cardiac tissues specimens  (OriGene)

Journal: iScience

Article Title: A defective mechanosensing pathway affects fibroblast-to-myofibroblast transition in the old male mouse heart

doi: 10.1016/j.isci.2023.107283

Figure Lengend Snippet: The aging phenotype of the male cardiac fibroblast is associated with a decreased expression of Kindlin-2 (A) Representative Western blot depicting the difference in α-SMA protein level between the young and old male cardiac fibroblasts (upper panel) and the diagram representing the quantification from separated experiments (N = 5–6 per group) (lower panel). (B) Representative Western blot for the actin (“F” depicting F-actin/Polymerized, and “G” for globular/monomeric). Actin quantity was estimated by densitometry for each sample, and the final F-to-G-actin ratio is shown in an individual-plotted diagram on the lower panel (N = 9–10 per group). (C) Representative F-actin staining for young and old male cardiac fibroblasts seeded on glass, scale bar: 20 μm. N = 10 individual samples from 4 separate experiments. (D) Representative Western blot depicting the difference in Kindlin-2 protein level in the lysate from young versus old cultured cardiac fibroblasts. Normalized densitometry was shown on the lower panel as an averaged value of 6–8 separated experiments. (E) Staining for Kindlin-2 (green) with a DAPI nucleus counterstaining (blue) for young and old cardiac fibroblasts, scale bar: 20 μm. (F–H) Representative staining for F-actin (F), α-SMA (G) and Kindlin-2/α-SMA (H) in cultured old male and old female cardiac fibroblasts, seeded on glass. Representative pictures from 3 to 4 biological repeats. Scale bar: 20 μm. (I) Western blot depicting the F-/G-actin fractions in the lysate from old female versus old male cardiac fibroblasts. The diagram (lower panel) shows the relative comparison of the F-to-G-actin. N = 4. (J) Western blot demonstrating the level of Kindlin-2 and α-SMA in the lysates from young and old female cardiac fibroblasts. Normalized densitometry is depicted as an individual-dotted diagram for both Kindlin-2 (lower left) and α-SMA (lower right). P-value was shown with an "N.S." below the bar, indicating a non-significant difference. M denotes a molecular weight marker; the corresponding molecular weight is indicated on the right side of the blot. (K) Dot-plots depicting the correlation between the level of Kindlin-2 or F-/G-actin ratio (y axis) versus α-SMA (x axis). The regression line shows a positive correlation in male cells (left panel) but not in female cells (middle panel). There is also a positive correlation between the F-/G-actin ratio and the level of α-SMA (right panel). Statistics: All the data are shown as an averaged mean ± SEM. P-value was calculated as an Unpaired t-test with Welch’s correction. p value <0.05 is considered statistically significant.

Article Snippet: To ensure Kindlin-2 overexpression, old cardiac fibroblasts were transfected with 2 μg of Fermt2 Mouse Untagged clone plasmid (Origene #MC202155) by using FuGene transfection reagent.

Techniques: Expressing, Western Blot, Staining, Cell Culture, Comparison, Molecular Weight, Marker

Journal: iScience

Article Title: A defective mechanosensing pathway affects fibroblast-to-myofibroblast transition in the old male mouse heart

