Foxd1

This assay was processed using the NovoNGS CUT&Tag High-Sensitivity Kit V2.0 (Novoprotein, Shanghai) according to the manufacturer’s instruction. 10⁶ cells/sample were harvested and washed in Wash Buffer. ConA magnetic bead-bound cells were resuspended in 50 μL precooled Primary Antibody Buffer containing the appropriate primary antibody (FOXD1, Santa Cruz). Mouse IgG (Invitrogen) was used as the control antibody. After removing the primary antibody, the diluted secondary antibody in 100 μl Secondary Antibody Buffer and cells were incubated at RT for 1 h. ChiTag™ 2.0 Transposome was used to resuspend the cells and the incubation was performed at RT for 1 h. After cells were incubated in Tagmentation Buffer on a rotating platform at 37 °C for 1 h, the DNA were extracted. For libraries amplification, DNA was mixed with i5 Primer, i7 Primer, and 5XAmpliMix were added and mixed, following cycling conditions: 72 °C for 3 min; 98 °C for 30 s; 22 cycles of 98 °C for 15 s, 60 °C for 20 s and 72 °C for 15 sec; final extension at 72 °C for 2 min and hold at 10 °C. Post-PCR clean-up was performed by adding NovoNGS® DNA Clean Beads, and libraries were incubated with beads for 5 min at RT, washed twice gently in 80% ethanol, and eluted in TE-Buffer. Libraries were sequenced by Genefund (Shanghai, China).

Aliquots of total protein (20–100 µg) were loaded into each well of an SDS gel, separated by electrophoresis and then transferred to PVDF membranes. Prior to the incubation with primary antibodies against Foxd1 (Santa Cruz), G3BP2 (Abcam), total/phosphorylated Rb (Cell Signaling) and GAPDH (AbFrontier) overnight at 4 °C, the membranes were incubated with blocking buffer (5% skim milk in TBS containing 0.1% Tween-20) for 2 h at room temperature. After several washes, the membranes were further incubated with a peroxidase-labeled secondary antibody for another 1 h at room temperature. Immunoreactive bands were finally visualized by using an enhanced chemiluminescence system (Amersham Biosciences, Tokyo, Japan). Uncut blots can be found at Figure S5.