Materials and Methods
Antibodies and DNA Vectors for Gene Expression and Knockdown
Cell Culture, Lentiviral Production, and Infection
- Rubinson D.A.
- Dillon C.P.
- Kwiatkowski A.V.
- Sievers C.
- Yang L.
- Kopinja J.
- Rooney D.L.
- Ihrig M.M.
- McManus M.T.
- Gertler F.B.
- Scott M.L.
- Van Parijs L.
Cell Cycle Profile Analysis
Clonogenic Assay and Cell Staining
Three-Dimensional Matrigel Culture of Breast Cells
Breast Cancer Xenograft Study
Real-Time RT-PCR Analysis
p27 Stability Determination
Cell-Based p27 Ubiquitination Assay
Protein Interaction Studies
YY1 Expression in Breast Cancer Cell Lines, TMA, and Breast Cancer Gene Arrays
- Kleer C.G.
- Cao Q.
- Varambally S.
- Shen R.
- Ota I.
- Tomlins S.A.
- Ghosh D.
- Sewalt R.G.
- Otte A.P.
- Hayes D.F.
- Sabel M.S.
- Livant D.
- Weiss S.J.
- Rubin M.A.
- Chinnaiyan A.M.
Manipulated YY1 Expression Affects the Migration, Invasiveness, Clonogenicity, and Cell Cycle Profiles of Mammary Cells
Manipulated YY1 Expression Changes Mammary Cell Architecture
YY1 Knockdown Reduces Tumor Formation by MDA-MB-231 Cells in a Xenograft Mouse Model
p27 Is a Potential Downstream Target of YY1 in Mediating Mammary Cell Tumorigenesis
- Santiago F.S.
- Ishii H.
- Shafi S.
- Khurana R.
- Kanellakis P.
- Bhindi R.
- Ramirez M.J.
- Bobik A.
- Martin J.F.
- Chesterman C.N.
- Zachary I.C.
- Khachigian L.M.
- Belletti B.
- Pellizzari I.
- Berton S.
- Fabris L.
- Wolf K.
- Lovat F.
- Schiappacassi M.
- D'Andrea S.
- Nicoloso M.S.
- Lovisa S.
- Sonego M.
- Defilippi P.
- Vecchione A.
- Colombatti A.
- Friedl P.
- Baldassarre G.
Effects of YY1 on Architecture and Proliferation of MCF-10A and MCF-7 Cells in Three-Dimensional Matrigel Culture Are Reversed by Adjusting p27 Expression
YY1 Negatively Regulates p27 Expression at the Posttranslational Level
- Dimri M.
- Naramura M.
- Duan L.
- Chen J.
- Ortega-Cava C.
- Chen G.
- Goswami R.
- Fernandes N.
- Gao Q.
- Dimri G.P.
- Band V.
- Band H.
- Supplemental Figure S1
YY1 expression in breast cells and tissues. A: Validation of specificity of YY1 antibody. MCF-7 cells were co-transfected with EGFP and either control shRNA or YY1 shRNA at a ratio of 1:5 (EGFP/shRNA). Three days after transfection, the cells were immunostained with YY1 (H-10) as the primary antibody. Yellow arrowheads indicate cells transfected with both EGFP and control shRNA, and pink arrowheads indicate cells containing both EGFP and YY1 shRNA. B and C: Immunohistochemical studies of MCF-10A and MCF-7 cells, and breast cancer tissues, respectively. Cultured MCF-10A cells, MCF-7 cells (B) and breast cancer tissues (C) were formalin-fixed and paraffin-embedded. The sections were subjected to immunohistochemical assays using YY1 (H-10) as the primary antibody (see Materials and Methods for details). D: Schema of the YY1 transcript. YY1 probes used in the gene array analyses are indicated on the top. The