doi: 10.1016/j.isci.2023.107283

Figure Lengend Snippet: The impact of cardiac ECM on Kindlin-2 expression (A) Representative immunostaining of Kindlin-2 (red) and α-SMA (green) for tridimensional (3-D) culture of cardiac fibroblasts seeded on cardiac matrices from age-matched or un-matched mouse donors for 72 h. Representative pictures from 3 to 4 experiments per group (young vs. old) and two donors (young vs. old) per experiment. Note that the old male matrices tend to exhibit a more intense autofluorescence. Scale bar: 20 μm. (B) Immunostaining for α-SMA (green) and F-actin (red) in old male cardiac fibroblasts seeded on glass (left), on matrix from old male cells (middle) or matrix from young male cells (right) (scale bar: 20 μm). Nuclei were counterstained with DAPI. (C) Representative Western blot of Kindlin-2 expressed by cells seeded on age-matched and unmatched matrices in the 2-D experiment. The normalized dataset is shown in an individual-plotted diagram on the right panel (N = 5–6 per group). (D) Representative Western blot showing the level of Kindlin-2 expressed by old male and old female fibroblasts seeded on sex-matched or unmatched matrices. The normalized dataset is shown in an individual-plotted diagram on the right panel (N = 4–5 per group). YM, OM, OF denote the young male, old male, and old female, respectively. M denotes a molecular weight marker; the corresponding molecular weight is indicated on the right side of the blot. (E) Scanning electron microscopy pictures of decellularized 3-D matrices from young or old mouse male hearts (upper panel), young or old female hearts (middle panel) or decellularized 2-D matrices deposited by young or old male cardiac fibroblasts (lower panel). Red arrows indicate cleavage sites in the old male cardiac ECM, and cyan circles depict aggregates in the old female cardiac ECM. Scale bar: 2 μm. Pictures in enlarged frames (yellow squares) depict the presence of cleavage in the old male ECM and aggregates in the old female ECM. All the data are shown as an averaged mean ± SEM. For the experimental dataset involving 4 groups, Brown-Forsythe and Welch one-way ANOVA was used to test the significant difference among the four groups, to determine the significant difference among groups. p value <0.05 is considered statistically significant.

Article Snippet: To ensure Kindlin-2 overexpression, old cardiac fibroblasts were transfected with 2 μg of Fermt2 Mouse Untagged clone plasmid (Origene #MC202155) by using FuGene transfection reagent.

Techniques: Expressing, Immunostaining, Western Blot, Molecular Weight, Marker, Electron Microscopy

Journal: iScience

Article Title: A defective mechanosensing pathway affects fibroblast-to-myofibroblast transition in the old male mouse heart

doi: 10.1016/j.isci.2023.107283

Figure Lengend Snippet: Kindlin-2 can facilitate the maturation of old fibroblasts by promoting actin polymerization and α-SMA expression (A) Western-blot depicting a decrease in Kindlin-2 protein levels 72 h post-Kindlin-2 siRNA (siK2) transfection. Averaged densitometry was shown as an individual-plotted diagram (lower panel). (B) Representative pictures of Kindlin-2 immunostaining and F-actin staining, scale bar: 30 μm. (C) Representative Western blot of actin fractions in siK2- compared with control (scrambled)-transfected cells. The F-G actin ratio was calculated for each experimental sample and depicted in a mean ± SEM diagram (N = 5 per each experimental group). (D) Staining of F-actin shown in false color using the OrientationJ plugin (ImageJ algorithm) depicting the changes in cell polarization and actin fiber orientation in control young male cells (left) and in Kindlin-2 deficient cells (right). Scale bar: 20 μm. (E) Representative Western blot showing the increase in α-SMA protein levels in response to Kindlin-2 overexpression (K-OE). Densitometry (lower panel) is provided in an individual-plotted diagram for both Kindlin-2 and a-SMA (N = 3–5 per group). For each experiment, cells were transfected with either K-OE or CMV control). (F) Immunostaining of Kindlin-2 in K-OE and CMV control clones (scale bar: 20 μm). Representative picture from 3 separate experiments. (G) Representative Western blot of F-/G-actin fractions showing the enrichment of F-actin in K-OE lysates. The F-to-G actin ratio was calculated and plotted in a diagram (right panel N = 3–5 per group). M denotes a molecular weight marker; the corresponding molecular weight is indicated on the right side of the blot. (H) Staining of F-actin shown in false color using the OrientationJ plugin (ImageJ algorithm) depicting the changes in cell polarization and actin fiber orientation in control old male cells (left) and in Kindlin-2 overexpressing cells (right). (I) Staining for F-actin (red) and α-SMA (green) in K-OE cells seeded on glass, or in 2-D matrices, compared with old control cells seeded on glass (scale bar: 20 μm). Representative picture from 3 separate experiments. See also Figure S1 . p value <0.05 is considered statistically significant.

Article Snippet: To ensure Kindlin-2 overexpression, old cardiac fibroblasts were transfected with 2 μg of Fermt2 Mouse Untagged clone plasmid (Origene #MC202155) by using FuGene transfection reagent.

