Journal: Oncogene

Article Title: PHLPP1 mediates melanoma metastasis suppression through repressing AKT2 activation

doi: 10.1038/s41388-017-0061-7

Figure Lengend Snippet: (A-E) Overexpression of activated AKT2 and AKT3 promotes metastasis in human A375p cells. Pan-AKT expression was analyzed by Western blot of A375p cells transfected with myr-AKT1, myr-AKT2 or myr-AKT3 (A). A375p cell transfected with the constructs described in (A) were analyzed by ELISA to determine phosphorylation of AKT1 (B), AKT2 (C) and AKT3 (D), and by experimental metastasis assay using tail vein injection to determine the gross pulmonary metastasis (E). c, empty vector control. Compared to vector control, total of Akt1, Akt2 and Akt3 were increased by 2.1, 1.7 and 1.9 fold respectively; phosphorylated Akt1, Akt2 and Akt3 were significantly enhanced by 2.9, 1.9 and 1.8 fold respectively. (F-J) Overexpression of activated AKT2 and AKT3 promotes metastasis in mouse B16F1 cells. Pan-AKT expression was analyzed by western blot of B16 cells transfected with myr-AKT1, myr-AKT2 or myr-AKT3 (F). B16 cells transfected with the constructs described in (F) were analyzed by ELISA to determine the phosphorylation of AKT1 (G), AKT2 (H) and AKT3 (I), and by experimental metastasis assay using tail vein injection to determine the gross pulmonary metastasis (J). c, empty vector control. Compared to vector control, total of Akt1, Akt2 and Akt3 were increased by 1.9, 2.5 and 2 fold respectively; phosphorylated Akt1, Akt2 and Akt3 were significantly enhanced by 1.6, 2 and 1.7 fold respectively. (K, L) Knockdown of AKT2 and AKT3 inhibits metastasis in human A375sm cells. A375sm cell transfected with shRNA for AKT1, AKT2 and AKT3 were analyzed by western blot with Akt isoform-specific anti-Akt1, Akt2 and Akt3 antibodies (K); gross pulmonary metastasis was determined by experimental metastasis assay using tail vein injection in NSG mice (L). shc, vector control; sh1 and sh2, different shRNAs of AKT1, AKT2 or AKT3. β-actin as an internal control. (M) A375sm cell cotransfected with CRISPR/cas9 and HDR plasmid for specifically knockout of AKT1, AKT2, and AKT3 were analyzed by western blot with Akt isoform-specific anti-AKT1, AKT2 and AKT3 antibodies. β-tubulin as an internal control. (N) Gross pulmonary metastasis was determined by experimental metastasis assay using tail vein injection of knockout of AKT1 (Akt1KO), AKT2 (Akt2KO) and AKT3 (Akt3KO) cells in NSG mice.

Article Snippet: Protein level of phospho-AKT 1 (Ser473), phospho-AKT 2(Ser474), phospho-AKT 3(Ser472), phospho-AKT (Thr308), total-AKT1, total-AKT2, total-AKT3, phospho-ERK 1/2 (Thr202/Tyr204) and total-ERK1/2 were measured by ELISA assays using PathScan® Phospho-AKT1 (Ser473), PathScan® Phospho -Akt2 (Ser474), PathScan® Phospho –Akt3 (Ser472), PathScan® Phospho -Akt (Thr308), PathScan® Total Akt1, PathScan® Total Akt2, PathScan® Total Akt3, PathScan® Phospho –Erk1/2 (Thr202/Tyr204) and PathScan® Total Erk1/2 Sandwich ELISA Kits purchased from Cell Signaling (Danvers, MA, USA).

Techniques: Over Expression, Expressing, Western Blot, Transfection, Construct, Enzyme-linked Immunosorbent Assay, Injection, Plasmid Preparation, shRNA, CRISPR, Knock-Out

Journal: Oncogene

Article Title: PHLPP1 mediates melanoma metastasis suppression through repressing AKT2 activation

doi: 10.1038/s41388-017-0061-7

Figure Lengend Snippet: (A-I) ELISA analysis of stable A375sm expressing PHLPP1, PHLPP1ΔC and PHLPP2 cells transfected with myr-Akt1, myr-AKT2 or myr-AKT3 plasmids was performed to determine protein levels of total Akt1 (A), total Akt2 (B) and total Akt3 (C), phosphorylated Akt1 (D), phosphorylated Akt2 (E), phosphorylated Akt3 (F), phosphorylated Akt-Thr-308 (G), phosphorylated Erk1/2 (H), total Erk1/2 (I); c, empty vector control; #, no statistical difference; *, p < 0.05; **, p < 0.01; ***, p < 0.001. (J) Gross pulmonary metastases of A375sm cells stably expressing PHLPP1, PHLPP1ΔC or PHLPP2 cells transfected with myr-AKT1, myr-AKT2 or myr-AKT3 constructs were determined using tail vein injection in SCID mice. c, empty vector control. (K) Representative histopathology with H&E staining of lung sections with metastases from mice inoculated with stable A375sm expressing PHLPP1, PHLPP1ΔC or PHLPP2 cells transfected with myr-AKT1, myr-AKT2 or myr-AKT3 constructs. c, empty vector control.

