The materials provided here are supplemental tables and figures to an
article to be published in 'Molecular and Cellular Biology.'(This refers to the article.) We previously reported that expression of
NRIF3 (Nuclear Receptor Interacting Factor-3) rapidly and selectively
leads to apoptosis of breast cancer cells. DIF-1 (a.k.a IRF-2BP2), the
cellular target of NRIF3, was identified as a transcriptional repressor
and DIF-1 knockdown leads to apoptosis of breast cancer cells but not
other cell types. Here, we identify IRF2BP1 (Interferon Regulatory
Factor-2 Binding Protein 1) and EAP1 (Enhanced At Puberty 1) as
important components of the DIF-1 complex mediating both complex
stability and transcriptional repression. This interaction of DIF-1,
IRF2BP1, and EAP1 occurs through the conserved C4 zinc-fingers of these
proteins. Microarray studies were carried out in breast cancer cell
lines engineered to conditionally and rapidly increase the levels of the
Death Domain region of NRIF3 (DD1). The DIF-1 complex was found to
repress FASTKD2, a putative pro-apoptotic gene, in breast cancer cells
and to bind to the FASTKD2 gene by chromatin immunoprecipitation.
FASTKD2 knockdown prevents apoptosis of breast cancer cells from NRIF3
expression or DIF-1 knockdown while expression of FASTKD2 leads to
apoptosis of both breast and non-breast cancer cells. Thus, regulation
of FASTKD2 by NRIF3 and the DIF-1 complex acts as a novel death switch
that selectively modulates apoptosis in breast cancer

Das, Sharmistha

Lomniczi, Alejandro

Neubert, Thomas A.

Ojeda, Sergio R.

Samuels, Herbert H.

Xu, Chong-Feng

Yeung, Kay T.

