Immunofluorescence Analysis of HEPG2 and H1581 Cells

For immunofluorescence (IF) of HEPG2 cells, 100,000 cells were grown on coverslips in 12-well plates and incubated with RSPO2-flag conditioned media for 1.5 h. For H1581 cells, 150,000 cells were grown on coverslips in 12-well plates and transfected with 50 nM siRNA for 48 h. HEPG2 and H1581 cells were fixed in 4% PFA for 15 min, blocked and permeabilized with 5% BSA in PBSTB for 1 h and treated with 1:250 diluted rabbit anti-FGFR4 (Cell Signaling Technology 8562 S), mouse anti-EEA1 (BD 610457), mouse anti-clathrin (BD 610499), rabbit anti-FGFR1 (Cell Signaling Technology 9740 S), mouse anti-Flag (Sigma F3156) and mouse anti-Lamp1 (Cell Signaling Technology 15665 S) overnight at 4 °C. 1:500 diluted donkey anti-mouse Alexa Fluor 647 (Invitrogen A31571), donkey anti-rat Alexa Fluor 488 (Invitrogen A21208), donkey anti-rabbit Alexa Fluor 546 (Invitrogen A10036) and goat anti-mouse Alexa Fluor 488 (Invitrogen A11029) and Hoechst dye (1:500) were applied for 2 h at room temperature. Quantification of IF was performed using FIJI (Image J) v1.51k software. Tyramide Signal Amplification to detect RSPO2-HRP was executed as previously described^{32 (link)}. Representative images were obtained using LSM 700 (Zeiss) and processed with Zeiss ZEN 2012 (black edition) version 2.5.