Genjet transfection reagent

To quantitatively track EGFP-YAP mechanotransduction with time, we transiently transfected NIH-3T3 cells with two plasmids, pEGFP-yap-C3-hYAP1 (Addgene, plasmid #17843) (17 (link)) and EBFP2-Nucleus-7 (nuclear localization signal, Addgene, plasmid #55249), using GenJet transfection reagent (SignaGen). Eighteen to twenty-four hours later, cells were seeded on fibronectin-coated PDMS, PAA, and glass substrates, and after cell attachment, they were transferred to a lab-built heated stage perfused with 5% CO₂ and mounted on a confocal microscope (Leica TCS SP8 with a 10× 0.4NA objective).
In order to examine the effects of the LINC complex on contractile and force-mediated nuclear compression and YAP translocation, we transfected the cells with two dominant-negative GFP-Nesprin1-KASH and GFP-Nesprin2-KASH (DNK1/2) plasmids which were kindly provided by Dr. Catherin Shanahan’s laboratory (King’s College, London) (30 (link), 83 (link)).

Genes for human RELA/P65 (NCBI Reference Sequence NM_021975.3) with a FLAG tag and human NFKBIA/IκBα (NCBI Reference Sequence NM_020529.2) with a FLAG tag were cloned into pcDNA3 (Invitrogen, Carlsbad, CA, USA). The plasmids (1–2 μg) were transiently transfected for 6 h using Genjet transfection reagent (SignaGen, Gaithersburg, MD, USA) according to the manufacturer's instructions. For the reporter assay, four copies of the NF-κB consensus site (5′-GGGAATTTCC-3′) were cloned into the pGL3 luciferase reporter vector (Promega, San Luis Obispo, CA, USA). In addition, 4T1 or MDA-MB-231 (Supplementary Figure 2) cells were transiently transfected in six-well culture plates with NF-κB-luciferase reporter vector (1 μg) for 6 h. The cells were treated with CXCL10 or JN-2 for 24 h after transfection. Luciferase activity was measured using the dual-luciferase assay system (Promega) according to the manufacturer's instructions.

For identification of the cellular partners interacting with CIB1, we transfected HEK239T cells (∼10⁷ cells per 10-cm dish) in 10 dishes with 12 µg plasmid encoding CIB1-FLAG or ORF75-FLAG (control) in the presence of GenJet transfection reagent (SignaGen). 36 h later, the cells were lysed with RIPA buffer supplemented with protease inhibitor cocktail and centrifuged at 13,000 rpm for 20 min at 4°C. Lysates were precleared with 100 µl Sepharose 4B (Sigma-Aldrich) to remove cell debris and then were mixed with a ∼50% slurry of anti-FLAG M2 Affinity Gel (Sigma-Aldrich) and incubated for 4 h at 4°C. The beads were thoroughly washed in lysis buffer, and the bound proteins were eluted by heating samples in 2× Laemmli SDS sample buffer for 5 min at 95°C before separation by electrophoresis on a NuPAGE 4–12% Bis-Tris gradient gel (Thermo Fisher Scientific). The coimmunoprecipitated proteins were stained by incubation with colloidal Coomassie solution (0.02% Coomassie Brilliant Blue G-250, 5% aluminum sulfate-(14–18)-hydrate, 10% ethanol, and 8% orthophosphoric acid [85%]; Kang et al., 2002 ) at room temperature overnight. Bands specifically present in the CIB1-FLAG sample but absent from the ORF75-FLAG sample were excised and analyzed by ion-trap mass spectrometry at the Taplin Biological Mass Spectrometry Facility (Harvard Medical School, Boston, MA).