Intraprep permeabilizaton reagent

FcγRI (CD64), FcγRIIA (CD32A), FcγRIIB (CD32B) and FcγRIII (CD16) expressions on monocytes (CD16+) were measured after the 2.5-hour incubation time of the ADCC/ADCP assay, without prior CFSE labeling of SKBR3 cells and without trastuzumab. As CD32A and CD32B can only be distinguished on their intracellular domain, the cells were fixed and permeabilized with the IntraPrep Permeabilizaton Reagent (Beckman Coulter) according to the manufacturer’s protocol. The cells were incubated with primary antibodies (online supplementary table S1) for 30 min at 4°C. Unconjugated antibodies were further stained with secondary antibodies for 30 min at 4°C (online supplementary table S1). FcγR expression levels were analyzed using a Gallios G flow cytometer (Beckman Coulter Inc.) and quantified with Kaluza Analysis Software V.2.1.1 (Beckman Coulter Inc.). Histogram overlays were created with FlowJo V.10.6.1 (Becton Dickinson, Franklin Lakes, New Jersey, USA).

SKBR3 cells were treated with different VPA and SAHA concentrations as described previously. The cells were further incubated with primary antibodies for HER2, CALR, CD47 or MCL1 (online supplementary table S1) for 30 min at 4°C. Unconjugated antibodies were further stained with secondary antibodies for 30 min at 4°C (online supplementary table S1). In the case of an intracellular protein staining, the cells were fixed and permeabilized with the IntraPrep Permeabilizaton Reagent (Beckman Coulter Inc.) according to the manufacturer’s protocol. Different protein expression levels were analyzed by the use of a Gallios G flow cytometer (Beckman Coulter Inc.) and quantified with Kaluza Analysis Software V.2.1.1 (Beckman Coulter Inc.). Histogram overlays were created with FlowJo V.10.6.1 (Becton Dickinson, Franklin Lakes, New Jersey, USA).