Article Snippet: Protein level of phospho-AKT 1 (Ser473), phospho-AKT 2(Ser474), phospho-AKT 3(Ser472), phospho-AKT (Thr308), total-AKT1, total-AKT2, total-AKT3, phospho-ERK 1/2 (Thr202/Tyr204) and total-ERK1/2 were measured by ELISA assays using PathScan® Phospho-AKT1 (Ser473), PathScan® Phospho -Akt2 (Ser474), PathScan® Phospho –Akt3 (Ser472), PathScan® Phospho -Akt (Thr308), PathScan® Total Akt1, PathScan® Total Akt2, PathScan® Total Akt3, PathScan® Phospho –Erk1/2 (Thr202/Tyr204) and PathScan® Total Erk1/2 Sandwich ELISA Kits purchased from Cell Signaling (Danvers, MA, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Transfection, Plasmid Preparation, Stable Transfection, Construct, Injection, Histopathology, Staining

Journal: Oncogene

Article Title: PHLPP1 mediates melanoma metastasis suppression through repressing AKT2 activation

doi: 10.1038/s41388-017-0061-7

Figure Lengend Snippet: (A) Whole cell lysates from A375sm cells and stable A375p-myr-AKT2 (A375p Akt2) cells treated with various doses of MK2206 were analyzed by western blot. (B-E) ELISA analysis of A375sm and stable A375p myr-AKT2 cells treated with different doses of MK2206 was performed to determine protein levels of phosphorylated AKT1 (B), phosphorylated AKT2 (C), phosphorylated AKT3 (D), and phosphorylated AKT-Thr-308 (E). #, no statistical difference; *, p < 0.05; **, p < 0.01; ***, p < 0.001. (F) Gross pulmonary metastases from mice treated with MK2206 or from pretreated cultured A375p Akt2 cells with either 12 nM or 65 nM MK2206 for 36 hours in cell culture (green color). Mice harboring A375p cells stably transfected with the myr-AKT2 expression vector (A375p Akt2) were treated with two different doses of MK2206 immediately (black color) or 7 days after cells injection (red color). After pretreatment with MK2206 for 36 hours, A375p Akt2 cells were injected into NSG mice by tail vein. c, mock control. (G-H) Tumor growth curve (G) and tumor weight (H) of A375sm cells in xenograft NSG mice with/without treatment of MK2206. (I) Representative histopathology (H&E staining) or immunohistochemical staining in metastases derived from mice bearing A375sm cells treated with MK2206. H&E, hematoxylin and eosin staining; t-Akt, immunohistochemical staining with the anti-AKT antibody; p-Akt, immunohistochemical staining with anti-p-AKT antibody 20X. (J) Immunoreactivity score of total AKT (T-Akt) and phosphorylated AKT (p-Akt) expression in metastases from mice treated with MK2206. Quantitative scores analyzed using ImageScope V10.0 software from Aperio Technologies, presenting the percentage of cells in strongly positive, positive, weakly positive and negative for immunohistochemical staining with an anti-AKT or phosphorylated AKT antibody. (K-L) Whole tissue lysates from tumors in mice treated with MK2206 were analyzed by ELISA to determine protein levels of phosphorylated AKT1 (p-Akt1), phosphorylated AKT2 (p-Akt2), phosphorylated AKT3 (p-Akt3), phosphorylated AKT-Thr-308 (p-AktThr308), and phosphorylated Erk1/2 (p-Erk1/2), total Erk1/2 (T-Erk1/2), total AKT (T-Akt1), total AKT2 (T-Akt2) and total AKT3 (T-Akt3).

