Mrtf b

Protein samples were extracted using ice-cold RIPA buffer (Thermo Scientific), supplemented with protease inhibitor and phosphatase inhibitor (Roche) when necessary. 20–40 μg of protein were fractionated by SDS-PAGE gel, transferred to PVDF membranes (Millipore) and blocked in 5% non-fat milk-PBS. Primary and HRP-conjugated secondary antibodies were applied followed by enhanced chemiluminescence substrate (Supersignal, Thermo Sci.) for detection. The antibody dilutions used were: SRF 1:2,000 (Santa Cruz, sc-13029), HSP90 1:2,000 (Cell signal, #4874), MRTF-A 1:500 (Sigma, HPA030782), MRTF-B 1:500 (Bethyl Lab., A302–768A), Vinculin 1:10,000 (Millipore, FAK100), C/EBPα 1:1,000 (Santa Cruz, sc-61), C/EBPβ 1:1,000 (Santa Cruz, sc-150), PRDM16 1:500 (Santa Cruz, sc-130243), GAPDH 1:50,000 (Millipore, MAB374), aP2 (R&D, AF1443), UCP1 1:500 (Millipore, AB3038), Smads 1:1,000 (Cell Signaling, #9963), α-SMA 1:1,000 (A5228, Sigma), α-tubulin 1:1,000 (Santa Cruz, sc-8035), TBP (Santa Cruz, sc-204).

Cells were grown in four-well cell culture chamber slides were fixed with 4% paraformaldehyde, followed by permeabilization in 0.5% Triton X-100 and blockade in 2% BSA prior to staining. Cells were incubated with primary antibodies: SRF (Abcam, ab155013, 1:200), MRTF-A (Sigma, HPA030782, 1:100) or MRTF-B (Bethyl Lab., A302–768A, 1:200) overnight at 4°C, followed by Alexa Fluor 594 conjugated anti-rabbit IgG antibody (Invitrogen, A11012, 1:200) for 1 hour at room temperature. Phalloidin (Invitrogen; A12379 or A12380, 1:1,000) or BODIPY® lipid probe (4,4-difluoro-3a,4adiaza-s-indacene, Invitrogen; D-3922, 1 mg/ml) were applied directly after Triton X-100 incubation. Slides were mounted with anti-fade mounting medium with DAPI. MitoTracker Deep Red (Invitrogen; M22426, 100 nM) incubation in live cells was used for mitochondria staining, as described (Nam et al., 2015 (link),Nam, Chatterjee, Yin et al., 2015 (link)). Immunofluorescent images were captured using a Nikon 80i microscope with a color camera and processed using Nikon NIS Elements acquisition software.

To quantify in vivo proliferation, mouse lungs bearing metastatic tumors were paraffin embedded, sectioned into 5 μm thick slices, stained for Ki67 (Cell Signaling Technologies, 9129) together with DAPI, and imaged using a 3DHISTECH Pannoramic Scanner with a 20× objective lens. For imaging of murine lungs, tissue was PFA perfused and fixed overnight at 4 °C, followed by paraffin embedding. Sections were stained for NKp46 (R&D Systems, Cat # AF2225), CD8 (Cell Signaling Technology, Cat # 98941), and MRTFB (Bethyl Laboratories, Cat # A302-768A). Metastatic lesions were analyzed and CD8⁺ and NKp46⁺ cells counted using CaseViewer (3D Histech) or Aperio Imagescope (Leica Biosystems) software. Patient samples of breast and lung cancer with matched brain metastases were immunostained using the Ventana Discovery with and anti-MRTFB antibody (Bethyl Laboratories, Cat # A302-768A), followed by imaging using CaseViewer.