Psgro hesc ipsc growth medium

Sphere-forming assay was performed as previously described [13 (link), 14 (link)]. Briefly, C9IV3 and HSC3 cells were transfected with plasmids containing siCon or siKRT17 for 24 h, or miR-Con, miR-485-5p, miR-485-5p + KRT17 for 48 h, or in combination with 1 μM dasatinib (Selleckchem, Houston, Texas, USA) treatment for 48 h prior to sphere forming assay. Transfected and/or treated cells were cultured in stem cell selective medium at cell density of 1 × 10⁵ cells per well with 3 ml of PSGro hESC/iPSC growth medium (System Biosciences, Palo Alto, CA, USA) in Costar ultra-low attachment 6-well plates (Corning, Oneonta, NY, USA). The total numbers of spheres formed were counted under microscope at day 10.

Monolayer cells of parental OVS1 cells were cultured in the stem cell selective condition by plating cells in Corning Costar ultra-low attachment 6-well plates (Sigma-Aldrich Inc., St Louis, MO, USA) at a density of 1 × 10⁵ cells per well with 3 mL of serum-free PSGro hESC/iPSC growth medium (System Biosciences, Palo Alto, CA, USA). Propagation of spheres was processed by gentle centrifugation, dissociation with trypsin-EDTA and repeated pipetting to obtain single-cell suspensions every 5–8 days, and then plating the cells in the above stem cell selective condition. The spheres were found under microscope after 10 days of culturing. Detailed procedures of total RNA isolation and RNA sequencing were described elsewhere [31 (link)].

We used Dulbecco’s modified eagle medium (DMEM; Invitrogen, Carlsbad, CA, USA) to grow human TNBC and breast epithelial cell lines, including MDA-MB-231, Hs578T and MCF10A cells, supplemented with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA). The cells were incubated at 37 °C in 5% CO₂. To obtain spheres, TNBC cells were cultured in a stem cell selective condition plating cells in Corning Costar ultra-low attachment 6-well plates (Sigma-Aldrich Inc., St Louis, MO, USA) at a density of 1 × 10⁴ cells per well with 1 mL of serum-free PSGro hESC/iPSC growth medium (System Biosciences, Palo Alto, CA, USA).