(A) Venn diagram displaying the overlap between ZNF165 and SMAD3 target genes in WHIM12 cells as identified by GREAT (ver 3.0.0). Significance was determined using the hypergeometric distribution. (B…
(A) Venn diagram displaying the peak overlap between ZNF165 binding sites identified in WHIM12 and SUM159 cells. P-value was calculated using the hypergeometric distribution. (B) Venn diagram …
(A) WHIM12 cells were transfected with siRNA for 48 hr, and qPCR was used to quantify relative expression (log2 fold change) of ZNF165-SMAD3 target genes upon depletion of ZNF165 (x-axis) or SMAD3 …
(A) Heatmap plotting the log2 fold-change in expression of 65/446 shared ZNF165-SMAD3 target genes (cutoff >0.25) following TGFβ exposure in WHIM12 cells. Expression data were obtained from GSE63986 …
(A) Forty-eight hours after transfection with HA-SMAD3 and/or ZNF165-myc cDNA, HEK293T cells were subjected to immunoprecipitation with HA antibody. Immunoblotting was performed with indicated …
(A) ZNF165 interactors that contain a SCAN domain as identified from a systematic yeast two-hybrid screening approach (interactome.dfci.harvard.edu). A domain map of ZNF165 is shown above for …
(A) Box-plot showing ZNF446 expression values (log2 median centered ratio) from normal breast and invasive lobular breast carcinoma. Data were obtained from TCGA and p-value was calculated using an …
(A) Heatmaps of ChIP-seq data for ZNF165 and ZNF446 in the indicated TNBC cell lines. All peaks within each heatmap are centered ±2.5 kb from the WHIM12 ZNF165 peaks (n = 322). (B) Normalized read …
(A) Browser tracks of ChIP-seq data showing peaks for ZNF165, SMAD3, and ZNF446 near the ITM2C and ZMPSTE24 loci in WHIM12 or SUM159 cells. (B) Venn diagram displaying the peak overlap between …
(A) Cartoon maps showing the domains of ZNF446 and TRIM27. The KRAB-binding domain of TRIM27 is highlighted for reference. (B) Forty-eight hours after transfection with indicated cDNA, HEK293T …
(A) WHIM12 cells were transfected with indicated siRNA for 72 hr. Whole cell lysates were collected and immunoblotted for the indicated proteins. Data are representative of two independent …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Strain, strain background (Mus musculus) | NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ; NSG | The Jackson Laboratory | Stock no. 005557; RRID:IMSR_JAX:005557 | |
Cell line (Homo sapiens) | HEK293T | ATCC | CRL-3216; RRID:CVCL_0063 | |
Cell line (H. sapiens) | MCF7 | ATCC | HTB-22; RRID:CVCL_0031 | |
Cell line (H. sapiens) | WHIM12 | Li et al., 2013 | ||
Cell line (H. sapiens) | SUM159 | Asterand | RRID:CVCL_5423 | |
Cell line (H. sapiens) | SUM159T-Luciferase | Westcott et al., 2015 | ||
Antibody | anti-HA (Rat monoclonal) | Roche | Cat #11867423001; RRID:AB_390918 | IB (1:1000), IP (1 μg) |
Antibody | anti-c-Myc (Rabbit polyclonal) | Santa Cruz Biotechnology | sc-789; RRID:AB_631274 | IB (1:1000) |
Antibody | anti-V5 (Rabbit monoclonal) | Cell Signaling Technology | Cat #13202; RRID:AB_2687461 | IB (1:1000) |
Antibody | anti-SMAD3 (Rabbit polyclonal) | Abcam | ab28379; RRID:AB_2192903 | IB (1:1000), PLA (1:200), ChIP-qPCR (1.5 μg), ChIP-seq (10 μg) |
Antibody | anti-phospho-SMAD3 (Rabbit monoclonal) | Cell Signaling Technology | Cat #9520; RRID:AB_2193207 | IB (1:1000), ChIP (5 μg) |
