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  • 95
    Selleck Chemicals mk2206
    Analysis of the selected senescence-associated genes in ULM cells induced by replication and <t>MK2206</t> induced senescence. (A) Venn diagrams demonstrate the number of unique and overlapping genes between the two groups (P2 vs. P0 in blue, and MK2206 vs. DMSO in yellow). Four genes shared by serial passaging and MK2206 treatment were SGIP1, SLITRK4, FAM108C1 and WIPI1. (B, C) Western blots of WIPI1 and SLITRK4 expression in replication senescent ULM cells (B, confirmed by upregulation of P21) and MK2206 induced senescence (C, confirmed by loss of pAKT). AKT and β-actin were used as loading control. The band density (right) was quantified (n=3) and expressed as the means ± SD. (D) SA-β-gal staining was performed after overexpression of WIPI1 or SLITRK4 in 2D and 3D primary ULM cells. ULM spheroids (n=5) are shown and color intensity was quantified with Image J for WIPI1 and SLITRK4. *p
    Mk2206, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 95/100, based on 875 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 95 stars, based on 875 article reviews
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    99
    Selleck Chemicals mk2206 2hcl
    Cell morphology by Coomassie blue staining (×400). A. Blank control group; B. TGF-β 1 group; C. TGF-β 1 inhibitor group (SB525334); D. AKT inhibitor group <t>(MK2206-2HCL);</t> E. Combined inhibitors group (SB525334+MK2206-2HCL).
    Mk2206 2hcl, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 99/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 22 article reviews
    Price from $9.99 to $1999.99
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    93
    BioVision mk2206
    Src/Akt/Erk signaling was constitutively activated and functionally involved in anoikis resistance in ID8-P1 cells A. pSrc, pAkt, pErk levels in ID8-P0 and ID8-P1 cells under different culture conditions by Western blot analyses. B. Survival of ID8-P1 cells under suspended conditions treated with Src siRNA (100 µM) and selective inhibitors: PP2 (10 µM), <t>MK2206</t> (1 µM), PD98059 (30 µM). C. Quantification of soft agar colony numbers of ID8-P1 cells treated with inhibitors. D. Number of floating ID8-P1 cells in mouse peritoneal cavities treated with the Src selective inhibitor PP2 (daily i.p. injection at a dose of 2 mg/kg, n=3). E–G . Ectopic expression of CA-Src in ID8-P0 increased anoikis resistance. E. pSrc levels in ID8-P0 and ID8-P0-CA-Src cells by Western blot analyses. F. Anoikis assays of ID8-P0 and ID8-P0-CA-Src cells in SF medium. G. Colony formation of ID8-P0 and ID8-P0-CA-Src cells in soft agar. H. Numbers of floating ID8-P0 and ID8-P0-CA-Src cells in mouse peritoneal cavities. * P
    Mk2206, supplied by BioVision, used in various techniques. Bioz Stars score: 93/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 93 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
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    92
    ApexBio mk 2206
    Exosomal miR-205 regulates the PTEN-AKT signalling pathway in HUVECs. ( A ) Interference efficiency was measured by western blotting. ( B ) Western blotting analysis of PTEN, p-AKT, AKT, and α-tubulin in HUVECs incubated with NC-Exos, miR-205-Exos, miR-205 inhibitor, NC and mimic groups. ( C ) Western blotting was used to detect PTEN, p-AKT and AKT expression in HUVECs after treatment with miR-205-Exos, miR-205-Exos+miR-205 inhibitor, NC, miR-205 mimic and miR-205 mimic+PTEN. ( D, E ) Effects of miR-205 inhibitor, NC, mimic, PTEN+inhibitor, PTEN, PTEN+mimic on HUVEC tube formation ability and migration were assessed by tube formation and Transwell migration assays. ( F, G ) Effects of miR-205 inhibitor, NC, mimic, siPTEN+inhibitor, siPTEN, siPTEN+mimic on HUVEC tube formation ability and migration were examined by tube formation and transwell migration assays. The scale bar in (D, F) represents 100 µm. The scale bar in (E, G) represents 50 µm. ( H ) Effects of GDC-0941 and <t>MK-2206</t> on tube formation and migration abilities induced by exosomal miR-205 in HUVECs. The scale bar in the upper panel represents 100 µm. The scale bar in the bottom panel represents 50 µm. ( I ) Western blot analysis shows the effects of GDC-0941 and MK-2206. All experiments were repeated three times, and the results are presented as the mean ± SEM. Statistical significance was determined by a two-tailed, unpaired Student's t test. ** P
    Mk 2206, supplied by ApexBio, used in various techniques. Bioz Stars score: 92/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Price from $9.99 to $1999.99
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    93
    Cayman Chemical mk 2206
    CXCL16 induction in L. donovani -infected macrophages is AKT/mTOR dependent but TLR independent. (A and B) WT, MyD88 KO, and UNC93B KO BMDM cultures were inoculated with L. donovani , treated with 250 ng/ml LPG, 100 ng/ml LPS, or 250 ng/ml poly(I:C), or left uninfected/untreated for 24 h. CXCL16 protein expression was assessed by Western blotting. β-Actin was used as a loading control. Parasite infection was monitored by probing for Leishmania IMPDH. Induction of COX-2 served as a surrogate for disrupted TLR signaling in MyD88 KO cells. (C) BMDM cultures were inoculated with L. donovani WT, lpg1 -KO, or lpg1 -KO+ LPG1 for 24 h or left uninfected, and the activity of AKT/mTOR signaling was monitored by Western blotting using phospho-specific and total antibodies against AKT and rpS6, respectively. CXCL16 expression and efficacy of infection were examined as for panel A. (D) BMDM cultures were pretreated with 2 μM <t>MK-2206,</t> 200 nM Torin-1, or an equal volume of dimethyl sulfoxide (DMSO) (vehicle) for 1 h and then infected with L. donovani promastigotes for 24 h. The phosphorylation status and total levels of AKT and rpS6 were assessed as for panel C. Results are representative of at least two independent biological replicates.
    Mk 2206, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Price from $9.99 to $1999.99
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    Image Search Results


    Analysis of the selected senescence-associated genes in ULM cells induced by replication and MK2206 induced senescence. (A) Venn diagrams demonstrate the number of unique and overlapping genes between the two groups (P2 vs. P0 in blue, and MK2206 vs. DMSO in yellow). Four genes shared by serial passaging and MK2206 treatment were SGIP1, SLITRK4, FAM108C1 and WIPI1. (B, C) Western blots of WIPI1 and SLITRK4 expression in replication senescent ULM cells (B, confirmed by upregulation of P21) and MK2206 induced senescence (C, confirmed by loss of pAKT). AKT and β-actin were used as loading control. The band density (right) was quantified (n=3) and expressed as the means ± SD. (D) SA-β-gal staining was performed after overexpression of WIPI1 or SLITRK4 in 2D and 3D primary ULM cells. ULM spheroids (n=5) are shown and color intensity was quantified with Image J for WIPI1 and SLITRK4. *p

