expression, Knockdown, Phospho-proteomics, Expressing, Chromatin Immunoprecipitation, Negative Control, Staining, Translocation Assay, Immunoprecipitation, Two Tailed Test

Journal: eLife

Article Title: Targeting mir128-3p alleviates myocardial insulin resistance and prevents ischemia-induced heart failure

doi: 10.7554/eLife.54298

Figure Lengend Snippet: ( A ) Cebpa transcript expression was not changed in 4w-MI myocardium, while ( B ) Mapk7 was decreased measured by qPCR (N = 6–8 mice). Data were normalized against 18S expression. Data presented as mean ± SEM. Groups were compared using two-tailed Student’s t-test. Figure 3—figure supplement 2—source data 1. Mapk7 and Cebpa expression in 4w-MI myocardium.

Article Snippet: Cells were transfected with 100 nmol/L of control siRNA (AGGUAGUGUAAUCGCCUUG, Sigma), rat Mapk7 siRNA (AAAGGGUGCGAGCCUAUAU, Sigma), rat Creb1 siRNA (Silencer Select s135439, Ambion), rat Cebpb siRNA (Silencer Select s127566, Ambion), rat Irs1 siRNA (Silencer Select s129870, Ambion), human Mapk7 siRNA (SignalSilencer 7301, Cell Signaling), human Mapk7 cDNA (a gift from Cathy Tournier’s lab), human Creb1 cDNA (ID #82203, Addgene), or human Cebpb cDNA (isoform of LAP, ID #15738, Addgene) following the manufacturer’s instructions.

Techniques: Expressing, Two Tailed Test

Journal: eLife

Article Title: Targeting mir128-3p alleviates myocardial insulin resistance and prevents ischemia-induced heart failure

doi: 10.7554/eLife.54298

Figure Lengend Snippet: MAPK7 activation was observed in neonatal rat cardiomyocytes (NRCMs) in response to short-term hypoxia, whereas MAPK7 was decreased by prolonged stress. Quantification is provided (N = 3–6 experiments). Data presented as mean ± SEM. One-way ANOVA followed by Bonferroni post hoc tests was employed to compare groups. Figure 3—figure supplement 4—source data 1. MAPK7 activation and expression in short and long-term hypoxia.

Article Snippet: Cells were transfected with 100 nmol/L of control siRNA (AGGUAGUGUAAUCGCCUUG, Sigma), rat Mapk7 siRNA (AAAGGGUGCGAGCCUAUAU, Sigma), rat Creb1 siRNA (Silencer Select s135439, Ambion), rat Cebpb siRNA (Silencer Select s127566, Ambion), rat Irs1 siRNA (Silencer Select s129870, Ambion), human Mapk7 siRNA (SignalSilencer 7301, Cell Signaling), human Mapk7 cDNA (a gift from Cathy Tournier’s lab), human Creb1 cDNA (ID #82203, Addgene), or human Cebpb cDNA (isoform of LAP, ID #15738, Addgene) following the manufacturer’s instructions.

Techniques: Activation Assay, Expressing

Journal: eLife

Article Title: Targeting mir128-3p alleviates myocardial insulin resistance and prevents ischemia-induced heart failure

doi: 10.7554/eLife.54298

Figure Lengend Snippet: Chromatin immunoprecipitation (ChIP) assay was performed on ARCMs DNA using anti-CEBPβ antibody (anti-IgG antibody as the negative control). The enrichment of CEBPβ binding to different regions in Pri-mir128-1 intronic promoter localized between exon1 and exon2 of R3hdm1 was examined by qPCR in the presence and absence of Mapk7 in response to hypoxia. TSS: transcription start site. Data were normalized to input chromatin (N = 3–5 experiments). Data presented as mean ± SEM. Two-way ANOVA followed by Bonferroni post hoc tests was employed to compare groups. Figure 3—figure supplement 5—source data 1. CEBPβ binding analysis to Pri-mir128-1 intronic promoter.

Article Snippet: Cells were transfected with 100 nmol/L of control siRNA (AGGUAGUGUAAUCGCCUUG, Sigma), rat Mapk7 siRNA (AAAGGGUGCGAGCCUAUAU, Sigma), rat Creb1 siRNA (Silencer Select s135439, Ambion), rat Cebpb siRNA (Silencer Select s127566, Ambion), rat Irs1 siRNA (Silencer Select s129870, Ambion), human Mapk7 siRNA (SignalSilencer 7301, Cell Signaling), human Mapk7 cDNA (a gift from Cathy Tournier’s lab), human Creb1 cDNA (ID #82203, Addgene), or human Cebpb cDNA (isoform of LAP, ID #15738, Addgene) following the manufacturer’s instructions.

