Decma 1

MEF^{Col1a1 4F2A Oct4-GFP} cells were subjected to 24 h of serum starvation followed by NTF1 treatment. The working concentrations of the four small molecular inhibitors used in the present study were as follows: Gefitinib, 10 μM; Lapatinib, 10 μM; PD0325901, 1 μM; WP1066, 10 μM. Regarding the application of inhibitors, cells were first treated with inhibitors for 15 min before NTF1 treatment.
Similarly, to examine the effect of the DECMA-1 antibody on NTF1 treatment, MEF cells were treated with NTF1, HAV peptide, HGV peptide, or DECMA-1 after 24-h serum starvation. After 2 h of treatment, cells were harvested and lysed for western blotting using anti-pSTAT3 (Y705), -STAT3, -pErk, and -Erk antibodies. The working concentrations of DECMA-1 (ThermoFisher Scientific, Waltham, MA, USA; 14-3249-82), HAV, and HGV peptide were 5 µg/mL, 5 mM, and 5 mM, respectively.

First, cells were fixed for $10\min$ with 4% PFA diluted in PBS. Next, the cell membrane was permeabilized with 0.5% Triton X-100 for $5\min$ . Cells were then washed twice with TBS and blocked at room temperature for ${\displaystyle 1\mathrm{h}\mathrm{r}}$ with a blocking buffer solution containing TBS, 1% bovine serum albumin (BSA, Sigma-Aldrich), and ${\displaystyle 50\mathrm{m}\mathrm{M}}$ glycine (Sigma-Aldrich). Then, cells were incubated for ${\displaystyle 2\mathrm{h}\mathrm{r}}$ in a dilution of primary antibodies with blocking buffer. For E-cadherin stainings, a 1:200 dilution of DECMA-1 (Thermo Fisher 14-3249-82) was used and for vinculin stainings a 1:400 dilution of hVIN-1 (Sigma-Aldrich V9131) was used. Cells were then washed three times with TBS for $10\min$ each. Then cells were incubated in a dilution of secondary antibodies, Alexa 555-conjugated phalloidin and DAPI in blocking buffer. For E-cadherin stainings, a 1:1000 dilution of Alexa 647-conjugated anti-rat (Sigma-Aldrich SAB4600186) was used; for vinculin stainings, a 1:1000 dilution of Alexa 647-conjugated anti-mouse (Thermo Fisher A-21235) and a 1:1000 dilution for phalloidin and DAPI. Fixed cells were then mounted with Mowiol 4-88 (Polysciences, Inc) onto glass slides and kept at $4\mathrm{°C}$ °C until imaging.