Techniques: Expressing, Western Blot, Transfection, Immunostaining, Staining, Control, Over Expression, Clone Assay, Molecular Weight, Marker

Journal: iScience

Article Title: A defective mechanosensing pathway affects fibroblast-to-myofibroblast transition in the old male mouse heart

doi: 10.1016/j.isci.2023.107283

Figure Lengend Snippet: The aging-associated deleterious activation of ERK can be rescued by Kindlin-2 overexpression in the old cells (A) Representative Western blot showing the difference in phosphorylated ERK1/2 (P-ERK) versus total ERK1/2 protein levels in the lysate from old and young male cardiac fibroblasts. Densitometry was presented on an individual-plotted diagram (N = 5–6 per group). (B) Representative Western blot depicting changes in F- and G-actin fraction enrichment in the cells cultured with 5 μM Temuterkib (ERK1/2 specific inhibitor, also known as LY3214996, “ERK-LY”) or DMSO (control, “-”) for 3 h. The F-to-G-actin ratio was calculated (lower panel) (N = 3–5 per group). (C–E). Representative Western blot showing the change in α-SMA and Kindlin-2 in the lysate of old male cells treated for 72 h with 5 μM LY3214996 (ERK-LY, “+”) or DMSO (control, “-“). For each protein, the normalized densitometry of each individual value is plotted in a diagram (lower panel) (N = 4–6 per group, “ERKinh” depicts cells treated with 5μM Temuterkib). Staining for F-actin (red)/α-SMA (green) (D.) and Kindlin-2 (green)/F-actin (red) (E.) on cells treated with 5 μM Temuterkib (ERK inhibitor) or DMSO (control) for 72 h. In E., arrows indicate the location of Kindlin-2 at the plasma membrane, and “N” its perinuclear location. Scale bar: 20 μm. Pictures in enlarged frames (yellow square) depicted the changes in Kindlin-2 localization between control and Temuterkib-treated cells. White arrows indicate focal adhesion localization, while “N” depicts nuclear/perinuclear localization of Kindlin-2 (Scale bar: 5 μm). (F) Representative Western blot depicting the level of phosphorylated ERK1/2 (P-ERK) and ERK1/2 in the lysate of pre-selected clones overexpressing Kindlin-2 (“K-OE + ”) or a control plasmid “K-OE -“). The P-ERK to ERK ratio was calculated and shown in an individual-plotted diagram (N = 6 per group). M denotes a molecular weight marker; the corresponding molecular weight is indicated on the right side of the blot.All the data are shown as an averaged mean ± SEM. Statistical significance was challenged by an unpaired t-test with Welch’s correction. p value <0.05 is considered statistically significant.

Article Snippet: To ensure Kindlin-2 overexpression, old cardiac fibroblasts were transfected with 2 μg of Fermt2 Mouse Untagged clone plasmid (Origene #MC202155) by using FuGene transfection reagent.

Techniques: Activation Assay, Over Expression, Western Blot, Cell Culture, Control, Staining, Membrane, Clone Assay, Plasmid Preparation, Molecular Weight, Marker

Journal: iScience

Article Title: A defective mechanosensing pathway affects fibroblast-to-myofibroblast transition in the old male mouse heart

doi: 10.1016/j.isci.2023.107283

Figure Lengend Snippet: The ECM/Kindlin-2/ERKs/actin axis in the mechanosensing-dependent maturation of the cardiac fibroblasts p value <0.05 is considered statistically significant.

Article Snippet: To ensure Kindlin-2 overexpression, old cardiac fibroblasts were transfected with 2 μg of Fermt2 Mouse Untagged clone plasmid (Origene #MC202155) by using FuGene transfection reagent.

Techniques:

Journal: iScience

Article Title: A defective mechanosensing pathway affects fibroblast-to-myofibroblast transition in the old male mouse heart

doi: 10.1016/j.isci.2023.107283

Figure Lengend Snippet:

Article Snippet: To ensure Kindlin-2 overexpression, old cardiac fibroblasts were transfected with 2 μg of Fermt2 Mouse Untagged clone plasmid (Origene #MC202155) by using FuGene transfection reagent.

