The Functional Role of Zinc Finger E Box-Binding Homeobox 2 (Zeb2) in Promoting Cardiac Fibroblast Activation

Figures

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  Figure 1. Zeb2 is localized to the nuclei of primary rat cardiac myofibroblasts. Zeb2 protein expression was enriched in the nuclear fraction from P1 rat cardiac myofibroblasts as compared to P0. Lamin and GAPDH were used as nuclear and cytoplasmic loading controls, respectively. The data shown are from n = 4 independent experiments, * p ≤ 0.05 vs. P0. Error bars represent SEM. Data were analyzed by performing Student’s t-test.

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  Figure 2. Zeb2 overexpression induces expression of proteins characteristic of the myofibroblast phenotype in primary rat cardiac fibroblasts. P1 cells were transduced with Ad-EGFP or Ad-HA-Zeb2 and incubated for 96 h prior to Western blot analysis. α-Tubulin was used as a loading control. Rabbit polyclonal anti-Zeb2 antibody was used to determine Zeb2 expression levels (Panel A). Ectopic expression of Zeb2 increased the expression of (A) α-SMA, (B) SMEmb and (C) ED-A fibronectin. Right panels are histographic representations of respective expression of markers relative to α-tubulin. The data shown are from n = 4 independent experiments for α-SMA and SMemb, and n = 3 for ED-A fibronectin. * p < 0.05 vs. Ad-EGFP. Error bars represent SEM. Data were analyzed by performing Student’s t-tests.

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  Figure 3. Zeb2 overexpression inhibits the migration of P1 myofibroblasts. (A) P1 myofibroblasts were transduced with Ad-EGFP or Ad-HA-Zeb2 and incubated for 96 h. After 96 h, culture inserts were removed and images were taken at the indicated time points (4× objective). (B) The number of cells in the wounded area was quantified using ImageJ software. Histographic representation shows the number of migrated cells in Ad-EGFP (200 MOI) and Ad-Zeb2 (200 MOI) infected plates. n = 3, * p ≤ 0.05 vs Ad-EGFP at 18 h. Error bars represent SEM. Data were analyzed by performing a Student’s t-test.

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  Figure 4. Zeb2 overexpression increases contraction of P1 myofibroblasts—a characteristic of mature myofibroblasts. (A) P1 cells were plated on collagen gels and transduced with either Ad-LacZ or Ad-HA-Zeb2 and incubated for 72 h. After 72 h, collagen gels were cut and allowed to contract for 14 h. Images were taken at the 0 h and 14 h timepoints (4× objective). (B) The gel size was quantified using Measuregel software. Histographic representation shows the percentage of change of area in case of Zeb2 infected gels compared to Ad-LacZ control. n = 3, * p ≤ 0.05 vs Ad-LacZ. Error bars represent SEM. Data were analyzed by performing a Student’s t-test.

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  Figure 5. Zeb2 knockdown in primary cardiac myofibroblasts. First-passage (P1) rat cardiac myofibroblasts were subjected to 24 h of serum deprivation prior to treatment with 100 nM of either scramble or FITC-tagged Zeb2-targeted siRNA in serum-free, antibiotic-free DMEM. Untreated cells served as negative controls; transfection efficiency was verified by fluorescent microscopy. Data shown is representative of n = 3 biological replicates. ** p < 0.01, * p < 0.05.