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Results: 8

1.
Figure 1

Figure 1. ERK signaling affects c-Jun expression. From: Re-wired ERK-JNK signaling pathways in melanoma.

A. Levels of P-ERK, P-JNK and proteins relevant to the PKC/JNK pathway in melanoma cell lines. Protein extracts (40μg) from melanoma cell lines were analyzed by Western blots using the indicated antibodies. Human melanocytes (HuMel) were used as a control. α-Tubulin reveals equal loading.
B. Inhibition of B-Raf reduces c-Jun levels. Lu1205 cells were treated with 300nM CHR-265 for 6h and 24h. Protein samples were analyzed by Western blots using the indicated antibodies. α-Tubulin reveals equal loading.
C. Inhibition of the MEK/ERK pathway reduces c-Jun levels. A375 cells were treated with 50μM PD98059 and 20μM U0126 for 24h. Protein samples were analyzed by Western blots using the indicated antibodies.

Pablo Lopez-Bergami, et al. Cancer Cell. ;11(5):447-460.
2.
Figure 8

Figure 8. Model of ERK cross-talk with JNK signaling. From: Re-wired ERK-JNK signaling pathways in melanoma.

Super-active ERK as seen in melanomas that carry mutant B-RAF or N-RAS genes increases stability of c-Jun via its phosphorylation-dependent inactivation of GSK3, and induces c-Jun transcription via its activation of CREB. In turn, c-Jun induces transcription of RACK1, which, in concert with active PKC and MKK4/7, augment the degree of JNK activity to further increase (and maintain) c-Jun stability and activity, constituting a feed-forward loop mechanism induced by ERK. Our data support the existence of this model in melanoma, but not in cells in which ERK activity is not sufficiently high.

Pablo Lopez-Bergami, et al. Cancer Cell. ;11(5):447-460.
3.
Figure 3

Figure 3. ERK regulates c-Jun transcription. From: Re-wired ERK-JNK signaling pathways in melanoma.

A. Inhibition of the MEK/ERK pathway affects c-Jun mRNA levels. A375 cells were treated with 50μM PD98059 for the indicated time periods. c-Jun mRNA levels were analyzed with Real-Time PCR. Data were normalized using levels of β-Actin mRNA. Results are shown as the mean (bar) ± standard deviation (SD) of the respective relative concentrations. A representative experiment (of three performed) is shown.
B. Inhibition of the MEK/ERK pathway affects CREB phosphorylation. A375 cells were treated with 50μM PD98059 for 12h. Protein samples were analyzed by Western blots using the indicated antibodies. α-Tubulin reveals equal loading.
C. CREB siRNA affects c-Jun protein levels in melanoma. Protein extracts from A375SM cells stably transfected with non-targeting vector (NT) or with a vector encoding CREB siRNA were analyzed by Western blots using the indicated antibodies. α-Tubulin reveals equal loading.
D. CREB siRNA affects c-Jun mRNA levels. RNA was extracted from A375SM cells stably transfected with non-targeting vector (NT) or with a vector encoding CREB siRNA. c-Jun mRNA levels were analyzed with Real-Time PCR. Data were normalized using levels of β-Actin mRNA. Results are shown as the mean (bar) ± SD of the respective relative concentrations. A representative experiment (of three performed) is shown.
E. CREB siRNA affects transcription driven by the c-Jun promoter. A375SM cells stably transfected with non-targeting vector (NT) or with a vector encoding CREB siRNA were transiently transfected with 0.2μM of luciferase reporter containing the c-Jun WT promoter or c-Jun promoters mutated on the Jun2 site (Jun2-M), the TRE site (TRE-M) or the double mutant. Results are shown as the mean ± SD. Data were standardized using β-galactosidase activity.

Pablo Lopez-Bergami, et al. Cancer Cell. ;11(5):447-460.
4.
Figure 6

Figure 6. ERK induces JNK activation as part of cross-talk and feed-forward mechanisms. From: Re-wired ERK-JNK signaling pathways in melanoma.

