X tremegene 9 reagent

Cells were seeded in regular growth medium into 24-well plates. The next day the growth medium was replaced with medium lacking serum and the transfection was performed using X-tremeGene9 reagent, as recommended by the manufacturer (Roche Diagnostics), with a mixture containing Cyclin-D1-luc, the internal control pRL-TK and miR-Ctrl, miR-338-3p m, alone or in presence of miR-338-3p i, shGPER, DN-Fos as indicated. The cells were treated overnight with 100 nM of E2 or G1. Luciferase activity was measured using the Dual Luciferase kit (Promega, Milan, Italy) according to the manufacturer’s instructions. Firefly luciferase values were normalized to the internal transfection control provided by the Renilla luciferase activity. The normalized relative light unit (RLU) values obtained from cells transfected with respective scrambled controls were set as 1-fold induction upon which the activity induced by the treatment was calculated.

HTL cells were split at 50 000 per well in a 24-well plate. After 1 day of growth, the cells were transfected with 65 ng total DNA using X-tremeGENE 9 Reagent (Roche Diagnostics, Indianapolis, IN, USA) with a ratio of 1:3 (1 µg DNA:3 µl reagent) according to the standard protocol. For coexpression with γ-secretase, 20 ng substrate-encoding DNA, 5 ng phRG-tk Renilla normalization standard and 10 ng of each of the γ-secretase subunit expression plasmids were transfected. For substrate only, 20 ng substrate, 5 ng phRG-tk Renilla and 40 ng pBSK mock plasmid were transfected with or without proper amount of inhibitors. For the control Tango assay, 10 ng Rho(4M)-TEV-site-rTA, 10 ng Arr(3A)-TEV, 5 ng phRG-tk Renilla and 40 ng pBSK mock plasmid were co-transfected. Cells were harvested and lysed the following day. Luminescence activities were measured using the Dual Luciferase Kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Renilla luciferase serves as transfection control. Relative activity is normalized activity using WT C99-T4L-rTA activity as 100.

The Wnt-induced luciferase activity was analyzed as described. (Koval et al., 2014 (link); Shaw et al., 2019 (link)) Briefly, 30 μl of BT-20 cells stably transfected with the TopFlash luciferase reporter (Koval et al., 2014 (link)) at 150,000 cells/ml were distributed in a white opaque 384-well plate. The cells were maintained in DMEM containing 10% FBS and incubated at 37°C, 5% CO₂ overnight for attachment. Afterwards, the BT-20 cells were additionally transfected with the plasmid constitutively (under the CMV promoter) expressing Renilla luciferase (Addgene, Cambridge, MA, United States). Transfection was carried out as described in manufacturer’s protocol using 12 µg/ml of DNA and 40 µl/ml XtremeGENE 9 reagent (Roche). Next day, the medium of each well was replaced by 30 μL fresh medium containing Wnt3a (500 ng/ml) [purified as described (Koval et al., 2011 (link)] or CHIR99021 (1 µM) and the compounds at 6-8 different concentrations (following 1h of pre-incubation with the compound). After overnight incubation, the supernatant in each well was removed and the cells were harvested and analyzed for activities of both firefly and Renilla luciferase as described (Koval et al., 2014 (link)).

HeLa cells were seeded into 96-well glass bottom plates (Greiner) at a density of 7000 cells per well 24 h prior to transfection. The cells were then transfected as described [25 (link)] with Rab11-GFP (Fig 4A–4C), 2XFYVE-GFP (Fig 3C–3E), galectin-3-mOrange (Figs 3C–3E, 4 and 5) or Rab11S25N-GFP, a Rab11 GDP-locked dominant negative (DN) (Fig 4D, kindly provided by B. Goud (Institut Curie)) using X-tremeGENE 9 reagent (Roche) for 24–48 h, according to the manufacturer’s instructions. For mutant library rupture timing screen (Fig 3B) cells were transfected with Actin-mOrange [30 (link)] and galectin-3-GFP [36 (link)] in an identical manner.

SkBr3 cells were serum deprived and transfected for 24 h with a control shRNA or shGPER using X-tremeGene9 reagent (Roche Molecular Biochemical), as recommended by the manufacturer, and then treated for 8 h with vehicle (–), E2 (10 nM) or G-1 (100 nM) before to be exposed for additional 8 h to conditioned medium from CAFs treated for 8 h with E2 or G-1. Then cells were fixed in 4% paraformaldehyde. After washed with PBS, images were acquired using the Cytation 3 Cell Imaging Multimode Reader (BioTek, Winooski, VT). For each individual cell, the polarity index (PI) was calculated dividing the length of the long migration-defined axis by the perpendicular axis passing by the centroid of the cell36 (link).

Cells were seeded in regular growth medium into 24-well plates. The next day the growth medium was replaced with medium lacking serum and the transfection was performed using X-tremeGene9 reagent, as recommended by the manufacturer (Roche Diagnostics), with a mixture containing CTGF-luc, the internal control pRL-TK, miR-Ctrl or miR-221, alone or in presence of LNA-i-miR-221, shRNA or shRel, alone or in combination with miR-221. Luciferase activity was measured after 48 h using the Dual Luciferase kit (Promega, Milan, Italy) according to the manufacturer’s instructions. Firefly luciferase values were normalized to the internal transfection control provided by the Renilla luciferase activity. The normalized relative light unit (RLU) values obtained from cells transfected with respective scrambled controls were set as 1-fold induction upon which the activity induced by miR-221 was calculated.