Millicell ez 8 well slides

Cells were grown to a confluency of 50–80% in millicell EZ 8-well slides (Millipore, Iberica, Madrid, Spain) and transfected with wild-type or p.Gly792Arg mutated TFR2-FLAG constructs by Genejuice (Merck Chemicals, Ltd, NJ, USA). At 48 h posttransfection, cells were fixed with 4% paraformaldehyde (Sigma Aldrich), permeabilized or not with Triton X-100 0.1%; incubated with the appropriate antibodies (mouse monoclonal anti-FLAG, [Sigma], rat monoclonal anti-E-cadherin [Millipore, CA, USA] or rabbit polyclonal anti-GRP78 BiP [Abcam, Cambridge, UK] as primary; AlexaFluor 488 rabbit-anti-rat, AlexaFluor 488 goat-anti-rabbit and AlexaFluor 568 goat-anti-mouse [Invitrogen Molecular Probes, OR, USA] as secondary); washed with PBS-Tween 0.02% and PBS and mounted in DAPI-containing mounting medium (Vector Laboratories, CA, USA). Epifluorescence images were acquired using a Leica DMI 6000B microscope at ×63 objective and analyzed by ImageJ software (Bethesda, MD).

MDA-MB-231 miR-NC, MDA-MB-231 miR-148a, and MDA-MB-231 miR-148a + PDK1 (1 × 10⁵ cells) were seeded in Millicell EZ 8-well slides (Millipore, Burlington, MA, USA). After 24 h, the cells were fixed with 4% formaldehyde and permeabilized with PBST containing 0.5% Triton X-100 for 20 min and blocked with 1% BSA/PBST for 2 h at room temperature. Next, 1% BSA/PBST containing antibodies against Vimentin (1:100) and E-cadherin (1:100) were added to the wells, and the slides were incubated at 4 °C overnight. Fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit secondary antibodies (1:200, Santa Cruz Biotechnology, Dallas, TX, USA) and tetramethyl rhodamine isothiocyanate (TRITC)-labeled goat anti-mouse secondary antibodies (1:200, Santa Cruz Biotechnology, Dallas, TX, USA) were used together to detect the two target proteins. Different colors of fluorescence were observed under a fluorescence microscope (ZEISS, Oberkochen, Baden-Württemberg, Germany).