Techniques: Chromatin Immunoprecipitation, Negative Control, Binding Assay

Journal: eLife

Article Title: Targeting mir128-3p alleviates myocardial insulin resistance and prevents ischemia-induced heart failure

doi: 10.7554/eLife.54298

Figure Lengend Snippet: Potential MAPK7 downstream effectors were analysed in ARCMs by immunoblots and quantification. CREB1 phosphorylation under short-term hypoxia was blocked by Erk5 deficiency (N = 4 experiments). Data presented as mean ± SEM. Two-way ANOVA followed by Bonferroni post hoc tests was employed to compare groups. Figure 3—figure supplement 6—source data 1. Analysis of MAPK7 downstream effectors after hypoxia.

Article Snippet: Cells were transfected with 100 nmol/L of control siRNA (AGGUAGUGUAAUCGCCUUG, Sigma), rat Mapk7 siRNA (AAAGGGUGCGAGCCUAUAU, Sigma), rat Creb1 siRNA (Silencer Select s135439, Ambion), rat Cebpb siRNA (Silencer Select s127566, Ambion), rat Irs1 siRNA (Silencer Select s129870, Ambion), human Mapk7 siRNA (SignalSilencer 7301, Cell Signaling), human Mapk7 cDNA (a gift from Cathy Tournier’s lab), human Creb1 cDNA (ID #82203, Addgene), or human Cebpb cDNA (isoform of LAP, ID #15738, Addgene) following the manufacturer’s instructions.

Techniques: Western Blot, Phospho-proteomics

Journal: eLife

Article Title: Targeting mir128-3p alleviates myocardial insulin resistance and prevents ischemia-induced heart failure

doi: 10.7554/eLife.54298

Figure Lengend Snippet: The impacts of Mapk7 knockdown on CREB1 activation and CEBPβ translocation to the nucleus in NRCMs in response to short-term hypoxia, which was determined by ( A ) immunoblots and ( B ) immunofluorescent detection of nuclear translocation of pCREB1 (green in upper panels) and CEBPβ (red in lower panels). DAPI (blue) stains nuclei (N = 3 experiments). Scale bar: 50 µm. Data presented as mean ± SEM. Two-way ANOVA followed by Bonferroni post hoc tests was employed to compare groups. Figure 3—figure supplement 7—source data 1. Hypoxia-induced CREB1 and CEBPβ nuclear translocation in NRCMs.

Article Snippet: Cells were transfected with 100 nmol/L of control siRNA (AGGUAGUGUAAUCGCCUUG, Sigma), rat Mapk7 siRNA (AAAGGGUGCGAGCCUAUAU, Sigma), rat Creb1 siRNA (Silencer Select s135439, Ambion), rat Cebpb siRNA (Silencer Select s127566, Ambion), rat Irs1 siRNA (Silencer Select s129870, Ambion), human Mapk7 siRNA (SignalSilencer 7301, Cell Signaling), human Mapk7 cDNA (a gift from Cathy Tournier’s lab), human Creb1 cDNA (ID #82203, Addgene), or human Cebpb cDNA (isoform of LAP, ID #15738, Addgene) following the manufacturer’s instructions.

Techniques: Knockdown, Activation Assay, Translocation Assay, Western Blot

Journal: eLife

Article Title: Targeting mir128-3p alleviates myocardial insulin resistance and prevents ischemia-induced heart failure

