Clc 3

Experiments were performed according to protocols previously described [31 (link)]. Samples were incubated overnight at 4⁰C with different dilutions for immunofluorescence with primary antibodies against ClC-3 (1:50, Abcam), β1 integrin (1:100, BD Pharmingen), α-tubulin (1:50, Abcam), vimentin (1:100, Boster), K18 (1:100, Boster) and K18 pS52 (1:50, Abcam). All antibodies were diluted to 1:1000 for immunoblotting.

Frozen tumor tissues or spheroids embedded in optimal cutting temperature (OCT) medium were cryosectioned into 10 µm and air-dried. For CDT1 staining, slides were fixed in Delaunay fixative and air-dried. Slides were rehydrated, fixed with 4% paraformaldehyde, permeabilized with 0.2% triton X-100 in PBS and blocked with 4% BSA, 5% normal serum and 0.1% Tween in PBS. We used the following antibodies diluted in 2% BSA, 2.5% normal serum in PBS, 0.1% Tween: pH3 (Abcam, ab5176, Cambridge, UK, 1:200), Cl.C3 (Cell signaling, Danvers, MA, USA, 1:400), pan-cytokeratin (Abcam, ab86734, Cambridge, UK, 1:300), CDT1 (Abcam, Ab202067, Cambridge, UK, 1:50), goat anti-rabbit IgG Alexa 488 (Thermo Scientific, A11008, Life Technology Europe BV, Zug, Switzerland, 1:500) and donkey anti-mouse IgG Alexa 555 (Thermo Scientific, A31570, Life Technology Europe BV, Zug, Switzerland, 1:500). We used an additional blocking step with anti-mouse IgG for the staining on mouse tissues (Thermo Scientific, A16074, Life Technology Europe BV, Zug, Switzerland, 1:25). Images were acquired by a Leica DM6000 (Leica Microsystems, Heerbrugg, Switzerland). We counted tumor cells (pan-cytokeratin+) positive for pH3 and Cl.C3 manually, and all tumor cells were quantified for CDT1 intensity using Image J 1.52a (Madison, WI, USA) software in at least 900 tumor cells.