Dm 5000 m fluorescence microscope

To assay for let-7 activity (and miR-1 activity as a control) we used miRNA sensors (pLVx-let-7s or pLVX-miR-1s^{29 (link)}), which were a gift from Dr. Tom Reh (University of Washington). HER10 cells were transfected in a 24-well plate with 0.5 μg miRNA sensor constructs and 0.5 μg pCAG-mCherry using Lipofectamine 2000 (Invitrogen) according to manufacturer’s protocols. After transfection, transfection media was replaced with maintenance media supplemented with 40 μM Nocodazole (Sigma-Aldrich) and incubated for 18 hours. After Nocodazole treatment, cells were immediately fixed in 4% paraformaldehyde for 10 minutes at RT and washed with PBS for subsequent analysis. The efficiency of Nocodazole treatment was confirmed by immunostaining for PH3, as described above. Fixed and stained cells were imaged on a Leica DM 5000 M fluorescence microscope with a Leica DFC 500 camera using the same exposure times to compare control and Nocodazole treated samples. Quantitation was performed by counting the number of cells that were both GFP- and mCherry+ from one image per well with 3 wells per condition (n = 3 separate experiments) using the Leica LAS X software. Images were assembled in Adobe Photoshop.

HER10 cells, mCSCs or primary cultures were appropriately transfected (described above) using Lipofectamine 2000 (Invitrogen) according to manufacturer’s protocol. 24 hours after transfection, cells were given a 30-min pulse of 10 μM EdU (5-ethynyl-2’-deoxyuridine; Invitrogen). After EdU incubation, cells were washed 2X with PBS, new maintenance media was added, and cells were incubated for an additional 18 hours before fixation in 4% paraformaldehyde for 10 min at RT. After fixation, EdU was visualized using the ClickIT EdU Alexa Fluor 488 Plus imaging kit (Invitrogen) according to manufacturer’s protocol. After EdU visualization, samples were blocked with 4% non-fat dry milk in TST (10 mM Tris, 150 mM NaCl with 0.1% Tween) and immunostained overnight at 4 °C with rabbit anti-Ki67 (Clone SP6; Thermo Scientific) diluted 1:200 in blocking solution. After incubation with an Alexa Fluor 647-conjugated donkey-anti-rabbit secondary antibody (Invitrogen) diluted 1:200 in 1% non-fat dry milk in TST, cells were washed and imaged on a Leica DM 5000 M fluorescence microscope with a Leica DFC 500 camera. Quantitation was performed by counting the number of transfected (mCherry+) cells that were EdU+ and Ki67+ from 3–5 images per experiment (n = 3 separate experiments) using ImageJ. Images were assembled in Adobe Photoshop.