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Fig. 6 | Experimental Hematology & Oncology

Fig. 6

From: ASAP2 interrupts c-MET-CIN85 interaction to sustain HGF/c-MET-induced malignant potentials in hepatocellular carcinoma

Fig. 6

ASAP2 is essential for HGF/c-MET signaling-induced malignant phenotypes in hepatocellular carcinoma (HCC). A Venn diagram showing the overlapping WikiPathways from the top 10 signaling pathways ranked by protein–protein interaction (PPI) strength and false discovery rate (FDR) value (Left); Histogram showing normalized enrichment score (NES) for the above mentioned three overlapping signaling pathways and the color indicated FDR value (right). B Effects of ASAP2 knockdown on the levels of total c-MET, p-c-MET, p-AKT, and p-ERK1/2 in SNU387 (left) and SNU182 (right) cells cultured in DMEM supplemented with 10% fetal bovine serum (FBS) were determined by western blot (WB) assays. C shControl or ASAP2-knockdown SNU182 (left)/SNU387 (right) cells were serum starved for 12 h; Afterwards, the culture medium was replaced with serum-free DMEM with or without hepatocyte growth factor (HGF, 20 ng/mL) plus Cycloheximide (CHX, 50 μg/mL), and cells were cultured for 60 min; Total protein was extracted, followed by WB analysis to determine the effects of ASAP2 knockdown on the levels of total c-MET, p–c-MET, p-AKT, and p-ERK1/2 in HCC cells upon HGF stimulation. D Association between ASAP2 and c-MET expression levels in clinical HCC samples. E shControl or ASAP2-knockdown SNU182 (left)/SNU387 (right) cells were serum starved for 12 h; Afterwards, the culture medium was replaced with DMEM containing 1% FBS with or without HGF (20 ng/mL), and cells were cultured for 3 days; At day 3, Cell Counting Kit-8 (CCK8) assays were conducted to evaluate the effects of ASAP2 knockdown on HCC cell proliferation rates following HGF stimulation. F shControl or ASAP2-knockdown SNU182 (upper)/SNU387(lower) cells were seeded into 6-well plates at a density of 1500 cells per well; After culturing in DMEM containing 10% FBS for 24 h to allow complete cell attachment to the plate, the medium was replaced with DMEM containing 2% FBS with or without HGF (20 ng/mL). The medium was replaced twice a week; At Day 14, the cells were stained with crystal violet to assess the influence of ASAP2 knockdown on the colony-formation capacity of HCC cells upon HGF stimulation. G shControl or ASAP2-konckdown SNU182 (upper)/SNU387 (lower) cells were seeded into Transwell chamber inserts (8 μm pore size, upper chamber) at a density of 5 × 104 cells per well; The upper chamber was supplemented with DMEM containing 1% FBS, while the lower chamber was supplemented with DMEM containing 1% FBS with or without HGF (20 ng/mL); Cells were cultured for 24 h to allow invasion; Afterwards, the upper chambers were collected and the invading cells were quantified with crystal violet staining to evaluate the influence of ASAP2 knockdown on invasion rates of HCC cells upon HGF stimulation. H Control and ASAP2-overexpression Hep3B cells were serum starved for 12 h, then the culture medium was replaced with serum-free DMEM with or without HGF (20 ng/mL); Cells were treated for 30 min and protein was extracted, then WB assays were conducted to evaluate the influence of ASAP2 overexpression on the levels of total c-MET, p–c-MET, p-AKT, and p-ERK1/2 in HCC cells upon HGF stimulation. I Control and ASAP2-overexpression Hep3B cells were seeded into Transwell chamber inserts (8 μm pore size, upper chamber) at a density of 5 × 104 cells per well; The upper chamber was supplemented with DMEM containing 1% FBS, while the lower chamber was supplemented with DMEM containing 1% FBS with or without HGF (20 ng/mL); Cells were cultured for 24 h to allow migration or invasion; Afterwards, the upper chambers were collected and the invading cells were quantified with crystal violet staining to evaluate the influence of ASAP2 overexpression on migration (left) and invasion (right) rates of HCC cells upon HGF stimulation

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