doi: 10.7554/eLife.54298

Figure Lengend Snippet: ( A ) Experimental design of male cardiac-specific Mapk7 -deleted mice subject to 1w-MI. ( B ) Echocardiography determined detrimental cardiac function in Mapk7 -cko mice post-MI, indicated by fractional shortening (%) and sLVID (N = 9–11 mice). ( C–E ) MAPK7 ablation led to increased serum cTnI (24 hr post-ligation) and LDH (one week post-ligation) concentration, TTC-detected infarct area (from one heart, scale bar: 1 mm), and interstitial fibrosis in the non-infarct region detected by Masson’s Trichrome (scale bar: 100 μm) (N = 8–10 mice). ( F ) Immunoblots analyses of phosphorylation and expression of CREB1 in the myocardium (N = 6 mice). ( G–H ) Real-time qPCR detected reduced Cebpb accompanied by upregulated mature mir128-3p due to Mapk7 deletion in response to MI (N = 5–8 mice). ( I–J ) Transcript and protein level of IRS1 was decreased by MAPK7 disruption (N = 5–8 mice). ( K ) Myocardial insulin sensitivity was assessed by phosphorylation of IRS1 and AKT after insulin injection (1 U/kg for 30 mins) (N = 5–6 mice). ( L–N ) In ARCMs, the effects of Mapk7 knockdown on Irs1 transcript, IRS1 protein level, and insulin-stimulated (0.1 µM for 30 mins) phosphorylation of IRS1 and AKT were observed upon 2 hr hypoxia (N = 4–6 experiments). ( O–P ) SLC2A4 plasma membrane translocation was detected by immunoblots of the membrane fraction and confocal images of MYC- Slc2a4 -mCherry immunofluorescence in H9C2 cells (N = 5 experiments). Fused mCherry (red) allows for SLC2A4 detection, while membrane translocated SLC2A4 is visualized by MYC (green). DAPI stains nuclear (blue). Scale bar: 25 µm. ( Q ) Insulin-dependent (0.1 µM for 30 mins) glucose uptake capacity under hypoxia was deferred in Mapk7 or Irs1 deficient ARCMs (N = 3 experiments). ( R ) ATP production was reduced in Mapk7 -knockdown ARCMs under hypoxic condition (N = 3 experiments). Data presented as mean ± SEM. Comparisons between two groups were performed using two-tailed Student’s t-test. Two-way ANOVA followed by Bonferroni post hoc tests was employed as appropriate. Figure 4—source data 1. Effect of MAPK7 deficiency on cardiac insulin resistance and in vivo and in vitro. Figure 4—source data 2. Echocardiographic parameters of Mapk7 -cko and control mice one week post-MI.

Article Snippet: Cells were transfected with 100 nmol/L of control siRNA (AGGUAGUGUAAUCGCCUUG, Sigma), rat Mapk7 siRNA (AAAGGGUGCGAGCCUAUAU, Sigma), rat Creb1 siRNA (Silencer Select s135439, Ambion), rat Cebpb siRNA (Silencer Select s127566, Ambion), rat Irs1 siRNA (Silencer Select s129870, Ambion), human Mapk7 siRNA (SignalSilencer 7301, Cell Signaling), human Mapk7 cDNA (a gift from Cathy Tournier’s lab), human Creb1 cDNA (ID #82203, Addgene), or human Cebpb cDNA (isoform of LAP, ID #15738, Addgene) following the manufacturer’s instructions.

Techniques: Ligation, Concentration Assay, Western Blot, Phospho-proteomics, Expressing, Disruption, Injection, Knockdown, Clinical Proteomics, Membrane, Translocation Assay, Immunofluorescence, Two Tailed Test, In Vivo, In Vitro, Control

Journal: eLife

Article Title: Targeting mir128-3p alleviates myocardial insulin resistance and prevents ischemia-induced heart failure

doi: 10.7554/eLife.54298

Figure Lengend Snippet: ( A ) Contrary to the analysis after 4w-MI, MAPK7 activation was observed after 1w-MI in Mapk7 flox mice (N = 4–6 mice). Cardiac hypertrophic growth in Mapk7- cko and Mapk7 flox mice was evaluated by ( B ) heart weight vs body weight ratio (N = 9–10 mice), and ( C ) cardiomyocyte size measured from H and E stained heart sections (N = 4–8 mice). Scale bar: 50 µm. Data presented as mean ± SEM. Comparisons between two groups were performed using two-tailed Student’s t-test. Two-way ANOVA followed by Bonferroni post hoc tests was employed as appropriate. Figure 4—figure supplement 1—source data 1. MAPK7 activation and hypertrophy in mouse myocardium one week post-MI.

Article Snippet: Cells were transfected with 100 nmol/L of control siRNA (AGGUAGUGUAAUCGCCUUG, Sigma), rat Mapk7 siRNA (AAAGGGUGCGAGCCUAUAU, Sigma), rat Creb1 siRNA (Silencer Select s135439, Ambion), rat Cebpb siRNA (Silencer Select s127566, Ambion), rat Irs1 siRNA (Silencer Select s129870, Ambion), human Mapk7 siRNA (SignalSilencer 7301, Cell Signaling), human Mapk7 cDNA (a gift from Cathy Tournier’s lab), human Creb1 cDNA (ID #82203, Addgene), or human Cebpb cDNA (isoform of LAP, ID #15738, Addgene) following the manufacturer’s instructions.

Techniques: Activation Assay, Staining, Two Tailed Test

Journal: eLife

Article Title: Targeting mir128-3p alleviates myocardial insulin resistance and prevents ischemia-induced heart failure

doi: 10.7554/eLife.54298

Figure Lengend Snippet: Real-time qPCR detection of ( A ) Pri-mir128-1 and Pri-mir128-2 , ( B ) mir144-3p and mir145-5p and ( C ) Cebpa in Mapk7 flox and Mapk7- cko subject to 1 week MI (N = 5–8 mice). ( D ) qPCR determined that Insr , Slc2a1 and Slc2a4 transcripts levels were not altered in Mapk7- cko one week post-MI (N = 5–8 mice). Data werenormalized against 18S or mir191-5p . Data presented as mean ± SEM. Two-way ANOVA followed by Bonferroni post hoc tests was employed to compare groups. Figure 4—figure supplement 2—source data 1. Gene expression comparison between Mapk7 flox and Mapk7- cko mice one week post-MI.

