For immunofluorescence, tumor cells were obtained as described in Rowald et al. (2016) (link). Cells were cultured on coverslips coated with Collagen I (Cultrex, 344.020-01). After 24 h, cells were fixed with 4% PFA for 10 min at room temperature or with a methanol/acetone mix for 10 min at −20°C and permeabilized with 0.2% Triton X-100 PBS for 10 min. Cells were treated with blocking buffer containing 5% donkey serum, 1% BSA, and 0.2% Triton X-100 in PBS for 1 h at RT. The following primary antibodies were used: rabbit anti-LC3 (1:100, Sigma, L7543), rabbit anti-Fibronectin (1:40, Abcam, ab23750), mouse anti-Ataxin1 (1:50, Santa Cruz, sc-365343), mouse anti-Myosin X (1:50, Santa Cruz, sc-166720), mouse anti-RhoA/RhoC (1:100, Thermo Scientific, 1B3-4A10), mouse anti-Vimentin (1:50, Sigma, V2258), and rabbit anti-AKT3 (1:800, Cell Signaling, E1Z3W). Donkey anti-mouse Alexa 488 and anti-rabbit Alexa 568 secondary antibodies were used (1:500, Invitrogen), and the DNA was stained with DAPI. Images were acquired with the Leica LAS 4.5 software on a Leica SP5 confocal microscope. Quantification of LC3 fluorescence intensity was performed with ImageJ software.