Lower transwell chamber

The lower Transwell chamber (Costar) contained cell appropriate medium (10% fetal calf serum as chemoattractants). Cells in appropriate medium (1% fetal calf serum) were seeded onto membranes of the upper Transwell chamber (6.5 mm diameter, 8 μm pores). After 48 h of transfection with NC or si-ATRX 105 LN229 cells were incubated for 24 h, cells were ethanol-fixed and stained (crystal violet). The non-invading cells were removed from the upper surfaces of the invasion membranes and the cells on four fields of the lower face were counted using an inverted microscope.

MDA-MB-231 and BT-549 cells at the concentration of 1×10⁵/ml were paved onto the upper Transwell chamber (Corning Costar, USA), and 600 μl DMEM medium that contained 10% FBS was poured into the lower transwell chamber (Corning Costar, USA). After routine culture for 24 h, the TNBC cells were stained by 0.1% crystal violet (Solarbio Life Sciences, China), thereafter photographs were taken under optical microscope (Olympus, USA). The experiments were undertaken with ≥ 3 replicates.

To determine whether CXCL9 and CXCL10 recruited CXCR3⁺ Tregs, chemotaxis assays were performed. MSC‐treated lungs were lavaged with 2 mL of RPMI 1640 medium supplemented with 10% FBS and penicillin and streptomycin. Then, the resulting BALF was centrifuged and the supernatant was collected and combined. CXCR3⁺ CD4 T‐cell isolation: lungs, mediastinal lymph nodes and spleens were collected from MSC‐treated mice. Single‐cell suspensions were prepared, and T cells were purified by antibody‐coupled magnetic beads. Then, CXCR3⁺ CD4 T cells were sorted out of the purified T cells by a FACSAria. Chemotaxis assay: medium alone and medium containing 200 ng mL⁻¹ of CXCL9 and CXCL10 (Proteintech, Illinois, United States) were used as negative and positive controls, respectively. 200 ng mL⁻¹ of anti‐CXCL9 or 1 μg mL⁻¹ anti‐CXCL10 antibodies (R&D) was added into the lower Transwell chamber (96‐well, Corning, New York, United States) that containing 150 μL of BALF. Then, about 1 × 10⁴ of CXCR3⁺ CD4 T cells were added into the upper Transwell chamber in a total volume of 100 μL. Plates were incubated at 37°C in 5% CO₂ for 2 h. The contents of the lower well were then collected, and cells were counted in a haemacytometer. Cell migration to medium alone was used as control. All assays were performed in triplicate.