1. Volume 11, Issue 6, Pages 1503-1515 (June 2003)
Identification of Transcription Factor KLF8 as a Downstream Target of Focal Adhesion Kinase in Its Regulation of Cyclin D1 and Cell Cycle Progression 
Jihe Zhao, Z.Christine Bian, Kristine Yee, Benjamin P.C Chen, Shu Chien, Jun-Lin Guan 
Molecular Cell 
Volume 11, Issue 6, Pages (June 2003)
DOI: /S (03)

2. Figure 1 Induction of KLF8 Expression by FAK and Cell Adhesion
(A) Mock, FAK, or ΔC14 cells were induced to express the corresponding proteins for 8 or 16 hr. Poly A-plus RNAs were then prepared as described in Experimental Procedures. They were separated by 1.2% agarose/formaldehyde gel, transferred to nylon membrane, and blotted with 32P-labeled KLF8 cDNA probe (top panel) or EF1α cDNA probe (bottom panel).
(B) The mean ± SE of relative KLF8 mRNA (normalized to Mock cells at 8 hr after induction) from three independent experiments obtained using the PhosphoImage Analyzer.
(C) Cell lysates were prepared from ΔC14 or FAK cells under uninduced (U) or induced (I) conditions, as indicated. They were also prepared from FAK−/− fibroblasts and FAK+/+ control cells, or from HEK293 cells transfected with the expression vector encoding KLF8 or the vector alone, as indicated. The lysates were subjected to Western blotting analysis with anti-KLF8 serum. The position of KLF8 is marked on the left.
(D) Quiescent FAK cells under induced conditions were suspended for 1 hr and then replated on FN or PLL for various times, as indicated. Cell lysates were prepared and used for Western blotting with anti-KLF8 antibody. “G,” lysates from growing FAK cells under induced condition.
(E and F) NIH 3T3 cells were transiently transfected with the indicated plasmid DNAs. 24 hr after transfection, cell lysates were prepared and used for Western blotting with anti-FAK (E) or anti-KLF8 (F).
(G) NIH 3T3 cells were treated with chemical inhibitors or DMSO control for 24 hr, as indicated. The lysates were then prepared and used for Western blotting with anti-KLF8.
Molecular Cell  , DOI: ( /S (03) )

3. Figure 2 KLF8 Is Localized Exclusively in the Nuclei
(A) Schematic structure of KLF8 protein with the PVDLS motif, nuclear localization signal (NLS) and zinc fingers (ZF) marked above the diagram.
(B–E) HEK293 or NIH3T3 cells were transfected with plasmids encoding GFP or GFP-KLF8 fusion protein, as indicated. Two days after transfection, the cells were viewed under a fluorescent microscope and photographed.
(F–I) FAK cells were incubated under uninduced (“U”) or induced (“I”) conditions, as indicated. They were then fixed and costained with Hoechst dye (F and G) or anti-KLF8 antibody followed by FITC-conjugated goat anti-rabbit IgG (H and I).
Molecular Cell  , DOI: ( /S (03) )

4. Figure 3 Regulation of Cell Cycle Progression by KLF8
Quiescent Mock cells or KLF8 cells with inducible expression of KLF8 were stimulated with 10% CS under uninduced (U, open bar) or induced (I, filled bar) conditions and in the presence of BrdU for 12 or 16 hr, as indicated. (A and B) The cells were then processed for immunofluorescent staining with α-BrdU and Hoechst dye staining and quantitation as described in Experimental Procedures. (C) and (D) show the induced expression of KLF8 (C) and cyclin D1 (D) at various times after induction, as detected by Western blotting using anti-KLF8 and anti-cyclin D1, respectively. The KLF8 band is marked by an arrow (C). Data are representatives for at least three independent experiments using more than five independent clones.
Molecular Cell  , DOI: ( /S (03) )

5. Figure 4
Specific Inhibition of FAK-Induced Cell Cycle Progression by KLF8 siRNA
(A–D and F–H) FAK cells were Mock transfected or transfected with KLF8 siRNA or the scramble control, as indicated. The effects on cell cycle progression upon induction of FAK expression for 12 hr (A) or 16 hr (F) were determined by measuring BrdU incorporation as described in Experimental Procedures. Aliquots of corresponding cells were used for Western blotting to determine protein levels of FAK (B and G), KLF8 (C and H), or cyclin D1 (D). Data are representatives for at least three independent experiments.
(E) FAK cells were cotransfected with a plasmid encoding β-gal and Mock control (1 and 4), KLF8 siRNA plus pRK5 vector (2 and 5), or KLF8 siRNA plus pRK5-D1 (3 and 6), as indicated. They were then analyzed for cell cycle progression under induced (“I”) or uninduced (“U”) conditions as described in the Experimental Procedures. The results show mean + SE for three independent experiments.
(I) FAK cells were Mock transfected (1, 2, 4, and 5) or transfected with KLF8 siRNA (3 and 6), as indicated. The effects on cell cycle progression upon stimulation with LPA (2, 3, 5, and 6) for 16 hr under uninduced (“U”) and induced (“I”) conditions were determined by measuring BrdU incorporation as described in Experimental Procedures.
Molecular Cell  , DOI: ( /S (03) )