Antibody | anti-SMURF2 (Mouse monoclonal) | Santa Cruz Biotechnology | sc-393848 | IB (1:500) |
Antibody | anti-RRAD (Goat polyclonal) | Thermo Fisher Scientific | PA537885; RRID:AB_2554493 | IB (1:1000) |
Antibody | anti-ZNF165 (Mouse monoclonal) | Novus Biologicals | H00007718-M02; RRID:AB_1717273 | IB (1:1000) |
Antibody | anti-ZNF165 (Rabbit polyclonal) | Atlas Antibodies | HPA007247; RRID:AB_2797217 | IB (1:1000), PLA (1:200), IP (5 μg) |
Antibody | anti-Tubulin (Rabbit monoclonal) | Cell Signaling Technology | Cat #2128; RRID:AB_823664 | IB (1:1000) |
Antibody | anti-ZNF446 (Rabbit polyclonal) | Proteintech | 16218–1-AP | IB (1:1000), PLA (1:200) |
Antibody | anti-TRIM27 (Rabbit monoclonal) | Cell Signaling Technology | Cat #15099; RRID:AB_2798707 | IB (1:1000) |
Antibody | anti-V5 (Mouse monoclonal) | Thermo Fisher Scientific | R960-25; RRID:AB_2556564 | PLA (1:500), IP (1 μg) |
Antibody | anti-SMAD3 (Mouse monoclonal) | Santa Cruz Biotechnology | sc-101154; RRID:AB_1129525 | PLA (1:200) |
Antibody | anti-SMAD4 (Mouse monoclonal) | Santa Cruz Biotechnology | sc-7966; RRID:AB_627905 | PLA (1:200) |
Antibody | anti-TRIM27 (Rabbit polyclonal) | Proteintech | 12205–1-AP; RRID:AB_2256660 | PLA (1:200), IHC (1:200) |
Antibody | anti-H3K27ac (Rabbit polyclonal) | Abcam | ab4729; RRID:AB_2118291 | ChIP-qPCR (2 μg) |
Antibody | Normal rabbit IgG (Rabbit polyclonal) | Cell Signaling Technology | Cat #2729; RRID:AB_1031062 | IP (1 μg), ChIP-qPCR (1.5–5 μg) |
Antibody | anti-V5 (Rabbit polyclonal) | Abcam | ab9116; RRID:AB_307024 | ChIP-seq (5 μg) |
Peptide, recombinant protein | Human Transforming Growth Factor-β1 | Cell Signaling Technology | Cat #8915 | |
Chemical compound, drug | SB-431542 | Tocris Bioscience | Cat #1614 | |
Commercial assay or kit | KAPA HyperPrep Kit | KAPA Biosystems | KK8502 | |
Commercial assay or kit | Duolink In Situ PLA Probe Anti-Mouse PLUS | Sigma-Aldrich | DUO92001; RRID:AB_2810939 | |
Commercial assay or kit | Duolink In Situ PLA Probe Anti-Rabbit MINUS | Sigma-Aldrich | DUO92005; RRID:AB_2810942 | |
Commercial assay or kit | Duolink In Situ Detection Reagent Red | Sigma-Aldrich | DUO92008 | |
Software, algorithm | ImageJ (Fiji) | Schindelin et al., 2012 | RRID:SCR_002285 | ver 2.0.0 |
Software, algorithm | Bowtie2 | Langmead and Salzberg, 2012 | RRID:SCR_005476 | ver 2.3.2 |
Software, algorithm | SAMtools | Li et al., 2009 | RRID:SCR_002105 | ver 1.6 |
Software, algorithm | DeepTools | Ramírez et al., 2016 | RRID:SCR_016366 | ver 2.3.5 |
Software, algorithm | Integrative Genomics Viewer | Robinson et al., 2011 | RRID:SCR_011793 | ver 2.3.93 |
Software, algorithm | HOMER | Heinz et al., 2010 | RRID:SCR_010881 | ver 4.9 |
Software, algorithm | GREAT | McLean et al., 2010 | RRID:SCR_005807 | ver 3.0.0 |
Software, algorithm | MEME | Bailey et al., 2009 | RRID:SCR_001783 | ver 5.1.1 |
Software, algorithm | GSEA | Subramanian et al., 2005 | RRID:SCR_003199 | ver 4.0.3 |
GREAT (ver. 3.0.0) analysis of the 118 ZNF165/SMAD3 co-bound regions determined using default association rules.
Genes included in the EPITHELIAL_MESENCHYMAL_TRANSITION gene set with corresponding enrichment values for WHIM12 cells depleted of ZNF165.
Interactome data for ZNF165 and ZNF446 obtained from the CCSB Interactome Database.
Sequence-based reagents used for gene expression analysis, ChIP, and knockdown experiments.