Fig. 5: sgSWSAP1 and sgSWS1 cells are sensitive to Olaparib inhibition, while breast, uterine/endometrial and prostate cancer mutations inhibit protein-protein interactions.

A Schematic of the SWSAP1 protein, which is 229 amino acids, highlighting variants identified in breast, uterine/endometrial, and prostate cancers from TCGA and cBioPortal. The Walker A and Walker B motifs are indicated in orange and blue, respectively. Summary of the yeast-2-hybrid results in (B) are highlighted with a red box (indicating a deficient yeast-2-hybrid interaction, 0-33%), yellow box (indicating a partial yeast-2-hybrid interaction, 34-67%), and a green box (indicating a proficient yeast-2-hybrid interactions, 68–100%) between SWSAP1 with SWS1. B Yeast-two-hybrid analysis of SWSAP1 variants from breast, uterine/endometrial, and prostate cancers identified in COSMIC and cBioPortal. Wild-type SWSAP1 or the cancer variant were expressed in the GAL4-DNA binding domain vector (BD) and their interaction with SWS1, expressed in the GAL4-DNA activating domain (AD) was assessed by yeast-two-hybrid by assessing growth on selective medium (SC medium minus leucine, tryptophan, and histidine; SC-LTH). Equal cell plating was assessed by plating the yeast on SC medium minus leucine and tryptophan (SC-LT) medium. pGAD and pGBD empty vectors were used as negative controls. Experiments were performed in triplicate, and colonies were quantified with ImageJ and normalized to the wild-type control. C AlphaFold models of SWSAP1 (purple) and SWS1 (pink) structures were fed into the HDOCK server to generate a combined model of SWSAP1-SWS1. The surface exposed cancer variants are highlighted in red. D Clonogenic survival assays of RPE-1, sgSWSAP1, and sgSWS1 RPE-1 cells exposed to the indicated dose of Olaparib for approximately 14 days. The cells were fixed and stained with crystal violet before being photographed. E Quantification of (D) where cellular survival area of RPE-1, sgSWSAP1, and sgSWS1 was normalized to untreated control and the colony area quantitated and the IC₅₀ was calculated in Graphpad PRISM. Significance was determined by two-way ANOVA from three experiments (SWS1-C1 p = <0.0001, SWS1-C2 p = <0.0001, SWSAP1-C1 p = <0.0001, SWSAP1-C2 p = <0.0001). F Quantification of (D) where colony intensity of RPE-1, sgSWSAP1, and sgSWS1 was normalized to untreated control and the colony intensity quantitated and the IC₅₀ was calculated in Graphpad PRISM. Note that two independent clones were analyzed for each knockout. Significance was determined by two-way ANOVA from three experiments (SWS1-C1 p = <0.0001, SWS1-C2 p = <0.0001, SWSAP1-C1 p = <0.0001, SWSAP1-C2 p = <0.0001).