6. Figure 5 Activation of Cyclin D1 Promoter by KLF8
(A) Schematic diagram of KLF8 and its mutants encoded by the pKH3 expression vectors.
(B) NIH3T3 cells were cotransfected with expression vectors encoding KLF8, its mutants, FAK, or vector control alone (pKH3) and with the cyclin D1 promoter reporter pGL3b-D1P or the basic luciferase control vector (pGL3b), as indicated. A plasmid encoding β-gal was also included to normalize transfection efficiency. The ratio of reporter luciferase activity to β-gal activity is shown. The assay was performed 24 hr after transfection. The results show mean ± SE of at least four independent experiments.
(C) Expression of the transfected constructs was monitored by Western blotting with anti-HA antibody. Molecular weight markers are shown in (C).
Molecular Cell  , DOI: ( /S (03) )

7. Figure 6
Specific Binding to the GT Box A by KLF8 Is Required for Its Activation of Cyclin D1 Promoter
(A) Schematic diagram of the cyclin D1 promoter in a luciferase reporter and deletion mutants with the removal of individual GT boxes. The sequences surrounding the GT boxes in cyclin D1 promoters are shown below.
(B) Nuclear extracts prepared from NIH3T3 cells were used for EMSA assays using oligonucleotides shown in (A) as probes. EMSA assays were conducted as described in Experimental Procedures. Arrows on the right mark the SP1 and GT box complexes, respectively.
(C) NIH3T3 cells were cotransfected with expression vectors encoding KLF8 or pKH3 vector control and cyclin D1 promoter reporter pGL3b-D1P (WT) or various mutants, as indicated. Luciferase assays were performed as described in Figure 5. The means ± SE of relative luciferase activities were obtained from three independent experiments. The relative activities were normalized to pKH3 control vector transfected cells.
(D) Nuclear extracts were prepared from KLF8 cells under uninduced (U; lanes 1–8) or induced (I; lanes 9–13) conditions, as indicated. They were used for EMSA with the 32P-labeled cyclin D1 GT box A oligonucleotide (lanes 1–7 and 9–13) or the labeled mutant GT box A oligonucleotide (“c,” lane 8). Increasing amounts of unlabeled GT box A (lanes 2–4) or mA (lanes 5–7) oligonucleotides were included as binding competitors. Anti-KLF8 antibody (lanes 10 and 11) or anti-GST (lanes 12 and 13) were included in the assays. The arrow on the right indicates the GT box A binding complex.
Molecular Cell  , DOI: ( /S (03) )

8. Figure 7
The KLF8 Binding to GT Box A Is Required for Activation of Cyclin D1 Promoter by FAK
(A–C) NIH3T3 cells were cotransfected with pGL3b or pGL3b-D1P and pKH3, pKH3-FAK, and Mock control, or pKH3-FAK with KLF8 siRNA, as indicated. Luciferase assays were performed as described in Figure 7. The mean ± SE of relative luciferase activities were obtained from three independent experiments. Aliquots of cell lysates were used for Western blotting with anti-FAK (B) or anti-KLF8 (C).
(D) NIH3T3 cells were cotransfected with pKH3 or pKH3-FAK and pGL3b-D1P (WT) or its mutants lacking GT box A or Ets B site, as indicated. Luciferase assays were performed as described in Figure 5. The means ± SE of relative luciferase activities were obtained from three independent experiments. The relative activities were normalized to pKH3 control vector transfected cells.
Molecular Cell  , DOI: ( /S (03) )

9. Figure 8
FAK Regulates Binding Activities of Ets and KLF8 on D1 Promoter in a Time-Dependent Manner
Quiescent FAK cells were stimulated with 10% CS under uninduced (U; [A and B]) or induced (I; [C and D]) conditions for various times, as indicated. Nuclear extracts were then prepared and used for EMSA with the radiolabeled oligonucleotide corresponding to the Ets B site (A and C) or GT box A site (B and D). The asterisk (*) and double asterisk (**) on the right indicate the Ets B and GT box A binding complexes, respectively.
Molecular Cell  , DOI: ( /S (03) )