New York University Faculty Digital Archive

  Yeung et al. 1 Supplemental Material A Novel Transcription Complex that Selectively Modulates Apoptosis of Breast Cancer Cells through Regulation of FASTKD2.  Kay T. Yeung, Sharmistha Das, Jin Zhang, Alejandro Lomniczi, Sergio R. Ojeda, Chong-Feng Xu, Thomas A. Neubert, and Herbert H. Samuels Contents 1) Legends to Supplemental Figures and Tables 2) Supplemental Figures (1-7) and Tables (1-3) ____________________________________________________________________________ 1) Supplemental Legends to Tables and Figures Supplemental Table 1. Components of the DIF-1 complex(es) identified by mass spectrometry and their Mascot confidence scores.  A Mascot score of 50 was applied as a cut-off for significantly confident (p<0.05) protein identification by mass spectrometry.  All ribosomal, keratin-related, and heat shock related proteins were not listed, as they are usually considered as contaminates in protein complex purification and mass spectrometry.  The protein Mascot scores are listed in parenthesis and listed with the highest Mascot scores at the top.   Supplemental Table 2. siRNA sequences.   Supplemental Table 3. Sequences of primers used in qRT-PCR for FASTKD2 expression studies or ChIP analyses.  Supplemental Figure 1. Gel electrophoresis of purified DIF-1 protein complexes from HeLa and T-47D cells.  This is a representative silver-stained gel comparing the pooled HA peptide purified eluates from HeLa or T-47D cells stably expressing FLAG-HA-DIF-1 or control (FLAG-HA tag only) cells.  The molecular weight markers are as shown on the left.    Yeung et al. 2 Supplemental Figure 2.   Localization of DIF-1, BP1, and EAP1 in the cell nucleus and in regions of the brain. Nuclear localization of DIF-1, BP1, and EAP1.  DIF-1 was previously shown to be a nuclear protein {Tinnikov, 2009 #463}.  (A) The expression of GFP-DIF-1, GFP-BP1, and GFP-EAP1 (in green) in HeLa cells indicates that all three proteins localize in the nucleus (in blue) with a distinct nucleoli exclusion pattern.  (B) Colocalization of EAP1 mRNA with the mRNAs encoding NRIF3, BP1 and DIF-1 in neuronal populations of the rat brain.   The transcripts were detected using a double in situ hybridization procedure in which an EAP1 cRNA probe was labeled with digoxygenin and NRIF3, BP1 and DIF-1 cRNAs were labeled with 35S-UTP.  Upper panels, Neurons of the piriform cortex (PIR CTX) containing EAP1 mRNA transcripts (purple color) that colocalize with NRIF3, BP1 and DIF-1 mRNAs (white grains).  Middle panels, Neurons of the arcuate (ARC) and ventromedial nucleus (VMH) of the hypothalamus showing a similar colocalization as in the PIR CTX.  Lower panel, Higher magnification image showing colocalization of EAP1 mRNA (purple color) with BP1 mRNA (white grains) in the VMH of the hypothalamus.  Bars in upper panels = 50µm; in middle panels = 100 µm; in lower panel = 20 µm. 3V = third ventricle. Supplemental Figure 3. Knockdown of BP1 or EAP1 leads to disruption of the DIF-1 complex(es).  (A) The affinity purified DIF-1 assoicated proteins prior to chromatography using (I) HeLa FLAG-HA-DIF1 or (II) T-47D FLAG-HA-DIF1 stable cells.  In addition, HeLa cells stably expressing FLAG-HA-DIF1 were transfected with 40 nM of either (B) control siRNA, (C) BP1 siRNA, (D) EAP1 siRNA, or (E) a combination of BP1 and EAP1 siRNA.  As described in Figure 2B, cells also received zVAD-fmk and zVDVAD-fmk to prevent apoptosis that might result from siRNA transfection.  Panel A shows the affinity purified DIF-1 associated proteins prior to chromatography.  The purified DIF-1 complex(es) was size-fractionated in a Superdex 200 gel filtration column as described in Figure 2B.  The collected fractions were analyzed by SDS-PAGE followed by silver staining.  The siRNA used in each panel is indicated in brackets [ ].  Position of protein standards for the size fractionation are indicated at the top.  The molecular weight markers for the SDS-PAGE are as shown on the left.  Supplemental Figure 4. HeLa DD1-ERT2 and T-47D DD1(S28A)-ERT2 stable cell lines do not undergo apoptosis after 24 h of 4OHT treatment. HeLa DD1-ERT2 or T-47D DD1(S28A)-ERT2 stable cells were seeded on coverslips in 48 spot multiwells 24 h before treatment with 1   Yeung et al. 3 uM 4OHT for 24 h.  Cells were fixed and permeabilized for immunofluorescence for FLAG (green) and for TUNEL assay (red).  Nuclei were stained with DAPI (blue).  Supplemental Figure 5.  siRNA knockdown of FASTKD2 protects SKBR3 cells from apoptosis resulting from GFP-DD1 expression or DIF-1 knockdown.  SKBR3 cells were transfected with FASTKD2 siRNA (40 nM) or control siRNA (40 nM).  Twenty-four h later, cells were transfected with GFP-DD1, GFP-DD1(S28A), or DIF-1 siRNA (40 nM).  Twenty-four h later, cells were fixed and processed for TUNEL (red) and GFP expression (green).  Nuclei were stained with DAPI (blue). Supplemental Figure 6.  ChIP studies of the FASTKD2 gene with primer sets A, C, and D. ChIP studies indicated that DIF-1 associates with a region downstream of the FASTKD2 promoter as determined with Primer set B.  This figure shows the results using Primer sets A, C, D.  Short fragments (150-300 bp) of chromatin from the stable cell lines expressing either FLAG-HA tag (T-47D or HeLa) or FLAG-HA-DIF-1 (T-47D DIF-1 and HeLa DIF-1) were used for ChIP.  ChIP was performed using FLAG-M2 agarose beads, and was followed by qRT-PCR using primer set A, C, or D to amplify the regions of the FASTKD2 gene (see Figure 7A for the Primer set positions).  The ChIP-qRT-PCR results shown represent the mean +/- SEM of three independent experiments. Supplemental Figure 7. BP1 or EAP1 mediated transcriptional repression is not dependent on DIF-1.  HeLa cells were transfected with either DIF-1 siRNA (40 nM) or control siRNA (40 nM).  Cells were incubated throughout the experiment with the caspase inhibitors zVAD-fmk and zVDVAD-fmk to prevent apoptosis resulting from decreased DIF-1 expression.  Forty h later cells were transfected with 100 ng of pG5-SV-BCAT and 200 ng of vectors expressing Gal4-BP1, Gal4-EAP1, Gal4-DIF-1 or 150 ng (equal molar amount of plasmid) of vector expressing only the Gal4-DBD.  Twenty-four h later the cells were harvested for CAT activity.  The results represent the mean +/- SEM of three independent experiments. 

