Table 1.

Fibrotic gene expression of treated human dermal fibroblasts assessed by RT-qPCR.

Gene			Treatment
	TGF-β (Comparison Group)		Galunisertib Only		TGF-β + Galunisertib
1 h
COL1A1	1.00 ± 0.04	↔	1.12 ± 0.12	↔	1.01 ± 0.02
COL3A1	1.01 ± 0.01	↔	1.30 ± 0.22	↔	1.02 ± 0.07
DCN	1.08 ± 0.14	↔	1.18 ± 0.13	↔	0.91 ± 0.00
ACTA2	1.02 ± 0.07	↔	1.04 ± 0.17	↔	0.80 ± 0.11
CTGF	1.55 ± 0.30	↓	1.10 ± 0.06 *	↓	1.07 ± 0.18 *
FN1	1.01 ± 0.06	↔	1.28 ± 0.09	↔	0.78 ± 0.20
MMP1	1.13 ± 0.16	↔	1.30 ± 0.13	↔	0.89 ± 0.02
MMP13	1.11 ± 0.12	↔	1.23 ± 0.17	↔	1.08 ± 0.24
Day 1
COL1A1	1.67 ± 0.12	↓	0.57 ± 0.07 *	↓	0.69 ± 0.06 *
COL3A1	1.17 ± 0.07	↓	0.71 ± 0.17*	↔	0.77 ± 0.23
DCN	0.38 ± 0.06	↔	0.79 ± 0.11	↔	0.79 ± 0.23
ACTA2	6.33 ± 2.41	↓	0.81 ± 0.42 *	↓	0.84 ± 0.51 *
CTGF	1.72 ± 0.55	↓	0.06 ± 0.02 *	↓	0.07 ± 0.03 *
FN1	2.93 ± 1.11	↓	0.76 ± 0.15 *	↓	0.94 ± 0.28 *
MMP1	0.63 ± 0.06	↔	0.84 ± 0.17	↔	0.66 ± 0.13
MMP13	0.85 ± 0.07	↔	0.78 ± 0.05	↔	0.79 ± 0.21
Day 3
COL1A1	2.02 ± 0.16	↓	0.47 ± 0.05 *	↓	0.48 ± 0.11 *
COL3A1	1.29 ± 0.17	↔	0.92 ± 0.32	↓	0.59 ± 0.12 *
DCN	0.21 ± 0.01	↑	1.39 ± 0.09 *	↑	1.00 ± 0.31 *
ACTA2	8.41 ± 2.60	↓	0.61 ± 0.27 *	↓	0.44 ± 0.18 *
CTGF	1.21 ± 0.15	↓	0.02 ± 0.01 *	↓	0.01 ± 0.01 *
FN1	4.28 ± 2.60	↓	0.69 ± 0.13 *	↓	0.55 ± 0.15 *
MMP1	0.57 ± 0.11	↔	2.90 ± 1.46	↔	1.17 ± 0.17
MMP13	0.89 ± 0.11	↔	1.31 ± 0.25	↔	1.01 ± 0.13
Day 7
COL1A1	1.24 ± 0.17	↓	0.35 ± 0.01 *	↓	0.43 ± 0.03 *
COL3A1	0.86 ± 0.15	↔	1.13 ± 0.31	↔	1.18 ± 0.32
DCN	0.83 ± 0.23	↑	1.71 ± 0.55 *	↑	1.66 ± 0.48 *
ACTA2	1.69 ± 0.74	↔	0.64 ± 0.17	↔	0.63 ± 0.32
CTGF	0.31 ± 0.10	↔	No expression	↔	No expression
FN1	3.03 ± 0.88	↓	0.57 ± 0.22 *	↓	0.61 ± 0.20 *
MMP1	1.54 ± 0.38	↑	11.33 ± 3.77 *	↑	11.49 ± 2.91 *
MMP13	1.20 ± 0.11	↑	1.88 ± 0.76 *	↔	1.50 ± 0.28

Galunisertib (10 µM) significantly reduced TGF-β-induced expression of αSMA (ACTA2), collagen-1a (COL1A1), fibronectin (FN1), connective tissue growth factor (CTGF), and decorin (DCN) after 1 day of treatment. Moreover, matrix metalloproteinases-1 (MMP1) and -13 (MMP13) gene expression were significantly increased by galunisertib in the presence of rhTGF-β after 7 days of treatment, indicating that galunisertib significantly alters TGF-β-induced fibrotic signaling. Comparison to the “Galunisertib only” group showed similar results, suggestive of a rescue effect. Arrows indicate significant differences in expression relative to the comparison group (human dermal fibroblasts induced to fibroproliferative dermal fibroblasts with rhTGF-β). Data are presented as mean expression fold changes ± SD from control calculated by the ΔΔCT method; fold change = 1 reflects basal expression of the target gene (* p < 0.05).