Article Snippet: Cells were transfected with 100 nmol/L of control siRNA (AGGUAGUGUAAUCGCCUUG, Sigma), rat Mapk7 siRNA (AAAGGGUGCGAGCCUAUAU, Sigma), rat Creb1 siRNA (Silencer Select s135439, Ambion), rat Cebpb siRNA (Silencer Select s127566, Ambion), rat Irs1 siRNA (Silencer Select s129870, Ambion), human Mapk7 siRNA (SignalSilencer 7301, Cell Signaling), human Mapk7 cDNA (a gift from Cathy Tournier’s lab), human Creb1 cDNA (ID #82203, Addgene), or human Cebpb cDNA (isoform of LAP, ID #15738, Addgene) following the manufacturer’s instructions.

Techniques: Gene Expression, Comparison

Journal: eLife

Article Title: Targeting mir128-3p alleviates myocardial insulin resistance and prevents ischemia-induced heart failure

doi: 10.7554/eLife.54298

Figure Lengend Snippet: ( A ) Immunoblots illustrated that insulin-stimulated (insulin IP injection, 1 U/kg for 30 mins) activation of IRS1 and AKT was at a comparable level in the myocardium from Mapk7 flox and Mapk7- cko under basal condition (N = 5–6 mice). ( B ) Glycogen in the hearts from 1w-MI stressed Mapk7 flox and Mapk7- cko mice was detected by Periodic Acid Schiff (PAS) staining. Diastase treatment was to discern unspecific staining (PAS-D) (N = 6–8 mice). Scale bar: 50 µm. Quantification of positive glycogen content is provided. Data presented as mean ± SEM. Groups were compared using two-tailed Student’s t-test. Figure 4—figure supplement 3—source data 1. Activation of the insulin cascade in sham mice and cardiac glycogen detection one week post-MI.

Article Snippet: Cells were transfected with 100 nmol/L of control siRNA (AGGUAGUGUAAUCGCCUUG, Sigma), rat Mapk7 siRNA (AAAGGGUGCGAGCCUAUAU, Sigma), rat Creb1 siRNA (Silencer Select s135439, Ambion), rat Cebpb siRNA (Silencer Select s127566, Ambion), rat Irs1 siRNA (Silencer Select s129870, Ambion), human Mapk7 siRNA (SignalSilencer 7301, Cell Signaling), human Mapk7 cDNA (a gift from Cathy Tournier’s lab), human Creb1 cDNA (ID #82203, Addgene), or human Cebpb cDNA (isoform of LAP, ID #15738, Addgene) following the manufacturer’s instructions.

Techniques: Western Blot, Injection, Activation Assay, Staining, Two Tailed Test

Journal: eLife

Article Title: Targeting mir128-3p alleviates myocardial insulin resistance and prevents ischemia-induced heart failure

doi: 10.7554/eLife.54298

Figure Lengend Snippet: ( A ) Insr , Slc2a1 and Slc2a4 levels were comparable in adult rat cardiomyocytes (ARCMs) with MAPK7 deficiency despite hypoxic stress (N = 6 experiments). ( B ) Irs1 was reduced in neonatal cardiomyocytes (NRCMs) due to Mapk7 knockdown, while the others remained at a similar level in response to hypoxia (N = 4–6 experiments). Data presented as mean ± SEM. Two-way ANOVA followed by Bonferroni post hoc tests was employed to compare groups. Figure 4—figure supplement 4—source data 1. Insulin-related gene expression in MAPK7-deficient rat cardiomyocytes under hypoxic stress.

Article Snippet: Cells were transfected with 100 nmol/L of control siRNA (AGGUAGUGUAAUCGCCUUG, Sigma), rat Mapk7 siRNA (AAAGGGUGCGAGCCUAUAU, Sigma), rat Creb1 siRNA (Silencer Select s135439, Ambion), rat Cebpb siRNA (Silencer Select s127566, Ambion), rat Irs1 siRNA (Silencer Select s129870, Ambion), human Mapk7 siRNA (SignalSilencer 7301, Cell Signaling), human Mapk7 cDNA (a gift from Cathy Tournier’s lab), human Creb1 cDNA (ID #82203, Addgene), or human Cebpb cDNA (isoform of LAP, ID #15738, Addgene) following the manufacturer’s instructions.

