Journal: PLoS ONE

Article Title: Mitogen-Activated Protein Kinase Kinase 4 (MAP2K4) Promotes Human Prostate Cancer Metastasis

doi: 10.1371/journal.pone.0102289

Figure Lengend Snippet: A ) MAP2K4 protein expression in MAP2K4 variant cell lines. The expression of MAP2K4 protein was measured in wild type, WT1, WT2, and constitutive active, CA1, CA2, CA3, MAP2K4 over expressing cell lines, and vector control, VC1, VC2, cell lines by Western blot. A representative Western blot is depicted, with quantification of the MAP2K4/GAPDH levels, normalized to VC1 cells, shown above each lane. B ) Invasion of MAP2K4 variant cell lines. Boyden chamber Matrigel cell invasion assays were performed, as described in . Data are from 5 independent experiments, each in replicates of N = 3. Data from the respective wild type, constitutive active and vector control cell lines were combined to give an average read out for WT-MAP2K4, CA-MAP2K4 and VC cells, respectively. Data are expressed as the mean ± SEM percentage of VC cells. C-E ) The effect of MAP2K4 expression upon metastatic spread and tumor growth. Equal numbers of mice were orthotopically implanted with VC1 or VC2 cells (forming the VC cohort), WT1 or WT2 cells (WT cohort), or with CA1, CA2 or CA3 cells (CA cohort). In (C) the resultant number of distant metastasis is expressed as the mean ± SEM percentage of VC mice, in (E) the number of mice developing circulating tumor cells is depicted, and in (F) tumor volume is graphed as the mean ± SEM percentage of VC mice. A representative image of a distinct metastasis is shown in (D). * denotes p≤0.05 between VC and an experimental group.

Article Snippet: The following were purchased: antibodies, SEK1/MKK4 (#9152), phospho-p38 MAPK (T180/Y182) (#4631), p38 MAPK (#9212), phospho-SAPK/JNK (T183/Y185) (#4668), SAPK/JNK (#9258), phospho-HSP27 (#2401), HSP27 (#2402), and GAPDH (#2118), all from Cell Signaling Technology, and horseradish peroxidase-conjugated anti-mouse and anti-rabbit, from GE Healthcare Biosciences; plasmids, wild type and constitutive active MAP2K4 (#14615 and #14813; Addgene) and GFP (Vivid Colors pcDNA 6.2/N-EmGFP-GW/TOPO, Invitrogen); chemical inhibitors, p38 MAPK inhibitor, SB203580 (Sigma-Aldrich) and associated negative control, SB202474, and JNK Inhibitor II (SP600125), and JNK Inhibitor II Negative Control (N 1 -Methyl-1,9-pyrazoloanthrone), all from EMD Millipore.

Techniques: Expressing, Variant Assay, Plasmid Preparation, Control, Western Blot

Journal: PLoS ONE

Article Title: Mitogen-Activated Protein Kinase Kinase 4 (MAP2K4) Promotes Human Prostate Cancer Metastasis

doi: 10.1371/journal.pone.0102289

Figure Lengend Snippet: Studies were conducted on MAP2K4 variant cell lines. A ) Knockdown of MAP2K4 protein by siRNA. After transfection of cells with siRNA targeting MAP2K4 (siMAP2K4) or non-targeting control (siCON), MAP2K4 expression was measured by Western blot. Percent knockdown is denoted above the blot. B ) Effect of MAP2K4 knockdown on cell invasion. MAP2K4 variant cell lines were treated with siRNA, as indicated, and cell invasion measured and depicted as in . Data are from four experiments, each in replicates of N = 2, and are expressed as the percentage of invading cells. C ) Effects on cell migration. Cell migration was measured as described in . Data from individual types of clones were combined, are from three independent experiments, each in replicates of N = 3, and are expressed as the percentage of migrating cells, normalized to VC. D ) MMP transcript expression. The levels of MMP-2, MMP-9, MMP-10 and MMP-13 transcript levels were measured by qRT/PCR, normalized to GAPDH, and expressed as the percentage of VC. Data are from four independent experiments, each in replicates of N = 2. E ) Cell growth. Cells were plated at the indicated concentrations, allowed to grow for 5 days, MTT added, and optical density determined. Data are from three independent experiments, each N = 3. F ) Colony formation. Colony formation after 14 days was determined in three independent experiments, each N = 2, and expressed as the percent of VC. Values in all graphs are the mean ± SEM. * denotes p≤0.05 between the indicated groups.

