Chip it express enzymatic

Chromatin was prepared according to the ChIP-IT Express Enzymatic protocol (Active Motif, Carlsbad, CA, USA). Immunoprecipitation was performed overnight using 2 μg of anti-NFATc1, anti-NFATc2, anti-IRF4 (Santa Cruz Biotechnology, Dallas, TX, USA), and anti-RNA Pol II and anti-IgG (Active Motif). Following incubation with antibody, DNA was eluted according to the Active Motif protocol and DNA was purified using the QIAquick PCR purification kit (Qiagen). Real-time qPCR was performed using iQSYBR green supermix (Bio-Rad) with the following primers: APOBEC3G ChIP-F 5′-GGG GAG GGG CTT GTG C-3′ and APOBEC3G ChIP-R 5′-AAG GCA ATT GCA AAG GGA A-3′. PCR was performed in triplicate with reaction conditions of 95 °C for 10 min followed by 40 cycles of 95 °C for 30 s, 60 °C for 1 min. Fold enrichment was calculated for each ChIP antibody used as quantity of ChIP DNA divided by amount of IgG DNA.

After treatment with MSP or vehicle for 4 h, cells were incubated with 1% formaldehyde in cell culture medium for 10 min at room temperature, and then the reaction was stopped with 0.12 M glycine for 5 min. DNA shearing and ChIP used the ChIP-IT Express Enzymatic (Active Motif) kit, following the recommended protocol. In the IP step, anti-histone H3, anti-H3acK9,K14 (Cell Signaling), and anti-RNA polymerase II (Active Motif) were incubated overnight at 4 °C. Additional IP experiments used antibodies to Sp1, Sp3, STAT3, HDAC8, HDAC1, and p300 (from sources listed above). After eluting pull-down DNA from magnetic beads, DNA purification used the QIAquick PCR Purification kit (Qiagen, Valencia, CA, USA). Quantification of immunoprecipitated and input (control) DNA was carried out by qPCR on a Light Cycler 480 II (Roche), with Light Cycle 480 SYBR Green I Kit master (Roche). The primer sequences are available on request.

Cells are prepared according to the truChIPTM Shearing Kit protocol (Covaris), collected, and rinsed using cold PBS 1 X. Cell pellets were fixed using buffer A and using 1% formaldehyde. After 10 min with stirring, we add buffer E then the cells are centrifuged (500 G, 5 min, room temperature). After two washes with PBS 1 X, buffer B is added to lyse plasma membranes (10 min with stirring at 4 °C). Then, cells are centrifuged, and the pellets resuspended in washing buffer C (10 min at 4 °C). The nuclei are recovered by centrifugation (1700 G, 5 min at 4 °C). The nuclear pellet is resuspended in the D3 buffer and transferred to milliTUBE AFA Fiber tubes (Covaris, Woburn, MA, USA). The samples are then ultrasound (3 sessions of 12 min). After this nuclear preparation, ChIP was conducted according to the manufacturer’s instruction: ChIP-IT^® Express Enzymatic (Active Motif, La Hulpe, Belgium) protocol. After dosing the chromatin, we carry out an incubation of 50 µg of chromatin with the magnetic beads, a supplied ChIP buffer, and the anti-VEGFR-2 (Cell Signaling, Danvers, MA, USA) or anti-VEGF (Cell signaling) antibodies overnight at 4 °C. The beads are then washed several times, and the chromatin is eluted using a buffer. The DNA obtained can be used immediately in PCR or stored at −20 °C. The sequences of the oligonucleotides used are described in Table 1.

MC3T3-E1 cells were plated into 15 cm dishes (1.5 × 10⁷ cells) at the densities described above in the Cell Culture section. ChIP was performed using antibodies against STAT5a (Cell Signaling Technology) (156 μg/mL) or rabbit IgG (Cell Signaling Technology) (2.5 mg/mL) using the ChIP-IT Express Enzymatic (Active Motif, Carlsbad, CA) according to the manufacturer’s instructions. DNA was amplified using primers flanking the putative STAT5a binding site (PROMO) within the GJA1 promoter.

ChIP assay was performed for antibodies listed in Supplementary Table S7 using the EZ-ChIP Chromatin Immunoprecipitation Kit (#17-371, Millipore, Billerica, MA, USA) or ChIP-IT Express Enzymatic (Active Motif) according to the manufacturer's instructions. DNA isolated from ChIP was quantified by qPCR using CA9 or VEGF promoter primers (Supplementary Table S4) (n=3).