Fig 2: TSPYL2 regulates cell growth in response to DNA damage.Charts representing crystal violet assays in TSPYL2 silenced H2228 (A) and A549 (B) cells in untreated condition and after etoposide treatment. Graphical representation of cell growth assays in TSPYL2 silenced U2OS (C) and MG-63 (D) cells in unstressed condition and upon etoposide treatment. P values were derived from Student’s t test between untreated and treated samples for each cell line.

Fig 3: SRY represses TSPYL2 accumulation in male cancer cells after DNA damage.A Agarose gel electrophoresis of SRY RT-PCR products derived from untreated and etoposide-treated control and SRY silenced MG-63 cells. Signals represent negative printing of ethidium bromide staining. B Western blot analysis of TSPYL2 induction at 24 h and 48 h after etoposide treatment in SRY-depleted MG-63. C Western blot analysis of TSPYL2 induction at 24 h and 48 h after etoposide treatment in ARPE-19, U2OS and HeLa cells transfected with MOCK or SRY expression vector. D Western blot analysis of MDM2 levels at 24 h and 48 h after etoposide treatment in SRY-depleted MG-63 cells.

Fig 4: Genotoxic stress increases TSPYL2 protein stability in female but not in male cancer cells.A Western blot analysis of TSPYL2 levels in U2OS and B MG-63 cells treated with etoposide for 18 h and added with cycloheximide (CHX) for 3, 5, 7 h. C Western blot analysis of TSPYL2 immunoprecipitates from U2OS and D MG-63 cells transfected with HA-Ubiquitin plasmid, in untreated condition and after 8 h of etoposide treatment. E Western blot analysis of TSPYL2 immunoprecipitates from U2OS cells transfected with FLAG-TSPYL2 and MYC-MDM2, in untreated condition and after 24 h of etoposide treatment. F Western blot analysis of TSPYL2 induction at 24 h after etoposide treatment in MDM2 depleted U2OS and G MG-63 cells. H Western blot analysis of TSPYL2 immunoprecipitates from MDM2 depleted U2OS and I MG-63 cells transfected with HA-Ubiquitin plasmid, in untreated condition and after 8 h of etoposide treatment.

Fig 5: TSPYL2 is more frequently mutated in female-specific cancers.A Distribution of mutations in TSPYL2 gene according to sex in sex-related and somatic cancers. Statistical significance was calculated using Fisher’s exact test. B TSPYL2 expression in tumor vs matched normal pairs according to TNMplot tool. C Kaplan-Meier plots showing the percentage of patients who are alive at a time point in lung adenocarcinoma and osteosarcoma according to TSPYL2 expression level (high in red and low in blue). Statistical significance was calculated using log-rank test. D Graphical scheme of our proposed model. In unstressed conditions, TSPYL2 is maintained at low levels by MDM2-dependent proteasome degradation. In response to DNA damage TSPYL2 protein is stabilized and accumulates in non-transformed and in female cancer cells, but not in male cancer cells where SRY expression promotes the MDM2-dependent proteasome degradation of TSPYL2. The accumulated protein promotes the arrest of damaged cell growth.