100 mm dish

Human subcutaneous fat tissue was isolated via liposuction after informed consent was obtained (Abion Inc., Seoul, Korea, IRB approval number 198034-161018-BR-001-02), and adipocyte-derived MSCs were isolated by digestion of 0.075% type I collagenase as described previously [49 (link)]. ASCs were cultured in low glucose DMEM supplemented with 10% FBS and 1% penicillin/streptomycin, or ASCs were established in STK1 medium (Abion Inc., Seoul, Korea). In the case of STK1, cells were washed twice at 4 days after seeding. After reaching 85% confluency, cells were digested with 0.05% TE and seeded (3 × 10⁵ cells) into 100 mm dish (Corning, Midland, NC, USA) containing STK2 (Abion Inc., Seoul, Korea).

Bone marrow cells were rinsed from the mouse femurs and tibias with phosphate buffered saline (PBS) solution containing 2% foetal bovine serum (FBS, BI). Next, the cells were seeded (1 × 10⁷ cells per dish) in a 100‐mm dish (Corning Glass Works) for incubation in a 37°C incubator containing 5% CO₂. After 2 days of culture, the adherent cells were cultured in α‐MEM (Invitrogen) supplemented with a combination of 20% FBS, 2 mM L‐glutamine (Invitrogen), 55 μm 2‐mercaptoethanol (Invitrogen), 100 μg/ml streptomycin and 100 U/ml penicillin (Invitrogen) for 14 days. Upon attaining 80%–90% confluence, the cells were subcultured for subsequent experimentation.

TNBC cell lines, MDA-MB-231 (HTB-22), HCC1395 (CRL-2324), and HCC1937 (CRL-2336), with different BRCA1 statuses were purchased from the American Type Culture Collection (Rockville, MD, USA). Cell lines were grown in Roswell Park Memorial Institute 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) and Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific) and 10,000 U/mL penicillin-streptomycin (Thermo Fisher Scientific). The cell lines were cultured at 37 °C in a 5% carbon dioxide (CO₂) incubator. Sub-culturing was performed when the cell confluence reached approximately 85–90% in a 100 mm dish (Corning, New York, NY, USA). Cells were cryopreserved in cell freezing medium containing 90% FBS + 10% dimethyl sulfoxide (Duchefa Biochemie, Amsterdam, The Netherlands).

To generate inducible MLCK-low-expression cells, H9C2 cells were plated on a 100 mm dish (Corning). At 50–70% confluence, cells were then transfected with 25 nM MLCK siRNA or siRNA for 48 h. These plasmids were obtained from Thermo Scientific, Rockford, IL, USA. Transfection was performed according to Lipofectamine RNAiMAX reagent protocol (Invitrogen).

6.5 × 10⁶ MIA PaCa-2 cells were plated to ~65% confluency in 100 mm dish (Corning Inc., Corning, NY, USA). Cells were treated with 400 nM gemcitabine (Selleckchem) for 0, 2, 18 and 48 h. Cells were then trypsinized and washed with DPBS (Corning Inc.). After lysis, immunoprecipitation was performed using anti-HuR and IgG antibodies (MBL International, Woburn, MA, USA) as described in the product manuscript. The RNA was quantified by RT-qPCR using an ABI StepOnePlus qPCR System. GAPDH and dCK mRNAs were included as a loading control.^{60 (link)}

Western blotting protocol was according to our previous reports [9 (link),34 (link)]. Cells were first seeded at a concentration of 1×10⁶ cells per 100-mm dish (Corning), incubated at 37 °c for certain days according to each experiment. For Western blotting analysis of NOX4, anti-NOX4 rabbit polyclonal antibody (ab154244; Abcam, Cambridge, MA, USA) was used. For analysis of Akt and p-Akt, blots were probed with their specific antibodies (diluted with 5% BSA to 1: 1000; all antibodies from Cell Signaling). Membranes were probed with horseradish peroxidase (HRP)–labeled anti-rabbit secondary antibody (diluted with 5% BSA to 1: 1000; all antibodies from Cell Signaling). Antibody binding was detected by enhanced enhanced chemiluminescence detection kit (ECL) (UK Amersham International plc).

We examined the effect of miR-375-3p in vivo growth. SAS-L1 cells (1.0 × 10⁶) were seeded in a 100 mm dish (Corning Life Sciences) and treated with 0.3% Lipofectamine RNAiMAX and 20 nM of a miRIDIAN microRNA human hsa-miR-375 mimic or a miRID-IAN microRNA mimic negative control #1 (Dharmacon, Horizon Discovery). After 24 h of reverse transfection, the cells were harvested and injected subcutaneously at two sites in the flanks of 5-week-old male Balb/c athymic nude mice (CLEA Japan, Tokyo, Japan) at a density of 1.5 × 10⁶ treated cells per 150 μL of plain DMEM. Tumor diameter was measured every three days starting one week after injection using digital calipers, and tumor volume (mm³) was calculated using the following formula: length × width × height × 0.523. Twenty-two days after injection, the xenografts were dissected and we measured the tumor weight by using an electronic balance (ATX224, Shimadzu, Kyoto, Japan).