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  • br O Bozickovic et al br Western blot immunostaining


    O. Bozickovic et al.
    2.4. Western blot, immunostaining and protein quantification
    Cells were washed, harvested in ice-cold PBS by scraping and lysed in RIPA lysis buffer (EMD Millipore, Temecula, CA, USA), supple-mented with 5 mM EDTA, 0.1 u M Sodium orthovanadate, Complete Mini EDTA-free protease inhibitor cocktail (Roche) and PhosStop phosphatase inhibitor cocktail (Roche). Cell lysates were dissolved by SDS-PAGE on a 4–20% gel (BioRad, Hercules, CA, USA) and transferred to nitrocellulose membranes (BioRad). The membranes were blocked with 5% BSA solution in TBS with 0.1% Tween (TBS-T) for 1 h, followed by sequential washing with TBS-T. Incubation with primary antibody was done for 1 h at 37 °C or overnight at 4 °C, followed by washing with TBS-T and incubation with secondary antibody for 30 min. All blots were detected using SuperSignal West Femto Maximum Sensitivity Substrate (ThermoFischer Scientific, Waltham, MA, USA) and the ChemiDoc XRS System (BioRad). The primary ATPγS tetralithium salt used were mouse monoclonal anti-TIF-2 (610985, BD Biosciences, San Jose, CA, USA), mouse monoclonal anti-AIB1 (611105, BD Biosciences), mouse monoclonal anti-Lyn (376100, Santa Cruz Biotechnology), rabbit polyclonal anti-P-Lyn (Tyr507) (2731, Cell Signaling, Danvers, MA, USA) and mouse monoclonal anti-β-Actin (AC15) (6276, Abcam). Target proteins were quantified relative to the protein level of β-Actin (ACTB) as a reference, using the QuantityOne analysis software (BioRad).
    shKTR, shSRC-2 and shSRC-3 cultures were trypsinated using TrypLE (ThermoFisher Scientific) and counted on Countess cell counter (Invitrogen). Cells were washed once in flow cytometry (FC) buffer (D-PBS with 0.5% BSA) prior to staining. 300 000 cells were used per staining reaction in 50 μl buffer. Cells were stained first with antibodies against cell surface markers (CD24, MUC-1) and live-dead markers for 30 min, washed, then fixed and permeabilized for 45 min and washed again in order to stain with antibodies against internal markers (KRT5, KRT8 and p63) for 30 min. All reactions were performed at room temperature. Anti-EpCAM staining was performed separately from the rest of the markers and did not include fixing or permeabilizing. Live dead discriminators used were 7-Aminoactinomycin D (7-AAD, BioLegend, 420404, 1:100) with anti-EpCAM staining, and LIVE/DEAD Fixable Aqua Dead Cell Stain (ThermoFisher Scientific, L34957, 1:20 000 AU). Clone information and catalogue numbers of antibodies used can be found in the Supplemental Table 2. Concentrations used were the following: anti-CD24-PerCP 1:50, anti-MUC-1 1:100, anti-KRT8 1:250, anti-KRT5 1:1000, anti-p63 1:1:100. Fixation and permeabili-zation were performed using Fixation and Permeabilization Buffer Set (eBioscience, 88-8824-00) and Permeabilization Buffer (e-Bioscience, 00-8333-56). Compensation matrix was created using AbC Total Antibody Compensation Beads (Thermo, Life Technologies A10513) and ArC Amine Reactive Compensation Bead Kit (Thermo, Life Technologies A10346). Fluorescence-minus-one (FMO) controls were used to distinguish between the positive and negative population should there be two separate populations found. Samples were analyzed on LSR Fortessa flow cytometer (BD Biosciences) one day after staining. Data was analyzed using FlowJo software v10.4 (FLOWJO LLC). Gating strategy can be found in Supplementary Fig. S1 (all markers except EpCAM) and Supplementary Fig. S2 (EpCAM). Geometric medians (MFIs) were extracted from the Live population, since only one popu-lation could be detected.
    2.6. Embedding and immunohistochemistry of 3D spheroid cultures
    For subsequent immunohistochemistry, 3D spheroid cultures were captured in a thrombin-gel and embedded in paraffin. Briefly, spheroids were first collected and fixed in 3.7% formalin overnight, and then counterstained with methylgreen for 2 min. Following a brief wash in