Journal: PLoS Genetics

Article Title: mTOR Directs Breast Morphogenesis through the PKC-alpha-Rac1 Signaling Axis

doi: 10.1371/journal.pgen.1005291

Figure Lengend Snippet: A-D. Rictor FL/FL PMECs and organoids were infected with the indicated adenoviruses. A-B. Western analysis of PMEC lysates. Quantitation was performed using Image J software and numbers represent P-PKC-alpha bands normalized to total PKC-alpha levels. C . Organoids photographed after 10 days in Matrigel culture. Average number of branches/organoid ±S.D. indicated below panels. N = 3 independent organoid isolates/condition analyzed in triplicate, Student’s T-test. D. Average colony size of organoids measured in pixel area ± S.D. N = 3 independent organoid isolates/condition, analyzed in triplicate, Student’s T-test . E. Western analysis of MCF10A parental and Rictor ZFN lysates. Quantitation was performed using Image J software and numbers represent P-PKC-alpha band normalized to actin for each sample. F-I. MCF10A parental and Rictor ZFN cells were infected with Ad.RFP or Ad.PKC-alpha. F. Western analysis of MCF10A parental and Rictor ZFN lysates, 48 hours post-infection. Quantitation was performed using Image J software and numbers represent P-PKC-alpha band normalized to actin for each sample. G. GST-Pak-PBD effector pull-downs followed by western analysis for Rac performed 48 hours post-infection. Quantitation was performed using Image J software. Numbers in upper panel represent Rac-GTP levels in MCF10A-Rictor ZFN compared to MCF10A parental control. Lower numbers represent Rac-GTP levels normalized to total Rac for each sample. H. Cells were assessed for invasion through Matrigel-coated transwell filters. Cells were stained with crystal violet after 24 hours, and then imaged. Number of cells invading was quantitated in Image J. Values shown represent the average ± S.D., Student’s T-test. N = 2 independent experiments, analyzed in triplicate. I. Cells were assessed for Annexin V-FITC. Number of Annexin V-FITC+ per total number of cells was quantitated in Image J, Student’s T-test. N = 2 independent experiments, analyzed in triplicate. J. MCF10A parental cells were assessed for invasion through Matrigel-coated transwell filters in the presence of PKC-alpha inhibitor GO6976. Cells were stained with crystal violet after 24 hours, and then imaged. Number of cells invading was quantitated in Image J. Values shown represent the average ± S.D., Student’s T-test. N = 2 independent experiments, analyzed in quadruplicate.

Article Snippet: PKC-alpha inhibitor GO6976 (Sigma-Aldrich, 2 nm) and adenoviral particles (Ad.RFP and Ad.PKC-alpha, Vector Biolabs) were purchased.

Techniques: Infection, Western Blot, Quantitation Assay, Software, Control, Staining

Journal: PLoS Genetics

Article Title: mTOR Directs Breast Morphogenesis through the PKC-alpha-Rac1 Signaling Axis

