Figure 2. Inhibition of fibroblast proliferation by romidepsin: IPF and normal fibroblasts were cultured for up to 144h in DMEM/FBS ± TGF-β1 (5ng/ml) and ± romidepsin.

Cells were formalin-fixed before being stained with methylene blue. Stained cells were eluted with a 1:1 ratio of 0.1% HCl and ethanol. Absorbance was measured at 630nm using a photometric plate reader. A. Dose response of IPF fibroblasts to romidepsin at 144h in the absence (solid symbols) or presence (open symbols) of TGF-β1. B. Comparison of the romidepsin concentration required to achieve 50% growth inhibition (IC50) of the IPF and normal fibroblasts at 144h. C. At 48h, after culturing as described, RNA was extracted using the Trizol method prior to cDNA synthesis and analysis by RTqPCR. Data were normalized to the housekeeping genes UBC/A2 using the ΔΔCT method. CDKN1A mRNA expression in response to increasing doses response of romidepsin in IPF fibroblasts without (solid bars) or with (open bars) TGF-β1. Data in B. & C. are shown as mean + SD (n = 3; two-way ANOVA with Dunnett's multiple comparisons). *P < 0.05, **P < 0.01. D. Cell cycle analysis of romidepsin treatment of 3 primary IPF fibroblasts cultures quantitatively determined by propidium iodide (PI) flow cytometric analysis. Solid bar = G1, grey bar = S, open bar = G2/M phase. Data are shown as mean + SD (n = 3; one-way ANOVA). **P < 0.01. Where there are no lines to indicate the statistical comparison, it is between the starred bar and its equivalent baseline non-romidepsin treated control.