    Journal: Laboratory investigation; a journal of technical methods and pathology

    Article Title: Application of ex-vivo spheroid model system for the analysis of senescence and senolytic phenotypes in uterine leiomyoma

    doi: 10.1038/s41374-018-0117-5

    Figure Lengend Snippet: Analysis of the selected senescence-associated genes in ULM cells induced by replication and MK2206 induced senescence. (A) Venn diagrams demonstrate the number of unique and overlapping genes between the two groups (P2 vs. P0 in blue, and MK2206 vs. DMSO in yellow). Four genes shared by serial passaging and MK2206 treatment were SGIP1, SLITRK4, FAM108C1 and WIPI1. (B, C) Western blots of WIPI1 and SLITRK4 expression in replication senescent ULM cells (B, confirmed by upregulation of P21) and MK2206 induced senescence (C, confirmed by loss of pAKT). AKT and β-actin were used as loading control. The band density (right) was quantified (n=3) and expressed as the means ± SD. (D) SA-β-gal staining was performed after overexpression of WIPI1 or SLITRK4 in 2D and 3D primary ULM cells. ULM spheroids (n=5) are shown and color intensity was quantified with Image J for WIPI1 and SLITRK4. *p

    Article Snippet: After two additional days, spheroids were treated with MK2206 (2–5μM, Selleck Chemicals), ABT-263 (2μM, AbbVie Inc, North Chicago, IL) or paraquat (PQ, Sigma-Aldrich) for 24 to 72 hours at the indicated concentrations.

    Techniques: Passaging, Western Blot, Expressing, Staining, Over Expression

    Molecular and cellular analysis of MK2206 induced senescence in primary ULM cells. (A) Immunohistochemistry analysis for pAKT in ULM spheroids (left) showed significant loss of pAKT after MK2206 treatment (right). (B) Primary ULM cells were treated with 5μM MK2206 or the vehicle (DMSO) and cells from 2D and 3D were stained for SA-β-gal and the dark blue (2D) and green (3D) stain were indicative of senescent cells. (C) Immunohistochemistry revealed increase of p16 and p21 expression in MK2206 treated ULM spheroids and a total of 8 spheroids were scored (right). (D) Western blot analysis for pAKT, total AKT, P21 and β-actin. Densitometric analysis was done (n=4). (E) Differentially expressed genes in MK2206 vs DMSO treated ULM were plotted in heatmap and Dendragram where upregulated genes are red and downregulated genes are blue. (F) GO analysis showed four highly upregulated functional pathways in ULM treated by MK2206. *p

    Journal: Laboratory investigation; a journal of technical methods and pathology

    Article Title: Application of ex-vivo spheroid model system for the analysis of senescence and senolytic phenotypes in uterine leiomyoma

    doi: 10.1038/s41374-018-0117-5

    Figure Lengend Snippet: Molecular and cellular analysis of MK2206 induced senescence in primary ULM cells. (A) Immunohistochemistry analysis for pAKT in ULM spheroids (left) showed significant loss of pAKT after MK2206 treatment (right). (B) Primary ULM cells were treated with 5μM MK2206 or the vehicle (DMSO) and cells from 2D and 3D were stained for SA-β-gal and the dark blue (2D) and green (3D) stain were indicative of senescent cells. (C) Immunohistochemistry revealed increase of p16 and p21 expression in MK2206 treated ULM spheroids and a total of 8 spheroids were scored (right). (D) Western blot analysis for pAKT, total AKT, P21 and β-actin. Densitometric analysis was done (n=4). (E) Differentially expressed genes in MK2206 vs DMSO treated ULM were plotted in heatmap and Dendragram where upregulated genes are red and downregulated genes are blue. (F) GO analysis showed four highly upregulated functional pathways in ULM treated by MK2206. *p

    Article Snippet: After two additional days, spheroids were treated with MK2206 (2–5μM, Selleck Chemicals), ABT-263 (2μM, AbbVie Inc, North Chicago, IL) or paraquat (PQ, Sigma-Aldrich) for 24 to 72 hours at the indicated concentrations.

    Techniques: Immunohistochemistry, Staining, Expressing, Western Blot, Functional Assay

    Depletion of senescent cells by ABT263, a senolytic reagent, in primary ULM spheroids. (A) Primary ULM cells were serially passaged (P2), prepared in 3D spheroids and treated with ABT263. Top panels showed cell death to form a rim of dissociated cells around spheroids. Bottom panels showed apoptosis cells in spheroids prepared in formalin-fixed and paraffin-embedded section. (B) P2 ULM spheroids were treated with DMSO and ABT263 and senescence were examined by SA-β-gal stain. Intensity of blue color represented the level of senescence. (C) ULM spheroids were treated with DMSO, ABT263, MK2206 and MK2206+ABT263 and examined by SA-β-gal stain and the level of senescence were measured based on the intensity of green color (right panel, n=3). (D) Senescent rate were measure in primary ULM spheroids from day 1 to day 3 based on intensity of SA-β-gal stain. (E). Cell viability analysis in 2D culture in primary ULM cells of control (DMSO), ABT263, MK2206 and MK2206+ABT263. Cell counts were measured and illustrated on right panel with the means ± rem. *p

    Journal: Laboratory investigation; a journal of technical methods and pathology

    Article Title: Application of ex-vivo spheroid model system for the analysis of senescence and senolytic phenotypes in uterine leiomyoma

    doi: 10.1038/s41374-018-0117-5

    Figure Lengend Snippet: Depletion of senescent cells by ABT263, a senolytic reagent, in primary ULM spheroids. (A) Primary ULM cells were serially passaged (P2), prepared in 3D spheroids and treated with ABT263. Top panels showed cell death to form a rim of dissociated cells around spheroids. Bottom panels showed apoptosis cells in spheroids prepared in formalin-fixed and paraffin-embedded section. (B) P2 ULM spheroids were treated with DMSO and ABT263 and senescence were examined by SA-β-gal stain. Intensity of blue color represented the level of senescence. (C) ULM spheroids were treated with DMSO, ABT263, MK2206 and MK2206+ABT263 and examined by SA-β-gal stain and the level of senescence were measured based on the intensity of green color (right panel, n=3). (D) Senescent rate were measure in primary ULM spheroids from day 1 to day 3 based on intensity of SA-β-gal stain. (E). Cell viability analysis in 2D culture in primary ULM cells of control (DMSO), ABT263, MK2206 and MK2206+ABT263. Cell counts were measured and illustrated on right panel with the means ± rem. *p

    Article Snippet: After two additional days, spheroids were treated with MK2206 (2–5μM, Selleck Chemicals), ABT-263 (2μM, AbbVie Inc, North Chicago, IL) or paraquat (PQ, Sigma-Aldrich) for 24 to 72 hours at the indicated concentrations.

    Techniques: Staining

    The BCR-ABL TKI imatinib and nilotinib or IL-10 inhibit phosphorylation of Akt in human moDC. Western blot analysis of total Akt levels and its phosphorylated form in purified immature CD209 + moDC (of four different donors). moDC were generated in vitro with GM-CSF and IL-4 alone (4/GM) or with additional (A) imatinib (3 μM), (B) nilotinib (3 μM), (C) Akt inhibitor MK2206 (Akt-inh., 300 nM) or (D) IL-10 (10 ng/mL). Indicated time refers to further treatment of cells prior to cell lysis (see Methods ). (E-G) GPNMB protein levels in moDC were analyzed by western blotting. GAPDH served as loading control. Exemplary results from at least three independent experiments using different donors are presented.