Article Snippet: Protein level of phospho-AKT 1 (Ser473), phospho-AKT 2(Ser474), phospho-AKT 3(Ser472), phospho-AKT (Thr308), total-AKT1, total-AKT2, total-AKT3, phospho-ERK 1/2 (Thr202/Tyr204) and total-ERK1/2 were measured by ELISA assays using PathScan® Phospho-AKT1 (Ser473), PathScan® Phospho -Akt2 (Ser474), PathScan® Phospho –Akt3 (Ser472), PathScan® Phospho -Akt (Thr308), PathScan® Total Akt1, PathScan® Total Akt2, PathScan® Total Akt3, PathScan® Phospho –Erk1/2 (Thr202/Tyr204) and PathScan® Total Erk1/2 Sandwich ELISA Kits purchased from Cell Signaling (Danvers, MA, USA).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Cell Culture, Stable Transfection, Transfection, Expressing, Plasmid Preparation, Injection, Histopathology, Staining, Immunohistochemical staining, Derivative Assay, Software

Journal: Science Advances

Article Title: Development of a physiological insulin resistance model in human stem cell–derived adipocytes

doi: 10.1126/sciadv.abn7298

Figure Lengend Snippet: ( A ) Schematic indicating the experimental setup to potentiate insulin response in functional assays. hPSCs are differentiated into adipocytes using a published protocol, after which an additional step was added and optimized to sensitize adipocytes. ( B and C ) Phosphorylation of AKT2 normalized to total AKT2 (B) and glucose uptake (C) after 100 nM insulin stimulation measured for all the medium compositions from our DoE model and the medium from a previously published protocol. RLU, relative light unit. ( D and E ) The parameter coefficient of each factor in our DoE model indicating its contribution to the AKT2 phosphorylation (D) and glucose uptake (E). ( F ) Time course of sensitization with the DoE-optimized media measuring glucose uptake at baseline and after insulin stimulation. Results are normalized to total protein for each sample. All bar graphs depict the mean with error bars representing SD, n = 2 biological replicates. OD 450 , optical density at 450 nm.

Article Snippet: PathScan Phospho-Akt2 (Ser474) and Total Akt2 Sandwich ELISA kits were used to quantify AKT2 levels (Cell Signaling Technology, #7048 and #7046, respectively) as per the manufacturer’s instructions, by colorimetric reading at 450 nm on a Thermo Fisher Multiskan Go plate reader.

Techniques: Functional Assay

Journal: Science Advances

Article Title: Development of a physiological insulin resistance model in human stem cell–derived adipocytes

doi: 10.1126/sciadv.abn7298

Figure Lengend Snippet: ( A ) Schematic indicating the experimental setup to measure insulin dose-response and induction of insulin resistance. ( B ) Insulin dose-response curve showing fold change in AKT2 phosphorylation compared to the unstimulated state. ( C ) Representative images of GLUT4 translocation to the cell membrane upon insulin stimulation. ( D ) TIRF measurement of GLUT4 signal intensity at the adipocyte cell membrane (** P < 0.01, unpaired two-tailed t test). ( E ) Insulin dose-response curve showing AKT2 phosphorylation fold change in three insulin preexposure conditions. Results are normalized to total AKT2 and plotted as fold change to unstimulated cells in that condition. ( F ) Insulin dose-response curve showing glucose uptake for sensitized and hyperinsulinemia-exposed adipocytes. Results are normalized to total protein content and plotted as fold change to unstimulated cells. ( G ) Glycerol release into the medium for published, sensitized, and hyperinsulinemia-exposed adipocytes showing basal or insulin-suppressed lipolysis, normalized to total protein. Bar graphs depict the mean with error bars representing SD, dose-response curves depict a nonlinear fit curve with error bars representing SD, and the scatterplot depicts individual cell values with mean and 95% confidence interval (CI) overlaid; n = 3 biological replicates unless otherwise indicated.

Article Snippet: PathScan Phospho-Akt2 (Ser474) and Total Akt2 Sandwich ELISA kits were used to quantify AKT2 levels (Cell Signaling Technology, #7048 and #7046, respectively) as per the manufacturer’s instructions, by colorimetric reading at 450 nm on a Thermo Fisher Multiskan Go plate reader.

Techniques: Translocation Assay, Two Tailed Test

Journal: Science Advances

Article Title: Development of a physiological insulin resistance model in human stem cell–derived adipocytes

doi: 10.1126/sciadv.abn7298

Figure Lengend Snippet: ( A ) Phosphorylated AKT2 measurements as a fraction of total AKT2 normalized to total protein per well for brown hPSC adipocytes, mouse 3T3-L1 differentiated adipocytes, and primary human SVF adipocytes. ( B ) Glucose uptake measurements normalized to total protein per well and displayed as fold change in glucose uptake versus the unstimulated condition for brown hPSC adipocytes, mouse 3T3-L1 differentiated adipocytes, and primary human SVF adipocytes. All bar graphs depict the mean with error bars representing SD, n = 4 biological replicates.

Article Snippet: PathScan Phospho-Akt2 (Ser474) and Total Akt2 Sandwich ELISA kits were used to quantify AKT2 levels (Cell Signaling Technology, #7048 and #7046, respectively) as per the manufacturer’s instructions, by colorimetric reading at 450 nm on a Thermo Fisher Multiskan Go plate reader.

Techniques: