Fig 2: The synthetic lethal interaction between STAG1 and STAG2 is manifested in disease-relevant bladder cancer and Ewing sarcoma cell lines.(A) The indicated bladder cancer cell lines were analyzed for STAG2 expression by immunoblotting. (B) The indicated bladder cancer cell lines were transfected with NTC, STAG1 and PLK1 siRNA duplexes. Viability was determined 7 or 10 days after transfection and normalized to the viability of NTC siRNA transfected cells (n = 2 independent experiments with 5 biological repeats each, error bars denote standard deviation). (C) STAG2 wild-type UM-UC-5 and STAG2 mutated UM-UC-3 cells were transfected with NTC, STAG1 and SGOL1 siRNA duplexes. Colony formation was analyzed 7 days after transfection by crystal violet staining. (D) 72 hr after siRNA transfection into UM-UC-5 and UM-UC-3 cells, Giemsa-stained chromosome spreads were prepared and analyzed for sister chromatid cohesion phenotypes (n = 100 spreads, error bars denote standard deviation of two independently analyzed slides). (E) The indicated Ewing sarcoma cell lines were analyzed for STAG2 protein expression by immunoblotting. (F) The indicated Ewing sarcoma cell lines were transfected with NTC, STAG1 and SGOL1 siRNA duplexes. Viability was measured 6 days after transfection and normalized to the viability of NTC siRNA transfected cells (n = 3 independent experiments with 3 biological replicates each, error bars denote standard deviation). (G) STAG2 mutated UM-UC-3 cells were transduced with a lentivirus encoding a FLAG-STAG2 transgene. Stably selected cell pools were subsequently transfected with NTC, STAG1 or SGOL1 siRNA duplexes. Viability was measured 7 days after transfection and normalized to the viability of NTC siRNA transfected cells (n = 4 biological replicates, error bars denote standard deviation).DOI: http://dx.doi.org/10.7554/eLife.26980.012

Fig 3: Loss of STAG1 function causes severe mitotic defects, abrogates sister chromatid cohesion and triggers apoptosis in STAG2- but not parental HCT 116 cells.(A) Parental and STAG2- 505c1 HCT 116 were transfected with NTC and STAG1 siRNA duplexes. Immunofluorescence analysis was performed 72 hr after transfection to determine the mitotic index by scoring the fraction of histone H3 phosphoSer10-positive (H3pS10+) cells (n = 1323 cells, error bars denote standard deviation of three independent experiments), and (B) to investigate mitotic spindle geometry and chromosome alignment. Cropped and magnified examples of chromosome alignment are shown in (B). Scale bars, 20 µm. (C) Giemsa-stained mitotic chromosome spreads were prepared 48 hr after transfection of parental and STAG2- HCT 116 cells with the indicated siRNA duplexes. The status of sister chromatid cohesion of individual metaphase spreads was categorized into normal, partial loss of cohesion or single chromatid phenotypes (n = 100 spreads, error bars denote standard deviation of two independently analyzed slides). Scale bar, 10 µm. (D) Caspase activity was tracked over time using a live-cell caspase 3/7 substrate cleavage assay in parental and STAG2- 505c1 HCT 116 cells after transfection with the indicated siRNAs or after treatment with 0.3 µM doxorubicin at t = 0 hr (n = 2 independent experiments with 4 biological repeats for NTC and STAG1 siRNA each and 1 biological repeat for doxorubicin each).DOI: http://dx.doi.org/10.7554/eLife.26980.009

Fig 4: Identification of STAG1 as a genetic vulnerability of STAG2 mutated cells.(A) Engineering of an isogenic HCT 116 cell model by CRISPR/Cas9-mediated inactivation of STAG2. The position of the sgRNAs used to create deleterious insertion and deletion mutations in the STAG2 coding sequence is indicated. (B) Parental HCT 116 cells and two STAG2 mutant clones (STAG2-) were subjected to an siRNA screen. Pools of 4 siRNA duplexes targeting 25 known cohesin subunits and regulators were transfected into the three cell lines. Cell viability was measured 7 days after transfection. Following normalization to non-target control (NTC) values, the average cell viability of siRNA pools in parental HCT 116 cells and two STAG2- clones was plotted against each other (n = 2 or more independent experiments with 2 biological repeats each). (C) HCT 116 parental cells and STAG2- clones were transfected with the indicated siRNAs. Protein lysates were prepared 72 hr after transfection and analyzed by immunoblotting. (D) Cell viability was assessed 8 days after siRNA transfection using a metabolic assay (n = 4 biological repeats, error bars denote standard deviation) and (E) using crystal violet staining. (F) Cas9-GFP expressing isogenic parental and STAG2- HCT 116 and KBM-7 cells were transduced with a lentivirus encoding mCherry and sgRNAs targeting the indicated genes. The percentage of mCherry-positive cells was determined over time by flow cytometry and normalized to the fraction of mCherry-positive cells at the first measurement and sequentially to a control sgRNA (n = 1 experimental replicate).DOI: http://dx.doi.org/10.7554/eLife.26980.003