    Journal: Cell Communication and Signaling : CCS

    Article Title: The transcription factor MITF is a critical regulator of GPNMB expression in dendritic cells

    doi: 10.1186/s12964-015-0099-5

    Figure Lengend Snippet: The BCR-ABL TKI imatinib and nilotinib or IL-10 inhibit phosphorylation of Akt in human moDC. Western blot analysis of total Akt levels and its phosphorylated form in purified immature CD209 + moDC (of four different donors). moDC were generated in vitro with GM-CSF and IL-4 alone (4/GM) or with additional (A) imatinib (3 μM), (B) nilotinib (3 μM), (C) Akt inhibitor MK2206 (Akt-inh., 300 nM) or (D) IL-10 (10 ng/mL). Indicated time refers to further treatment of cells prior to cell lysis (see Methods ). (E-G) GPNMB protein levels in moDC were analyzed by western blotting. GAPDH served as loading control. Exemplary results from at least three independent experiments using different donors are presented.

    Article Snippet: Immature moDC were generated in vitro with GM-CSF and IL-4 alone (4/GM) or with additional Akt inhibitor MK2206 (300 nM) or Erk inhibitor FR180204 (300 nM) and analyzed for GPNMB mRNA expression by qRT-PCR.

    Techniques: Western Blot, Purification, Generated, In Vitro, Lysis

    Akt inhibition reduces the capacity of human moDC to induce T cell responses. moDC generated in vitro with GM-CSF and IL-4 alone (4/GM) or with imatinib (3 μM) or Akt inhibitor MK2206 (300 nM) were used as stimulators in MLR with allogeneic T cells. Increasing concentration (0.0 μg/mL - 20 μg/mL) of blocking soluble recombinant T cell ligand SD-4 were added with recombinant Klotho β serving as control. T cell proliferation was measured by [ 3 H]thymidine incorporation. CCPM = corrected counts per minute. The mean (±SD) of quadruple measurements is shown. Exemplary result from three independent experiments using different donors is presented.

    Journal: Cell Communication and Signaling : CCS

    Article Title: The transcription factor MITF is a critical regulator of GPNMB expression in dendritic cells

    doi: 10.1186/s12964-015-0099-5

    Figure Lengend Snippet: Akt inhibition reduces the capacity of human moDC to induce T cell responses. moDC generated in vitro with GM-CSF and IL-4 alone (4/GM) or with imatinib (3 μM) or Akt inhibitor MK2206 (300 nM) were used as stimulators in MLR with allogeneic T cells. Increasing concentration (0.0 μg/mL - 20 μg/mL) of blocking soluble recombinant T cell ligand SD-4 were added with recombinant Klotho β serving as control. T cell proliferation was measured by [ 3 H]thymidine incorporation. CCPM = corrected counts per minute. The mean (±SD) of quadruple measurements is shown. Exemplary result from three independent experiments using different donors is presented.

    Article Snippet: Immature moDC were generated in vitro with GM-CSF and IL-4 alone (4/GM) or with additional Akt inhibitor MK2206 (300 nM) or Erk inhibitor FR180204 (300 nM) and analyzed for GPNMB mRNA expression by qRT-PCR.

    Techniques: Inhibition, Generated, In Vitro, Concentration Assay, Blocking Assay, Recombinant

    Imatinib, nilotinib, IL-10 or Akt inhibitor prevent phosphorylation of GSK3ß in human moDC. Western blot analysis of total GSK3ß and GSK3α, as well as their phosphorylated forms in purified immature CD209 + moDC (of four different donors). moDC were generated in vitro with GM-CSF and IL-4 alone (4/GM) or with additional (A) imatinib (3 μM), (B) nilotinib (3 μM), (C) Akt inhibitor MK2206 (Akt-inh., 300 nM) or (D) IL-10 (10 ng/mL). Indicated time refers to further treatment of cells prior to cell lysis (see Methods ). GAPDH served as loading control. Exemplary results from at least three independent experiments using different donors are presented.

    Journal: Cell Communication and Signaling : CCS

    Article Title: The transcription factor MITF is a critical regulator of GPNMB expression in dendritic cells

    doi: 10.1186/s12964-015-0099-5

    Figure Lengend Snippet: Imatinib, nilotinib, IL-10 or Akt inhibitor prevent phosphorylation of GSK3ß in human moDC. Western blot analysis of total GSK3ß and GSK3α, as well as their phosphorylated forms in purified immature CD209 + moDC (of four different donors). moDC were generated in vitro with GM-CSF and IL-4 alone (4/GM) or with additional (A) imatinib (3 μM), (B) nilotinib (3 μM), (C) Akt inhibitor MK2206 (Akt-inh., 300 nM) or (D) IL-10 (10 ng/mL). Indicated time refers to further treatment of cells prior to cell lysis (see Methods ). GAPDH served as loading control. Exemplary results from at least three independent experiments using different donors are presented.

    Article Snippet: Immature moDC were generated in vitro with GM-CSF and IL-4 alone (4/GM) or with additional Akt inhibitor MK2206 (300 nM) or Erk inhibitor FR180204 (300 nM) and analyzed for GPNMB mRNA expression by qRT-PCR.

    Techniques: Western Blot, Purification, Generated, In Vitro, Lysis

    Upon treatment of moDC with imatinib, nilotinib, IL-10 or MK2206, MITF translocates into the nucleus. Western blot analysis of MITF level and phosphorylation status in the cytoplasmic or nuclear fraction of purified immature CD209 + moDC. moDC were generated in vitro with GM-CSF and IL-4 alone (4/GM) or with (A) imatinib (3 μM), (B) nilotinib (3 μM), (C) Akt inhibitor MK2206 (Akt-inh.; 300 nM) or (D) IL-10 (10 ng/mL). (E) Cells were treated with nilotinib (3 μM). Indicated time refers to further treatment of cells prior to cell lysis (see Methods ). GAPDH served as loading control. Exemplary results from at least three independent experiments using different donors are presented.

    Journal: Cell Communication and Signaling : CCS

    Article Title: The transcription factor MITF is a critical regulator of GPNMB expression in dendritic cells

    doi: 10.1186/s12964-015-0099-5

    Figure Lengend Snippet: Upon treatment of moDC with imatinib, nilotinib, IL-10 or MK2206, MITF translocates into the nucleus. Western blot analysis of MITF level and phosphorylation status in the cytoplasmic or nuclear fraction of purified immature CD209 + moDC. moDC were generated in vitro with GM-CSF and IL-4 alone (4/GM) or with (A) imatinib (3 μM), (B) nilotinib (3 μM), (C) Akt inhibitor MK2206 (Akt-inh.; 300 nM) or (D) IL-10 (10 ng/mL). (E) Cells were treated with nilotinib (3 μM). Indicated time refers to further treatment of cells prior to cell lysis (see Methods ). GAPDH served as loading control. Exemplary results from at least three independent experiments using different donors are presented.