Techniques: Recombinant, Transfection, Blocking Assay, Staining, Protease Inhibitor, BIA-KA, Protein Concentration, Mass Spectrometry, Negative Control, Plasmid Preparation, Clone Assay, Software

Journal: iScience

Article Title: A defective mechanosensing pathway affects fibroblast-to-myofibroblast transition in the old male mouse heart

doi: 10.1016/j.isci.2023.107283

Figure Lengend Snippet: The aging phenotype of the male cardiac fibroblast is associated with a decreased expression of Kindlin-2 (A) Representative Western blot depicting the difference in α-SMA protein level between the young and old male cardiac fibroblasts (upper panel) and the diagram representing the quantification from separated experiments (N = 5–6 per group) (lower panel). (B) Representative Western blot for the actin (“F” depicting F-actin/Polymerized, and “G” for globular/monomeric). Actin quantity was estimated by densitometry for each sample, and the final F-to-G-actin ratio is shown in an individual-plotted diagram on the lower panel (N = 9–10 per group). (C) Representative F-actin staining for young and old male cardiac fibroblasts seeded on glass, scale bar: 20 μm. N = 10 individual samples from 4 separate experiments. (D) Representative Western blot depicting the difference in Kindlin-2 protein level in the lysate from young versus old cultured cardiac fibroblasts. Normalized densitometry was shown on the lower panel as an averaged value of 6–8 separated experiments. (E) Staining for Kindlin-2 (green) with a DAPI nucleus counterstaining (blue) for young and old cardiac fibroblasts, scale bar: 20 μm. (F–H) Representative staining for F-actin (F), α-SMA (G) and Kindlin-2/α-SMA (H) in cultured old male and old female cardiac fibroblasts, seeded on glass. Representative pictures from 3 to 4 biological repeats. Scale bar: 20 μm. (I) Western blot depicting the F-/G-actin fractions in the lysate from old female versus old male cardiac fibroblasts. The diagram (lower panel) shows the relative comparison of the F-to-G-actin. N = 4. (J) Western blot demonstrating the level of Kindlin-2 and α-SMA in the lysates from young and old female cardiac fibroblasts. Normalized densitometry is depicted as an individual-dotted diagram for both Kindlin-2 (lower left) and α-SMA (lower right). P-value was shown with an "N.S." below the bar, indicating a non-significant difference. M denotes a molecular weight marker; the corresponding molecular weight is indicated on the right side of the blot. (K) Dot-plots depicting the correlation between the level of Kindlin-2 or F-/G-actin ratio (y axis) versus α-SMA (x axis). The regression line shows a positive correlation in male cells (left panel) but not in female cells (middle panel). There is also a positive correlation between the F-/G-actin ratio and the level of α-SMA (right panel). Statistics: All the data are shown as an averaged mean ± SEM. P-value was calculated as an Unpaired t-test with Welch’s correction. p value <0.05 is considered statistically significant.

Article Snippet: To induce Kindlin-2 knockdown, young cardiac fibroblasts were transfected with 27-mer siRNAs (Origene) by using Lipofectamine RNAimax (ThermoFisher #13778030) following suppliers' recommendations.

Techniques: Expressing, Western Blot, Staining, Cell Culture, Comparison, Molecular Weight, Marker

Journal: iScience

Article Title: A defective mechanosensing pathway affects fibroblast-to-myofibroblast transition in the old male mouse heart