Techniques: Knockdown, Gene Expression

Journal: eLife

Article Title: Targeting mir128-3p alleviates myocardial insulin resistance and prevents ischemia-induced heart failure

doi: 10.7554/eLife.54298

Figure Lengend Snippet: ARCMs cell death under hypoxia was augmented by Mapk7 or Irs1 knockdown, as evidenced by increased ( A ) lactate dehydrogenase (LDH) released to the culture medium (N = 3 experiments), ( B ) cleaved caspase three level (N = 3 experiments), and ( C ) the number of TUNEL positive nuclei (green). DAPI (blue) detects nuclear and actinin (red) is cardiomyocyte indicator (N = 3 experiments). Scale bar: 100 µm. Data presented as mean ± SEM. Two-way ANOVA followed by Bonferroni post hoc tests was employed to compare groups. Figure 4—figure supplement 5—source data 1. Effect of Mapk7 and Irs1 knockdown in ARCMs cell death after hypoxia.

Article Snippet: Cells were transfected with 100 nmol/L of control siRNA (AGGUAGUGUAAUCGCCUUG, Sigma), rat Mapk7 siRNA (AAAGGGUGCGAGCCUAUAU, Sigma), rat Creb1 siRNA (Silencer Select s135439, Ambion), rat Cebpb siRNA (Silencer Select s127566, Ambion), rat Irs1 siRNA (Silencer Select s129870, Ambion), human Mapk7 siRNA (SignalSilencer 7301, Cell Signaling), human Mapk7 cDNA (a gift from Cathy Tournier’s lab), human Creb1 cDNA (ID #82203, Addgene), or human Cebpb cDNA (isoform of LAP, ID #15738, Addgene) following the manufacturer’s instructions.

Techniques: Knockdown, TUNEL Assay

Journal: eLife

Article Title: Targeting mir128-3p alleviates myocardial insulin resistance and prevents ischemia-induced heart failure

doi: 10.7554/eLife.54298

Figure Lengend Snippet: ( A ) MAPK7 expression in myocardium was restored to physiological level in Mapk7- cko mice using AAV9- Mapk7 injection (AAV9- Gfp as control) (N = 4–6 mice). Cardiac hypertrophic response was observed in Mapk7- cko hearts with restored MAPK7 expression 1 week after MI, indicated by ( B ) heart weight vs. body weight ratio (N = 5–7 mice) and ( C ) cardiomyocyte size (N = 4–7 mice). Data presented as mean ± SEM. One or two-way ANOVA followed by Bonferroni post hoc tests were employed as appropriate. Figure 5—figure supplement 1—source data 1. MAPK7 expression and cardiac hypertrophy in AAV9- Mapk7 -injected mice one week post-MI.

Article Snippet: Cells were transfected with 100 nmol/L of control siRNA (AGGUAGUGUAAUCGCCUUG, Sigma), rat Mapk7 siRNA (AAAGGGUGCGAGCCUAUAU, Sigma), rat Creb1 siRNA (Silencer Select s135439, Ambion), rat Cebpb siRNA (Silencer Select s127566, Ambion), rat Irs1 siRNA (Silencer Select s129870, Ambion), human Mapk7 siRNA (SignalSilencer 7301, Cell Signaling), human Mapk7 cDNA (a gift from Cathy Tournier’s lab), human Creb1 cDNA (ID #82203, Addgene), or human Cebpb cDNA (isoform of LAP, ID #15738, Addgene) following the manufacturer’s instructions.

Techniques: Expressing, Injection, Control

Journal: eLife

Article Title: Targeting mir128-3p alleviates myocardial insulin resistance and prevents ischemia-induced heart failure