doi: 10.1371/journal.pgen.1005291

Figure Lengend Snippet: A. In situ detection of GTP-bound Rac via IF detection of GST-PBD (red; nuclei stained with DAPI, blue) on mammary gland sections from 6-week old mice. N = 6 independent fields analyzed in sections from 3 independent mammary gland sections/genotype. B . Quantitation of average percent red fluorescence (Rac-GTP) relative to blue (DAPI) on mammary gland sections from 6 week old virgin Rictor WT and Rictor MGKO mice, ± S.D., Student’s T-test. C. Cells were probed with GST-PBD (red) and counterstained with DAPI (blue). GST alone (not conjugated to PBD) was used as a negative control. GST-PBD+ fraction of total PMECs was counted and average ± S.D. shown. N = 15 fields/condition, Student’s T-test. D-I Rictor FL/FL PMECs infected with Ad.Cre or Ad.LacZ (±Ad.caRac1) and analyzed. D. Western analysis of PMEC lysates. E. Cells were probed with GST-PBD (green) and counterstained with phalloidin (red). F. Transwell invasion assays were performed. Invading cells were visualized with crystal violet and counted ±S.D. N = 6 independent isolates, analyzed in duplicate, Student’s T-test. G. PMECs were assessed by TUNEL analysis. N = 3 independent cell isolates, analyzed in duplicate. Representative images shown. Average percent TUNEL+ nuclei per total epithelial nuclei quantified. Midline values indicate average, whiskers indicate S.D., Student’s T-test. H. Rictor FL/FL organoids were infected Ad.Cre or Ad.LacZ (±Ad.caRac1) prior to embedding, and were photographed after 10 days in Matrigel culture. Right panel shows average number of branches per organoid. Each data point is the average branches per colony from individual isolates, analyzed in triplicate. Midlines are the average of all isolates, whiskers indicate S.D., Student’s T-test. N = 6. I. WT organoids were cultured ±Rac inhibitor for 10 days. Number of branches per organoid quantified. Each data point is the average branches per colony from individual isolates, analyzed in triplicate. Midline values indicate average, whiskers indicate S.D., Student’s T-test. Student’s T-test. Right panel shows average organoid size ±S.D. J-K. PMECs from Rictor FL/FL mice were tranduced with control Ad.GFP versus Ad.Cre in the presence or absence of Ad.PKC-alpha, Ad.caRac, or Ad.Akt myr . PMECs were transplanted into the cleared inguinal fat pads of 4 week old recipient female mice. Mammary glands were harvested six weeks post-transplantation and epithelial architecture and branching morphogenesis in whole-mount preparations was assessed. J. Whole-mount preparations from indicated transplanted glands. K. Quantitation of average number of branches ± S.D., Student’s T-test. Representative images shown from N = 2 independent experiments.

Article Snippet: PKC-alpha inhibitor GO6976 (Sigma-Aldrich, 2 nm) and adenoviral particles (Ad.RFP and Ad.PKC-alpha, Vector Biolabs) were purchased.

Techniques: In Situ, Staining, Quantitation Assay, Fluorescence, Negative Control, Infection, Western Blot, TUNEL Assay, Cell Culture, Control, Transplantation Assay

Journal: PLoS Genetics

Article Title: mTOR Directs Breast Morphogenesis through the PKC-alpha-Rac1 Signaling Axis

doi: 10.1371/journal.pgen.1005291

Figure Lengend Snippet: A-G. WT PMECs and organoids were cultured in DMSO vehicle or rapamycin. PMECs were cultured 0, 0.5, or 24 hours. Organoids were cultured 10 days in Matrigel in the presence of DMSO or rapamycin. A . Western analysis of PMEC lysates. Quantitation was performed using Image J software and numbers represent P-Akt, P-S6, or P-PKC-alpha bands normalized to total Akt, S6, or PKC-alpha levels. B . WT organoids photographed after 10 days in Matrigel culture. Average organoid size scored as average pixel area ± S.D., Student’s T-test, N = 6 epithelial isolates, each analyzed in triplicate. C . TUNEL analysis of PMECs. Average percent TUNEL+ nuclei per total PMEC nuclei ± S.D. is shown, Student’s T-test. N = 3 independent cell isolates, analyzed in duplicate. D. PMECs were labeled with BrdU after initial pre-treatments with rapamycin for 0.5 hours or 24 hours. Average percent BrdU+ nuclei per total nuclei ± S.D. is shown, One-way ANOVA, N = 3 independent cell isolates, analyzed in triplicate. E-F. Organoids were infected with Ad.LacZ or Ad.PKC-alpha (panel E) or Ad.caRac1 (panel F) prior to embedding in Matrigel plus rapamycin or DMSO. Average number of branches/colony is shown below each image. Average organoid size scored as average pixel area ± S.D., Student’s T-test (E) and one way ANOVA (F), N = 6 epithelial isolates, each analyzed in triplicate. G. Confluent PMEC monolayers were scratch-wounded, cultured in rapamycin or DMSO, and imaged at 24 hours. Total wounded area remaining after 24 hours was measured. Values shown are the average wound area remaining ± S.D. N = 3 independent cell isolates, analyzed in triplicate.

