Rock inhibitor y2763

Fresh tissue was collected into RPMI 1640 medium supplemented with 2% human serum (Sigma), cut into 1 mm² pieces, and enzymatically digested for 20min at 37°C using the Human Tumor Dissociation Kit (Miltenyi Biotec) in the presence of 10μM ROCK inhibitor Y-2763 (Sigma). Cell suspension was passed through 70μm cell strainers and centrifuged for 7min at 450g at 4°C. Supernatant was removed and cells were subject to ACK Lysing Buffer (Life Technologies) for 2min on ice, centrifuged for 7min at 450 g at 4°C, and resuspended in RPMI 1640 supplemented with 2% human serum (Sigma). The single cell suspension was stained with Zombie Violet in PBS (Invitrogen) for 10min on ice and subsequently with antibodies against human CD326, CD45, and CD235a (Biolegend) in RPMI 1640 medium supplemented with 1% human serum in the presence of 10μM Y-2763 for 15 min on ice. Zombie Violet⁻ CD235a⁻ CD45⁻ CD326⁺ cells were bulk sorted into 1.5ml Eppendorf tube containing 1× TD buffer, 2.5μl Tn5 (Illumina), 0.1 % NP40, 0.3× PBS in a 50μl reaction volume for ATAC-seq as described above. Using the identical gating scheme, single cells were sorted into Eppendorf twin-tec PCR plates containing 10μl TCL lysis buffer (Qiagen) supplemented with 1% beta-Mercaptoethanol and processed for scRNAseq as described above.

Antibodies against p127YAP (D9W2I), YAP (D8H1X), MST1, pMST (E7UD1), LATS1 (C66B5), pLATS1, Lamin B1 (D9V6H) and GAPDH were from CST. Antibodies of Ezrin, GNA12, GNA13, RhoA, flag, TEAD1, GST and GFP were from Santa Cruz Biotechnology. RGS12 antibody (ab1) was from Sigma. The secondary fluorescent antibodies were from Abcam. PDZ domain peptides of RGS12 were purchased from GenScript. GPCR activator LPA, GPCR inhibitor Ki6425, Rho GTPases inhibitor C3 and Rock inhibitor ‎Y2763 were obtained from Sigma. Doxorubicin hydrochloride (DOX), methotrexate 4-Amino-10-methylfolic acid hydrate (MTX) and EDTA-free cocktail inhibitor tablets were all obtained from Fisher Scientific^™. TEAD1 siRNA and controls were from Santa Cruz Biotechnology. FuGENE^® HD Transfection Reagent was purchased from Promega Corporation. The primers used for the quantification are listed in Supplementary Table 1.
The following plasmids: pRL-TK was generously provided by Dr. Zhen Zhang (University of Pennsylvania, Philadelphia, PA, USA); pcDNA3.1, shYAP1/2, pcDNA3.1-GFP-YAP, PET-GST-YAP and pcDNA3.1-RhoA were obtained from Addgene. Three human shRGS12 lentivectors (shRGS12–1, shRGS12–2 and shRGS12–3; Catalog # i019000) were ordered from ABM. pcDNA3.1-flag-RGS12 (flag-RGS12) and the pcDNA3.1-flag-RGS12 mutant with the deletion of the PDZ domain vectors (flag-RGS12ΔPDZ) were constructed in our lab.