length (in nucleotides) is shown at the bottom. Sequences of these YY1 probes are as follows: 213494_S_AT, 5′-GAAGGGGCACACATAGGGCCTGTCTCCGGTATGGATTCGCACATGTGTGNGCAAANTTGAAGTCCAGTGAAAAGCGTTTCCCACAGCCTTCGAACGTGCACTGAAAGGGCTTCTCTCCAGTATGAACCAGTTGGTGTCGTTTTAGTTTTGAACTCTCAACAAAAGCTTTGCCACATTCTGCACAGACGTGGACTCTGGGACCGTGGGTGTGCAGATGTTTTCTCATGGCCGAGTTATCCCTGAACATCTTTGTGCAGCCTTTATGAGGGCAAGCTATTGTTCTTGGAGCATCATCTTCTTTAATTTTTCTTGGCTTCATTCTAGCAAATTCTGCCAGTTGTTTGGGATCTGAGAGGTCAATGCCAGGTATTCCTCCAGGAGGAAGTTTCTTTCCTGTCATATATTCTGAATAATCAGGAGGTGAGTTCTCTCCAATGATCTGTTCTTCAACCACTGTCTCATGGTCAATATCTTTTTTTTCATCTGAGGACCACATGGTGACCGAGAACTCGCCCTC-3′; 201901_S_AT, 5′-AGCTTGCCCTCATAAAGGCTGCACAAAGATGTTCAGGGATAACTCGGCCATGAGAAAACATCTGCACACCCACGGTCCCAGAGTCCACGTCTGTGCAGAATGTGGCAAAGCTTTTGTTGAGAGTTCAAAACTAAAACGACACCAACTGGTTCATACTGGAGAGAAGCCCTTTCAGTGCACGTTCGAAGGCTGTGGGAAACGCTTTTCACTGGACTTCAATTTGCGCACACATGTGCGAATCCATACCGGAGACAGGCCCTATGTGTGCCCCTTCGATGGTTGTAATAAGAAGTTTGCTCAGTCAACTAACCTGAAATCTCACATCTTAACACATGCTAAGGCCAAAAACAACCAGTGAAAAGAAGAGAGAAGACCCTTCTCGACCACGGGAAGCATCTTCCAGAAGTGTGATTGGGAATAAATATGCCTCTCCTTTGTATATTATTTCTAGGAAGAATTTTAAAAATGAATCCTACACACCTAAGGGACATG-3′; and 200047_S_AT, 5′-GGTTTTGTTTGCTATCTTAATTTTGGTTGTATTCTTTGATGTTAACACATTTTGTATAATTGTATCGTATAGCTGTATTGAATCATGTAGTATCAAATATTAGATGTGATTTAATAGTGTTAATCAATTTAAACCCATTTTAGTCACTTTTTTTTTCCAAAAAAATACTGCCAGATGCTGATGTTCAGTGTAATTTCTTTGCCTGTTCAGTTACAGAAAGTGGTGCTCAGTTGTAGAATGTATTGTACCTTTTAACACCTGATGTGTACATCCCATGTA-3′. E: Relative gene expression in samples from the Uppsala breast cancer cohort (258 patient samples). Relative expression of analyzed genes including YY1, Ezh2, HER2, ER-α, BRCA1, and Ki-67 are shown. A probe recognizing a sequence in Bacillus subtilis was used as a negative control, and probes for β-actin and GAPDH were positive controls. Average values of the lowest expressed 10% genes (low 10th percentile) and the highest expressed 10% genes (high 10th percentile) are shown.
- Supplemental Figure S2
In vitro study to test the effects of YY1 knockdown on MDA-MB-231 cells. A: Migration assays of MDA-MB-231 cells with inducible-YY1 shRNA at days 1 and 3. B and C: Boyden chamber assay of MDA-MB-231 cells with inducible-H1 shRNAs. Average cell numbers in three random views at the indicated conditions are shown in B (*P < 0.05 versus other three groups). Representative images of trans-well migrated cells are shown in C. D: Representative Western blots of YY1 expression in these four cell groups.
- Supplemental Figure S3
Clonogenic assays to determine the effects of YY1 knockdown on breast cells. MCF-10A cells (A) and MCF-7 cells (B) were infected with puromycin-resistant lentiviruses carrying control shRNA or YY1 shRNA. The puromycin-selected cells were seeded as indicated for clonogenic studies. Representative images of the stained dishes are shown under the graphs. Insets show Western blots indicating efficient YY1 knockdown.
