Ab5706

Cocultures were completed and macrophages were fixed as described above. Coverslips were washed once with PBS prior to staining with SYTOX Green (final concentration, 10 μM) (ThermoFisher) for double-stranded DNA (dsDNA), and Hoechst 33342 (final concentration, 5 μM ) (ThermoFisher) for condensed chromatin (nuclei). Additional staining for histones and MMPs was accomplished by blocking cells in 1% bovine serum albumin in PBS for 30 min at 37°C followed by a 1-h incubation at 37°C with antibodies for histone H3 (ab5103; Abcam, Cambridge, MA), neutrophil elastase (ab68672; Abcam), myeloperoxidase (ab9535; Abcam), matrix metalloproteinase 1 (MMP-1) (ab551168; Abcam), MMP-7 (ab5706; Abcam), MMP-8 (ab81286; Abcam), MMP-9 (ab38898; Abcam), or MMP-12 (ab137444; Abcam). Cells were then washed three times with 1% BSA in PBS, followed by a 30-min incubation with an Alexa Fluor 594-conjugated goat anti-rabbit secondary antibody (ThermoFisher) and two additional washes with 1% BSA in PBS prior to mounting coverslips onto glass microscope slides with Aqua Poly/Mount (Polysciences Inc., Warrington, PA). Macrophages were visualized with a Zeiss LSM 710 META inverted laser scanning confocal microscope, and extracellular traps were identified by dsDNA staining that extended into the extracellular environment.

The commercially available mitochondrial division inhibitor, Mdivi-1, was from Sigma-Aldrich (Saint-Louis, MO, USA) and utilized for treating SK-HEP-1 and Huh7 cells, with DMSO (Sigma-Aldrich) as the control group. Translation inhibitor cycloheximide chase was also from Sigma-Aldrich for assessing protein stability. The primary antibodies against cleaved (c)-caspase-3 (ab2302, Abcam), total (t)-caspase-3 (ab13847, Abcam), c-caspase-6 (ab2326, Abcam), t-caspase-6 (ab185645, Abcam), Bax (ab32503, Abcam), Bcl-2 (ab32124, Abcam), E-cadherin (ab40772, Abcam), N-cadherin (ab76057, Abcam), MMP2 (ab37150, Abcam), MMP7 (ab5706, Abcam), DRP1 (ab184247, Abcam), PDI1 (ab4644, Abcam), CDK1 (ab18, Abcam), H-RAS (G12V) (ab140571, Abcam), LZTR1 (ab106655, Abcam), c-Myb (ab109127, Abcam), and GAPDH (ab245356, Abcam), as well as secondary antibodies were all obtained from Abcam (Cambridge, MA, USA). Antibodies against p-DRP1 (S616) (#3455S, Cell Signaling Technology, Danvers, MA, USA), p-ERK1/2 (#4370S, Cell Signaling Technology), and ERK1/2 (#4695S, Cell Signaling Technology) were obtained from Cell Signaling Technology (Danvers, MA, USA).

Total proteins were extracted through a Total Protein Extraction Kit (KeyGen Biotech, Nanjing, China). Concentrations of protein were examined under BCA commercial kit (KeyGen). Proteins were separated using SDS-PAGE and then moved onto PVDF membranes (Millipore, Carlsbad, USA). Membranes were sealed in non-fat milk and then incubated with given primary antibodies: anti-MMP2 (1:1000, ab37150, Abcam, Cambridge, USA), anti-MMP7 (1:1000, ab5706, Abcam), anti-Bcl-2 (1:1000, ab32124, Abcam), anti-bax (1:1000, ab32503, Abcam), anti-caspase 3 (1:1000, ab13847, Abcam), anti-cleaved caspase-3 (1:1000, ab2302, Abcam), anti-caspase 8 (1:1000, ab25901, Abcam), anti-cleaved caspase-8 (1:1000, ab25901, Abcam), anti-caspase 9 (1:1000, ab32539, Abcam), anti-cleaved caspase-9 (1:1000, ab2324, Abcam), anti-E-cadherin (1:1000, ab15148, Abcam), anti-N-cadherin (1:1000, ab76057, Abcam) and anti-GAPDH (1:1000, ab8245, Abcam). GAPDH was used as a measurement control for other proteins. Moreover, the membranes were co-cultured with goat anti-mouse IgG H&L (Cy3^®) preadsorbed (1:2000, ab97035, Abcam) secondary antibodies for 1 h darkness. Chemiluminescence system (Invitrogen) was employed to observe the protein bands.

RIPA buffer (Beyotime, Shanghai, China) was applied to extract protein from transfected Saos-2 or HOS cells. The protein concentration was quantified by BCA kit (Beyotime). Proteins in same amount were loaded on 10% SDS–PAGE gel (Bio-Rad) and shifted to PVDF membranes (Millipore, Bedford, MA, U.S.A.). Following incubation for 1 h in a closed buffer, blots were incubated with primary antibodies against MMP2 (ab97779, Abcam, Cambridge, MA, U.S.A.), MMP7 (ab5706, Abcam), MMP9 (ab38898, Abcam), E-cadherin (ab76319, Abcam), N-cadherin (ab18203, Abcam), Vimentin (ab92547, Abcam), FUS (ab70381, Abcam), LDHB (ab75167, Abcam), and GAPDH (ab9485, Abcam). Later, secondary antibodies were added and the ECL PLUS/KIT (GE Healthcare, Milwaukee, WI, U.S.A.) was applied for chemiluminescence detection.

RPMI 1640 and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). Antibodies against DKK1 (ab109416), β-catenin (ab32572) and MMP7 (ab5706) were purchased from Abcam (Cambridge, MA, USA). β-actin antibody (sc-47778), goat anti-rabbit IgG-HRP (sc-2004) and goat anti-mouse IgG-HRP (sc-2005) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). MMP7 siRNA (sc-41553) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).