A. Inhibition of the MEK/ERK pathway affects RACK1 and P-JNK levels. A375 cells were treated with 50μM PD98059 (PD) or 10μM of JNK inhibitor for the indicated times. Protein samples were analyzed by Western blots using the indicated antibodies. α-Tubulin reveals equal loading.
B. Inhibition of the MEK/ERK pathway affects c-Jun, P-JNK and cyclin D1 levels. The indicated melanoma cell lines were treated with 50μM PD98059 for 16h. Protein samples were analyzed by Western blots using the indicated antibodies. α-Tubulin reveals equal loading.
C. Inhibition of c-Jun attenuates JNK activity in SW1 mouse melanoma cells. SW1 cells were transfected to establish stable clones expressing the dominant negative form of c-Jun, TAM67. Cells were used to monitor JNK phosphorylation on aa 183/5, which reflect JNK activity.
D. c-Jun regulates cyclin D1 expression. Protein extracts from Lu1205 and SW1 cells stably transfected with FLAG-TAM67 were blotted with the indicated antibodies. α-Tubulin antibody was used to monitor equal protein loading.
E. LiCl restores c-Jun and cyclin D1 levels that are reduced by MEK/ERK inhibitors. Lu1205 cells were treated with 50μM PD98059 (PD) in the presence or absence of 10mM LiCl for 12h. Protein samples were analyzed by Western blots using the indicated antibodies. α-Tubulin reveals equal loading.
F. CREB siRNA affects both c-Jun and cyclin D1 protein levels in melanoma. Protein extracts from A375SM cells stably transfected with non-targeting vector (NT) or with a vector encoding CREB siRNA were analyzed by Western blots using the indicated antibodies. α-Tubulin reveals equal loading.

Pablo Lopez-Bergami, et al. Cancer Cell. ;11(5):447-460.
5.
Figure 4

Figure 4. c-Jun regulates RACK1 transcription. From: Re-wired ERK-JNK signaling pathways in melanoma.

A. Go6976 inhibits JNK activation. A375 cells were treated with Go6976 for 2h before protein extracts were prepared. Levels of P-JNK and total JNK were assessed by Western blot. β-actin was used to monitor equal loading.
B. Inhibition of PKC reduces RACK1-driven luciferase activity. The luciferase activity driven by the RACK1 promoter (2Kb of the proximal RACK1 promoter cloned into a pGL2 vector) was assessed in Lu1205 cells in the presence of Go6976 (3μM for 8h). TRE and TOP vectors were used as positive and negative controls, respectively. Results are shown as the mean ± SD. Data were standardized on the basis of β-galactosidase activity.
C. c-Jun regulates RACK1 expression. Protein extracts from SW1 cells stably transfected with FLAG-TAM67 were blotted with RACK1 and FLAG antibodies. β-actin antibody was used to monitor equal protein loading.
D. RACK1 expression is reduced in c-Jun-/- fibroblasts. Protein extracts from c-Jun -/- and control fibroblasts were blotted with RACK1 antibody. β-actin and α-Tubulin antibodies were used to monitor equal protein loading.
E. Decrease in relative mRNAs levels of RACK1 in c-Jun -/- MEF and in SW1-TAM67 cells. Relative levels of RACK1 mRNA were determined by Real-Time quantitative PCR. Reactions were run in triplicate. β-actin was used as a control. Results are shown as the mean (bar) ± SD of the respective relative concentrations. A representative experiment (of three performed) is shown.
F. c-Jun regulates RACK1-driven transcription. The luciferase activity driven by the RACK1 promoter (2Kb of the proximal RACK1 promoter cloned into a pGL2 vector) was assessed in SW1 cells expressing TAM67 as well as in wt and c-Jun mutant fibroblasts. Results are shown as the mean ± SD. Data were standardized on the basis of β-gal activity.
G. Activation of the JNK/c-Jun pathway increases RACK1 expression. HEK293T cells were transfected with ΔMEKK1 or empty pEF plasmid. Protein extracts were obtained 48h post-transfection and blotted with the indicated antibodies. β-actin antibody was used to monitor loading. Arrow indicates position of ΔMEKK1.
H. ΔMEKK1 increases RACK1-driven transcription. The luciferase activity driven by the RACK1 promoter (2Kb of the proximal RACK1 promoter cloned into a pGL2 vector) was assessed in HEK293T cells transfected with ΔMEKK1. Results are shown as the mean ± SD. Data were standardized on the basis of β-gal activity.

Pablo Lopez-Bergami, et al. Cancer Cell. ;11(5):447-460.
6.
Figure 2

Figure 2. ERK regulates c-Jun stability. From: Re-wired ERK-JNK signaling pathways in melanoma.

