Fig 2: STC1 promotes breast cancer cell proliferation. (A–D) Detection of cell proliferation by Cell Counting Kit-8 (CCK8). Breast cancer cell lines transduced with STC1 cDNA (MCF-7 or ZR-7530) or shRNA [MDA-MB-231 (231) or MDA-MB-231 HM (231 HM)] were incubated in 96-well culture plates for 12 h to allow for attachment; after which, a 0-time point measurement was determined. After culturing for 1, 2, 3, 4, or 5 days, cell growth was detected using CCK-8. Absorbance at 450 nm was measured using a microplate reader. (E–H) Colony formation assay. MCF-7 STC1 and ZR-7530 STC1 cells or 231 shSTC1 and 231 HM shSTC1 cells and their corresponding control cells were seeded in six-well plates, and the cells were incubated for 12–14 days, fixed with methanol (30 min, RT), stained with 0.1% Giemsa (30 min, RT), and quantified. Data are shown as mean ± SEM from three independent experiments. P-value was determined by two-tailed unpaired t-test (*P < 0.05; **P < 0.01; ***P < 0.001; N.S., no significance).

Fig 3: Stc1 influences the migration of breast cancer cells. (A–D) Migration assays. MCF-7 STC1 or ZR-7530 STC1 cells or 231 shSTC1 or 231 HM shSTC1 cells and their corresponding control cells were added in each upper chamber of a high-throughput screening multi-well insert 24-well two-chamber plate and allowed to migrate at 37°C for 6–12 h toward a lower reservoir containing a medium plus 2.5% fetal bovine serum. The invaded cells were fixed with ice-cold methanol for 30 min, stained with 0.1% crystal violet for 15 min, photographed, and counted at × 200 magnification under a microscope. (A,C) Representative images of migrated cells; (B,D) quantitative analysis of the number of migrated cells. Data are shown as mean ± SEM from three independent experiments. P-value was determined by two-tailed unpaired t-test (**P < 0.01; ***P < 0.001).

Fig 4: STC1 decreases cell apoptosis after irradiation. Cells were treated with a dose of 8-Gy X-rays, and apoptosis was then measured by flow cytometry in MCF-7 STC1, ZR-7530 STC1, or 231 shSTC1 cells and their corresponding control cells. (A) Representative images of apoptosis assay. (B) The percentage of apoptotic cells. Data are shown as mean ± SEM. P-value was determined by two-tailed unpaired t-test (*P < 0.05). IR +, with radiation treatment; IR-, without radiation treatment.

Fig 5: STC1 facilitates homologous recombination in a BRCA1-mediated manner. Representative images (A) and quantification of ?H2AX foci in MCF-7 STC1 cells and their corresponding control cells (B) or U2OS cells stably expressing STC1 shRNA and their control cells (C). Cells were treated with IR (2 Gy) at different time points and then were subjected to immunofluorescence with ?H2AX antibody. The number of ?H2AX was determined by ImageJ. (D) Immunoblot analysis of phosphorylation of ATM and H2AX in 231 shSTC1 cells and their control cells with or without IR. (E) Quantification of BRCA1 foci in MCF-7 STC1 cells and their corresponding control cells treated with IR (2 Gy) at 4-h time point. (F) The 293T cells were transfected with Flag-STC1 or vector and then treated with IR (10 Gy). One hour later, the cells were lysed and immunoprecipitated with anti-Flag agarose beads. The beads were boiled and blotted with indicated antibodies. P-value was determined by two-tailed unpaired t -test (*P < 0.05).