doi: 10.7554/eLife.54298

Figure Lengend Snippet: ( A ) Experimental design of cardiac MAPK7 restoration in Mapk7 -cko mice via tail vein administration of AAV9- Mapk7 (1 × 10 11 viral particles), followed by LAD ligation for one week. ( B ) Echocardiography determined improved cardiac function by AAV9- Mapk7 injection, measured by fractional shortening (%) and sLVID (N = 5–8 mice). ( C–F ) MAPK7 restoration decreased serum cTnI (24 hr post-ligation) and LDH (one week post-ligation), infarct area (scale bar: 1 mm), interstitial fibrosis in non-infarct area (scale bar: 100 μm), and apoptotic cells detected by TUNEL assay by triple staining (scale bar: 30 µm) of TUNEL (green), DAPI (blue for nuclear) and α-actinin (red for marking cardiomyocytes). Arrows indicate TUNEL positive nuclei (N = 4–8 mice). ( G ) qPCR showed that Cebpb , Pri-mir128-1 , and mature mir128-3p were retained at the normal range by AAV9- Mapk7 injection (N = 6–8 mice). ( H–I ) Transcript and protein level of IRS1 was maintained by MAPK7 restoration (N = 4–8 mice). ( J ) Irs1 transcript in ARCMs was preserved by MAPK7 overexpression ( Mapk7 o/e) (N = 4 experiments). ( K ) Glucose uptake capacity of ARCMs was rescued by MAPK7 overexpression in response to insulin stimulation (0.1 µM for 30 mins) during long-term hypoxia (N = 3 experiments). Data presented as mean ± SEM. Comparisons between two groups were performed using two-tailed Student’s t-test. Two-way ANOVA followed by Bonferroni post hoc tests was employed as appropriate. Figure 5—source data 1. Functional and transcriptional effects of MAPK7 restoration in Mapk7 -cko mice after MI and rat cardiomyocytes under hypoxia. Figure 5—source data 2. Echocardiographic parameters of Mapk7 -cko mice injected with AAV9-Gfp or AAV9-Mapk7 one week post-MI.

Article Snippet: Cells were transfected with 100 nmol/L of control siRNA (AGGUAGUGUAAUCGCCUUG, Sigma), rat Mapk7 siRNA (AAAGGGUGCGAGCCUAUAU, Sigma), rat Creb1 siRNA (Silencer Select s135439, Ambion), rat Cebpb siRNA (Silencer Select s127566, Ambion), rat Irs1 siRNA (Silencer Select s129870, Ambion), human Mapk7 siRNA (SignalSilencer 7301, Cell Signaling), human Mapk7 cDNA (a gift from Cathy Tournier’s lab), human Creb1 cDNA (ID #82203, Addgene), or human Cebpb cDNA (isoform of LAP, ID #15738, Addgene) following the manufacturer’s instructions.

Techniques: Ligation, Injection, TUNEL Assay, Staining, Over Expression, Two Tailed Test, Functional Assay

Journal: eLife

Article Title: Targeting mir128-3p alleviates myocardial insulin resistance and prevents ischemia-induced heart failure

doi: 10.7554/eLife.54298

Figure Lengend Snippet: Infarct area detected by triphenyltetrazolium chloride (TTC) staining in (N = 8 mice). Mapk7 flox and Mapk7- cko mice with control injection and Mapk7- cko mice with AAV9- Mapk7 injection one week post-MI. Scale bar: 1 mm. Figure 5—figure supplement 2—source data 1. Infarct area comparison between Mapk7 flox and Mapk7- cko injected with AAV9- Mapk7 seven days after MI.

Article Snippet: Cells were transfected with 100 nmol/L of control siRNA (AGGUAGUGUAAUCGCCUUG, Sigma), rat Mapk7 siRNA (AAAGGGUGCGAGCCUAUAU, Sigma), rat Creb1 siRNA (Silencer Select s135439, Ambion), rat Cebpb siRNA (Silencer Select s127566, Ambion), rat Irs1 siRNA (Silencer Select s129870, Ambion), human Mapk7 siRNA (SignalSilencer 7301, Cell Signaling), human Mapk7 cDNA (a gift from Cathy Tournier’s lab), human Creb1 cDNA (ID #82203, Addgene), or human Cebpb cDNA (isoform of LAP, ID #15738, Addgene) following the manufacturer’s instructions.

Techniques: Staining, Control, Injection, Comparison

Journal: eLife

Article Title: Targeting mir128-3p alleviates myocardial insulin resistance and prevents ischemia-induced heart failure

doi: 10.7554/eLife.54298

Figure Lengend Snippet: ( A ) Experimental design of CEBPβ overexpression in Mapk7 -cko myocardium via tail vein injection of AAV9- Cebpb (1 × 10 11 viral particles), followed by MI for one week. ( B ) Echocardiography showing preserved cardiac function by AAV9- Cebpb injection (N = 5–7 mice). ( C–F ) CEBPβ overexpression reduced serum cTnI (24 hr post-ligation) and LDH (one week post-ligation), infarct area, interstitial fibrosis in the non-infarct region (scale bar: 100 μm), and TUNEL positive apoptotic cells (green, scale bar: 30 µm), co-stained with DAPI (blue) and α-actinin (red). Arrows indicate TUNEL positive nuclei (N = 5–7 mice). ( G ) Cardiomyocyte size was measured from H and E stained sections (N = 4–7 mice). ( H ) Pri-mir128-1 and mature mir128-3p levels were examined in the myocardium by qPCR (N = 3–7 mice). ( I–J ) qPCR and immunoblotting analyses showed the maintenance of IRS1 expression by CEBPβ overexpression after MI despite MAPK7 depletion (N = 3–7 mice). Data presented as mean ± SEM. Comparisons between two groups were performed using two-tailed Student’s t-test. Two-way ANOVA followed by Bonferroni post hoc tests was employed as appropriate. Figure 6—source data 1. CEBPβ overexpression effect on mir128-3p and Irs1 expression in Mapk7 -cko post-MI. Figure 6—source data 2. Echocardiographic parameters of Mapk7 -cko mice injected with AAV9-Gfp or AAV9- Cebpb one week post-MI.

