shRNA and generation of stable cells. MDA-MB-231, T47D, and 293T cells were purchased from ATCC. We generated lentiviruses (biosettia) with a single shRNA targeting p53 or Elk-1 (Table S1) to knock down p53 and Elk-1 in MDA-MB-231 and T47D cells. The infected MDA-MB-231 and T47D cells were selected using a medium supplemented with 10 mg/mL puromycin (Sigma Aldrich).
qRT-PCR and ChIP-qPCR. Total RNA was isolated with Trizol reagent (Invitrogen, USA). Then, the RNA (1µg) was synthesized into cDNA using a reverse transcription kit (Qiagen). The ChIP-DNA was prepared using an anti- ELK-1 antibody in MDA-MB-231 as described. All gene expression data were normalized with an internal control gene (GADPH). All ChIP-qPCR data was normalized with target amplification site in input. Primer sequences were listed in Supplementary Table S2& Table S3.
Antibody. Antibodies were p53(#48818, Cell Signaling Technology), FRA-1(#5281, Cell Signaling Technology), ELK-1(#9182, Cell Signaling Technology), E-cadherin (#3195, Cell Signaling Technology), andβ-actin (#4970, Cell Signaling Technology).
Immunofluorescent staining. Fix cells with 4% formaldehyde/PBS. After overnight incubation with anti-E-cadherin (1:200) at 4°C, the target protein was detected by anti-rabbit IgG PE-conjugated secondary antibody. Nuclei were stained with DAPI.
Wound-healing assay. Cells in 6-well plates were grown to confluence. Use a 200 ml pipette tip to draw a line from the monolayer to remove part of the cells. The area of migrated cells was estimated after 24 hours and analyzed with ImageJ software.
Transwell assay. For transwell migration assays, we seeded cells into the top chamber of a 24-well cell culture insert (Corning, 3422) with 1% FBS medium and then added 10% FBS medium to the bottom chamber. Cells transferred to the bottom of the membrane were fixed with 4% formaldehyde, stained with 0.05% crystal violet several hours later (MDA-MB-231, 17 hours; T47D, 45 hours), and counted. Measurements were performed in triplicates.
GSEA enrichment analysis. Gene Set Enrichment Analysis (GSEA) using the expression matrix of differential genes in control and Tp53 knockdown RNA-seq data, the selected reference gene sets were c2.cp.reactome.v7.5.1.symbols.gmt and c2.cp. kegg.v7.5.symbols.gmt. The ggplot2 package is used to visualize GSEA collections.
Analysis of TCGA gene expression data. To assess whether FRA-1 expression is induced in cancers with TP53 mutations, we used data from The Cancer Genome Atlas (TCGA). First, using XenaPython, we downloaded data from all samples in TCGA for BRCA, LUAD, and PAAD cancer types and classified the data, including wild-type p53 and p53GOF (missense mutations, including six hotspots ((R175, G245, R248, R249, R273, R282) together with R280K and L194F). GraphPad generated dot plots representing the distribution of gene expression values. Dot plots represent three genes in Tp53WT and Tp53GOF tumors from each cancer. The number of samples in each group was as follows: BRCA-p53WT = 675, Tp53GOF = 56; LUAD-p53WT = 276, Tp53GOF = 22; PAAD-p53WT = 57, Tp53GOF = 21.