Ab70378

All cell lines were cultured in DMEM medium with 10% fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin (Millipore-Sigma) in a humidified incubator with 5% CO₂ at 37 °C.
Initial experiments were performed on primary MEFs derived from 13.5 days NM1 WT and KO embryos^{46 (link)}. In other experiments were used immortalized MEFs (ATCC® CRL-2752) or cell lines derived from these immortalized cells.
The antibodies against H3K9me3 (ab8898), H3K27ac (ab4729), H3K4me1 (ab8895), H3K4me3 (ab8580), H3K9ac (ab10812), H3 (ab1791), γH2AX (ab2893), p53-K370 (ab183544), PCAF (ab12188), Set1 (ab70378), GAPDH (ab8245), and nonspecific rabbit IgG isotype control (ab37415) were purchased from Abcam (Cambridge, MA, USA). The antibody against NM1 has been previously characterized^{2 (link)}. Alexa Fluor 555 Goat Anti-Rabbit (ab150078) and Alexa Fluor 488 Goat Anti-Mouse (ab150117) were used as secondary antibodies for immunofluorescence, and horseradish peroxidase (HRP)-fused Goat Anti-Rabbit (ab6721) and Rabbit Anti-Mouse (ab6728) secondary antibodies for western blottings were purchased from Abcam. Hoechst 43222 (H1399) and ProLong Gold Antifade Mountant with 4′,6-diamidino-2-phenylindole (DAPI; P36931) were purchased from Invitrogen, Waltham, MA, USA. All antibodies have been used according to the manufacturers’ protocols.

IHC was conducted to evaluate the hSETD1A expression in tissue samples acquired from 159 TNBC cases. Briefly, tissue samples were fixed in 10% buffered formalin and embedded in paraffin for a routine histologic examination. Tissue sections were cut from a paraffin block of each specimen and applied to slides for IHC. Slides were stained with hematoxylin and eosin with additional immunostains for ER, PR, HER2, and hSETD1A. Primary antibodies (rabbit monoclonal antibody; 1:100 dilution; ab70378, Abcam, MA) were used to evaluate hSETD1A. Staining was performed using goat anti-rabbit biotinylated IgG (Abcam, MA) and incubating in phosphate-buffered saline containing 1% bovine serum albumin for 30 min at ambient temperature, followed by incubation with the ABC reagent (Vectorlabs, CA) for an additional 30 minutes. Immunostaining was visualized using 3,30-diaminobenzidine (Sigma-Aldrich, Darmstadt, Germany).
Two pathologists evaluated all histologic and IHC tumor slides separately for nuclear grade. HER2 status was evaluated using IHC, and ER and PR were considered positive if there were at least 1% positive invasive tumor nuclei in the sample. HSETD1A was considered positive if 10% or more of tumor cells showed positive membrane expression (see Figure 1).

After treatment with anlotinib for the designated times, the cells were lysed, and the protein concentration was determined using a BCA Protein Assay Kit (Pierce, Rockford, IL, USA) based on the methods previously reported25 (link). Whole cell lysates (50 μg for each sample) were subjected to western blot analysis using the indicated primary antibodies and secondary HRP-conjugated antibodies (CA7074, 1:1000, Cell Signaling Technology, MA, USA). Blots were detected by visualization using the ECL Western Blotting Detection Kit (GeneFlow, Staffordshire, UK). The primary antibodies used were anti-β-actin (CA4970S, 1:1000, Cell Signaling Technology, MA, USA), anti-BAX (CA5023S, 1:1000, CST), anti-MCL1 (CA2538S, 1:1000, CST), anti-BAK (CA12105 1:1000, CST), anti-MYC (CA5605S 1:1000, CST), anti-PARP (CA9532, 1:1000, CST), anti-cleaved PARP (CA5625, 1:1000, CST), anti-caspase-3 (CA9662, 1:1000, CST), anti-cleaved caspase-3 (CA9661, 1:1000, CST), anti-p-p53 (Ser15) (CA9286, 1:1000, CST), anti-MDM2 (CA86934, 1:1000, CST), anti-γH2A.X (CA2577s, 1:1000, CST), and anti-SETD1A (ab70378, Abcam, Cambridge, UK) antibodies.