Article Snippet: PKC-alpha inhibitor GO6976 (Sigma-Aldrich, 2 nm) and adenoviral particles (Ad.RFP and Ad.PKC-alpha, Vector Biolabs) were purchased.

Techniques: Cell Culture, Western Blot, Quantitation Assay, Software, TUNEL Assay, Labeling, Infection

Journal: Oncogene

Article Title: Proteomic analysis reveals a role for PAX8 in peritoneal colonization of high grade serous ovarian cancer that can be targeted with micelle encapsulated thiostrepton

doi: 10.1038/s41388-019-0842-2

Figure Lengend Snippet: (A) Representative immunoblot of OVCAR8 RFP and OVCAR8 RFP PAX8 −/− clone 1 and clone 2 demonstrates PAX8 deletion reduces PKCα levels. (B) Wound closure assay performed with OVCAR8 RFP and OVCAR8 RFP PAX8 −/− clone 1 and clone 2 exposed to DMSO (control) or 5nM Go6976 (experimental) inhibitor against PKCα. (C) Wound closure assay performed over 24 hours with OVCAR8 RFP and OVCAR8 RFP PAX8 −/− clone 1 and clone 2 transfected with neomycin vector (control) or PKCαCAT vector (experimental) containing the PKCα catalytic site. Error bars for all assays represent standard errors for three replicates (n=3, error bars = SEM).

Article Snippet: Transient transfection of OVCAR8 RFP cell lines was performed with either 2.5μg of CMV-PAX8 vector (Transomic, Huntsville, AL, Catalog No. TCM1204) or 2.5μg of PKCαCAT vector (generated by Dr. Bernard Weinstein, Addgene plasmid #21234).

Techniques: Western Blot, Wound Closure Assay, Control, Transfection, Plasmid Preparation

Journal: International journal of oncology

Article Title: Identification of a GαGβγ, AKT and PKCα signalome associated with invasive growth in two genetic models of human breast cancer cell epithelial-to-mesenchymal transition.

doi: 10.3892/ijo.2012.1457

Figure Lengend Snippet: Figure 3. Invasive phenotype induced by MCF-7 cell EMT and oncogenic pathways downstream of GαGβγ subunits. (A), EMT cells were tested for their ability to invade collagen type I gels for 24 h in the presence or absence of chemical inhibitors of GPR signaling (BIM-46187: 1 µM, PTx: 0.1 µg/ml, gallein: 5 µM), PKA (KT5720, 1 µM), PLC (U73122, 1 µM), PKCα/βI (Gö6976, 40 nM and MT477, 1 µM) and PI3K (wortmannin, 1 nM). The pan inhibitor of MMPs 1-2-7 and -14 marimastat was tested at 10 µM. Data are means ± SEM of 3 experiments. ***P<0.005 or better. (B), Immunoblot analysis of total and phosphorylated forms of PKCs (isoforms α, βII, δ) and AKT in parental, control and EMT cells. Total protein loading was 40 µg and α-tubulin was used as a loading control. The PKCα and AKT phosphorylation levels were assessed using relative densitometric units (RDU) according to the densitometry of the phosphorylated form signals (RDU) versus the corresponding total PKCα and AKT signals (control = 1). (C), Quantitative RT-PCR analysis of the transcripts encoding PKCα. (D), Relative density of the Rac-GTP and Rho-GTP immunoreactive bands was quantified, according to total Rac and Rho levels detected by direct immunoblotting (arbitrary units). Data are means ± SEM of 3 experiments performed in duplicate (qRT-PCR and invasion assays) or are representative of two other experiments (immunoblots).

Article Snippet: Human PKCα sh-RNA expression vectors (pGFP-V-RS, OriGene, Rockville, MD) were used for stable silencing of PRKCA (PKCα) in MCF-7-SNAIL-6SA by Lipofectamine.

