Type 1 collagen

Mouse primary PEC were plated and rested overnight. Cells were incubated in RPMI-1640 containing 1% serum and supplemented with different concentrations of DDR1 inhibitor (0.01, 0.1 and 1 μM) for 1 h followed by additional treatment with type I collagen 100 μg/mL (Nitta Gelatin, Japan). After 6 or 24 h of type I collagen stimulation, cells were lysed for phospho DDR1 ELISA (Cell Signaling Technology) and total RNA isolation. Total RNA was isolated and amplified using an RNeasy Mini kit (Qiagen) and Transcriptor Universal cDNA Master (Roche) according to manufacturer’s instructions. Quantitative RT-PCR was performed on a LightCycler LC480 (Roche) for C3, Mmp2, Mmp14 and Vcam1 using the following primers: C3 (Mm01232779_m1, TaqMan^® Gene Expression Assays, Applied Biosystems), Mmp2 (Mm00439498_m1), Mmp14 (Mm00485054_m1), and Vcam1 (Mm01320970_m1). Relative gene expression was calculated with the 2-∆Ct method using GAPDH as an endogenous control.

Cell lysates were prepared and western blot analysis was performed as described previously [22 (link)]. Antibodies against the following proteins were used: phosphorylated EGFR (p-EGFR,Y1068, #2234), Total EGFR (T-EGFR, #2232),phosphorylated Signal transducer and activator of transcription 3(p-STAT3,Y705, #9145), Total STAT3 (T-STAT3, #4904), phosphorylated v-Akt Murine Thymoma Viral Oncogene (p-AKT, Ser473, #9271), Total AKT (T-AKT, #9272), phosphorylated Extracellular Signal-regulated Kinase (p-ERK, Thr202/Tyr204, #9101), Total ERK (T-ERK, #9102), heat shock protein 60(HSP 60, #12165), Vimentin(#5741), phosphorylated focal adhesion kinase (p-FAK, Y397, #3283), Total FAK(T-FAK, #3285), α-tubulin(#2144) (all antibodies except type I collagen were purchased from Cell Signalling Technology, Danvers, MA, USA, 1:1000). type I collagen (abcam, Cambridge, UK, #34710). The band densitometric analysis was performed by using the public software Image J (Wayne Rasband National Institutes of Health, Bethesda, MD, USA). Expression of α-tubulin was used to correct for variation in sample loading.