1. A Self-Enabling TGFβ Response Coupled to Stress Signaling
Yibin Kang, Chang-Rung Chen, Joan Massagué 
Molecular Cell 
Volume 11, Issue 4, Pages (April 2003)
DOI: /S (03)
Copyright © 2003 Cell Press Terms and Conditions

2. Figure 1
Transcriptional Repression of Id Family Genes by TGFβ Is a Common Feature of TGFβ Cytostasis Program in Epithelial Cells
(A) Relative transcript level of genes that have roles in cell cycle control, before and after 3 hr of TGFβ treatment in three human epithelial cell lines. Data are absolute values from the DNA microarray profiling experiment.
(B) HaCaT, MCF-10A, and HPL1 cells were incubated with TGFβ or BMP2 for 3 hr. Total RNA was isolated and subjected to Northern blot analysis using the indicated probes.
(C) HaCaT cells were treated with TGFβ for the indicated times. Total RNA was subjected to Northern blot analysis. The Id1 mRNA signals were quantified and normalized against the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) values.
(D) HaCaT-tetMyc cells were transfected with pId1-Luc reporter construct, cultured for 20 hr in the presence (Myc-Off) or absence (Myc-On) of tetracycline, followed by an additional 20 hr of treatment with or without TGFβ. Luciferase activity (top) and c-myc, Id1, and GAPDH mRNA levels were determined.
(E) HaCaT cells were incubated in the absence (−) or presence (+) of cycloheximide for 1 hr, following by incubation with TGFβ for the indicated times. Total RNA was subjected to Northern blot analysis. The Id1 mRNA signals were quantified and normalized against the GAPDH values.
Molecular Cell  , DOI: ( /S (03) )
Copyright © 2003 Cell Press Terms and Conditions

3. Figure 2
Identification of a Minimal TGFβ Responsive Region on the Id1 Promoter
(A and C–E) HaCaT cells were transfected with the indicated Id1 reporter constructs (López-Rovira et al., 2002), treated with or without TGFβ for 16–20 hr prior to lysis, and analyzed for luciferase activity. A schematic representation of each promoter construct is shown in the left side of each panel. DNA fragments containing the 120 bp minimal TGFβ responsive region were highlighted as a yellow box. Data are the average of triplicate determinations ±SD.
(B) Nucleotide sequence of the minimal TGFβ-responsive region on the Id1 promoter. Nucleotide sequence positions are indicated relative to the transcription start site. The YY1 site (green box), SP1 site (blue box), Egr-1 site (brown box), ATF/CREB site (purple box), three consensus Smad binding sites (green arrows), and the mutations targeting these sites are indicated.
(F) HaCaT cells were transfected with the indicated Id1 reporter constructs, treated with or without TGFβ or BMP for 16–20 hr prior to lysis, and analyzed for luciferase activity.
(G) Schematic representation of the minimal TGFβ-responsive region with major transcriptional factor binding sites indicated by colored boxes as in (B). The requirement of each site for the Id1 transcriptional responses to TGFβ and BMP is indicated below.
Molecular Cell  , DOI: ( /S (03) )
Copyright © 2003 Cell Press Terms and Conditions

4. Figure 3 ATF3 Is a Direct Transcriptional Activation Target of TGFβ
(A and B) HaCaT cells were incubated with TGFβ for the indicated times. Total RNA or cell lysates were harvested and subjected to Northern blot (A) or Western blot (B) analysis, respectively.
(C) HaCaT cells were incubated in the presence (+) or absence (−) of TGFβ or BMP2 for 3 hr. Total RNA was isolated and subjected to Northern blot analysis.
(D) HaCaT cells were left untreated (−) or treated (+) with TGFβ for 2 hr, chromatin immunoprecipitations were performed with the indicated antibodies, and PCR was performed with primers specific for the ATF3 and β-actin promoters.
Molecular Cell  , DOI: ( /S (03) )
Copyright © 2003 Cell Press Terms and Conditions