    Article Snippet: Immature moDC were generated in vitro with GM-CSF and IL-4 alone (4/GM) or with additional Akt inhibitor MK2206 (300 nM) or Erk inhibitor FR180204 (300 nM) and analyzed for GPNMB mRNA expression by qRT-PCR.

    Techniques: Western Blot, Purification, Generated, In Vitro, Lysis

    PI3K/Akt-inhibition upregulates GPNMB gene expression in human moDC. Immature moDC were generated in vitro with GM-CSF and IL-4 alone (4/GM) or with additional TKI (3 μM imatinib or 3 μM nilotinib) or inhibitors of signal transduction (300 nM Akt inhibitor MK2206 (Akt-inh.), 300 nM Erk inhibitor FR180204 (Erk-inh.), 100 nM PI3K inhibitor LY294002 (PI3K-inh.), 20 nM c-Raf inhibitor 553003 (c-Raf-inh.)) and analyzed for GPNMB expression. Exemplary results from at least three independent experiments using different donors are presented. (A) qRT-PCR analysis: relative level of GPNMB mRNA. The mean (±SD) of duplicate measurements is shown. (B, C) GPNMB protein level of CD209 + moDC (of three different donors) was analyzed by flow cytometry. Where indicated, maturation of moDC was induced by LPS. Data were analyzed using FlowJo software and Difference in Median Fluorescence Intensity (DMFI) of CD209 + cells is shown in the upper right quadrants. (D) Phenotypic changes of immature moDC in the absence (4/GM) or presence of nilotinib or Akt inhibitor were analyzed by flow cytometry. Double stainings were performed with monoclonal antibodies recognizing CD209, CD1a or CD14. DMFI of CD209 + cells is shown in the upper right quadrants.

    Journal: Cell Communication and Signaling : CCS

    Article Title: The transcription factor MITF is a critical regulator of GPNMB expression in dendritic cells

    doi: 10.1186/s12964-015-0099-5

    Figure Lengend Snippet: PI3K/Akt-inhibition upregulates GPNMB gene expression in human moDC. Immature moDC were generated in vitro with GM-CSF and IL-4 alone (4/GM) or with additional TKI (3 μM imatinib or 3 μM nilotinib) or inhibitors of signal transduction (300 nM Akt inhibitor MK2206 (Akt-inh.), 300 nM Erk inhibitor FR180204 (Erk-inh.), 100 nM PI3K inhibitor LY294002 (PI3K-inh.), 20 nM c-Raf inhibitor 553003 (c-Raf-inh.)) and analyzed for GPNMB expression. Exemplary results from at least three independent experiments using different donors are presented. (A) qRT-PCR analysis: relative level of GPNMB mRNA. The mean (±SD) of duplicate measurements is shown. (B, C) GPNMB protein level of CD209 + moDC (of three different donors) was analyzed by flow cytometry. Where indicated, maturation of moDC was induced by LPS. Data were analyzed using FlowJo software and Difference in Median Fluorescence Intensity (DMFI) of CD209 + cells is shown in the upper right quadrants. (D) Phenotypic changes of immature moDC in the absence (4/GM) or presence of nilotinib or Akt inhibitor were analyzed by flow cytometry. Double stainings were performed with monoclonal antibodies recognizing CD209, CD1a or CD14. DMFI of CD209 + cells is shown in the upper right quadrants.

    Article Snippet: Immature moDC were generated in vitro with GM-CSF and IL-4 alone (4/GM) or with additional Akt inhibitor MK2206 (300 nM) or Erk inhibitor FR180204 (300 nM) and analyzed for GPNMB mRNA expression by qRT-PCR.

    Techniques: Inhibition, Expressing, Generated, In Vitro, Transduction, Quantitative RT-PCR, Flow Cytometry, Cytometry, Software, Fluorescence

    SAV1 is necessary for MERTK inhibition-mediated suppression of Akt activation in RCC cells. a , b IB analysis of WCL derived from HEK293 cells transfected with Flag-SAV1 and HA-Akt1. Where indicated, cells were treated with indicated doses of MK2206 ( a ) or GDC0068 ( b ) for 10 h before cell collection. c , d Cell viability assays were performed with WT- and SAV1-depleted RCC4 cells treated with either indicated doses of MK2206 ( c ) or GDC0068 ( d ) for 3 days (mean ± SD, n = 3). 1000 cells were plated in each well on 96-well plates. * P

    Journal: Nature Communications

    Article Title: MERTK mediated novel site Akt phosphorylation alleviates SAV1 suppression

    doi: 10.1038/s41467-019-09233-7

    Figure Lengend Snippet: SAV1 is necessary for MERTK inhibition-mediated suppression of Akt activation in RCC cells. a , b IB analysis of WCL derived from HEK293 cells transfected with Flag-SAV1 and HA-Akt1. Where indicated, cells were treated with indicated doses of MK2206 ( a ) or GDC0068 ( b ) for 10 h before cell collection. c , d Cell viability assays were performed with WT- and SAV1-depleted RCC4 cells treated with either indicated doses of MK2206 ( c ) or GDC0068 ( d ) for 3 days (mean ± SD, n = 3). 1000 cells were plated in each well on 96-well plates. * P

    Article Snippet: Akt kinase inhibitors MK2206 (Selleck S1078) and GDC0068 (Selleck S2808), PI3Kα inhibitor BKM120 (Selleck S2247), mTOR inhibitor Torin 2 (Selleck S2817), PI3Kγ inhibitor IPI549 (Selleck S8330), c-Met inhibitor cabozantinib (Selleck S1119), Syc inhibitor dasatinib (Selleck S1021), VEGFR inhibitor pazopanib (Selleck S3012), MERTK inhibitors UNC2025 and UNC4241 (produced by UNC drug discovery unit) were used at the indicated dose(s).

    Techniques: Inhibition, Activation Assay, Derivative Assay, Transfection

    OMT induced autophagy in SW982 cells through the HMGB1/Akt/mTOR pathway. ( a ) Cells were treated with 2 mM OMT for the indicated times, and the protein expression of Akt, p-Akt, mTOR, p-mTOR and HMGB1 was detected by western blotting. ( b ) After SW982 cells were untreated or pretreated with 5 μM MK-2206 2HCL, an Akt inhibitor, for 2 h, they were transfected with HMGB1 3#siRNA or control siRNA, followed by 2 mM OMT treatment for another 48 h. Western blotting was used to determine the levels of LC3, Akt and p-Akt expression. The cells were untreated or pretreated with 100 nM Rapa for 2 h and then transfected with HMGB1 3#siRNA or control siRNA, followed by 2 mM OMT treatment for another 48 h. The expression of LC3, mTOR and p-mTOR was determined via western blotting ( c ). Loading control was performed by evaluating β-actin expression in the same filter. *p

