Decma 1

Anti-RHBDL2 and anti-E-cadherin antibodies were purchased from Proteintech (Rosemont, IL, USA). Anti-VE-cadherin (clone BV-9) and anti-HA (clone F-7) antibodies were supplied by Santa Cruz Biotechnology. EGFR phosphorylation was detected by a phospho-specific antibody (directed against p-Tyr¹⁰⁶⁸) from Abcam (Cambridge, UK). The total and phosphorylated forms of p44/42 MAPK(Erk1/2) and AKT (against pAKT-S⁴⁷³ and pMAPK-Thr²⁰²/Tyr²⁰⁴) were detected with antibodies from Cell Signaling Technology (Danvers, MA, USA). Other antibodies that were applied in this study were: for Western Blotting, anti-vinculin (Sigma), anti-N-cadherin (Abcam), anti-Thrombomodulin (D-3, Santa Cruz Biotechnology, Dallas, CA, USA), and anti-RFP (Rockland Immunochemicals Inc., Limerick, PA, USA), for immunofluorescence anti-E-cadherin (clone 36/E, BD Transduction Laboratories) and anti-VE-cadherin (clone F-8, Santa Cruz Biotechnology). The secondary antibodies were purchased from Jackson Laboratories (Bar Harbor, ME, USA). The E-cadherin function-blocking antibody DECMA-1 was purchased from Sigma Aldrich. Santa Cruz Biotechnology supplied the IKK inhibitor IKK-16, while the metalloproteases inhibitors BB94 and Marimastat and the serine protease inhibitor 3,4-dichloroisocoumarin (DCI) were from Sigma. Human recombinant TNFα was purchased from Peprotech (Rocky Hill, NJ, USA).

To disassemble the adherens junctions in monolayers, the cells were incubated with DMEM (no serum or antibiotics) containing 4 mM EGTA for 1 hour at 37 °C, 95% humidity and 5% CO₂. Cells were then returned to regular growth media (calcium repletion) and fixed at various time points of the calcium switch experiment (steady state, low calcium, 1, 2 and 3 hours after calcium repletion). For live-cell microscopy, imaging was performed after returning the cells to regular growth media composed of: FluoroBrite DMEM (Life Technologies), FBS (10% v/v), penicillin (100 I.U./mL), streptomycin (100 μg/mL), OxyFluor (1% v/v, Oxyrase) and Sodium dl – Lactate (20 mM, Sigma Aldrich). For fixed cells and live cell imaging E-cadherin function blocking antibody (100 μg/ml, DECMA-1, Sigma Aldrich) was added during calcium repletion.

Torin 1 (S2827, Selleckchem), PD98059 (A1663, ApexBio) and danusertib (A4116, ApexBio) were injected via intraperitoneal (i.p.) injection, daily, at the dosage of 6.8, 10 and 15 mg kg⁻¹, respectively. EPZ6438 (A8221, ApexBio) was injected via i.p. injection once every 2 days at 50 mg kg⁻¹. For antibody therapy, 4 mg kg⁻¹ DECMA-1 (U3254; Sigma) or rat immunoglobulin G (I4131; Sigma) was injected via i.p.injection twice a week. In the experiments using MCF-7 cells, oestradiol pellets were prepared and transplanted into nude mice before cancer cell injection, according to a previously published protocol50 (link), unless otherwise noted.