Matrix Mechanics Influence Fibroblast-Myofibroblast Transition by Directing the Localization

Supplemental InformationLi et al.

Matrix mechanics influence fibroblast-myofibroblast transition by directing the localization of histone deacetylase 4

Yanfen Li1, Claire B. Tang2, and Kristopher A. Kilian1

1Department of Bioengineering, 2Department of Molecular and Cellular Biology, University of Illinois at Urbana-Champaign

Urbana, Illinois, 61801

Figure S1. Western blot analysis confirms change in class I HDAC expression for fibroblasts cultured on substrates of different stiffnesses. N=4; Error bars represent ±SEM, *p-value<0.05.

Figure S2. Correlation plot of nuclear area and HDAC2 intensity of fibroblasts cultured on soft vs stiff substrates show no significant correlation.

Figure S3. Fibroblasts cultured on soft hydrogels 4 hours after initial adhesion show higher expression of HDAC4 compared to fibroblasts cultured on stiff hydrogels. Left: Example image of immunofluorescence staining of HDAC4, right: quantification of HDAC4 staining.N=3; Error bars represent ±SEM.Scale bar = 90 µm.

Figure S4. HDAC4 localization is confirmed by confocal microscopy and exhibit higher expression on softer substrates. Imaged by Leica Microsystems DMi8 confocal.

Figure S5. Fibroblasts cultured on soft hydrogels at high density show higher expression of HDAC4, and increased cytoplasmic localization, compared to fibroblasts cultured on soft hydrogels at low density and to fibroblasts cultured on stiff hydrogels. Left: Example image of immunofluorescence staining of HDAC4, right: quantification of HDAC4 staining. N=3; Error bars represent ±SEM.

Figure S6. Fibroblasts cultured on soft hydrogels show higher expression of HDAC4, and increased cytoplasmic localization, compared to fibroblasts cultured on stiff hydrogels. Treatment with inhibitors of c-Jun N-terminal Kinase (JNK) decreases HDAC4 expression. Left: Example image of immunofluorescence staining of HDAC4, right: quantification of HDAC4 staining.N=3; Error bars represent ±SEM. **p-value < 0.001; **p-value < 0.05.

Figure S7. Western blot analysis confirms changes in pHDAC4 and PP2a levels for fibroblasts cultured on substrates of different stiffnesses. N=4, Error bars represent ±SEM; **p-value < 0.001; *p-value < 0.05.

.

Figure S8. Quantification of western blot of focal adhesion proteins and αSMA of fibroblasts N=4, Error bars represent ±SEM.

1