A. Regulation of c-Jun by the MEK/ERK pathway is via transcriptional and post-transcriptional mechanisms. A375 cells were treated with 50μM PD98059 (PD) at the indicated time points in the presence or absence of Actinomycin D (Act D) (see Methods). Protein samples were analyzed by Western blots using the indicated antibodies. α-Tubulin reveals equal loading. ns: non-specific band. Quantification of c-Jun level is shown. For all quantifications, c-Jun levels were normalized using α-Tubulin levels. The experiment shown is representative of the four experiments performed.
B. Inhibition of the MEK/ERK pathway affects the levels of exogenously expressed c-Jun. Lu1205 cells were transfected with either 0.5μg Jun-HA (an expression vector containing the EF [Elongation Factor] promoter or 1μg CMVHA-Jun (an expression vector using a CMV promoter). ATF2-Flag (1μg) was used to monitor transfection efficiency. Twenty-four hours after transfection, cells were treated with 50μM PD98059 (PD) for 8h. Protein samples were analyzed by Western blots using HA or Flag antibody as indicated. α-Tubulin reveals equal loading.
C. LiCl restores c-Jun levels reduced by MEK/ERK inhibitors. Lu1205 cells were transfected with c-Jun-HA and ATF2-Flag as indicated in B. Twenty four hours after transfection, cells were treated with 50μM PD98059 (PD) in the presence or absence of 10mM LiCl for 12h. Protein samples were analyzed by Western blots using HA and Flag antibodies. α-Tubulin reveals equal loading.
D. LiCl and Kenpaullone attenuate the effect of MEK/ERK inhibitors on endogenous c-Jun levels. A375 cells were treated with 50μM PD98059 (PD) for 12h in the presence or absence of 10mM LiCl or 10μM Kenpaullone (Ken). Protein samples were analyzed by Western blots using the indicated antibodies. α-Tubulin reveals equal loading. c-Jun protein levels were normalized to α-Tubulin levels. The experiment shown is representative of the four experiments performed.
E. Phosphorylation of GSK3(S9/S21) is inhibited by PD98059. A375 cells were treated with 50μM PD98059 (PD) for 12h. Protein samples were analyzed by Western blots using the indicated antibodies. α-Tubulin reveals equal loading.
F. T239 and S243 are required for regulation of c-Jun by MEK/ERK. Lu1205 cells were transfected with 0.5μg of Flag-tagged c-Jun WT, c-Jun T239A, c-Jun S243A or c-Jun T239A/S243A. ATF2-Flag (1μg) was used to monitor transfection efficiency. Twenty four hours after transfection, cells were treated with 50μM PD98059 for 12h. Protein samples were analyzed by Western blots using Flag antibody. α-Tubulin reveals equal loading. Graph shows c-Jun protein levels normalized to α-Tubulin levels. One experiment representative of three performed is shown.
G. ERK regulates c-Jun levels in melanoma. The indicated melanoma cell lines were treated with 50μM PD98059 (PD) or 10μM LY294002 (LY) for 12h in the absence of serum. Protein samples were analyzed by Western blots using the indicated antibodies. α-Tubulin reveals equal loading.

Pablo Lopez-Bergami, et al. Cancer Cell. ;11(5):447-460.
7.
Figure 7

Figure 7. Analysis of RACK1, c-Jun, cyclin D, P-ERK and P-JNK levels in melanoma tumor samples. From: Re-wired ERK-JNK signaling pathways in melanoma.

