Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Insulin Resistance in Macrophages Alters Their Metabolism and Promotes an M2-Like Phenotype.

doi: 10.4049/jimmunol.1800065

Figure Lengend Snippet: FIGURE 1. Macrophages become insulin resistant and do not uptake glucose upon stimulation. (A) Western blot analysis of p-Akt (Ser473) in nonstimulated and after 30 min of stimulation with 100 nM insulin for control and macrophages from short-term HFD–fed mice. Summary graph of average phosphorylation of Akt in all conditions (basal and after insulin stimulation) normalized to total Akt. (B) Expression of IR in control and macrophages from short-term HFD–fed mice, measured by real-time PCR and Western blot. Akt2 macrophages are insulin resistant, as demonstrated by (C) Western blot analysis of Akt phosphorylation for WT (control) and Akt22/2 macrophages before and after insulin stimulation and (D) Akt2 phosphorylation p-Akt2 (Ser474) and (E) Akt1 phosphorylation p-Akt1 (Ser473) in macrophages from control and HFD-fed mice before and after 30 min of stimulation with 100 nM insulin. All graphs are representative of three to six independent experiments and show mean 6 SD. *p , 0.05, **p , 0.01, ***p , 0.001, insulin stimulation versus no stimulation. #p , 0.05, ##p , 0.01, ###p , 0.001, insulin-resistant versus control macrophages.

Article Snippet: Briefly, after blocking with 5% BSA PBS (pH 7.4) for an hour at room temperature, the membranes were incubated with rabbit polyclonal anti-mouse p-Akt (Ser473) Ab (Cell Signaling Technology), rabbit polyclonal anti-mouse Akt Ab (Cell Signaling Technology), rabbit polyclonal anti-mouse p-Akt2 (Ser474) Ab (Cell Signaling Technology), rabbit polyclonal anti-mouse Akt2 Ab (Cell Signaling Technology), rabbit polyclonal anti-mouse p-S6 (235/236) Ab (Cell Signaling Technology), rabbit polyclonal anti-mouse p–4E-BP1(Thr37/46) Ab (Cell Signaling Technology), rabbit polyclonal anti-mouse inducible NO synthase (iNOS) Ab (Abcam), goat polyclonal anti-mouse arginase 1 (Abcam), goat polyclonal anti-mouse Fizz1 Ab (Abcam), rabbit polyclonal anti-mouse IGF-1Rb Ab (Cell Signaling Technology), rabbit monoclonal anti-mouse IR-b (Cell Signaling Technology), or mouse monoclonal anti-mouse b-actin (Abcam) at 4 ̊C overnight.

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

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Insulin Resistance in Macrophages Alters Their Metabolism and Promotes an M2-Like Phenotype.

doi: 10.4049/jimmunol.1800065

Figure Lengend Snippet: FIGURE 2. mTORC1 pathway is more active in insulin-resistant macrophages. Western blot analysis of p-S6 (Ser235/236) and p–4E-BP1 (Thr37/46) in macrophages from Akt22/2 (A) and from short-term HFD–fed mice (B) compared with control before and after 30 min of stimulation with 100 nM insulin. Evaluation of Akt2 phosphorylation (C) and mTORC1 pathway activation (D) by Western blot analysis in control and Igf1R2/2 macrophages under basal conditions and after 30 min of stimulation with 100 nM insulin. (E) Western blot analysis of Akt phosphorylation in WT (control) and Igf1R2/2 macrophages. (F) Western blot analysis of Akt2 phosphorylation in control, HFD, Igf1R2/2, and Akt22/2 macrophages before and after 30 min of stimulation with 2.6 nM IGF1. (G) Akt1 is activated in insulin-resistant macrophages. Western blot analysis of p-Akt1 (Ser473) for control, HFD, Akt22/2 Igf1R2/2, and Akt12/2 macrophages under basal conditions. All graphs are representative of three to six independent experiments and show mean 6 SD. *p , 0.05, **p , 0.01, ***p , 0.001, insulin or IGF1 stimulation versus no stimulation. #p , 0.05, ##p , 0.01, ###p , 0.001, insulin-resistant versus control macrophages.

Article Snippet: Briefly, after blocking with 5% BSA PBS (pH 7.4) for an hour at room temperature, the membranes were incubated with rabbit polyclonal anti-mouse p-Akt (Ser473) Ab (Cell Signaling Technology), rabbit polyclonal anti-mouse Akt Ab (Cell Signaling Technology), rabbit polyclonal anti-mouse p-Akt2 (Ser474) Ab (Cell Signaling Technology), rabbit polyclonal anti-mouse Akt2 Ab (Cell Signaling Technology), rabbit polyclonal anti-mouse p-S6 (235/236) Ab (Cell Signaling Technology), rabbit polyclonal anti-mouse p–4E-BP1(Thr37/46) Ab (Cell Signaling Technology), rabbit polyclonal anti-mouse inducible NO synthase (iNOS) Ab (Abcam), goat polyclonal anti-mouse arginase 1 (Abcam), goat polyclonal anti-mouse Fizz1 Ab (Abcam), rabbit polyclonal anti-mouse IGF-1Rb Ab (Cell Signaling Technology), rabbit monoclonal anti-mouse IR-b (Cell Signaling Technology), or mouse monoclonal anti-mouse b-actin (Abcam) at 4 ̊C overnight.