Article Snippet: Cells were transfected with 100 nmol/L of control siRNA (AGGUAGUGUAAUCGCCUUG, Sigma), rat Mapk7 siRNA (AAAGGGUGCGAGCCUAUAU, Sigma), rat Creb1 siRNA (Silencer Select s135439, Ambion), rat Cebpb siRNA (Silencer Select s127566, Ambion), rat Irs1 siRNA (Silencer Select s129870, Ambion), human Mapk7 siRNA (SignalSilencer 7301, Cell Signaling), human Mapk7 cDNA (a gift from Cathy Tournier’s lab), human Creb1 cDNA (ID #82203, Addgene), or human Cebpb cDNA (isoform of LAP, ID #15738, Addgene) following the manufacturer’s instructions.

Techniques: Over Expression, Injection, Ligation, TUNEL Assay, Staining, Western Blot, Expressing, Two Tailed Test

Journal: eLife

Article Title: Targeting mir128-3p alleviates myocardial insulin resistance and prevents ischemia-induced heart failure

doi: 10.7554/eLife.54298

Figure Lengend Snippet: ( A ) Construction of antimir128-TuD built in the intronic region Gfp of AAV9- Gfp capsid. The sequence of antimir128-TuD is provided. ( B ) pmiRGlo- mir128-3p (four repeated complementary sequences of mir128-3p built in the 3’ region of pmiRGlo) luciferase reporter activity was inhibited by mir128-3p mimic in H9C2 cardiomyocytes, but recovered by antimir128-TuD (N = 3 experiments). ( C ) Antimir128-TuD increased IRS1 protein level in cardiomyocytes (N = 3 experiments). ( D ) Experimental design of AAV9-delivered antimir128-TuD into Mapk7 -cko mice by tail vein administration of AAV9- antimir128 (1 × 10 11 viral particles), followed by MI. ( E ) Cardiac function was evaluated by echocardiography (N = 6–7 mice). ( F–I ) AAV9- antimir128 inhibition of endogenous mir128-3p upregulated due to MAPK7 deficiency led to less serum cTnI (24 hr post-ligation) and LDH (one week post-ligation), infarct area, fibrosis (scale bar: 100 μm), and apoptosis is detected by TUNEL assay (green, scale bar: 30 µm), co-stained with DAPI (blue) and α-actinin (red). Arrows indicate TUNEL positive nuclei (N = 6–7 mice). ( J ) Cardiomyocyte size was measured according to H and E staining (N = 6–7 mice). ( K ) Mature mir128-3p was silenced by AAV9-antimir128 injection; however, Pri-mir128-1 upregulation was not affected (N = 6 mice). ( L–M ) qPCR and immunoblotting analyses illustrated that expression and activation of IRS1 and AKT were preserved by AAV9- antimir128 application in the absence of MAPK7 despite MI stress (N = 6–7 mice). Data presented as mean ± SEM. Comparisons between two groups were performed using two-tailed Student’s t-test. One-way or two-way ANOVA followed by Bonferroni post hoc tests were employed as appropriate. Figure 7—source data 1. Functional effect of silencing mir128-3p post-MI. Figure 7—source data 2. Echocardiographic parameters of Mapk7 -cko mice injected with AAV9-Control or AAV9- Antimir128 one week post-MI.

Article Snippet: Cells were transfected with 100 nmol/L of control siRNA (AGGUAGUGUAAUCGCCUUG, Sigma), rat Mapk7 siRNA (AAAGGGUGCGAGCCUAUAU, Sigma), rat Creb1 siRNA (Silencer Select s135439, Ambion), rat Cebpb siRNA (Silencer Select s127566, Ambion), rat Irs1 siRNA (Silencer Select s129870, Ambion), human Mapk7 siRNA (SignalSilencer 7301, Cell Signaling), human Mapk7 cDNA (a gift from Cathy Tournier’s lab), human Creb1 cDNA (ID #82203, Addgene), or human Cebpb cDNA (isoform of LAP, ID #15738, Addgene) following the manufacturer’s instructions.

