Ma1 41017

RH4-BRD4-mGFP were seeded at 85,000 per well into chambered cover glass (ThermoFisher #155379PK, Waltham, MA, USA). The following day, they were treated with 100 nM ARV-771 or an equal volume of DMSO (0.1%). Then, 6 hours later, they were fixed with 1 mL of 4% formaldehyde for 14 min at RT, and quenched with 200 µL of glycine for 5 min at RT. They were subsequently washed with PBS 3x, permeabilized with 0.5% Triton X-100 in TE pH 7.4 for 15 min at RT, washed with PBS 3x, and blocked with 4% FBS in PBS-T (PBS + 0.1% Triton X-100) for 1 h at RT. The following primary antibodies and dilutions were then applied and incubated overnight at 4 °C: rabbit anti-BRD4 (Abcam #ab128874, Cambridge, UK) 1:200, mouse anti-MyoD (Invitrogen #MA1-41017, Waltham, MA, USA) 1:200 in PBS-T. The next day, slides were washed 3x with PBS-T, incubated for 1 h at RT protected from light with secondary antibodies, and washed again. Secondaries included anti-rabbit 594 (Cell Signaling #8889S, Boston, MA, USA) and anti-mouse 488 (ThermoFisher #A-21121) diluted 1:1000 in PBS-T. Slides were then stored in PBS at 4 °C until imaging on a Leica TCS SP8 gated STED microscope.

Immunohistochemistry (IHC) was performed using the DAB (3,3′-diaminobenzidine) method (K500711-2, Dako REAL EnVision Detection System, Agilent Technologies, Santa Clara, CA, USA). The procedures suggested by the manufacturer were followed. Briefly, the sections were fixed followed by blocking as mentioned for the IF above. Then, they were incubated overnight at 4 °C with the primary antibodies, including mouse anti-MyoD (MA1-41017, Invitrogen, 1:500) and mouse anti-MyoG (MA5-11486, Invitrogen, 1:500). After washing, the sections were incubated at room temperature with the secondary antibody and the DAB substrate of the DAB kit for 30 and 1 min, respectively. At least three animals from each group were included in this analysis.
A negative control for the immunostainings (IF and IHC) was included using staining without the primary antibodies.