A.ERK activity is associated with JNK, c-Jun and RACK1 levels and activity in human tumor samples. Protein samples (80μg) obtained from melanoma tumors were analyzed by Western blot using the indicated antibodies. For the RACK1, Bcl-2 and α-Tubulin blots, 20μg of protein was used. The status of B-RAF and N-RAS genes is shown above the tumor ID. RAS indicates a Q61K or Q61R mutation. RAF indicates a V599E mutation. WT indicates the absence of Q61 and V599 mutation in N-RAS or B-RAF.
B. Stack graph represents relative levels of P-ERK, c-Jun, RACK1 and P-JNK that were quantified (Fig. S16) and converted into numbers (0, 1, 2, 3) to reflect protein expression level qualitatively.
C. Hierarchical clustering of the samples shown in A was performed with the aid of HCE program (Seo et al., 2002), using average linkage and Euclidean distance measure between expression levels. Different expression levels are shown in different colors: 0- light green, 1- green, 2- black and 3- red. The order of linking corresponds to the proximity of expression levels in different cell types. Minimal similarity values (computed in HCE program using Euclidean distance and normalized to a scale from 0 (no similarity) to 1 (identical profile)) are shown on the tree to reflect qualitatively similarities in expression patterns. The analysis revealed significant relations between P-ERK and c-Jun (0.672), c-Jun and RACK1 (0.617) and between P-ERK and RACK1 (0.680) levels. These values are significant with 99% confidence (the critical value for 1% significance level for 22 degrees of freedom (24-2) is 0.515, using Student’s t-statistics). Bcl2 levels, which are not directly associated with the other proteins in this analysis, served as an outlier for the statistical analysis.

Pablo Lopez-Bergami, et al. Cancer Cell. ;11(5):447-460.
8.
Figure 5

Figure 5. Mapping the c-Jun response element on the RACK1 promoter. From: Re-wired ERK-JNK signaling pathways in melanoma.

A. Structure of the RACK1 promoter. Putative c-Jun response elements and fragments of the promoter that were cloned are depicted (black boxes, A-E).
B. Mapping of the functional AP-1 site of the mouse RACK1 promoter. DNA fragments containing various response elements (shown in A) were cloned into pGL2 and their luciferase activity was assayed in Lu1205 cells in the presence of Go6976 (3μM for 8h). TOP vector was used as negative control. Results are shown as the mean ± SD.
C. c-Jun binds to the AP-1 element located at -60 on the RACK1 promoter. Ten micrograms of nuclear extracts from control cells were incubated with a doublestranded DNA fragment probe corresponding to the –67 to –54 region of the RACK1 promoter carrying a single AP-1 motif (lanes 4 to 12). Unlabeled competitors (RACK1, wt oligonucleotide (lane 5), RACK1-M, RACK1 oligonucleotide containing two bp changes in the AP-1 element (lane 6) and LRP6, non-related competitor (lane 7)) were added at a 10-fold excess as indicated (lanes 5 and 7 were run in duplicates). For supershift assays, 0.4 (lanes 8 and 10) or 2 μg (lanes 9 and 11) of c-Jun Ab or control IgG (Santa Cruz Biotechnology, Santa Cruz, CA) were included in the reaction. Lanes 4 and 12 show the shift in the absence of antibodies or competitors. The lower arrow (A) indicates a specific retarded band corresponding to c-Jun in complex with the probe. The upper arrow (B) indicates the supershifted complex. Reactions using nuclear extracts from c-Jun siRNA treated cells were included as a control (lane 2 and 3, 20 and 10μg respectively). The lower panel shows changes in c-Jun protein levels in cells transfected with control (sc) and c-Jun siRNA. A picture of the complete gel is shown in Fig. S13.
D. Mutation of c-Jun element at -60 inhibits reporter activity. The AP-1 response element D was mutated and the relative luciferase activity of the WT and mutant construct was assessed in Lu1205 cells. Results are shown as the mean ± SD. Data were standardized on the basis of β-galactosidase activity.
E. Chromatin immunoprecipitation assay. Sheared chromatin from SW1 cells or SW1 stably transfected with TAM67 was immunoprecipitated with appropriate antibody (anti-c-Jun or control IgG). Immunoprecipitated DNA was used as the template in PCR using primers corresponding to the proximal region of the RACK1 promoter. GAPDH primers were used as control.
F. c-Jun is not present at the RACK1 promoter in cells treated with c-Jun siRNA. Sheared chromatin from SW1 cells or SW1 transfected with c-Jun siRNA was immunoprecipitated with appropriate antibody (anti-c-Jun or control IgG). Samples were processed as in Fig. 5E.
G. MEK inhibitor affects c-Jun binding to RACK1 promoter. Sheared chromatin from SW1 cells or SW1 treated with 50μM PD98059 (PD) in the presence or absence of 10mM LiCl was immunoprecipitated with appropriate antibody (anti-c-Jun or control IgG). Samples were processed as in Fig. 5E.

Pablo Lopez-Bergami, et al. Cancer Cell. ;11(5):447-460.

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