Rabbit anti mlc2

Suit2-007 cells (metastatic PDAC cell line) were kindly donated by Prof. Malte Buchholz from Philipps-Universität Marburg. Suit2 cells were cultured in Dulbecco’s Modified Eagle’s Media (Merck, Dorset, UK, D8437) supplemented with 10% v/v foetal bovine serum (FBS; Merck, F7524), 2 mM l-glutamine (Merck, G7513), 1% v/v penicillin/streptomycin (Merck, P4333) and 1% v/v fungizone Amphotericin B (Gibco, Thermo Fisher Scientific, UK, 15290-026). Cells were incubated at 37 °C, with 5% CO₂. For all RAR-β agonist treatments, cells were exposed to 1 μM RAR-β agonist (CD 2314, Tocris, Abingdon, UK, 3824) 24 h prior to experiments. Gene transfection was performed 48 h prior to experiments, utilising the Neon transfection system (Thermo Fisher Scientific) with 2 μg MLC-2 plasmid (pEGFP-MRLC1, a gift from Tom Egelhoff, Addgene, #35680), 10 μg RAR-β siRNA (Santa Cruz, SC-29466) or 10 μg control siRNA (siRNA-Scr, Santa Cruz, SC-37007). Tissue micro arrays (TMAs) were obtained from Biomax (RAR-β: PA803, MLC-2: PA242e). Primary antibodies used were rabbit anti-RAR-β (Abcam, Cambridge, UK, ab53161), rabbit anti-MLC-2 (Cell Signaling Technology, 3672S), mouse anti-PAN Cytokeratin (Abcam, ab6401), rabbit anti-YAP (Cell Signaling Technology, 4912S) and rabbit anti-laminin (Sigma-Aldrich, L9393). See Supplementary Methods for immunostaining, tissue microarray, ChIP-seq and RT qPCR details.

Whole cells were lysed with RIPA lysis buffer (Beyotime, China, Cat No.P0013B). After protein quantification with the BCA Protein Assay Kit (Thermo Fisher Scientific, CA, Cat No.23225), equal amounts of proteins were separated on SDS-PAGE, and transferred to a PVDF membrane. Antibodies against the following proteins were used: Rabbit-anti-NRF2, rabbit-anti-MLC2, rabbit-anti-phospho-MLC2 (Thr18/Ser19), rabbit-anti-FAK, rabbit-anti-phospho-FAK (Tyr397), and rabbit-anti-ERR1, all purchased from Cell Signaling (Cat No. 12721, 3672, 3674, 3285, 8556 and 13826, respectively). Mouse-anti-RhoA was purchased from Cytoskeleton (Cat No. ARH03, CA), and mouse-anti-actin was purchased from ProteinTech (Cat No. 60008-1-Ig, CA).