Techniques: Western Blot, Control, Phospho-proteomics, Quantitative RT-PCR

Journal: International journal of oncology

Article Title: Identification of a GαGβγ, AKT and PKCα signalome associated with invasive growth in two genetic models of human breast cancer cell epithelial-to-mesenchymal transition.

doi: 10.3892/ijo.2012.1457

Figure Lengend Snippet: Figure 6. Functional consequences of PRKCA silencing on the invasive phenotype induced by MCF-7 cell EMT. (A), Left: Expression and phosphorylation levels of PKCα in Snail-6SA transformed cells and their corresponding PRKCA (PKCα) silenced counterparts. These clonal derivative cells were compared to parental SNAIL-6SA cells by immunoblot analysis to confirm their reduced levels of total and phosphorylated PKCα forms. Right: Negative impact of PKCα silencing on the invasive phenotype of SNAIL-6SA transformed MCF-7 cells in EMT (top). Dose-effect of the PKCα inhibitor MT477 on the invasive potential of SNAIL-6SA cells transfected by the control empty vector pGFP-V-RS (bottom). Control vector and sh-Scr cells, PKCα silenced cells, and SNAIL-6SA cells were tested for their ability to invade collagen type I gels for 24 h. Data are means ± SEM of 3 experiments (invasion assays) or are representative of one other experiment (immunoblots). (B), Phase-contrast microscopy of control sh-Scr and PKCα silenced cells (clones C5 and C8) cultured for 24 h in the collagen type I invasion assay. Scale bar, 100 µm. Significantly different at ***P<0.005. (C), MCF-7 SNAIL-WT cells were compared to SNAIL-6SA cells (sh-Scr and silenced sh-PKCα MCF-7 cells, Clone 5) for their ability to invade collagen type I gels in the 14-day assays. Data collected from each experimental condition are recorded from 10 microscopic fields. Significantly different at **P<0.01 or better.

Article Snippet: Human PKCα sh-RNA expression vectors (pGFP-V-RS, OriGene, Rockville, MD) were used for stable silencing of PRKCA (PKCα) in MCF-7-SNAIL-6SA by Lipofectamine.

Techniques: Functional Assay, Expressing, Phospho-proteomics, Transformation Assay, Western Blot, Transfection, Control, Plasmid Preparation, Microscopy, Clone Assay, Cell Culture, Invasion Assay

Journal: International journal of oncology

Article Title: Identification of a GαGβγ, AKT and PKCα signalome associated with invasive growth in two genetic models of human breast cancer cell epithelial-to-mesenchymal transition.

doi: 10.3892/ijo.2012.1457

Figure Lengend Snippet: Figure 7. Molecular signature of the EMT induced by Snail activation and WISP-2 silencing in MCF-7 cells. This tentative signalome take into account the consequences of the EMT on: a) cancer cell proliferation and invasion; b) implication of signaling networks using heterotrimeric G-proteins Gα/Gβγ, c) PKCα- p27kip1-CDK3 cascade targeting the c-Jun/AP1 oncogenic pathway; d) the PI3K/Rac/AKT cascade; e) the independence of the EMT-associated invasive phenotype versus MAPK/SAPK, HER family pathways and other critical operators including the loss of ERα, PRA/PRB, HER and GPR30 signaling addition as molecular criteria of triple-negative breast cancers. This signalome is fully valid in two separate genetic models of MCF-7 induced to EMT and TN phenotypes. Such a simi larity and convergence is well explained by the common implication of several genetic protagonists, including the expression of Slug, Twist and Zeb1/2 and WISP-2 depletion in the two models. This coincidence led us to anticipate the functional and clinical significance of these signaling elements in breast cancer patients.

Article Snippet: Human PKCα sh-RNA expression vectors (pGFP-V-RS, OriGene, Rockville, MD) were used for stable silencing of PRKCA (PKCα) in MCF-7-SNAIL-6SA by Lipofectamine.

