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. 2022 Dec 28;120(1):e2211832120. doi: 10.1073/pnas.2211832120

Fig. 5.

Fig. 5.

Characterization of mutant AR-V7. (A) AR-V7 3C-A transactivation. COS7 cells were transfected with 0.25 µg GRE-LUC, 0.025 µg AR-V7, AR-V7 3C-A, or vector, and cmv-renilla-LUC. Cells were maintained in DME + 10% FBS and were harvested 48 h after transfection and dual luciferase assay was performed (n = 3). (B) UT-143 inhibits AR-V7, but not AR-V7 3C-A transactivation. COS7 cells were transfected with AR-V7 or AR-V7 3C-A mutant and treated in DME + 10% FBS as indicated in the figure. Dual luciferase assay was performed 24 h after treatment (n = 3). (C) Single-point mutation of cysteines in AF-1 inhibits AR-V7 transactivation but does not reverse UT-143 antagonism. COS7 cells were transfected with AR-V7 wildtype, AR-V7 C406A, or AR-V7 C327A and treated with UT-143 in DME + 10% FBS. Dual luciferase assay was performed 24 h after treatment (n = 3). (D) Mutating three cysteines in AF-1 results in its failure to interact with SRC-3. AF-1 or AF-1 3C-A-purified recombinant protein was incubated with recombinant GST-SRC-3 at 4 °C overnight. SRC-3 was immunoprecipitated with GST antibody, and western blot for AF-1 was performed. (E) AR-V7 3C-A fails to form molecular condensate. AR-V7 3C-A was transfected into COS7 cells. Cells were fixed, immunostained with AR-V7 antibody, and images obtained using confocal microscope. Experiments were performed at least three independent times. AR – androgen receptor; V7 3C-A – AR-V7 with C267, C327, and C406 mutated to alanine; C406A – Cysteine 406 mutated to alanine; C327A – Cysteine 327 mutated to alanine; C267A – Cysteine 267 mutated to alanine.