    Journal: Scientific Reports

    Article Title: HMGB1-mediated autophagy decreases sensitivity to oxymatrine in SW982 human synovial sarcoma cells

    doi: 10.1038/srep37845

    Figure Lengend Snippet: OMT induced autophagy in SW982 cells through the HMGB1/Akt/mTOR pathway. ( a ) Cells were treated with 2 mM OMT for the indicated times, and the protein expression of Akt, p-Akt, mTOR, p-mTOR and HMGB1 was detected by western blotting. ( b ) After SW982 cells were untreated or pretreated with 5 μM MK-2206 2HCL, an Akt inhibitor, for 2 h, they were transfected with HMGB1 3#siRNA or control siRNA, followed by 2 mM OMT treatment for another 48 h. Western blotting was used to determine the levels of LC3, Akt and p-Akt expression. The cells were untreated or pretreated with 100 nM Rapa for 2 h and then transfected with HMGB1 3#siRNA or control siRNA, followed by 2 mM OMT treatment for another 48 h. The expression of LC3, mTOR and p-mTOR was determined via western blotting ( c ). Loading control was performed by evaluating β-actin expression in the same filter. *p

    Article Snippet: Anti-β-actin antibodies were purchased from Biosen (Beijing, China), E64d, pepstatin A and MK-2206 2HCL were obtained from Selleck (Shanghai, China).

    Techniques: Expressing, Western Blot, Transfection

    The Akt’s activation state impacts the phosphorylation of the HRR-associated Akt-target protein MERIT40. ( A ) TrC1 stably expressing the constitutively active Akt1-E17K, phosphorylation-deficient Akt1-TASA or Akt1-WT were exposed to irradiation with 5 Gy. Akt1-WT expressing TrC1 were additionally treated with 4 µM of MK-2206 2 h prior to IR. Phosphorylation status (S473) of the Akt1 mutants, as well as the expression and phosphorylation status of the assumed Akt-target protein MERIT40, at 0.5 h after irradiation depicted by western blot analysis. For S473 and Akt: lower bands (60 kDa) show endogenous Akt and upper bands (87 kDa) depict eGFP-fused Akt1-mutants. ( B ) The quantification of pMERIT40 western blots of 3 independent experiments shows volume intensity normalized to the background. Volume intensity of phosphorylated Akt was normalized to the volume intensity of the total amount of Akt. Bars represent means ± SD from 3 independent experiments. * p

    Journal: International Journal of Molecular Sciences

    Article Title: Restraining Akt1 Phosphorylation Attenuates the Repair of Radiation-Induced DNA Double-Strand Breaks and Reduces the Survival of Irradiated Cancer Cells

    doi: 10.3390/ijms19082233

    Figure Lengend Snippet: The Akt’s activation state impacts the phosphorylation of the HRR-associated Akt-target protein MERIT40. ( A ) TrC1 stably expressing the constitutively active Akt1-E17K, phosphorylation-deficient Akt1-TASA or Akt1-WT were exposed to irradiation with 5 Gy. Akt1-WT expressing TrC1 were additionally treated with 4 µM of MK-2206 2 h prior to IR. Phosphorylation status (S473) of the Akt1 mutants, as well as the expression and phosphorylation status of the assumed Akt-target protein MERIT40, at 0.5 h after irradiation depicted by western blot analysis. For S473 and Akt: lower bands (60 kDa) show endogenous Akt and upper bands (87 kDa) depict eGFP-fused Akt1-mutants. ( B ) The quantification of pMERIT40 western blots of 3 independent experiments shows volume intensity normalized to the background. Volume intensity of phosphorylated Akt was normalized to the volume intensity of the total amount of Akt. Bars represent means ± SD from 3 independent experiments. * p

    Article Snippet: Pretreatment with the inhibitor MK-2206 (purchased from Selleckchem, Houston, TX, USA) diluted in culture medium was performed at indicated times before irradiation.

    Techniques: Activation Assay, Stable Transfection, Expressing, Irradiation, Western Blot

    Expression of phosphorylation-deficient Akt1 mutants reduced cancer cell radiosensitivity. TrC1 were exposed to irradiation with 5 Gy. ( A ) The phosphorylation status (S473, T308) of the Akt1 mutants at 0.5 h after irradiation depicted by western blot analysis. Lower bands (60 kDa) show endogenous Akt; upper bands (87 kDa) depict eGFP-fused Akt1-mutants. ( B ) The quantification of pS473 and pT308 western blots of 3 independent experiments shows the volume intensity normalized to the background. The volume intensity of phosphorylated Akt was normalized to the volume intensity of total amount of Akt. ( C , D ) Long-term survival (survival fraction, SF) altered by Akt1 mutants upon IR (0–10 Gy). Akt1-TASA showed significantly reduced survival upon IR. Pictures depict a standard 6-well cell culture plate. ( E ) Long-term survival in Akt1-WT expressing cells treated with 4 µM MK-2206 for 16 h before IR (WT + MK) compared to the effect evoked by Akt1-WT and Akt1-TASA expression without additional treatment. Data represent SF upon 8 Gy. * p

    Journal: International Journal of Molecular Sciences

    Article Title: Restraining Akt1 Phosphorylation Attenuates the Repair of Radiation-Induced DNA Double-Strand Breaks and Reduces the Survival of Irradiated Cancer Cells

    doi: 10.3390/ijms19082233

    Figure Lengend Snippet: Expression of phosphorylation-deficient Akt1 mutants reduced cancer cell radiosensitivity. TrC1 were exposed to irradiation with 5 Gy. ( A ) The phosphorylation status (S473, T308) of the Akt1 mutants at 0.5 h after irradiation depicted by western blot analysis. Lower bands (60 kDa) show endogenous Akt; upper bands (87 kDa) depict eGFP-fused Akt1-mutants. ( B ) The quantification of pS473 and pT308 western blots of 3 independent experiments shows the volume intensity normalized to the background. The volume intensity of phosphorylated Akt was normalized to the volume intensity of total amount of Akt. ( C , D ) Long-term survival (survival fraction, SF) altered by Akt1 mutants upon IR (0–10 Gy). Akt1-TASA showed significantly reduced survival upon IR. Pictures depict a standard 6-well cell culture plate. ( E ) Long-term survival in Akt1-WT expressing cells treated with 4 µM MK-2206 for 16 h before IR (WT + MK) compared to the effect evoked by Akt1-WT and Akt1-TASA expression without additional treatment. Data represent SF upon 8 Gy. * p

    Article Snippet: Pretreatment with the inhibitor MK-2206 (purchased from Selleckchem, Houston, TX, USA) diluted in culture medium was performed at indicated times before irradiation.

