Mln4924

MLN4924 (Pevonedistat, # HY-70062) was purchased from MedChemExpress (MCE). Paraformaldehyde (#J19943-K2) and Prolonged Diamond DAPI (#P36966) were purchased from Thermofisher. Finally, 4-hydroxytamoxifen was purchased from Sigma (#H6278) and SAG was purchased from Abcam (#ab142160). The working concentration of: (a) SAG is 1 µg/ml, (b) 4-hydroxytamoxifen for the ex vivo experiments is 2 µg/ml, and (c) MLN4924 is 0.25 or 0.5 μM.

MG-132 (HY-13259, MedChemExpress) is a proteasome inhibitor that can effectively prevent the hydrolysis of the 26S proteasome, which was added at a concentration of 18.5 μM for 24 h. MLN4924 (HY-70062, MedChemExpress) can selectively inhibit the activity of the NEDD8-activating enzyme, which is present at the C-terminus of CUL3 and can activate CRL by various modification pathways [36 (link)]; it was added at a concentration of 0.3 μM for 24 h. LY294002 (HY-10108, MedChemExpress) is a broad-spectrum inhibitor of PI3K, which was added at a concentration of 40 mM for 24 h. Rapamycin (HY-10219, MedChemExpress) is a specific mTOR inhibitor; it was added at a concentration of 15 nM for 12 h.

QCA570 was synthesized as reported previously (Qin et al., 2018 (link)). JQ1, MLN4924 and MG132 were purchased from Med Chem Express (Monmouth Junction, NJ).

MLN4924 (1mM, DMSO) (MedChemExpress, USA) was dissolved in DMEM/F12 culture medium to achieve a final concentration of 1 Μm (Fig. S1A) [69 (link)]. The control group received an equivalent volume of DMSO treatment. For MLN4924 treatments, when the cell density reached 70-80%, MLN4924 (1 µM) was added into the medium and incubated for 24 h, then the cells were harvested.

A total of 300,000 MDA-MB-231 cells (ATCC) per technical replicate were treated with 1 μM of drug (or 10 μM in the case of less toxic compounds—DS20, DS21, DS25, 919278, DS50 and roscovitine) for 6 h. Pretreatment of relevant conditions with 0.1 μM MLN4924 (MedChemExpress) was performed for 2 h before compound treatment. RNA extraction was performed using TRIzol (Life Technologies Company) and quantified using Qubit (Thermo Fisher Scientific). RNA was then treated with DNase I, quantified using Qubit and evaluated using a High Sensitivity RNA kit on a TapeStation (Agilent). ERCC spike-in (Thermo Fisher Scientific) was then added. Total RNA prep, complementary DNA synthesis and library preparation was performed on the basis of Illumina Stranded Total RNA prep, Ligation with Ribo-Zero protocol (Illumina). RNA was rRNA depleted and bead purified using RNAClean XP beads (Beckman Coulter). RNA was then fragmented followed by cDNA synthesis and bead purification using AMPure XP beads (Beckman Coulter). cDNA was then dual indexed, amplified, bead purified, evaluated using a DNA 1000 kit (Agilent) on a TapeStation and quantified using a Qubit. Finally, libraries were pooled and sequenced on a NovaSeqS4 (Illumina), using 150 bp paired-end reads.

MLN4924 (HY‐10484, MedChemExpress USA) was purchased from MedChem Express and dissolved in dimethyl sulphoxide (DMSO) (Sigma‐Aldrich, St. Louis, MO, USA) to make a 10mM to 100 mM solution and stored at −80℃. In animal experiments, the drug was dissolved in 10% HP‐β‐CD (Sangon Biotech Inc, Shanghai, China). U0126 (HY‐12031, MedChemExpress USA) was purchased from MedChemExpress and dissolved in DMSO (Sigma‐Aldrich, St. Louis, MO, USA) to generate a 1 mM solution and stored at −40℃. Tenofovir (HY‐13782A, MedChemExpress USA) was purchased from MedChemExpress and dissolved in DMSO (Sigma‐Aldrich, St. Louis, MO, USA) to generate a 1 mM solution and stored at −40℃. Tetracycline (HY‐B0474, MedChemExpress USA) was purchased from MedChemExpress and dissolved in DMSO (Sigma‐Aldrich, St. Louis, MO, USA) to generate a 100mg/ml solution and stored at −40℃.