- Supplemental Figure S4
Cell cycle profile studies of mammary cells with manipulated YY1 expression. A: MCF-10A cells were infected with lentiviruses produced by pSL5 vector and pSL5/YY1. B: MCF-7 cells were infected with lentiviruses expressing control shRNA and YY1 shRNA, C: MDA-MB-231 cells integrated by inducible-YY1 shRNA were cultured in the absence and presence of Dox. Three days after these treatments, the cells were collected and treated for FACS analysis (see Materials and Methods). Western blots of YY1 expression in these cells are shown on the right. The experiments were repeated multiple times, and representative results are shown.
- Supplemental Figure S5
Xenograft tumor volumes and images. A: Tumor volumes of xenografted MDA-MB-231 cells with inducible shRNAs in the absence and presence of Dox. B: Images at 4 weeks of xenograft tumors from Dox-treated mice. Mouse identifying numbers are labeled. L, left; R, right.
- Supplemental Figure S6
Expression of YY1 and p27 in different breast cell lines. Cell lysates of breast cell lines were analyzed using Western blot analysis using YY1 (H-10), p27, and GAPDH antibodies. Two levels of exposure of the p27 immunoblot are shown.
- Supplemental Figure S7
3-D Matrigel culture and cell proliferation rates of breast cell lines with manipulated YY1 expression. A: MCF-10A and MCF-7 cells cultured in 3-D Matrigel system. Magnified examples of MCF-10A cells forming spherical architecture are also shown. B: 3-D Matrigel culture of MCF-10A cells transduced with lentiviruses of pSL5 vector, pSL5/YY1, and pSL5/YY1 plus pSL9/p27. C and D: 3-D Matrigel culture of MCF-7 cells and MDA-MB-231 cells, respectively, transduced by lentiviruses with Dox-inducible YY1 shRNA in the absence and presence of Dox and with or without pLu-Neo-U6/p27 shRNA infection.
- Supplemental Figure S8
Cell growth studies of MCF-10A and MCF-7 cells with manipulated YY1 and p27 expression. A and B: WST-1 assay of MCF-10A and MCF-7 cells, respectively, with manipulated YY1 and p27 expression. A: MCF-10A cells were individually infected with pSL5 and pSL5/YY1 lentiviruses, or pSL5/YY1 transduced cells were infected with pSL9/p27. B: MCF-7 cells transduced by inducible-YY1 shRNA were cultured either in the absence or presence of Dox or were further infected with lentivirus expressing p27 shRNA. Cell proliferation was determined using WST-1 assays. *P < 0.05. C and D: Proliferation rates of the cells in A and B plotted with vertical axes in a logarithmic scale.
- Supplemental Figure S9
Reporter assay of YY1 on p27 promoter activity. A: Schema of p27 promoter reporter construct. B: Effects of ectopic YY1 expression on p27 promoter. The reporter plasmid was constructed by subcloning the EcoRI-SacI fragment of the p27 promoter (corresponding to the −1991 to +315, with the p27 transcription start site designated as +1) in front of Gaussia luciferase (Gluc). Data were derived from three independent experiments. *P = 0.02.
- Supplemental Figure S10
Densitometric quantitation of p27 stability in the conditions of manipulated YY1 expression of Figures 8A and 8B. A: Relative p27 levels in MCF-10A cells transduced by pSL5 vector and pSL5/YY1, respectively. *P = 0.0066. The half-life of p27 in MCF-10A cells was reduced from approximately 45 hours to 12 hours by ectopic YY1 expression. B: Relative p27 levels in MCF-7 cells transduced by pLu-Puro-U6/control shRNA and pLu-Puro-U6/YY1 shRNA, respectively. *P = 0.0052. With YY1 depletion, the half-life of p27 in MCF-7 cells was increased from approximately 25 hours to a level that cannot be determined using this assay. MCF-10A cells and MCF-7 cells were treated with 60 μg/mL and 45 μg/mL cycloheximide, respectively, and collected at different times. Densitometric quantitation of Western blots was performed using Quantity One 4.2.2 software (Bio-Rad Laboratories). The relative p27 levels were normalized against the corresponding β-actin expression. The coordinate with a logarithmic y-axis (relative p27 levels) has been previously used.59
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Supported by the Golfers Against Cancer Fund, the Kulynych Interdisciplinary Cancer Research Fund, the Startup Fund of Wake Forest University School of Medicine (G.S.), and grant RSG-09-082-01-MGO from the American Cancer Society (G.S.).
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