E3 Ubiquitin Ligase CHIP Inhibits the Interaction between Hsp90β and MAST1 to Repress Radiation Resistance in Non-Small-Cell Lung Cancer Stem Cells

Figure 5

CHIP inhibits the NSCLC stem cell properties and radiation resistance through the inhibition of MAST1 protein stability. (a) Western blot assay to determine the expression of CHIP, MAST1, and Hsp90β in CD133⁺ A549 cells in response to overexpression of CHIP and MAST1 alone or in combination. (b) RT-qPCR analysis for expression of stemness-related transcription factors (Oct4, SOX2, and Nanog) in irradiated/unirradiated (4 Gy) CD133⁺ A549 cells in response to overexpression of CHIP and MAST1 alone or in combination. (c) Sphere formation analysis after 7 days of sphere culture of irradiated/unirradiated (4 Gy) CD133⁺ A549 cells in response to overexpression of CHIP and MAST1 alone or in combination. (d) Cell survival analysis of CD133⁺ A549 cells exposed to different doses of radiation by colony formation assay after 9 days of culture. (e) Annexin V/PI double staining for detection of apoptosis of irradiated (4 Gy) CD133⁺ A549 cells in response to overexpression of CHIP and MAST1 alone or in combination. (f) Immunofluorescence detection of the number of γ-H2AX foci in irradiated (4 Gy) CD133⁺ A549 cells in response to overexpression of CHIP and MAST1 alone or in combination. (g) The quantitation of tumor volume in nude mice subcutaneously injected with CD133⁺ A549 cells in response to overexpression of CHIP and MAST1 alone or in combination as well as irradiation (4 Gy) (). (h) Statistics of tumor weight of nude mice in response to overexpression of CHIP and MAST1 alone or in combination as well as irradiation (4 Gy) (). . Cell experiments were independently repeated three times.

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