Techniques: Sequencing, Luciferase, Activity Assay, Inhibition, Ligation, TUNEL Assay, Staining, Injection, Western Blot, Expressing, Activation Assay, Two Tailed Test, Functional Assay, Control

Journal: eLife

Article Title: Targeting mir128-3p alleviates myocardial insulin resistance and prevents ischemia-induced heart failure

doi: 10.7554/eLife.54298

Figure Lengend Snippet: ( A ) Transcripts of Mapk7 , Cebpb, and Irs1 were reduced in human iPSC-CMs in response to long-term hypoxia (N = 6 experiments). ( B ) Immunoblots and quantifications showed that phosphorylation of MAPK7 and CREB1 were accompanied by increased CEBPβ under short-term hypoxia (N = 3 experiments). ( C ) Mapk7 knockdown using its siRNA in iPSC-CMs was validated by qPCR (N = 6 experiments). ( D ) Phosphorylated CREB1 nuclear accumulation (red) induced by short-term hypoxia. However, Mapk7 knockdown blocked its nuclear deposition, counterstained with DAPI (blue) (N = 4 experiments, more than 50 nuclei measured per group per experiment). Scale bar: 20 µm. ( E–F ) qPCR and immunofluorescent staining (scale bar: 20 µm) determined that MAPK7 deficiency reduced expression and nuclear localization of CEBPβ (N = 4–6 experiments, more than 50 nuclei measured per group per experiment). ( G–H ) Augmented mature mir128-3p and decreased Irs1 were detected by qPCR in Mapk7 -knockdown iPSC-CMs under hypoxia (N = 3–6 experiments). ( I–J ) mir128-3p mimic abrogated insulin stimulation (0.1 µM for 30 mins)-induced glucose uptake in iPSC-CMs. On the contrary, mir128-3p antagomiR rescued insulin-dependent glucose uptake capability, albeit prolonged hypoxia circumstance (N = 3 experiments). Data presented as mean ± SEM. Comparisons between two groups were performed using two-tailed Student’s t-test. One-way or two-way ANOVA followed by Bonferroni post hoc tests were employed as appropriate. Figure 8—source data 1. Analysis of the MAPK7-CEBPβ- mir128-3p axis in human iPSC-CMs under hypoxia.

Article Snippet: Cells were transfected with 100 nmol/L of control siRNA (AGGUAGUGUAAUCGCCUUG, Sigma), rat Mapk7 siRNA (AAAGGGUGCGAGCCUAUAU, Sigma), rat Creb1 siRNA (Silencer Select s135439, Ambion), rat Cebpb siRNA (Silencer Select s127566, Ambion), rat Irs1 siRNA (Silencer Select s129870, Ambion), human Mapk7 siRNA (SignalSilencer 7301, Cell Signaling), human Mapk7 cDNA (a gift from Cathy Tournier’s lab), human Creb1 cDNA (ID #82203, Addgene), or human Cebpb cDNA (isoform of LAP, ID #15738, Addgene) following the manufacturer’s instructions.

Techniques: Western Blot, Phospho-proteomics, Knockdown, Staining, Expressing, Two Tailed Test

Journal: eLife

Article Title: Targeting mir128-3p alleviates myocardial insulin resistance and prevents ischemia-induced heart failure

doi: 10.7554/eLife.54298

Figure Lengend Snippet: Short-term ischemia/hypoxia activates MAPK7-CREB1, which facilitates CEBPβ expression and its nucleus translocation to act as transcriptional repressor on Pri-mir128-1 , consequently maintaining IRS1 in the myocardium. However, prolonged stress induces MAPK7 loss and defective CEBPβ repression on mir128-3p , which results in IRS1 loss and impaired insulin pathway. Targeting this signaling cascade manifests the beneficial effects on maintenance of cardiac insulin sensitivity in response to ischemic/hypoxic stress, which is evidenced by AAV9-delivered cardiac MAPK7 restoration, cardiac-specific Cebpb overexpression, or silencing endogenous mir128-3p . List of source data files.

Article Snippet: Cells were transfected with 100 nmol/L of control siRNA (AGGUAGUGUAAUCGCCUUG, Sigma), rat Mapk7 siRNA (AAAGGGUGCGAGCCUAUAU, Sigma), rat Creb1 siRNA (Silencer Select s135439, Ambion), rat Cebpb siRNA (Silencer Select s127566, Ambion), rat Irs1 siRNA (Silencer Select s129870, Ambion), human Mapk7 siRNA (SignalSilencer 7301, Cell Signaling), human Mapk7 cDNA (a gift from Cathy Tournier’s lab), human Creb1 cDNA (ID #82203, Addgene), or human Cebpb cDNA (isoform of LAP, ID #15738, Addgene) following the manufacturer’s instructions.

Techniques: Expressing, Translocation Assay, Over Expression