5. Figure 4 TGFβ-Induced Binding of Smad3-Smad4 and ATF3 to Id1 Promoter
(A–C) HaCaT cells were incubated in the absence (−) or presence (+) of TGFβ or BMP for 2 hr. Cell lysates were incubated with the indicated wild-type or mutant (m) biotinylated oligonucleotides. DNA-bound proteins were precipitated by streptavidin-agarose and detected by immunoblotting. Total HaCaT cell extract (Input) was included as a reference. Schematic representation of three biotinylated double-stranded oligonucleotides (GC-rich, ATF, and SBE) covering different regions of the 120 bp minimal TGFβ-responsive region of the Id1 promoter is shown at the top of (A).
(D) HaCaT cells were left untreated (−) or treated (+) with TGFβ for 2 hr, and chromatin immunoprecipitations were performed with the indicated antibodies. PCR was performed with primers specific for the Id1 and β-actin.
(E) HaCaT cells were left untreated (0) or treated with TGFβ for the indicated times, and chromatin immunoprecipitations were performed with the indicated antibodies and primers specific for the Id1 promoter.
(F) Signals were quantified and illustrated.
Molecular Cell  , DOI: ( /S (03) )
Copyright © 2003 Cell Press Terms and Conditions

6. Figure 5 Protein-Protein Interaction between Smad3 and ATF3
(A) COS-1 cells were transfected with vectors encoding the indicated Flag epitope-tagged Smad proteins, HA epitope-tagged ATF1, and ATF3. The cells were treated with (+) or without (−) TGFβ for 2 hr, and lysates were precipitated with the anti-FLAG antibody. The immunoprecipitates were subjected to immunoblotting with anti-HA antibody. Protein expression was assessed by direct immunoblotting of total lysates (Input).
(B) HaCaT cells were treated with (+) or without (−) TGFβ for 2 hr prior to lysis, and immunoprecipitations were performed with the indicated antibodies.
(C) GST control or GST-Smad3 bound to glutathione beads was mixed with purified maltose binding protein (MBP)-fusion proteins. MBP-fusion proteins bound to the beads were detected by immunoblotting with anti-MBP antibodies. The amount of each MBP-fusion protein used in the GST pull-downs was monitored by immunoblot analysis of total cell extracts (Input).
(D) HaCaT cells were incubated in the presence (+) or absence (−) of TGFβ or BMP for 2 hr and then subjected to immunofluorescence analysis with anti-Smad3 or anti-ATF3 antibodies to localize endogenous Smad3 and ATF3, respectively. Nuclei were visualized by DNA staining with DAPI.
(E) (Top) HaCaT cell cultures were transfected with pHA-ATF3. Two days after transfection, cells were subjected to immunofluorescence analysis with anti-ATF3 antibody to localize transfected ATF3. (Bottom) Quantitative analysis of the effect of FAST and ATF3 on the nucleocytoplasmic shuttling activity of Smad2 and Smad3. Fusion proteins consisting of the MS2 coat protein linked to Smad2, Smad3, or HIV-1 Rev were assayed in 293T cells for their ability to support nuclear export of unspliced reporter RNA containing the MS2 operator RNA and chloramphenicol acetyl transferase (CAT) reporter gene. Indicated effector plasmids encoding MS2-fusion proteins were cotransfected with pDM128/8xMS2 reporter plasmid (8xMS2-CAT) (green bars) or negative control parental plasmid pDM128/CAT (black bars). Induced CAT activity was measured at 48 hr posttransfection. Effects of FAST (brown bars) and ATF3 (blue bars) Smad2-interacting proteins on the nucleocytoplasmic shuttling activity of Smad2 were measured by cotransfecting FAST- or ATF3-expressing plasmids in addition to the indicated reporter and effector plasmids. Data are the average of triplicate determinations ±SD.
Molecular Cell  , DOI: ( /S (03) )
Copyright © 2003 Cell Press Terms and Conditions

