All suspension cells were plated in 96-well plates in triplicate at 5,000–10,000 cells per well and treated for 72 hours with vehicle or the indicated concentrations of STM2457 and STM2120 (0.04-50 μM). On day 4, an equal volume for all wells was split using fresh media and compound, such that the resulting cell density in each well matched the initial seeding density. Plates were measured on day 6 using CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (Promega) in order to calculate the relative cell proliferation. All the compounds were dissolved in DMSO.
For the rescue experiments, cDNA was obtained by reverse transcription of MOLM13 cell RNA with Supercript III (ThermoFisher Scientific), then the SP1 full-length coding sequence was amplified by PCR and cloned into pHIV-ZsGreen plasmid (Addgene 18121) by Gibson assembly (Gibson Assembly Cloning Kit, NEB), using the HpaI site. MOLM13 cells were transduced with the SP1 or empty lentiviral vectors, then GFP+ cells were isolated by flow cytometry sorting after 4 days and employed in proliferation and western blot assays as descripted above. All data in this section were plotted using GraphPad Prism (Version 9).
For the rescue experiments, cDNA was obtained by reverse transcription of MOLM13 cell RNA with Supercript III (ThermoFisher Scientific), then the SP1 full-length coding sequence was amplified by PCR and cloned into pHIV-ZsGreen plasmid (Addgene 18121) by Gibson assembly (Gibson Assembly Cloning Kit, NEB), using the HpaI site. MOLM13 cells were transduced with the SP1 or empty lentiviral vectors, then GFP+ cells were isolated by flow cytometry sorting after 4 days and employed in proliferation and western blot assays as descripted above. All data in this section were plotted using GraphPad Prism (Version 9).