Article Snippet: The following were purchased: antibodies, SEK1/MKK4 (#9152), phospho-p38 MAPK (T180/Y182) (#4631), p38 MAPK (#9212), phospho-SAPK/JNK (T183/Y185) (#4668), SAPK/JNK (#9258), phospho-HSP27 (#2401), HSP27 (#2402), and GAPDH (#2118), all from Cell Signaling Technology, and horseradish peroxidase-conjugated anti-mouse and anti-rabbit, from GE Healthcare Biosciences; plasmids, wild type and constitutive active MAP2K4 (#14615 and #14813; Addgene) and GFP (Vivid Colors pcDNA 6.2/N-EmGFP-GW/TOPO, Invitrogen); chemical inhibitors, p38 MAPK inhibitor, SB203580 (Sigma-Aldrich) and associated negative control, SB202474, and JNK Inhibitor II (SP600125), and JNK Inhibitor II Negative Control (N 1 -Methyl-1,9-pyrazoloanthrone), all from EMD Millipore.

Techniques: Variant Assay, Knockdown, Transfection, Control, Expressing, Western Blot, Migration, Clone Assay, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Mitogen-Activated Protein Kinase Kinase 4 (MAP2K4) Promotes Human Prostate Cancer Metastasis

doi: 10.1371/journal.pone.0102289

Figure Lengend Snippet: A ) MAP2K4 protein expression in LNCaP and 1542CPTX cell lines by Western Blot. B ) Relative cellular invasion of LNCaP and 1542CPTX (42C) cell lines as measured by a Matrigel coated Boyden chamber. Data are from four independent experiments, each in replicates of N = 2. * denotes p≤0.05 between the indicated groups. C ) Relative MMP-2 mRNA expression in LNCaP and 42C cell lines as measured by qRT/PCR. Data are from five independent experiments, each in replicates of N = 2. * denotes p≤0.05 between the indicated groups. D ) Phosphorylated and total HSP27 expression in LNCaP and 1542CPTX cell lines by Western Blot.

Article Snippet: The following were purchased: antibodies, SEK1/MKK4 (#9152), phospho-p38 MAPK (T180/Y182) (#4631), p38 MAPK (#9212), phospho-SAPK/JNK (T183/Y185) (#4668), SAPK/JNK (#9258), phospho-HSP27 (#2401), HSP27 (#2402), and GAPDH (#2118), all from Cell Signaling Technology, and horseradish peroxidase-conjugated anti-mouse and anti-rabbit, from GE Healthcare Biosciences; plasmids, wild type and constitutive active MAP2K4 (#14615 and #14813; Addgene) and GFP (Vivid Colors pcDNA 6.2/N-EmGFP-GW/TOPO, Invitrogen); chemical inhibitors, p38 MAPK inhibitor, SB203580 (Sigma-Aldrich) and associated negative control, SB202474, and JNK Inhibitor II (SP600125), and JNK Inhibitor II Negative Control (N 1 -Methyl-1,9-pyrazoloanthrone), all from EMD Millipore.

Techniques: Expressing, Western Blot, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Mitogen-Activated Protein Kinase Kinase 4 (MAP2K4) Promotes Human Prostate Cancer Metastasis

doi: 10.1371/journal.pone.0102289

Figure Lengend Snippet: A ) Knockdown of HSP27 protein by siRNA. After transfection of cells with siRNA targeting HSP27 (siHSP27) or non-targeting control (siCON), HSP27 expression was measured by Western blot. B ) Effect of HSP27 knockdown on cell invasion. MAP2K4 variant cell lines were treated with siRNA, as indicated, and cell invasion measured and depicted as in . Data are from four experiments, each in replicates of N = 2, and are expressed as the percentage of invading cells. C ) Knockdown of MMP-2 mRNA by siRNA. After transfection of cells with siRNA targeting MMP-2 (siMMP2) or non-targeting control (siCON), MMP-2 expression was measured by qRT/PCR. D ) Effect of MMP-2 knockdown on cell invasion. MAP2K4 variant cell lines were treated with siRNA, as indicated, and cell invasion measured and depicted as in . Data are from four experiments, each in replicates of N = 2, and are expressed as the percentage of invading cells.