Techniques: Activation Assay, Expressing, Functional Assay

Journal: PLoS ONE

Article Title: Functional Role of mTORC2 versus Integrin-Linked Kinase in Mediating Ser473-Akt Phosphorylation in PTEN-Negative Prostate and Breast Cancer Cell Lines

doi: 10.1371/journal.pone.0067149

Figure Lengend Snippet: (A) Western blot analysis of the effect of si/shRNA-mediated knockdown of ILK, rictor, MK2, DNA-PK, ATM, PKCα, PKCβII, PAK1, or PAK2 on the expression of individual target proteins and the phosphorylation of Akt at Ser473 versus Thr308. (B) Effects of siRNA-mediated ILK knockdown, alone or in combination with Ku-0063794 (0.4 µM, 24 h), on Akt phosphorylation at Ser473 versus Thr308. (C) Dose-dependent effect of ectopic expression of ILK, alone (left panel) or in combination with Ku-0063794 (0.4 µM, 24 h), on the phosphorylation of Ser473-Akt and GSK3β. All immunoblots are representative of three independent experiments.

Article Snippet: Antibodies against various target proteins were purchased from the following commercial sources: Akt, p- 473 S-Akt, p- 308 T-Akt, GSK-3β, p- 9 S-GSK-3β, ILK, p70S6K, p- 389 T-p70S6K, mTOR, rictor, raptor, ATM, DNA-PK, PAK1, PAK2 (Cell Signaling Technology, Inc., Danvers, MA); MK2, PKCα, PKCβII (Santa Cruz Biotechnology, Inc, Santa Cruz, CA); β-actin (MP Biomedicals, Irvine, CA). shRNA for ILK in the pLKO.1 vector was purchased from Sigma-Aldrich.

Techniques: Western Blot, shRNA, Knockdown, Expressing, Phospho-proteomics

Journal: PLoS ONE

Article Title: Functional Role of mTORC2 versus Integrin-Linked Kinase in Mediating Ser473-Akt Phosphorylation in PTEN-Negative Prostate and Breast Cancer Cell Lines

doi: 10.1371/journal.pone.0067149

Figure Lengend Snippet: (A) Western blot analysis of the effect of si/shRNA-mediated knockdown of ILK, rictor, MK2, DNA-PK, ATM, PKCα, PKCβII, PAK1, or PAK2 on the expression of individual target proteins and the phosphorylation of Akt at Ser473 versus Thr308. (B) Effects of shRNA-mediated rictor knockdown, alone or in combination with T315 (2 µM, 24 h), on Akt phosphorylation at Ser473 versus Thr308. (C) Western blot analysis of the effect of shRNA-mediated repression of ILK (left) or rictor (right) on the phosphorylation of Akt at Ser473 versus Thr308, GSK3β (ILK target) and p70S6K (mTOR target) in MDA-MB-468 cells. For induction of ILK shRNA, cells stably transfected with a lentiviral vector encoding tetracycline-inducible ILK shRNA (TRE-ILKi) were exposed to 2 µg/ml doxycycline for 5 days. All immunoblots are representative of three independent experiments.

Article Snippet: Antibodies against various target proteins were purchased from the following commercial sources: Akt, p- 473 S-Akt, p- 308 T-Akt, GSK-3β, p- 9 S-GSK-3β, ILK, p70S6K, p- 389 T-p70S6K, mTOR, rictor, raptor, ATM, DNA-PK, PAK1, PAK2 (Cell Signaling Technology, Inc., Danvers, MA); MK2, PKCα, PKCβII (Santa Cruz Biotechnology, Inc, Santa Cruz, CA); β-actin (MP Biomedicals, Irvine, CA). shRNA for ILK in the pLKO.1 vector was purchased from Sigma-Aldrich.

Techniques: Western Blot, shRNA, Knockdown, Expressing, Phospho-proteomics, Stable Transfection, Transfection, Plasmid Preparation