1. JNK Regulates Autocrine Expression of TGF-β1
Juan-Jose Ventura, Norman J Kennedy, Richard A Flavell, Roger J Davis 
Molecular Cell 
Volume 15, Issue 2, Pages (July 2004)
DOI: /j.molcel

2. Figure 1
JNK Deficiency Deregulates TGF-β1 Expression and Causes Constitutive Activation of the TGF-β-Stimulated Smad Signaling Pathway
(A and B) The expression of TGF-β1, TGF-β2, TβR-I, TβR-II, and ribosomal protein L32 mRNA was examined using total RNA isolated from wild-type and Jnk−/− fibroblasts and a ribonuclease protection assay. The protected fragments were detected by autoradiography and quantitated by Phosphorimager analysis. The expression of TGF-β1, TGF-β2, TβR-I, and TβR-II mRNA in the wild-type and Jnk−/− cells was normalized using L32 mRNA and was arbitrarily assigned a relative expression value of 1.0 for wild-type cells. The altered expression of TGF-β1, TGF-β2, TβR-I, and TβR-II mRNA is presented graphically. The increased secretion of TGF-β1 by Jnk−/− cells was confirmed by immunoblot analysis of culture medium conditioned by the cells for 3 days.
(C) The expression of phospho-Smad-2 (P-Smad-2) and Smad-2, -3, -4, -6, and -7 in extracts prepared from wild-type and Jnk−/− fibroblasts was examined by immunoblot analysis.
(D) Nuclear extracts were prepared from wild-type and Jnk−/− fibroblasts and examined using an electrophoretic mobility shift assay (EMSA) using an oligonucleotide probe that contains a consensus Smad binding site (CAGAC). The specific binding was quantitated by Phosphorimager analysis, and the number below the panel indicates the relative binding.
Molecular Cell  , DOI: ( /j.molcel )

3. Figure 2 Tgf-β1 Promoter Activity in Wild-Type and Jnk−/− Cells
(A) Tgf-β1 promoter (−1799/+55) activity was assessed in a CAT reporter gene assay. Transfection efficiency was monitored using the Renilla luciferase expression vector pRL null. Wild-type cells, Jnk−/− cells, and Jnk−/− cells transduced with a retroviral vector that expresses JNK1 or JNK2 were examined.
(B and C) ChIP assays were performed to detect the binding of Smad and AP-1 proteins to the distal regulatory region of the Tgf-β1 promoter in wild-type and Jnk−/− cells using 45 cycles of PCR amplification. Control studies to confirm the specificity of the immunoprecipitation were performed using primers to amplify Gapdh.
(D and E) ChIP assays were performed to detect the binding of Smad-2/3, Smad-4, cJun, and JunD to the distal regulatory region of the Tgf-β1 promoter. PCR amplificaction was performed using 30, 45, and 60 cycles. Control studies to confirm the specificity of the immunoprecipitation were performed using primers to amplify Gapdh.
(F) The binding of HDAC3 to the the distal regulatory region of the Tgf-β1 promoter was examined by ChIP assays. The specificity of the immunoprecipitation was confirmed by control studies using a nonimmune antibody and by amplification of Gapdh.
(G) Complementation analysis of Jnk−/− cells. JNK1 or JNK2 were expressed in Jnk−/− cells. The binding of Smad-2/3 and cJun to the distal regulatory region of the Tgf-β1 promoter was examined by ChIP assays.
Molecular Cell  , DOI: ( /j.molcel )

4. Figure 3
Expression of the p15/Ink4b Gene Is Induced in Wild-Type Cells, but Is Constitutively Expressed by JNK-Deficient Cells
(A) Electrophoretic mobility shift assays were performed using a double-stranded oligonucleotide probe containing the SBE site from the p15/Ink4b promoter. Binding activity present in nuclear extracts prepared from wild-type cells, Jnk−/− cells, and Jnk−/− cells transduced with retroviral vectors that express JNK1 or JNK2. Competition analysis was performed using a 100-fold excess of unlabeled probe with nuclear extracts prepared from Jnk−/− cells. The binding activity was quantitated by Phosphorimager analysis and is presented as relative binding below the panel.
(B) Oligonucleotide precipitation assays were performed using the SBE site from the p15/Ink4b promoter. Control experiments were performed using an oligonucleotide with a scrambled sequence. Bound Smad-2, Smad-3, and Smad-4 were detected by immunoblot analysis.
(C) p15/Ink4b promoter (−751/+70) activity was assessed in a firefly luciferase reporter gene assay. Transfection efficiency was monitored using the Renilla luciferase expression vector pRL null. Control experiments were performed using the empty firefly luciferase vector pGL2-basic. The cells were treated without and with 1 ng/ml TGF-β1.
(D) The expression of p15/INK4b, c-Myc, Smad-2, phospho-Smad-2, and Tubulin by wild-type and Jnk−/− cells was examined by immunoblot analysis. The effect of treatment with 1 ng/ml TGF-β1 was investigated.
Molecular Cell  , DOI: ( /j.molcel )

5. Figure 4
Dominant-Negative TβR-I Inhibits the Constitutive TGF-β Signaling Caused by JNK Deficiency
(A and B) Jnk−/− cells were transduced with retroviral vectors that express JNK1, JNK2, or dn-TβR-I. Immunoblot analysis was performed with an antibody to JNK1/2 or to the HA epitope to detect dn-TβR-I.
(C) The expression of TGF-β1 and ribosomal protein L32 mRNA was examined in a ribonuclease protection assay.
(D) The expression of p15/INK4b, Smad-2, and phospho-Smad-2 was examined by immunoblot analysis.
Molecular Cell  , DOI: ( /j.molcel )

6. Figure 5
Activated Ras Potentiates TGF-β1 Expression by JNK-Deficient Cells and Increases Tumorigenesis
(A) The expression of TGF-β1 and ribosomal protein L32 mRNA was examined in a ribonuclease protection assay. Wild-type and Jnk−/− cells were compared, and the effect of expression of activated H-Ras and dn-TβR-I was examined.
(B) Lung tumor formation in nude mice caused by wild-type and Jnk−/− fibroblasts expressing activated H-Ras (L61) was investigated, and the effect of dn-TβR-I was examined. Control experiments performed using wild-type and Jnk−/− cells without activated H-Ras demonstrated no tumor formation. The tumor burden in the nude mice was examined by measurement of lung mass as a percentage of total body mass. The data shown represent the mean ± SE (n = 15).
Molecular Cell  , DOI: ( /j.molcel )

7. Figure 6
JNK Deficiency Causes Increased TGF-β-Dependent Invasion and Proliferation
(A) The population of cells in the G1, S, and G2 phases of the cell cycle was examined by flow cytometry by staining with an antibody to BrdU and with propidium iodide (PI).
(B) Cells (4 × 104) were incubated in 11 mm wells with DMEM/10% serum. Relative cell number was measured by staining with crystal violet.
(C and D) Boyden chamber assays were performed to measure chemotaxis (cell migration) from serum-free medium to medium supplemented with 10% serum. Invasion assays were performed in similar experiments using Matrigel. The cells were stained with 4′-6′- diamino-2-phenylindole and visualized by fluorescence microscopy. The relative cell migration and invasion was quantitated by counting the number of cells. Wild-type and Jnk−/− fibroblasts were compared, and the effect of expression of dn-TβR-I was examined.
Molecular Cell  , DOI: ( /j.molcel )