Fig 2: Schematic Diagram Illustrating GPR50 Function in the Notch Signaling Pathway in HCCGPR50 regulates ADAM17 transcription via the AKT/SP1 axis and directly interacts with ADAM17 to induce ADAM17 enzymatic activity, which subsequently activates ligand-independent Notch signaling, mediating HCC progression.

Fig 3: Silencing of GPR50 Expression Downregulates Notch Signaling in HCC Cell Lines(A) mRNA expression levels of different signaling target genes (DKK1 for Wnt signaling, HES1 for Notch signaling, GL1 for Hedgehog signaling, and YAP1 for Hippo signaling) were analyzed using qRT-PCR and normalized to that of GAPDH. (B) mRNA expression levels of NOTCH1, MAML1, RBPjK, and HES1 were analyzed using RT-PCR; GAPDH was used as an internal standard. (C) Protein levels of NICD and HES1 were assessed using western blotting; actin was used as a loading control. (D and E) Notch signaling transcriptional activity was analyzed using HES1 (D) and HES5 (E) luciferase assays. (F) Overexpression of FLAG-NICD was confirmed by western blot analysis using FLAG antibody; actin was used as a loading control. (G and H) Fold change of Notch signaling transcriptional activity was analyzed after overexpression of NICD in the indicated cells using HES1 (G) and HES5 (H) luciferase assays. (I and J) Cell viability (I) and sphere formation (J) were analyzed after overexpression of NICD in the indicated cells. ns, no significance; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Fig 4: GPR50 Knockdown Led to Suppressed Cancer Properties in HCC(A) GPR50 expression in scramble and shGPR50-transduced cells was analyzed by RT-PCR and western blotting in the indicated cancer cell lines. GAPDH/actin was used as a loading control. (B) Cell proliferation was analyzed using trypan blue. Cells were counted over 5 days. (C) Wound-healing assay to test migration of the indicated cells. Cell migration was observed at the indicated time points and presented as percentage (%) wound enclosure (right panel). Photos were acquired using inverted light microscopy. (D) Cell-sphere formation assay was performed using noncoated culture dishes. Spheres were counted after 5 days of culture with crystal violet staining and presented as the percent (%) of colonies. Photos were acquired by inverted light microscopy. (E) mRNA expression of stemness markers was analyzed by RT-PCR. GAPDH was used as an internal standard. (F) Effect of GPR50 knockdown on drug resistance was measured by cell counting after 48 h of doxorubicin (DOX) treatment (0.5 μM). (G) mRNA expression levels of drug-resistance marker genes were analyzed by RT-PCR using GPR50-knockdown cells. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Fig 5: 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.