7. Figure 6
The Smad3 Interacting Region of ATF3 Functions as a Dominant-Negative Inhibitor of Id1 Transcriptional Repression by TGFβ
(A) COS-1 cells were transfected with vectors encoding HA-tagged ATF3 together with the deletion constructs of Flag-Smad3 as diagrammed at the top of each panel. Cell lysates were immunoprecipitated with anti-Flag or anti-HA antibody. Immunoprecipitates were analyzed by immunoblotting with the antibodies indicated on the left. Expression of the proteins was monitored by immunoblotting of total cell extracts (Input).
(B) Schematic representation of ATF3 protein, its splicing variant, ATF3Δzip, and deletion mutants, with amino acid number annotated and two functional domains of ATF3, the basic domain and the leucine zipper domain, highlighted with green and blue boxes, respectively.
(C) COS-1 cells were transfected with vectors encoding Flag-Smad3, together with HA-tagged ATF3 or its deletion mutants. Cell lysates were immunoprecipitated with anti-Flag antibody. Immunoprecipitates were analyzed by immunoblotting with anti-HA or anti-Flag antibody. Expression of the proteins was monitored by immunoblotting of total cell extracts (Input). Results were summarized in the right side of (B).
(D) A schematic representation of protein-protein interactions between Smad3 and ATF3. The interacting domains are connected with a purple line.
(E) HaCaT cells were transfected with 1 μg of pId1-Luc reporter plasmid, together with 0.5, 1, or 2 μg (indicated with darker hue of blue bars) of indicated expression plasmid, treated with (+) or without (−) TGFβ, and analyzed for luciferase activity.
(F) HaCaT cells were transduced with MSCV-ATF3-Δ4-IRES-GFP or the control parental MSCV-IRES-GFP retroviruses, and sorted for positive GFP expression. Total RNA was isolated from GFP-positive cells after 3 hr of incubation in the presence (+) or absence (−) of TGFβ, and subjected to Northern blot analysis.
Molecular Cell  , DOI: ( /S (03) )
Copyright © 2003 Cell Press Terms and Conditions

8. Figure 7
TGFβ and Stress Signals Converge on ATF3 and Id1 to Regulate Cell Cycle
(A) HaCaT cell cultures were incubated with TNFα or thapsigargin for the indicated times. Total RNA was subjected to Northern blot analysis with the indicated cDNA probes. The Id1 mRNA signals were quantified and normalized against the GAPDH values.
(B) HaCaT were incubated in the presence (+) or absence (−) of p38 inhibitor SB for 1 hr, followed by incubation in the presence (+) or absence (−) of TGFβ or TNFα for 3 or 1 hr, respectively. Total RNA was isolated and subjected to Northern blot analysis.
(C) HaCaT cells were incubated with TGFβ for the indicated times, in the presence (+) or absence (−) of TNFα. Total RNA was subjected to Northern blot analysis. The ATF3 and Id1 mRNA signals were quantified and normalized against the GAPDH values.
(D) HaCaT cells were transduced with MSCV-Id1-IRES-GFP or the control parental MSCV-IRES-GFP retroviruses, and FACS sorted for positive GFP expression. Total RNA was isolated from GFP-positive cells after 3 hr of incubation in the presence (+) or absence (−) of TGFβ and subjected to Northern blot (top panel) or Western blot analysis.
(E) Inhibition of 125I-deoxyuridine incorporation into DNA by TGFβ in HaCaT cells transduced with MSCV-Id1-IRES-GFP or the control parental MSCV-IRES-GFP. Exponentially growing cultures were incubated for 20 hr with the indicated concentrations of TGFβ with (+) or without (−) TNFα. Data are the average of triplicate determinations ±SD.
(F) HaCaT transduced with MSCV-Id1-IRES-GFP or the control parental MSCV-IRES-GFP were treated with TGFβ or TNFα, individually or simultaneously. Twenty-four hours after treatment, flow cytometry was performed on methanol-fixed cells stained with propidium iodide to determine DNA content. Quantification of the various cell cycle phases for each population was determined as indicated.
(G) A schematic model for Id1 transcriptional response to TGFβ and stress signals. Binding of TGFβ to its receptors results in phosphorylation of Smad3, which activates transcription of stress response factor ATF3. Subsequently, interaction of Smad3 and ATF3 enables the formation of a transcriptional repression complex on the Id1 promoter, leading to Id1 downregulation. Stress signals, acting through p38 kinase, can also induce ATF3 and repress Id1. TGFβ and stress signals synergize in achieving rapid and sustained induction of ATF3 and downregulation of Id1, jointly contributing to the stress response. In contrast, BMP-activated Smads bind to the TGFβ/BMP response element to activate Id1 transcription (Korchynskyi and ten Dijke, 2002; López-Rovira et al., 2002).
Molecular Cell  , DOI: ( /S (03) )
Copyright © 2003 Cell Press Terms and Conditions