Spectrin-Based Regulation of Cardiac Fibroblast Cell-Cell Communication

Figures

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  Figure 1. (A) Summary data for the number of WT cardiac fibroblasts (CFs) at 24, 48 and 72 h following treatment with either WT or qv4J culture conditioned media (CCM). All groups had an initial seeding at a density of 1 × 105 cells/well. N = 3 independent preparations; * p < 0.05 with two-way ANOVA (with timepoint and genotype as factors) and Holm-Sidak post hoc pairwise comparison. (B) Summary data on collagen gel area (left) and representative collagen gel images (right) following 72 h of treatment of WT CFs with WT or qv4J CCM. N = 3 independent preparations; * p < 0.05 with unpaired two-tailed t-test. Scale bars = 2 mm.

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  Figure 2. Summary data for the number of WT cardiac fibroblasts at 24, 48 and 72 h following treatment with small MW fraction or large MW fraction of WT or qv4J culture conditioned media (CCM). All groups had an initial seeding at a density of 1 × 105 cells/well. N = 3 independent preparations; * p < 0.05 with two-way ANOVA (with timepoint and CCM group as factors) and Holm-Sidak post hoc pairwise comparison. There was a significant (p < 0.05) difference between the 24 h and 48 h timepoints and 48 h and 72 h timepoints for all groups (not indicated in figure for simplicity).

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  Figure 3. Representative dot blots (noncontiguous images from 2 separate assays for WT and qv4J) and quantitative estimation on top candidates from proteome profiler mouse cytokine array performed on conditioned culture media (CCM) from WT and qv4J cardiac fibroblasts following 24 h in serum-free media. Quantitative estimation was performed by normalizing densitometry value for each sample to a control [CC motif chemokine ligand 3 (CCL3)] and then expressing as percent change in qv4J compared to WT CCM. N = 2 independent preparations for each condition; Bars indicate mean value (data points superimposed). Abbreviations are as follows: CCL17 = CC motif chemokine ligand 17; CD142 = coagulation factor III; CX3CL1 = C-X3-C motif chemokine ligand 1 (or fractalkine); CXCL16 = C-X-C motif chemokine ligand 16; MMP3 = matrix metallopeptidase 3; OPG = osteoprotegerin.

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  Figure 4. Size distribution of extracellular vesicles isolated from WT or qv4J conditioned culture medium (CCM) (left) and summary data (right) on area under the curve (AUC) in the 30–150 nm size range. N = 7 independent preparations for each condition; * p < 0.05 vs. WT with Whitney-Mann rank-sum test.

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  Figure 5. Dot blots (noncontiguous images from 2 separate assays for vehicle and S3I-201) from proteome profiler mouse cytokine array performed on conditioned culture media (CCM) from qv4J cardiac fibroblasts pretreated for 72 h with STAT3 inhibitor S3I-201 (100 μM) or vehicle control (N = 2 independent preparation for each condition). Abbreviations are as follows: CC motif chemokine ligand 3 = CCL3; CCL17 = CC motif chemokine ligand 17; CD142 = coagulation factor III; CX3CL1 = C-X3-C motif chemokine ligand 1 (or fractalkine); CXCL16 = C-X-C motif chemokine ligand 16; MMP3 = matrix metallopeptidase 3; OPG = osteoprotegerin.

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  Figure 6. Size distribution of extracellular vesicles isolated from conditioned culture medium (CCM) collected from qv4J fibroblasts ± STAT3 inhibitor S3I-201 (100 M) (left) and summary data (right) on area under the curve (AUC) in the 30–150 nm size range. N = 4 independent preparations for each condition; * p < 0.05 with Whitney-Mann rank-sum test.