Article Snippet: The following were purchased: antibodies, SEK1/MKK4 (#9152), phospho-p38 MAPK (T180/Y182) (#4631), p38 MAPK (#9212), phospho-SAPK/JNK (T183/Y185) (#4668), SAPK/JNK (#9258), phospho-HSP27 (#2401), HSP27 (#2402), and GAPDH (#2118), all from Cell Signaling Technology, and horseradish peroxidase-conjugated anti-mouse and anti-rabbit, from GE Healthcare Biosciences; plasmids, wild type and constitutive active MAP2K4 (#14615 and #14813; Addgene) and GFP (Vivid Colors pcDNA 6.2/N-EmGFP-GW/TOPO, Invitrogen); chemical inhibitors, p38 MAPK inhibitor, SB203580 (Sigma-Aldrich) and associated negative control, SB202474, and JNK Inhibitor II (SP600125), and JNK Inhibitor II Negative Control (N 1 -Methyl-1,9-pyrazoloanthrone), all from EMD Millipore.

Techniques: Knockdown, Transfection, Control, Expressing, Western Blot, Variant Assay, Quantitative RT-PCR

Journal: Journal of Cell Communication and Signaling

Article Title: Rsu1-dependent control of PTEN expression is regulated via ATF2 and cJun

doi: 10.1007/s12079-018-00504-4

Figure Lengend Snippet: The Rsu1 is necessary for activation of p38 Mapk and MKK4 but not MKK3/6 in response to EGF. (a, b) Cell lysates were isolated from control siRNA and Rsu1 siRNA transfected MCF10A cells following EGF treatment (100 ng/ml) for the indicated time. Immunoblot analysis was performed with equal amounts of protein samples as indicated in Materials and Methods and the levels of phospho-p38 (T180/Y182), phospho-MKK4 (S257 and T261), phospho-MKK3/6 (S189/S207), phospho-JNK (T183/Y185), were detected. Total p38, JNK, MKK4 and MKK3/6 were measured and α-tubulin and Rsu1 antibodies were used for loading controls and depletion levels, respectively. c MCF10A cells stably expressing control vector (pBabe), Rsu1-myc (Rsu1) or Rsu1-N92D-myc (Rsu1-N92D) were transfected with negative control siRNA or endogenous Rsu1 specific siRNA and treated with EGF (100 ng/ml) for 10 min as described in Materials and Methods. The lysates were subjected by western blot and analyzed with phosphorylation specific antibodies indicated. d Lysates from MCF10A cells depleted of MKK4, MKK3 or MKK6 were examined for levels of phospho-p38, phospho-JNK and phospho-Erk. Quantitation is expressed as fold stimulation compared to T = 0 for control siRNA transfected cells set as 1

Article Snippet: Rsu1 depletion in MCF10A cells was described previously (Gonzalez-Nieves et al. 2013 ). p38α (MapK14), p38β1 (MapK11), p38γ (MapK12), MKK3 (Map2K3), MKK6 (Map2K6), MKK4 (Map2K4), MEKK1 (Map3K1) and ASK1 (Map3K5) siRNA were purchased from Qiagen (Valencia, CA).

Techniques: Activation Assay, Isolation, Control, Transfection, Western Blot, Stable Transfection, Expressing, Plasmid Preparation, Negative Control, Phospho-proteomics, Quantitation Assay

Journal: Journal of Cell Communication and Signaling

Article Title: Rsu1-dependent control of PTEN expression is regulated via ATF2 and cJun