    Techniques: Expressing, Irradiation, Western Blot, Cell Culture

    The genetic or pharmacologic inhibition of Akt1-phosphorylation affects DNA repair upon IR. TrC1 stably overexpressing Akt1-WT, pre-treated for 2 h with 0 or 4 µM MK-2206, or the phosphorylation-deficient Akt1-TA, -SA or -TASA mutants were exposed to irradiation with 0 Gy, 3 Gy ( A,B ) or 40 Gy ( C,D ) as indicated. ( A , B ) Cells were fixed in 3% para-formaldehyde (PFA), permeabilized with 0.2% Triton X-100 in phosphate-buffered saline (PBS) at distinct time points between 0 h and 24 h upon irradiation with 3 Gy, and stained with Hoechst33342, to visualize the nuclei (blue), and γH2A.X (magenta), to visualize sites of DNA DSB. ( A ) The number of γH2A.X foci at 2–24 h after irradiation with 3 Gy using the Focinator v 2.2. software [ 20 ] was normalized to the number of foci detected at 0.5 h time point. ( C , D ) Cells were processed by applying neutral comet assay to quantify the amount of damaged DNA in the form of DSB at a fixed time-point. The quantification was performed by the OpenComet software and depicts the comet tail length of each indicated Akt1 mutant 4 h upon 40 Gy. ( A , C ) Data show means ± SD from 3 independent experiments with 50 analyzed nuclei per trial and condition. * p

    Journal: International Journal of Molecular Sciences

    Article Title: Restraining Akt1 Phosphorylation Attenuates the Repair of Radiation-Induced DNA Double-Strand Breaks and Reduces the Survival of Irradiated Cancer Cells

    doi: 10.3390/ijms19082233

    Figure Lengend Snippet: The genetic or pharmacologic inhibition of Akt1-phosphorylation affects DNA repair upon IR. TrC1 stably overexpressing Akt1-WT, pre-treated for 2 h with 0 or 4 µM MK-2206, or the phosphorylation-deficient Akt1-TA, -SA or -TASA mutants were exposed to irradiation with 0 Gy, 3 Gy ( A,B ) or 40 Gy ( C,D ) as indicated. ( A , B ) Cells were fixed in 3% para-formaldehyde (PFA), permeabilized with 0.2% Triton X-100 in phosphate-buffered saline (PBS) at distinct time points between 0 h and 24 h upon irradiation with 3 Gy, and stained with Hoechst33342, to visualize the nuclei (blue), and γH2A.X (magenta), to visualize sites of DNA DSB. ( A ) The number of γH2A.X foci at 2–24 h after irradiation with 3 Gy using the Focinator v 2.2. software [ 20 ] was normalized to the number of foci detected at 0.5 h time point. ( C , D ) Cells were processed by applying neutral comet assay to quantify the amount of damaged DNA in the form of DSB at a fixed time-point. The quantification was performed by the OpenComet software and depicts the comet tail length of each indicated Akt1 mutant 4 h upon 40 Gy. ( A , C ) Data show means ± SD from 3 independent experiments with 50 analyzed nuclei per trial and condition. * p

    Article Snippet: Pretreatment with the inhibitor MK-2206 (purchased from Selleckchem, Houston, TX, USA) diluted in culture medium was performed at indicated times before irradiation.

    Techniques: Inhibition, Stable Transfection, Irradiation, Staining, Software, Neutral Comet Assay, Mutagenesis

    The basal Akt phosphorylation is not required for its nuclear localization . TrC1 Akt1-TASA, Akt1-WT and Akt1-WT expressing cells pretreated with solvent or 4 µM MK-2206 (2 h before IR) were exposed to IR with 0 Gy or 5 Gy. 30 min after the irradiation cells were fixed in 3% paraformaldehyde, permeabilized with 0.2% Triton X-100 in phosphate-buffered saline (PBS), and subjected to immunofluorescence analysis. ( A ) Representative photomicrographs showing subcellular localization of the eGFP-coupled Akt1 variants Akt1-TASA and Akt1-WT upon the indicated treatments. The overview pictures were done using 63-fold magnification. Detailed pictures were obtained using 63-fold magnification. ( B ) Quantification of the integrated eGFP-intensity in nuclei of TrC1 expressing Akt1-TASA and Akt1-WT mutants with and without pre-treatment with the MK-2206 inhibitor. Quantification involves the analysis of 50 cells per condition and was performed by a CellProfiler software [ 19 ]. Data show means ± SD from 3 independent experiments; ANOVA test with Tukey correction and showed no significant differences.

    Journal: International Journal of Molecular Sciences

    Article Title: Restraining Akt1 Phosphorylation Attenuates the Repair of Radiation-Induced DNA Double-Strand Breaks and Reduces the Survival of Irradiated Cancer Cells

    doi: 10.3390/ijms19082233

    Figure Lengend Snippet: The basal Akt phosphorylation is not required for its nuclear localization . TrC1 Akt1-TASA, Akt1-WT and Akt1-WT expressing cells pretreated with solvent or 4 µM MK-2206 (2 h before IR) were exposed to IR with 0 Gy or 5 Gy. 30 min after the irradiation cells were fixed in 3% paraformaldehyde, permeabilized with 0.2% Triton X-100 in phosphate-buffered saline (PBS), and subjected to immunofluorescence analysis. ( A ) Representative photomicrographs showing subcellular localization of the eGFP-coupled Akt1 variants Akt1-TASA and Akt1-WT upon the indicated treatments. The overview pictures were done using 63-fold magnification. Detailed pictures were obtained using 63-fold magnification. ( B ) Quantification of the integrated eGFP-intensity in nuclei of TrC1 expressing Akt1-TASA and Akt1-WT mutants with and without pre-treatment with the MK-2206 inhibitor. Quantification involves the analysis of 50 cells per condition and was performed by a CellProfiler software [ 19 ]. Data show means ± SD from 3 independent experiments; ANOVA test with Tukey correction and showed no significant differences.

    Article Snippet: Pretreatment with the inhibitor MK-2206 (purchased from Selleckchem, Houston, TX, USA) diluted in culture medium was performed at indicated times before irradiation.

    Techniques: Expressing, Irradiation, Immunofluorescence, Software

    GRAMD1B inhibition increases Akt signaling. ( a ) Inhibitory effects of GRAMD1B on PI3K/Akt signaling. Gramd1b . ( b ) The morphology changes of Gramd1b knockdown cells are negated in the presence of the pan-Akt inhibitor MK-2206. (Scale bar = 20 μm). ( c -c’) MK-2206 treatment suppresses the GRAMD1B inhibition-enhanced cell migration. (Scale bar = 100 μm). Data is represented as mean ± SEM of n = 3. ( d ) Treatment of cells with MK-2206 significantly inhibits the expression of Rac1 and Cdc42 induced by Gramd1b knockdown. Data is represented as mean ± SEM of n = 3. * P

    Journal: Scientific Reports

    Article Title: GRAMD1B regulates cell migration in breast cancer cells through JAK/STAT and Akt signaling

    doi: 10.1038/s41598-018-27864-6

    Figure Lengend Snippet: GRAMD1B inhibition increases Akt signaling. ( a ) Inhibitory effects of GRAMD1B on PI3K/Akt signaling. Gramd1b . ( b ) The morphology changes of Gramd1b knockdown cells are negated in the presence of the pan-Akt inhibitor MK-2206. (Scale bar = 20 μm). ( c -c’) MK-2206 treatment suppresses the GRAMD1B inhibition-enhanced cell migration. (Scale bar = 100 μm). Data is represented as mean ± SEM of n = 3. ( d ) Treatment of cells with MK-2206 significantly inhibits the expression of Rac1 and Cdc42 induced by Gramd1b knockdown. Data is represented as mean ± SEM of n = 3. * P

    Article Snippet: Cells were treated with the JAK2 inhibitor AG490 (Sigma-Aldrich) and the Akt inhibitor MK-2206 (Selleck, USA) for 24 hours.

