Figures & data
Figure 1 Evaluation of the activation of Hippo/YAP signaling under irradiation exposure. U251 cells were treated with 0, 2, 4, 6 or 8 Gy irradiation, then the cells were collected 24 hrs after irradiation and submitted for the following assays. (A) CCK-8 assay to detect cell viability. (B, C) Flow cytometry assay with Annexin V/PI double staining was used to assess cell apoptosis. (D, E) Western blotting assay was performed to test the protein expression of caspase 3 and Bax. (F) Real-time qPCR was carried out the mRNA levels of Hippo/YAP1 signaling-related genes (n=3, *P<0.05, **P<0.01, ***P<0.001).
Abbreviations: CCK-8, Cell counting kit-8; YAP, Yes-associated protein.
Figure 2 Irradiation decreased YAP1 expression by increasing its ubiquitination degradation. (A, B) The protein expression of YAP1 was determined by Western blotting assay after 24 hrs of U251 cells treatment with 4 Gy irradiation. (C) The interaction between Ub and YAP1 proteins was detected by Co-IP assay. (D, E) After 24 hrs of 4 Gy exposure, U251 cells were incubated with 100 μg/mL CHX for 0, 1, 2, 4, 8 and 24 hrs, then the cells were collected for Western blotting assay to detect YAP1 expression (n=3, *P<0.05).
Abbreviations: YAP, Yes-associated protein; CHX, cycloheximide; Co-IP, co-immunoprecipitation.
Figure 3 Evaluation of the effects of irradiation on the subcellular location of YAP1 protein and the interactions of YAP1 protein with nuclear transcription factors. U251 cells were exposed to 4 Gy irradiation, then the cells were collected after 24 hr and subjected to the following assays. (A, B) Western blotting assay was used to detect the expression of YAP1 in nuclear and cytoplasm. (C) Immunofluorescence technology was used to assess the effect of 4 Gy irradiation on the subcellular location of YAP1 protein in U251 cells. (D) Co-IP assay was used to detect the interaction between YAP1 protein and TEAD, CREB, P73 and RUNX2 proteins, IgG was used as negative controls. (E.) Western blotting analysis of the expression of p-YAP1 after cells was treated with 4 Gy irradiation (n=3, *P<0.05).
Abbreviations: CREB, cAMP responsive element binding protein 1; RUNX2, runt-related transcription factor 2; TEAD, TEA/ATTS domain; YAP, Yes-associated protein; Co-IP, co-immunoprecipitation.
Figure 4 Downregulation of YAP1 weakened the effect of irradiation on cell apoptosis induction. (A, B) The knockdown efficiency of si-YAP1 was assessed by Western blotting assay after 48 hrs of cell transfection. (C) Cell proliferation was detected by CCK-8 assay after 24 hrs of irradiation exposure. (D, E) Cell apoptosis was tested by flow cytometry assay after 24 hrs of irradiation exposure (n=3, *P<0.05).
Abbreviations: YAP, Yes-associated protein; CCK-8, Cell counting kit-8; Co-IP, co-immunoprecipitation.
Figure 5 The ubiquitination of YAP1 protein was regulated by RCHY1. U251 cells were treated with 4 Gy irradiation, then the following assays were carried out after 24 hrs of irradiation exposure. (A, B) The expression of TrCP, CDC4, RCHY1, MDM2, SKP2, UBE3A and SMURF1 was detected by Western blotting assay. (C) Co-IP assay was used to detect the effects of RCHY1 overexpression on the ubiquitination of YAP1 protein (n=3, *P<0.05).
Abbreviation: YAP, Yes-associated protein.
Figure 6 Downregulation of RCHY1 weakened the effect of irradiation on YAP1 downregulation and cell apoptosis induction. (A) The knockdown efficiency of si-RCHY1 was assessed by Western blotting assay after 48 hrs of cell transfection. Next, cells were transfected with si-RCHY1, following by irradiation exposure, then the following experiments were carried out. (B) The expression of YAP1 protein was assessed by Western blotting assay after 24 hrs of irradiation exposure. (C) Cell viability was detected by CCK-8 assay after 24 hrs of irradiation exposure. (D) Cell apoptosis was tested by flow cytometry assay after 24 hrs of irradiation exposure (n=3, *P<0.05).
Abbreviations: YAP, Yes-associated protein; CCK-8, Cell counting kit-8.