doi: 10.1007/s12079-018-00504-4

Figure Lengend Snippet: Rsu1-dependent changes in AKT signaling pathway. a MCF10A, T47D and MDA-MB-231 cells were infected with adenoviral vector encoding HA-tagged Rsu1 or empty vector. At 72 h post infection cell lysates were prepared and analyzed by western blot with phosphorylation specific antibodies, phospho-AKT (T308) and phospho-AKT (S473). Antibodies directed against total AKT, ILK, Rac1 and Rsu1were used to determine expression levels of those proteins. Quantitation shows the level of phosphorylated AKT normalized to total AKT and the level set in the control lane = 1. b MCF10A lysates were isolated from control siRNA and Rsu1 siRNA transfected MCF10A cells after EGF treatment for the indicated times. Immunoblot analysis was performed with equal amounts of protein samples and separated by SDS-PAGE for the levels of phospho-MKK4 (S80), phospho-AKT (p S473), total MKK4, AKT and PTEN. α-tubulin and Rsu1 antibodies were used for internal and depletion controls. c MCF10A RNA from control siRNA and Rsu1 siRNA transfected cells was used to determination level of PTEN RNA by quantitative real-time PCR. Quantitation is expressed as fold stimulation or relative amount compared to T = 0 for control siRNA transfected cells set as 1

Article Snippet: Rsu1 depletion in MCF10A cells was described previously (Gonzalez-Nieves et al. 2013 ). p38α (MapK14), p38β1 (MapK11), p38γ (MapK12), MKK3 (Map2K3), MKK6 (Map2K6), MKK4 (Map2K4), MEKK1 (Map3K1) and ASK1 (Map3K5) siRNA were purchased from Qiagen (Valencia, CA).

Techniques: Infection, Plasmid Preparation, Western Blot, Phospho-proteomics, Expressing, Quantitation Assay, Control, Isolation, Transfection, SDS Page, Real-time Polymerase Chain Reaction

Journal: Journal of Cell Communication and Signaling

Article Title: Rsu1-dependent control of PTEN expression is regulated via ATF2 and cJun

doi: 10.1007/s12079-018-00504-4

Figure Lengend Snippet: PTEN gene regulation by MKK4-p38 Mapk signaling and Rsu1-dependent expression of PTEN in MCF10A cells. a Determination of PTEN RNA levels was measured by quantitative real-time PCR following transfection with siRNAs for the following: Rsu1, p38α, MKK3, MKK4 and MKK6. The 18S RNA was used for normalization. Bar graphs show the fold change of PTEN expression quantified as the ratios to the control siRNA level. The data show means ± SD, n = 3. b RNA levels of PTEN were measured by quantitative real-time PCR following SB 203580 treatment of MCF10A cells (0, 5 or 20 μM). The 18S RNA was used for normalization. Bar graphs show the fold change of PTEN expression quantified as the ratios to the control, the means ± SD, n = 3. c Immunoblot analysis was performed with equal amounts of protein samples for the levels of phospho-AKT (S473) and phospho-p38 (T180/Y182), total AKT, p38, PTEN expression and α-tubulin was for internal controls

Article Snippet: Rsu1 depletion in MCF10A cells was described previously (Gonzalez-Nieves et al. 2013 ). p38α (MapK14), p38β1 (MapK11), p38γ (MapK12), MKK3 (Map2K3), MKK6 (Map2K6), MKK4 (Map2K4), MEKK1 (Map3K1) and ASK1 (Map3K5) siRNA were purchased from Qiagen (Valencia, CA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Transfection, Control, Western Blot

Journal: Journal of Cell Communication and Signaling

Article Title: Rsu1-dependent control of PTEN expression is regulated via ATF2 and cJun

doi: 10.1007/s12079-018-00504-4

Figure Lengend Snippet: Schematic model of the regulation of PTEN promoter in MCF10A cells by Rsu1 and IPP complex. Rsu1 plays a role in the regulation of PTEN gene transcription via MKK4-p38-ATF2 and MKK4-JNK-cJun. p38 MAPK can be constitutively activated by detachment or by blocking Rsu1-PINCH1 interaction and this is mediated by MKK3/6 signaling. However, EGF-induced p38 MAPK phosphorylation is dependent on Rsu1 and occurs through MKK4 and possibly MEKK1. In MCF10A cells, PTEN gene transcription is regulated through the ATF2/AP1 by the binding of AFT2 and cJun

Article Snippet: Rsu1 depletion in MCF10A cells was described previously (Gonzalez-Nieves et al. 2013 ). p38α (MapK14), p38β1 (MapK11), p38γ (MapK12), MKK3 (Map2K3), MKK6 (Map2K6), MKK4 (Map2K4), MEKK1 (Map3K1) and ASK1 (Map3K5) siRNA were purchased from Qiagen (Valencia, CA).

Techniques: Blocking Assay, Phospho-proteomics, Binding Assay