    Techniques: Inhibition, Migration, Expressing

    SC79-mediated RPE cytoprotection against UV requires Akt activation ARPE-19 cells were pre-treated with MK-2206 (5 μM) for 1h, followed by SC79 (5 μg/mL) treatment for 1 h, p-Akt (Ser-473) and Akt1 expression was tested by Western blot assay ( A ). ARPE-19 cells were pre-treated with MK-2206 (5 μM) for 1 h, followed by UV radiation (30 mJ/cm 2 ), or plus SC79 (5 μg/mL, 30 min prior UV), cells were further cultured for applied time; Cell viability (( B ) MTT assay) and cell apoptosis (( C ) Histone DNA ELISA assay) were tested. The stably ARPE-19 cells expressing scramble control shRNA (“Scr shRNA”) or Akt1 shRNA were treated with UV (30 mJ/cm 2 ) radiation, or plus SC79 (5 μg/mL, 30 min prior UV); Cells were further cultured for applied time, cell viability ( E ) and cell apoptosis ( F ) were tested. SC79 (5 μg/mL, 1 h)-induced Akt activation in above cells was tested by Western blot assay (D). Akt phosphorylation (vs. regular Akt1) was quantified (A), Akt1 expression (vs. Tubulin) was quantified ( D ). “dmso” stands for 0.1% DMSO vehicle control (B and C). For each assay, n = 5. Experiments in this figure were repeated three times to insure consistency of results. * p

    Journal: Oncotarget

    Article Title: SC79 protects retinal pigment epithelium cells from UV radiation via activating Akt-Nrf2 signaling

    doi: 10.18632/oncotarget.11164

    Figure Lengend Snippet: SC79-mediated RPE cytoprotection against UV requires Akt activation ARPE-19 cells were pre-treated with MK-2206 (5 μM) for 1h, followed by SC79 (5 μg/mL) treatment for 1 h, p-Akt (Ser-473) and Akt1 expression was tested by Western blot assay ( A ). ARPE-19 cells were pre-treated with MK-2206 (5 μM) for 1 h, followed by UV radiation (30 mJ/cm 2 ), or plus SC79 (5 μg/mL, 30 min prior UV), cells were further cultured for applied time; Cell viability (( B ) MTT assay) and cell apoptosis (( C ) Histone DNA ELISA assay) were tested. The stably ARPE-19 cells expressing scramble control shRNA (“Scr shRNA”) or Akt1 shRNA were treated with UV (30 mJ/cm 2 ) radiation, or plus SC79 (5 μg/mL, 30 min prior UV); Cells were further cultured for applied time, cell viability ( E ) and cell apoptosis ( F ) were tested. SC79 (5 μg/mL, 1 h)-induced Akt activation in above cells was tested by Western blot assay (D). Akt phosphorylation (vs. regular Akt1) was quantified (A), Akt1 expression (vs. Tubulin) was quantified ( D ). “dmso” stands for 0.1% DMSO vehicle control (B and C). For each assay, n = 5. Experiments in this figure were repeated three times to insure consistency of results. * p

    Article Snippet: Reagents, chemicals and antibodies MK-2206 (S1078) and SC79 (S7863) were from Selleck (Shanghai, China).

    Techniques: Activation Assay, Expressing, Western Blot, Cell Culture, MTT Assay, Enzyme-linked Immunosorbent Assay, Stable Transfection, shRNA

    SC79 activates Nrf2 signaling in RPE cells ARPE-19 cells were treated with applied concentration of SC79 (0.1–10 μg/mL) for 4 h, mRNA expression of HO-1, NQO-1 and Nrf2 was tested by RT-qPCR assay ( A ). Stably ARPE-19 cells with scramble-shRNA (“Scr shRNA”) or Akt1 shRNA were treated with SC79 (5 μg/mL) or plus MK-2206 (5 μM), HO-1 and NQO-1 mRNA expression was tested by RT-qPCR assay ( B and C ). ARPE-19 cells, pretreated with SC79 (5 μg/mL) for 30 min, were subjected to UV radiation (30 mJ/cm 2 ), cells were further cultured and relative ROS production was tested ( D ). The stably ARPE-19 cells with scramble-shRNA (“Scr shRNA”) or Nrf2 shRNA (“−1/−2”, with non-overlapping sequences) were treated with SC79 (5 μg/mL) for indicated time, expressions of listed proteins and mRNA were tested by Western blot assay ( E ) and RT-qPCR assay ( F and G ) respectively. Above cells were treated with UV (30 mJ/cm 2 ) radiation, or plus SC79 (5 μg/mL, 30 min prior UV), cells were further cultured for 24h and cell viability was evaluated by MTT assay ( H ). HO-1 protein expression (vs. Tubulin) was quantified (E). For each assay, n = 5. Experiments in this figure were repeated three times to insure consistency of results. * p

    Journal: Oncotarget

    Article Title: SC79 protects retinal pigment epithelium cells from UV radiation via activating Akt-Nrf2 signaling

    doi: 10.18632/oncotarget.11164

    Figure Lengend Snippet: SC79 activates Nrf2 signaling in RPE cells ARPE-19 cells were treated with applied concentration of SC79 (0.1–10 μg/mL) for 4 h, mRNA expression of HO-1, NQO-1 and Nrf2 was tested by RT-qPCR assay ( A ). Stably ARPE-19 cells with scramble-shRNA (“Scr shRNA”) or Akt1 shRNA were treated with SC79 (5 μg/mL) or plus MK-2206 (5 μM), HO-1 and NQO-1 mRNA expression was tested by RT-qPCR assay ( B and C ). ARPE-19 cells, pretreated with SC79 (5 μg/mL) for 30 min, were subjected to UV radiation (30 mJ/cm 2 ), cells were further cultured and relative ROS production was tested ( D ). The stably ARPE-19 cells with scramble-shRNA (“Scr shRNA”) or Nrf2 shRNA (“−1/−2”, with non-overlapping sequences) were treated with SC79 (5 μg/mL) for indicated time, expressions of listed proteins and mRNA were tested by Western blot assay ( E ) and RT-qPCR assay ( F and G ) respectively. Above cells were treated with UV (30 mJ/cm 2 ) radiation, or plus SC79 (5 μg/mL, 30 min prior UV), cells were further cultured for 24h and cell viability was evaluated by MTT assay ( H ). HO-1 protein expression (vs. Tubulin) was quantified (E). For each assay, n = 5. Experiments in this figure were repeated three times to insure consistency of results. * p

    Article Snippet: Reagents, chemicals and antibodies MK-2206 (S1078) and SC79 (S7863) were from Selleck (Shanghai, China).

