MM cell lines were treated for 72h with pomalidomide (Selleck), bortezomib (Selleck), and dexamethasone (MilliporeSigma) individually or in combination (half IC10 concentration for bortezomib and half IC10 for MM1S for dexamethasone and pomalidomide, half IC50 for OPM2 and MOLP2) at the indicated concentrations. To investigate the effects of drug removal on CXCR4 expression, cells were treated for 72h, washed, and incubated in RPMI-1640 media without drug for a further 72h. Cells were stained with PE/Cy7 anti-human CXCR4 (12G5, BioLegend, 1:100) and FITC Annexin V (BioLegend) and were analyzed using a BD Accuri C6 Flow Cytometer. The mean fluorescence index of each sample was calculated using FlowJo software v10.
For bulk ATAC sequencing, MMCLs were treated with inhibitors for 72h and cells were ficolled. 50,000 cells were pelleted and ATAC sequencing was performed using the Omni-ATAC protocol as described by Corces et al.42 (link).
To assess sensitivity to CXCR4 inhibitors following pre-treatment with PVD, CXCR4 inhibitors plerixafor (Selleck) and BKT140 (Bachem) were added at IC50 concentrations after 72h. Cell viability was measured after a further 72h using the CellTiterGlo Luminescent Viability Assay (Promega).
For bulk ATAC sequencing, MMCLs were treated with inhibitors for 72h and cells were ficolled. 50,000 cells were pelleted and ATAC sequencing was performed using the Omni-ATAC protocol as described by Corces et al.42 (link).
To assess sensitivity to CXCR4 inhibitors following pre-treatment with PVD, CXCR4 inhibitors plerixafor (Selleck) and BKT140 (Bachem) were added at IC50 concentrations after 72h. Cell viability was measured after a further 72h using the CellTiterGlo Luminescent Viability Assay (Promega).