Fig 1: LIFR–YAP1 signaling is critical for LIF-mediated invasion of NPC cells. a Endogenous protein expression of LIFR, p-YAP1(S127), and YAP1 in three cancer cell lines. b Immunostaining for YAP1 and LIFR in three cancer cell lines. Scale bars, 10 µm. c Western blot analysis of LIFR and p-YAP1 (S127) protein levels in WT and cLIF cancer cells transfected with SMARTpool LIFR siRNA or control siRNA. The p-YAP1 (S127) expressions with respect to total YAP1 levels were quantified and presented as mean ± SEM (n = 3). At least three independent experiments were performed. d Western blot analysis of expression of focal adhesion molecules and SRC in WT and LIF+/- cancer cells transfected with YAP1 siRNA. GAPDH was used as the loading control. e Representative images for p-PXN (Y118) expression in WT or LIF+/- cancer cells transfected with YAP1 or control siRNA (n = 3). Alexa Fluor 488 phalloidin (green) was used to stain F-actin. Scale bars, 10 µm. f Representative images of the HUVEC layer replacement assay. Fixed numbers of WT or LIF+/- cancer cells transfected with YAP1 or control siRNA were plated onto the confluent HUVEC layer and co-cultivated for 24 h. Cancer cells were labeled with antibody against pan-cytokeratin (red) and HUVEC cells with antibody against VE-cadherin (green). Scale bars, 20 µm. g, h Quantification of displaced areas depicted in f. WT cancer cells (g). LIF+/- cancer cells (h). Invaded areas were calculated using CellSens imaging software (Olympus). Data are presented with scatter dot plot (mean ± SEM). Each black dot represents one captured image. Mann–Whitney test. i Immunohistochemistry for LIFR and YAP1 expression (brown) in consecutive NPC biopsy sections derived from primary or bone marrow metastatic lesions
Fig 2: LIF regulates focal adhesion molecules. a Detection of endogenous focal adhesion kinases in cancer cells via western blot using GAPDH as a loading control. b LIF regulates the spatial distribution of activated focal adhesion kinases. Cells were labeled with antibodies against phospho-PXN (Y118) or phospho-FAK (Y397). Alexa Fluor 488 phalloidin (green) was used to stain F-actin. Blue, nuclear staining. Scale bars, 10 µm. c Live imaging of focal adhesion during transendothelial invasion. Cancer cells expressing LifeAct-RFP were pre-labeled with Talin-GFP and plated onto the HUVEC layer. Images were captured 24 h post-plating. Blue, nuclei labeled with Hoechst33342. Scale bars, 20 µm. The white arrow indicates the damaged area caused by Talin-rich elongated protrusion. d Representative images of LIF, p-PXN (Y118), and p-FAK (Y397) expression in paraffin-embedded consecutive NPC tissue sections. Scale bars, 50 µm. e, f Correlation analyses based on IHC scores (Spearman’s correlation test). Correlations were evident between LIF and p-FAK (Y397) (e) and LIF and p-PXN (Y118) (f)
Fig 3: AZD0530 treatment suppresses LIF-mediated tumor invasion. a Western blot analysis of YAP1 and focal adhesion proteins in cancer cells treated with AZD0530 (5 µM). Protein lysates were harvested at 24 h post treatment. b Immunostaining for YAP1 (red) in cancer cells treated with AZD0530. Alexa Fluor 488 phalloidin (green) was used to stain F-actin. Blue, nuclear staining. Scale bars, 10 µm. c–e Representative images of YAP1 (c), p-PXN (Y118) (d), and p-FAK (Y397) (e) expression in mouse WT and cLIF xenografts treated with AZD0530 or vehicle. Scale bars, 50 µm. f Quantification of mouse NPC xenografts with events of local invasion based on results of hematoxylin and eosin staining. The AZD0530 treatment procedure in the mouse model is described in Methods
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