Figure 3. The SFK member LCK regulates YAP tyrosine phosphorylation and transcriptional activity in cholangiocarcinoma.

(A) Cell lysates from HuCCT-1 cell lines transfected with targeting and non-targeting siRNA were subjected to immunoblot for SRC, YES, LCK, FYN, LYN. GAPDH was used as a loading control. (B) Cell lysates from HuCCT-1 cell lines transfected with non-targeting siRNA and siRNA for five SFKs were subjected to immunoblot for phosphorylated YAP (pYAP^Y357). Total YAP and GAPDH were used as loading controls. (C) CTGF and CYR61 mRNA expression in HuCCT-1 cell line transfected with non-targeting siRNA (NC1) and siRNA for five SFKs. Fold expression targeting/non-targeting is depicted as geometric mean +/−geometric standard deviation (n=3). *p<0.05 (D) Relative cell proliferation in HuCCT-1 cell lines transfected with targeting and non-targeting siRNA for five SFKs, quantitation. *p<0.05 (E) HuCCT-1 cell lines transfected with a doxycycline inducible CRISPR/Cas9 construct targeting LCK were incubated with doxycycline (7.5 µg/mL) for the noted time. Cell lysates were subjected to immunoblot for pYAP^Y357 (total YAP as loading control) and LCK (GAPDH as loading control). (F) HuCCT-1 cell lines transfected with a doxycycline inducible CRISPR/Cas9 construct targeting LCK or empty vector were incubated with doxycycline (7.5 µg/mL) for the noted time. Immunofluorescence for YAP was undertaken and representative images are displayed (upper panel) with quantification of YAP localization (lower panel). Original magnification 40×. Scale bar = 10 microns. (G) Phosphorylation of recombinant YAP in a cell free system by recombinant LCK was evaluated by luminescence and immunoblot (inset). (H) Heat map representation of FPKM for LCK and ARID3a from PDX derived from patients undergoing curative intent liver resection with recurrence in less than 10 months (L10) versus recurrence in greater than 10 months (G10).