doi: 10.1016/j.isci.2023.107283

Figure Lengend Snippet: The impact of cardiac ECM on Kindlin-2 expression (A) Representative immunostaining of Kindlin-2 (red) and α-SMA (green) for tridimensional (3-D) culture of cardiac fibroblasts seeded on cardiac matrices from age-matched or un-matched mouse donors for 72 h. Representative pictures from 3 to 4 experiments per group (young vs. old) and two donors (young vs. old) per experiment. Note that the old male matrices tend to exhibit a more intense autofluorescence. Scale bar: 20 μm. (B) Immunostaining for α-SMA (green) and F-actin (red) in old male cardiac fibroblasts seeded on glass (left), on matrix from old male cells (middle) or matrix from young male cells (right) (scale bar: 20 μm). Nuclei were counterstained with DAPI. (C) Representative Western blot of Kindlin-2 expressed by cells seeded on age-matched and unmatched matrices in the 2-D experiment. The normalized dataset is shown in an individual-plotted diagram on the right panel (N = 5–6 per group). (D) Representative Western blot showing the level of Kindlin-2 expressed by old male and old female fibroblasts seeded on sex-matched or unmatched matrices. The normalized dataset is shown in an individual-plotted diagram on the right panel (N = 4–5 per group). YM, OM, OF denote the young male, old male, and old female, respectively. M denotes a molecular weight marker; the corresponding molecular weight is indicated on the right side of the blot. (E) Scanning electron microscopy pictures of decellularized 3-D matrices from young or old mouse male hearts (upper panel), young or old female hearts (middle panel) or decellularized 2-D matrices deposited by young or old male cardiac fibroblasts (lower panel). Red arrows indicate cleavage sites in the old male cardiac ECM, and cyan circles depict aggregates in the old female cardiac ECM. Scale bar: 2 μm. Pictures in enlarged frames (yellow squares) depict the presence of cleavage in the old male ECM and aggregates in the old female ECM. All the data are shown as an averaged mean ± SEM. For the experimental dataset involving 4 groups, Brown-Forsythe and Welch one-way ANOVA was used to test the significant difference among the four groups, to determine the significant difference among groups. p value <0.05 is considered statistically significant.

Article Snippet: To induce Kindlin-2 knockdown, young cardiac fibroblasts were transfected with 27-mer siRNAs (Origene) by using Lipofectamine RNAimax (ThermoFisher #13778030) following suppliers' recommendations.

Techniques: Expressing, Immunostaining, Western Blot, Molecular Weight, Marker, Electron Microscopy

Journal: iScience

Article Title: A defective mechanosensing pathway affects fibroblast-to-myofibroblast transition in the old male mouse heart

doi: 10.1016/j.isci.2023.107283

Figure Lengend Snippet: Kindlin-2 can facilitate the maturation of old fibroblasts by promoting actin polymerization and α-SMA expression (A) Western-blot depicting a decrease in Kindlin-2 protein levels 72 h post-Kindlin-2 siRNA (siK2) transfection. Averaged densitometry was shown as an individual-plotted diagram (lower panel). (B) Representative pictures of Kindlin-2 immunostaining and F-actin staining, scale bar: 30 μm. (C) Representative Western blot of actin fractions in siK2- compared with control (scrambled)-transfected cells. The F-G actin ratio was calculated for each experimental sample and depicted in a mean ± SEM diagram (N = 5 per each experimental group). (D) Staining of F-actin shown in false color using the OrientationJ plugin (ImageJ algorithm) depicting the changes in cell polarization and actin fiber orientation in control young male cells (left) and in Kindlin-2 deficient cells (right). Scale bar: 20 μm. (E) Representative Western blot showing the increase in α-SMA protein levels in response to Kindlin-2 overexpression (K-OE). Densitometry (lower panel) is provided in an individual-plotted diagram for both Kindlin-2 and a-SMA (N = 3–5 per group). For each experiment, cells were transfected with either K-OE or CMV control). (F) Immunostaining of Kindlin-2 in K-OE and CMV control clones (scale bar: 20 μm). Representative picture from 3 separate experiments. (G) Representative Western blot of F-/G-actin fractions showing the enrichment of F-actin in K-OE lysates. The F-to-G actin ratio was calculated and plotted in a diagram (right panel N = 3–5 per group). M denotes a molecular weight marker; the corresponding molecular weight is indicated on the right side of the blot. (H) Staining of F-actin shown in false color using the OrientationJ plugin (ImageJ algorithm) depicting the changes in cell polarization and actin fiber orientation in control old male cells (left) and in Kindlin-2 overexpressing cells (right). (I) Staining for F-actin (red) and α-SMA (green) in K-OE cells seeded on glass, or in 2-D matrices, compared with old control cells seeded on glass (scale bar: 20 μm). Representative picture from 3 separate experiments. See also Figure S1 . p value <0.05 is considered statistically significant.

Article Snippet: To induce Kindlin-2 knockdown, young cardiac fibroblasts were transfected with 27-mer siRNAs (Origene) by using Lipofectamine RNAimax (ThermoFisher #13778030) following suppliers' recommendations.

