Fig 2: GPR50 Directly Interacts with ADAM17 and Activates the Ligand-Independent Notch Signaling Pathway via ADAM17 in HCC(A) Protein expression levels of ADAM17 were analyzed using western blotting; actin was used as a loading control. (B) Fold change in ADAM17 activity was analyzed using the ADAM17 ELISA assay. (C) Lysates from the indicated cells were used for immunoprecipitation using Protein A/G Sepharose, as well as antibodies specific for GPR50, ADAM17, and normal IgG. The immunoprecipitates were analyzed by western blotting with the indicated antibodies. For inputs, lysates were analyzed by western blotting with the indicated antibodies. (D) Overexpression of FLAG-ADAM17 was confirmed by western blot analysis using FLAG antibody; actin was used as a loading control. (E and F) Fold change in Notch signaling transcriptional activity was analyzed using HES1 (E) and HES5 (F) luciferase assays after overexpression of ADAM17 and treatment with/without marimastat (4 μM). (G) Fold change in ADAM17 enzymatic activity was analyzed using the ADAM17 ELISA assay after overexpression of ADAM17 and treatment with/without marimastat (4 μM). (H) Cell viability was analyzed using the EZ-cytox 4-[3-(4iodophenyl)-2-(4- nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) assay after overexpression of ADAM17 and treatment with/without marimastat (4 μM) after 24 h. (I) Sphere formation assay was performed using the noncoated culture dishes after overexpression of ADAM17 and treatment with/without marimastat (4 μM). Spheres were counted after 5 days of culture using crystal violet staining and presented as fold change in colonies. ns, no significance; ∗∗p < 0.01, ∗∗∗∗p < 0.0001.

Fig 3: GPR50 Regulates ADAM17 Transcription via the AKT/SP1 Axis(A) Expression levels of Notch signaling regulating genes ADAM9, ADAM10, ADAM12, and ADAM17 were analyzed using qRT-PCR; GAPDH was used as an internal standard. (B) Protein expression levels of AKT, phosphorylated (p)-AKT, GSK3β, p-GSK3β, ERK, p-ERK, p38, and p-p38 were analyzed using western blotting; actin was used as a loading control. (C) Scramble and shGPR50-HepG2 cells were treated with/without LY294002 (20 μM), and then protein levels of PI3K, AKT, p-AKT, GSK3β, and p-GSK3β were assessed by western blotting; actin was used as a loading control. (D) Scramble and shGPR50-HepG2 cells were treated with LY294002 (20 μM), and then mRNA expression levels of SP1, SP2, SP3, and SP4 were analyzed using qRT-PCR and normalized to that of GAPDH. (E and F) Scramble and shGPR50-HepG2 cells were treated with LY294002 (20 μM), and then cell viability (E) and wound healing (F) were analyzed at the indicated time points. (G) Scramble and shGPR50-HepG2 cells were treated with LY294002 (20 μM) in the presence of DOX (0.5 μM), and then drug resistance was measured by cell counting after 48 h. (H) Scramble and shGPR50-HepG2 cells were treated with LY294002 (20 μM), and then a sphere-formation assay was performed using the noncoated culture dishes. Spheres were counted after 5 days of culture using crystal violet staining and represented as the percent (%) of colonies. (I) Scramble and shGPR50-HepG2 cells were treated with LY294002 (20 μM) and then mRNA expression levels of ADAM9, ADAM10, ADAM12, and ADAM17 were analyzed using qRT-PCR and normalized to that of GAPDH. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Fig 4: Overexpression of GPR50 Augments Notch Signaling via the Ligand-Independent ADAM17 Axis in HCC Cells(A) GPR50 overexpression was confirmed by RT-PCR and western blot analysis and normalized to that of GAPDH or actin. (B) Cell viability was analyzed over 3 days after overexpression of GPR50 using the EZ-cytox WST-1 assay. (C) Cell-sphere formation assay was performed using the noncoated culture dishes after GPR50 overexpression. Spheres were counted after 5 days of culture using crystal violet staining and presented as the percent (%) of colonies. Photos were acquired by inverted light microscopy. (D) Protein expression levels of ADAM17, NICD, and HES1 were analyzed by western blot analysis after GPR50 overexpression. (E and F) Notch signaling transcriptional activity was analyzed after GPR50 overexpression using the HES1 (E) and HES5 (F) luciferase assays. (G) Fold change in ADAM17 enzymatic activity was analyzed using the ADAM17 ELISA assay after GPR50 overexpression. (H) Protein expression levels of GPR50, ADAM17, NICD, and HES1 were analyzed by western blot analysis after GPR50 overexpression and treatment with/without DAPT (Notch signaling inhibitor) and/or marimastat (ADAM17 inhibitor). (I) Fold change in ADAM17 activity was analyzed using the ADAM17 ELISA assay after GPR50 overexpression and treatment with/without DAPT and/or marimastat. (J and K) Notch signaling transcriptional activity was analyzed after GPR50 overexpression and treatment with/without DAPT and/or marimastat using the HES1 (J) and HES5 (K) luciferase assay. ns, no significance; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.