Evaluation of RNA

Total RNA was extracted from the cells by Trizol (Invitrogen) according to the manufacturer's instructions. Reverse transcription (RT) for analysis of gene expression or miRNA expression was carried out by high capacity cDNA RT kit (Applied Biosystems, Carlsbad, CA, USA; 10-ng RNA fractions).

All RT reactions were carried out using the StepOnePlus Real-Time PCR System (Applied Biosystems).

The reactions were conducted using SYBR Green dye (Applied Biosystems) according to the manufacturer's protocol. The following primers were used for the analyses:

ErbB2 (forward primer: 5'-GGTCCTGGAAGCCACAAGG-3', reverse primer: 5'-GGTTTTCCCACCACATCCTCT-3')

ER (forward primer: 5'-TGATGAAAGGTGGGATACGA-3', reverse primer: 5'-AGCTCTCATGTCTCCAGCAG-3')

Fyn (forward primer: 5'-GGACATGGCAGCACAGGTG-3', reverse primer: 5'-TTTGCTGATCGCAGATCTCTATG-3')

Akt (forward primer: 5'- ACGTGGCTATTGTGAAGGAG-3', reverse primer: 5'- CATTCTTGAGGAGGAAGTAGCG-3')

FAK (forward primer: 5'- AAATACGGCGATCATACTGGG-3', reverse primer: 5'- TTGGCCTTGACAGAATCCAG-3')

Hypoxanthine phosphoribosyltransferase 1 (HPRT1) as endogenous control (forward primer: 5'-TGACACTGGCAAAACAATGCA-3', reverse primer: 5'-GGTCCTTTTCACCAGCAAGCT-3').

miR-125a-3p (Assay ID: 2199), miR-125a-5p (Assay ID: 2198), and U6-snRNA (Assay ID: 001973) were measured by the TaqMan miRNA kit (Applied Biosystems) according to the manufacturer's instructions. Mature miRNAs were normalized to U6-snRNA. Relative expression was calculated using comparative ΔCt.

Samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as previously described (13) and immunoblotted with the appropriate primary antibodies: anti-ErbB2 (#29D8; Cell Signaling Technology, MA, USA) or anti-actin (#MAB1501; Millipore, Temecula, CA, USA). Blots were visualized using ECL (Pierce Pico ECL kit).

Cells were suspended in FACS buffer (5% FCS, 0.05% sodium azide in PBS) and incubated with FITC anti-human ErbB2 IgG1 (#324404, BioLegend) or FITC mouse IgG1 isotype control (#400109, BioLegend) antibodies for 30 min at 4°C in the dark and then washed prior to analysis. Samples were collected with FACScanto (BD PharMingen) and analyzed with FCS Express software (De Novo Software); 50,000 total events were recorded per sample.

Immunofluorescence was performed as described previously (12). Briefly, cells were stained with FITC-conjugated anti-ErbB2 (#ab31891; Abcam, Cambridge, UK), washed, stained with Lysotracker Deep Red (#{"type":"entrez-nucleotide","attrs":{"text":"L12492","term_id":"289562","term_text":"L12492"}}L12492; Thermo Fisher Scientific, Waltham, MA, USA) and with Hoechst 33342 (1 mg/ml; Sigma-Aldrich, Rehovot, Israel), and analyzed on an Zeiss LSM 510 laser confocal scanning microscope (Carl Zeiss, Oberkochen, Germany) or a Leica TCS STED (Stimulated Emission Depletion) microscope (Leica, Wetzlar, Germany).

Migration assays were performed as previously described (13). Briefly, samples of MDA-MB-231-overexpressing miR-125a-3p or scrambled RNA, treated/untreated with trastuzumab, were stained with Trypan blue, and their viability was examined using a Countess automated cell counter (Invitrogen). For migration assay, live cells (2^*105) were pre-incubated in FCS-free DMEM in the upper wells of a Transwell plate (24 wells, 8 μm pore size membranes, Corning 3422; Corning, NY, USA). After 6 h, 350 μl of DMEM with 20% FCS was added to the lower well, and cells were allowed to migrate during a 12 h incubation period at 37°C in 5% CO₂ in air. The migrating cells at the bottom of the membrane were visualized by fluorescence microscopy, photographed, and counted using Image J software.

Cells were trypsinized, washed 3 times in cold PBS, re-suspended for 15 min at room temperature in 100 μl buffer (MEBCYTO Apoptosis Kit, #4700; MBL International, MA, USA) containing 10 μl Annexin V-FITC and 5 μl PI. The cells were subjected to FACS analysis, and the readings were analyzed using FCS software.

MDA-MB-231 cells (1.5 × 10⁶), stably expressing miR-125a or scrambled miRNA, were mixed with an equal volume of Matrigel (BD Matrigel, BD Biosciences, CA, USA) and injected into the mammary fat pad of 6 week-old female athymic nude mice. Tumor size was measured twice a week with a caliper, in addition to a weekly CT scan by TomoScope®. Tumor volume was calculated using the formula: 1/2^*[length (mm)] × [width (mm)]². When tumors reached a volume of 100 mm³, the mice were randomly allocated to experimental and control groups and were injected intraperitoneally (i.p.) twice a week with 10 mg/kg trastuzumab or vehicle (control) for 28 additional days.

All in vivo procedures were performed in compliance with the guidelines of the Sackler School of Medicine, Tel Aviv University; protocols were approved by the Institutional Animal Care and Use Committee (IACUC).