Techniques: Expressing, Western Blot, Transfection, Immunostaining, Staining, Control, Over Expression, Clone Assay, Molecular Weight, Marker

Journal: iScience

Article Title: A defective mechanosensing pathway affects fibroblast-to-myofibroblast transition in the old male mouse heart

doi: 10.1016/j.isci.2023.107283

Figure Lengend Snippet: The aging-associated deleterious activation of ERK can be rescued by Kindlin-2 overexpression in the old cells (A) Representative Western blot showing the difference in phosphorylated ERK1/2 (P-ERK) versus total ERK1/2 protein levels in the lysate from old and young male cardiac fibroblasts. Densitometry was presented on an individual-plotted diagram (N = 5–6 per group). (B) Representative Western blot depicting changes in F- and G-actin fraction enrichment in the cells cultured with 5 μM Temuterkib (ERK1/2 specific inhibitor, also known as LY3214996, “ERK-LY”) or DMSO (control, “-”) for 3 h. The F-to-G-actin ratio was calculated (lower panel) (N = 3–5 per group). (C–E). Representative Western blot showing the change in α-SMA and Kindlin-2 in the lysate of old male cells treated for 72 h with 5 μM LY3214996 (ERK-LY, “+”) or DMSO (control, “-“). For each protein, the normalized densitometry of each individual value is plotted in a diagram (lower panel) (N = 4–6 per group, “ERKinh” depicts cells treated with 5μM Temuterkib). Staining for F-actin (red)/α-SMA (green) (D.) and Kindlin-2 (green)/F-actin (red) (E.) on cells treated with 5 μM Temuterkib (ERK inhibitor) or DMSO (control) for 72 h. In E., arrows indicate the location of Kindlin-2 at the plasma membrane, and “N” its perinuclear location. Scale bar: 20 μm. Pictures in enlarged frames (yellow square) depicted the changes in Kindlin-2 localization between control and Temuterkib-treated cells. White arrows indicate focal adhesion localization, while “N” depicts nuclear/perinuclear localization of Kindlin-2 (Scale bar: 5 μm). (F) Representative Western blot depicting the level of phosphorylated ERK1/2 (P-ERK) and ERK1/2 in the lysate of pre-selected clones overexpressing Kindlin-2 (“K-OE + ”) or a control plasmid “K-OE -“). The P-ERK to ERK ratio was calculated and shown in an individual-plotted diagram (N = 6 per group). M denotes a molecular weight marker; the corresponding molecular weight is indicated on the right side of the blot.All the data are shown as an averaged mean ± SEM. Statistical significance was challenged by an unpaired t-test with Welch’s correction. p value <0.05 is considered statistically significant.

Article Snippet: To induce Kindlin-2 knockdown, young cardiac fibroblasts were transfected with 27-mer siRNAs (Origene) by using Lipofectamine RNAimax (ThermoFisher #13778030) following suppliers' recommendations.

Techniques: Activation Assay, Over Expression, Western Blot, Cell Culture, Control, Staining, Membrane, Clone Assay, Plasmid Preparation, Molecular Weight, Marker

Journal: iScience

Article Title: A defective mechanosensing pathway affects fibroblast-to-myofibroblast transition in the old male mouse heart

doi: 10.1016/j.isci.2023.107283

Figure Lengend Snippet: The ECM/Kindlin-2/ERKs/actin axis in the mechanosensing-dependent maturation of the cardiac fibroblasts p value <0.05 is considered statistically significant.

Article Snippet: To induce Kindlin-2 knockdown, young cardiac fibroblasts were transfected with 27-mer siRNAs (Origene) by using Lipofectamine RNAimax (ThermoFisher #13778030) following suppliers' recommendations.

Techniques:

Journal: iScience

Article Title: A defective mechanosensing pathway affects fibroblast-to-myofibroblast transition in the old male mouse heart

doi: 10.1016/j.isci.2023.107283

Figure Lengend Snippet:

Article Snippet: To induce Kindlin-2 knockdown, young cardiac fibroblasts were transfected with 27-mer siRNAs (Origene) by using Lipofectamine RNAimax (ThermoFisher #13778030) following suppliers' recommendations.

Techniques: Recombinant, Transfection, Blocking Assay, Staining, Protease Inhibitor, BIA-KA, Protein Concentration, Mass Spectrometry, Negative Control, Plasmid Preparation, Clone Assay, Software