Gata2

MiRIDIAN miRNA hairpin inhibitors and negative control #1 were obtained from Dharmacon (Lafayette, CO). These miRNA inhibitors bind to miRNAs by complementary sequences and thus block their capacity to silence mRNA target without affecting the level of miRNAs^{43 (link)}. Lentiviral particles containing shRNAs against c-Jun (TRCN0000039589, TRCN0000039590), c-Fos (TRCN0000016004, TRCN000016007) and GATA2 (TRCN0000019264, TRCN0000019265) were obtained from Sigma (St Louis, MO).

Whole cell extracts were separated by SDS-PAGE using 4–15% precast TGX gels (BioRad), and transferred onto PVDF membranes using the Trans-Blot® Turbo™ system (Biorad). Blots were blocked and incubated with rabbit monoclonal antibodies targeting PARP (1:1000, Cell Signaling Technologies), p21 (1:1000, Abcam), p16 (1:1000, Abcam), N-Cadherin (1:1000, Abcam), GATA2 and GATA3 (Sigma, 1:1000); rabbit polyclonal antibody targeting LC3 (1:1000, Abcam); and mouse monoclonal antibodies targeting E-Cadherin (1:500, Abcam), Rb (1:1000, Abcam), ST3GAL4 (Abcam, 1:500) and MGAT5 (Abcam, 1:400). The membranes were then incubated with a secondary goat anti-mouse or anti-rabbit antibody (1:5000, Abcam). The blots were developed using TMB for enzymatic colourimetric detection. To analyse protein loading, the mouse monoclonal, α-Tubulin antibody (1:10,000; Santa Cruz, CA, USA) was used. Western blots were quantified using ImageJ^TM software (FIJI).

Short hairpin RNAs (shRNAs) against FOS, GATA2, MAFK, TEAD4, TFAP2C, NCOR2, RCOR1 and ZNF362 were purchased from Sigma-Aldrich (Supplementary Table S1). A total of 7.3 × 10⁶ HEK293T cells were transfected with 2.5 μg of the pLKO construct with a specific shRNA, 1.7 μg of Δ8.9 and 0.8 μg of VSVG using 15 μl of a GenJet transfection reagent (SignaGen) to generate lentiviruses expressing a specific shRNA. After 24 h, the medium was replaced with the TSC medium. After cultivating at 37°C for 24 h, the viral supernatant was collected, filtered with a syringe filter (0.45 μm) and used to infect TSCs. To knock down individual TFs, 2.5 × 10⁵ TSCs were infected with pLKO-shRNA viral particles in a well of a 12-well plate. Infected cells were incubated overnight, and the medium was replaced with fresh TSC medium supplemented with puromycin to select out the infected cells. To determine the KD efficiency of each TF and profile global gene expression, we harvested the cells after 3 days of selection to get complete KD of an individual TF.