Supplements to article: A novel transcription complex that selectively
modulates apoptosis of breast cancer cells through regulation of FASTKD2

DSpace at New York University

  Yeung et al. 
1 
Supplemental Material 
A Novel Transcription Complex that Selectively Modulates Apoptosis of Breast Cancer 
Cells through Regulation of FASTKD2. 
 
Kay T. Yeung, Sharmistha Das, Jin Zhang, Alejandro Lomniczi, Sergio R. Ojeda, Chong-
Feng Xu, Thomas A. Neubert, and Herbert H. Samuels 
Contents 
1) Legends to Supplemental Figures and Tables 
2) Supplemental Figures (1-7) and Tables (1-3) 
____________________________________________________________________________ 
1) Supplemental Legends to Tables and Figures 
Supplemental Table 1. Components of the DIF-1 complex(es) identified by mass spectrometry 
and their Mascot confidence scores.  A Mascot score of 50 was applied as a cut-off for 
significantly confident (p<0.05) protein identification by mass spectrometry.  All ribosomal, 
keratin-related, and heat shock related proteins were not listed, as they are usually considered as 
contaminates in protein complex purification and mass spectrometry.  The protein Mascot scores 
are listed in parenthesis and listed with the highest Mascot scores at the top.   
Supplemental Table 2. siRNA sequences.   
Supplemental Table 3. Sequences of primers used in qRT-PCR for FASTKD2 expression 
studies or ChIP analyses.  
Supplemental Figure 1. Gel electrophoresis of purified DIF-1 protein complexes from HeLa 
and T-47D cells.  This is a representative silver-stained gel comparing the pooled HA peptide 
purified eluates from HeLa or T-47D cells stably expressing FLAG-HA-DIF-1 or control 
(FLAG-HA tag only) cells.  The molecular weight markers are as shown on the left.  
  Yeung et al. 
2 
Supplemental Figure 2.   Localization of DIF-1, BP1, and EAP1 in the cell nucleus and in 
regions of the brain. Nuclear localization of DIF-1, BP1, and EAP1.  DIF-1 was previously 
shown to be a nuclear protein {Tinnikov, 2009 #463}.  (A) The expression of GFP-DIF-1, GFP-
BP1, and GFP-EAP1 (in green) in HeLa cells indicates that all three proteins localize in the 
nucleus (in blue) with a distinct nucleoli exclusion pattern.  (B) Colocalization of EAP1 mRNA 
with the mRNAs encoding NRIF3, BP1 and DIF-1 in neuronal populations of the rat brain.   The 
transcripts were detected using a double in situ hybridization procedure in which an EAP1 cRNA 
probe was labeled with digoxygenin and NRIF3, BP1 and DIF-1 cRNAs were labeled with 35S-
UTP.  Upper panels, Neurons of the piriform cortex (PIR CTX) containing EAP1 mRNA 
transcripts (purple color) that colocalize with NRIF3, BP1 and DIF-1 mRNAs (white grains).  
Middle panels, Neurons of the arcuate (ARC) and ventromedial nucleus (VMH) of the 
hypothalamus showing a similar colocalization as in the PIR CTX.  Lower panel, Higher 
magnification image showing colocalization of EAP1 mRNA (purple color) with BP1 mRNA 
(white grains) in the VMH of the hypothalamus.  Bars in upper panels = 50µm; in middle panels 
= 100 µm; in lower panel = 20 µm. 3V = third ventricle. 
Supplemental Figure 3. Knockdown of BP1 or EAP1 leads to disruption of the DIF-1 
complex(es).  (A) The affinity purified DIF-1 assoicated proteins prior to chromatography using 
(I) HeLa FLAG-HA-DIF1 or (II) T-47D FLAG-HA-DIF1 stable cells.  In addition, HeLa cells 
