Calyculin a

Briefly, THP-1 human monocytes (TIB-202; ATCC) were cultured in RPMI-1640 (189-02025; Wako) supplemented with 10% fetal bovine serum (175012; Nichirei), penicillin (100 U/mL) (168-23191; Wako), and streptomycin (100 mg/mL) (168-23191; Wako). F. nucleatum was cultured as previously described (43 (link)). THP-1 monocytes were pretreated with 100 μM FAC (RES20400-A7; Sigma-Aldrich), 100 μM DFO (D9533; Sigma-Aldrich), or the indicated concentration of calyculin A (038-14453; Wako) for the indicated time, followed by stimulation with 100 ng/mL LPS (tlrl-eblps; InvivoGen) for the indicated time. THP-1 monocytes were differentiated into macrophages using 6 ng/mL phorbol 12-myristate 13-acetate (AG-CN2-0010; AdipoGen) for 48 hours. Differentiated cells were pretreated with 100 μM FAC, 100 μM DFO, or 5 μM TAK-242 (13871; CAYMAN) for the indicated times, followed by treatment with F. nucleatum at a multiplicity of infection of 10 for 3 hours.

The invasive breast cancer MDAMB231 cells, non-invasive tumorigenic breast cancer MCF7 cells, and normal mammary epithelial MCF10A cells were used. MCF10A cells were cultured in growth medium composed of Dulbecco’s modified Eagle’s medium/Ham’s F-12 containing HEPES and L-glutamine (DMEM/F12, Invitrogen) supplemented with 5% horse serum (Invitrogen), 1% penicillin/streptomycin (Invitrogen), 10 μg/mL insulin (Sigma), 0.5 μg/mL hydrocortisone (Sigma), 20 ng/mL EGF (Peprotech) and 0.1 μg/mL cholera toxin (Sigma) and maintained under humidified conditions at 37 °C and 5% CO₂. MDAMB-231 and MCF7 cells were cultured in growth medium composed of Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen), supplemented with 10% fetal bovine serum (Invitrogen) and 1% penicillin/streptomycin (Invitrogen) and maintained under humidified conditions at 37 °C and 5% CO₂. Cells were passaged regularly by dissociating confluent monolayers with 0.25% trypsin-EDTA (Invitrogen). Cells were passaged at 1:4 in growth medium. Blebbistatin (Sigma) dissolved in dimethylsulfoxide (DMSO) was used at 5 μM in growth media for the myosin II inhibition experiments. Calyculin A (Wako) dissolved in dimethylsulfoxide (DMSO) was used at 0.1 nM in growth media for the myosin II enhancement experiments.

After treatment, chromosome spreads were obtained following 30 min incubation in 30 μM calyculin-A (Wako, Germany) [68 (link)]. Spreads of these prematurely condensed chromosomes (PCC) were prepared by a standard procedure, consisting of treatment with a hypotonic solution (75 mM KCl) followed by fixation in freshly prepared Carnoy solution. Q-FISH staining was performed as described by Berardinelli et al. [69 (link)] with minor modifications. Briefly slides and probes (Cy3 linked telomeric and chromosome 2 centromeric Peptide Nucleic Acid PNA probes; PANAGENE, Republic of Korea) were co-denatured at 80 °C and hybridized for 2 h at room temperature in a humidified chamber. Slides were counterstained with DAPI in Vectashield. Images were captured at a 63× magnification with Axio Imager M1 microscope equipped with a CCD camera. The telomere size was analyzed with ISIS software (MetaSystems, Altlußheim, Germany). In particular, the software calculates telomere lengths as the ratio between the total telomeres fluorescence (T) and the fluorescence of the centromere of the two chromosomes 2 (C), thus data were expressed as a percentage (T/C%). Experiments were repeated at least three times, and at least 10 metaphases were scored for each experiment.