Techniques: Western Blot, Control, Phospho-proteomics, Activation Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Insulin Resistance in Macrophages Alters Their Metabolism and Promotes an M2-Like Phenotype.

doi: 10.4049/jimmunol.1800065

Figure Lengend Snippet: FIGURE 3. Insulin-resistant macrophages show reduced M1 responses. (A) Western blot analysis of p-Akt2 (Ser474) in naive and stimulated with LPS (100 ng/ml) for 6 h, WT (control), and insulin-resistant macrophages. (B and C) Levels of IL-6 and TNF-a secreted in the supernatant of cultures of TEPMs and alveolar macrophages (AMs). (D) mRNA expression levels of IL-12 and iNOS of WT (control) and insulin-resistant macrophages before (nonstimulated) and after 6 h stimulation with LPS. (E) Expression levels of iNOS analyzed by Western blot in control and insulin-resistant macrophages before and after 6 h stimulation with LPS. (F) ROS production was evaluated by flow cytometry for control and insulin-resistant TEPMs 6 h after LPS stimulation. Graphs are representative of three to six independent experiments and show mean 6 SD. Box shows 5th to 95th percentiles, horizontal line represents median, and whiskers represent minimum and maximum. *p , 0.05, **p , 0.01, ***p , 0.001, LPS-stimulated versus nonstimulated macrophages. #p , 0.05, ##p , 0.01, ###p , 0.001, insulin-resistant versus control macrophages. MFI, mean fluorescence intensity of TEPMs.

Article Snippet: Briefly, after blocking with 5% BSA PBS (pH 7.4) for an hour at room temperature, the membranes were incubated with rabbit polyclonal anti-mouse p-Akt (Ser473) Ab (Cell Signaling Technology), rabbit polyclonal anti-mouse Akt Ab (Cell Signaling Technology), rabbit polyclonal anti-mouse p-Akt2 (Ser474) Ab (Cell Signaling Technology), rabbit polyclonal anti-mouse Akt2 Ab (Cell Signaling Technology), rabbit polyclonal anti-mouse p-S6 (235/236) Ab (Cell Signaling Technology), rabbit polyclonal anti-mouse p–4E-BP1(Thr37/46) Ab (Cell Signaling Technology), rabbit polyclonal anti-mouse inducible NO synthase (iNOS) Ab (Abcam), goat polyclonal anti-mouse arginase 1 (Abcam), goat polyclonal anti-mouse Fizz1 Ab (Abcam), rabbit polyclonal anti-mouse IGF-1Rb Ab (Cell Signaling Technology), rabbit monoclonal anti-mouse IR-b (Cell Signaling Technology), or mouse monoclonal anti-mouse b-actin (Abcam) at 4 ̊C overnight.

Techniques: Western Blot, Control, Expressing, Cytometry

Journal: Cancer biology & therapy

Article Title: PTEN suppression of YY1 induces HIF-2 activity in von-Hippel-Lindau-null renal-cell carcinoma.

doi: 10.4161/cbt.8.14.8880

Figure Lengend Snippet: Figure 6. Akt regulates YY1 protein expression. (A) The 786-0 cells were treated with either vehicle (DMSO) alone, the PI3K inihibitor, LY294002 [10 μM], the Akt inhibitor, triciribine [20 μM, 40 μM], or the mTOR inhibitor, rapamycin [20 nM] in serum-free conditions over- night. Whole cell lysates were purified and immunoblotted for P-Akt (ser473), YY1 and HIF- 2α. Actin was used as a loading control. (B) The 786-0 cells were transfected with 50 nM of control siRNA or siRNAs specifically targeting both Akt1 and Akt2 isoforms. Whole cell lysates were harvested 24 h later and immunoblotted for total Akt, YY1, HIF-2α and actin as a loading control. The 786-0 cells were co-transfected with the HRE-luc and either treated with 20 μM triciribine (C) or co-transfected with 50 nM of control or Akt1, 2 siRNAs (D). Transfectants were harvested after 24 h and analyzed for luciferase activity. Results represent three independent experiments of triplicate samples. Columns, mean; bars, SD; p < 0.0001 (***). (E) 786-0 cells were transfected with 50 nM of control or Akt2 specific siRNAs for 24 h. Whole cells lysates were then purified and analyzed by immunoblotting for total Akt2, YY1, HIF-2α and actin as a loading control.

Article Snippet: For Akt siRNA experiments, 786-0 cells were transiently transfected with 50 nM of either non-targeting control siRNA (Cell Signaling, 6201), SignalSilence Akt siRNA (Cell Signaling, 6211), or SignalSilence Akt2 siRNA 1400 Cancer Biology & Therapy 2009; Vol.

Techniques: Expressing, Purification, Control, Transfection, Luciferase, Activity Assay, Western Blot

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, Phospho-proteomics, Injection, Plasmid Preparation, Control, Knockdown, 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, Control, 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, Control, Histopathology, Staining, Immunohistochemical staining, Derivative Assay, Software