stably expressing FLAG-HA-DIF1 were transfected with 40 nM of either (B) control siRNA, (C) 
BP1 siRNA, (D) EAP1 siRNA, or (E) a combination of BP1 and EAP1 siRNA.  As described in 
Figure 2B, cells also received zVAD-fmk and zVDVAD-fmk to prevent apoptosis that might 
result from siRNA transfection.  Panel A shows the affinity purified DIF-1 associated proteins 
prior to chromatography.  The purified DIF-1 complex(es) was size-fractionated in a Superdex 
200 gel filtration column as described in Figure 2B.  The collected fractions were analyzed by 
SDS-PAGE followed by silver staining.  The siRNA used in each panel is indicated in brackets [ 
].  Position of protein standards for the size fractionation are indicated at the top.  The molecular 
weight markers for the SDS-PAGE are as shown on the left.  
Supplemental Figure 4. HeLa DD1-ERT2 and T-47D DD1(S28A)-ERT2 stable cell lines do 
not undergo apoptosis after 24 h of 4OHT treatment. HeLa DD1-ERT2 or T-47D DD1(S28A)-
ERT2 stable cells were seeded on coverslips in 48 spot multiwells 24 h before treatment with 1 
  Yeung et al. 
3 
uM 4OHT for 24 h.  Cells were fixed and permeabilized for immunofluorescence for FLAG 
(green) and for TUNEL assay (red).  Nuclei were stained with DAPI (blue).  
Supplemental Figure 5.  siRNA knockdown of FASTKD2 protects SKBR3 cells from apoptosis 
resulting from GFP-DD1 expression or DIF-1 knockdown.  SKBR3 cells were transfected with 
FASTKD2 siRNA (40 nM) or control siRNA (40 nM).  Twenty-four h later, cells were 
transfected with GFP-DD1, GFP-DD1(S28A), or DIF-1 siRNA (40 nM).  Twenty-four h later, 
cells were fixed and processed for TUNEL (red) and GFP expression (green).  Nuclei were 
stained with DAPI (blue). 
Supplemental Figure 6.  ChIP studies of the FASTKD2 gene with primer sets A, C, and D. 
ChIP studies indicated that DIF-1 associates with a region downstream of the FASTKD2 
promoter as determined with Primer set B.  This figure shows the results using Primer sets A, C, 
D.  Short fragments (150-300 bp) of chromatin from the stable cell lines expressing either 
FLAG-HA tag (T-47D or HeLa) or FLAG-HA-DIF-1 (T-47D DIF-1 and HeLa DIF-1) were used 
for ChIP.  ChIP was performed using FLAG-M2 agarose beads, and was followed by qRT-PCR 
using primer set A, C, or D to amplify the regions of the FASTKD2 gene (see Figure 7A for the 
Primer set positions).  The ChIP-qRT-PCR results shown represent the mean +/- SEM of three 
independent experiments. 
Supplemental Figure 7. BP1 or EAP1 mediated transcriptional repression is not dependent on 
DIF-1.  HeLa cells were transfected with either DIF-1 siRNA (40 nM) or control siRNA (40 
nM).  Cells were incubated throughout the experiment with the caspase inhibitors zVAD-fmk 
and zVDVAD-fmk to prevent apoptosis resulting from decreased DIF-1 expression.  Forty h 
later cells were transfected with 100 ng of pG5-SV-BCAT and 200 ng of vectors expressing 
Gal4-BP1, Gal4-EAP1, Gal4-DIF-1 or 150 ng (equal molar amount of plasmid) of vector 
expressing only the Gal4-DBD.  Twenty-four h later the cells were harvested for CAT activity.  
The results represent the mean +/- SEM of three independent experiments. 


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Supplements to article: A novel transcription complex that selectively modulates apoptosis of breast cancer cells through regulation of FASTKD2

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