    Techniques: Concentration Assay, Expressing, Quantitative RT-PCR, Stable Transfection, shRNA, Cell Culture, Western Blot, MTT Assay

    SC79 activates Nrf2 signaling in primary murine RPE cells and its retinal protection activity in vivo Primary murine RPE cells were treated with SC79 (5 μg/mL) for indicated time, expressions of listed mRNAs ( A ) and proteins ( B ) were tested. Primary murine RPE cells were treated with UV (30 mJ/cm 2 ) radiation, or plus SC79 (5 μg/mL, 30 min prior UV), cells were further cultured before ROS content was analyzed ( C ). Primary murine RPE cells were pre-treated with SC79 (5 μg/mL), or plus MK-2206 (“MK”, 5 μM)/ZnPP (10 μM), followed by UV (30 mJ/cm 2 ) radiation, cells were further cultured for 24 h before cell viability was tested ( D ). After the light exposure in mice retina, ERG was measured, quantified amplitudes of a- and b-waves were presented ( E and F ). For each assay, n = 5. * p

    Journal: Oncotarget

    Article Title: SC79 protects retinal pigment epithelium cells from UV radiation via activating Akt-Nrf2 signaling

    doi: 10.18632/oncotarget.11164

    Figure Lengend Snippet: SC79 activates Nrf2 signaling in primary murine RPE cells and its retinal protection activity in vivo Primary murine RPE cells were treated with SC79 (5 μg/mL) for indicated time, expressions of listed mRNAs ( A ) and proteins ( B ) were tested. Primary murine RPE cells were treated with UV (30 mJ/cm 2 ) radiation, or plus SC79 (5 μg/mL, 30 min prior UV), cells were further cultured before ROS content was analyzed ( C ). Primary murine RPE cells were pre-treated with SC79 (5 μg/mL), or plus MK-2206 (“MK”, 5 μM)/ZnPP (10 μM), followed by UV (30 mJ/cm 2 ) radiation, cells were further cultured for 24 h before cell viability was tested ( D ). After the light exposure in mice retina, ERG was measured, quantified amplitudes of a- and b-waves were presented ( E and F ). For each assay, n = 5. * p

    Article Snippet: Reagents, chemicals and antibodies MK-2206 (S1078) and SC79 (S7863) were from Selleck (Shanghai, China).

    Techniques: Activity Assay, In Vivo, Cell Culture, Mouse Assay

    Acarbose ameliorated EPC function and suppressed intracellular ROS levels via an Akt dependent pathway in vitro. Acarbose (1 μ M) and p-Akt inhibitor and MK-2206 (1 μ M) were added to the high glucose medium for 24 h. Measurement of tube formation (a), migration (b), and adhesion (c) capacity of BM-EPCs. Determination of intracellular NO level (d) and O 2 − level (e). ∗∗∗ P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Acarbose Accelerates Wound Healing via Akt/eNOS Signaling in db/db Mice

    doi: 10.1155/2017/7809581

    Figure Lengend Snippet: Acarbose ameliorated EPC function and suppressed intracellular ROS levels via an Akt dependent pathway in vitro. Acarbose (1 μ M) and p-Akt inhibitor and MK-2206 (1 μ M) were added to the high glucose medium for 24 h. Measurement of tube formation (a), migration (b), and adhesion (c) capacity of BM-EPCs. Determination of intracellular NO level (d) and O 2 − level (e). ∗∗∗ P

    Article Snippet: The Akt inhibitor MK-2206 2HCl was obtained from Selleck (Shanghai, China).

    Techniques: In Vitro, Migration

    The PI3K/Akt axis regulates BCG-induced IL-10 secretion in macrophages. Human TDMs and mice J774A.1 macrophages were mock-treated or infected with BCG at an MOI = 10 for 3 h, washed then treated or not with the PI3K inhibitor, Wortmannin (Wort, 100 nM), or with the Akt inhibitor, MK-2206 (5 μM) for 24 h. (A) Inactivation of PI3k/Akt axis was determined, 24 h post infection/treatment, in whole cell lysate of TDMs by western blotting using the indicated anti-phospho specific antibodies. Total Akt, FOXO3, and β-actin were used as loading controls. Shown are representative images of three independent experiments with similar results. (B) FOXO3 mRNA expression were quantified by qRT-PCR in both TDMs and J774A.1 macrophages. Levels of mRNA were normalized to GAPDH and fold induction was calculated relative to uninfected and untreated cells. (C) Assessment of IL-10 transcription and secretion levels, after the indicated treatments, were quantified by qRT-PCR and sandwich ELISA, respectively. For all panels, results are represented in box and whiskers plot format min to max and are representative of three independent experiments, each one carried out in triplicate. Asterisks indicate statistical significance (* p

    Journal: Frontiers in Immunology

    Article Title: FOXO3 Transcription Factor Regulates IL-10 Expression in Mycobacteria-Infected Macrophages, Tuning Their Polarization and the Subsequent Adaptive Immune Response

    doi: 10.3389/fimmu.2019.02922

    Figure Lengend Snippet: The PI3K/Akt axis regulates BCG-induced IL-10 secretion in macrophages. Human TDMs and mice J774A.1 macrophages were mock-treated or infected with BCG at an MOI = 10 for 3 h, washed then treated or not with the PI3K inhibitor, Wortmannin (Wort, 100 nM), or with the Akt inhibitor, MK-2206 (5 μM) for 24 h. (A) Inactivation of PI3k/Akt axis was determined, 24 h post infection/treatment, in whole cell lysate of TDMs by western blotting using the indicated anti-phospho specific antibodies. Total Akt, FOXO3, and β-actin were used as loading controls. Shown are representative images of three independent experiments with similar results. (B) FOXO3 mRNA expression were quantified by qRT-PCR in both TDMs and J774A.1 macrophages. Levels of mRNA were normalized to GAPDH and fold induction was calculated relative to uninfected and untreated cells. (C) Assessment of IL-10 transcription and secretion levels, after the indicated treatments, were quantified by qRT-PCR and sandwich ELISA, respectively. For all panels, results are represented in box and whiskers plot format min to max and are representative of three independent experiments, each one carried out in triplicate. Asterisks indicate statistical significance (* p

    Article Snippet: For experimental setups, BMDMs were seeded in a 24 well plate at a density of 5 × 105 cells per well, infected with BCG at an MOI of 1:10 and subsequently overlaid with medium without or with 5 μM of MK-2206.

    Techniques: Mouse Assay, Infection, Western Blot, Expressing, Quantitative RT-PCR, Sandwich ELISA

    Activation of Nrf2 signaling, downstream of VEGFR2-Akt, is required for gremlin-induced cytoprotection against UV Primary skin keratinocytes were treated with gremlin (1-100 ng/mL) for 8 hours, mRNA expression of listed genes was tested by RT-qPCR assay A. Primary skin keratinocytes were pretreated with 1 μM of SU5416 or 10 μM of MK-2206 for 30 min, cells were then treated with gremlin (25 ng/mL) for 8 hours, HO1 mRNA expression was tested B. Primary skin keratinocytes expressing the Nrf2 shRNA (“shNrf2”), dominant negative Nrf2 (S40T, “dnNrf2”, Flag-tagged) or empty vector (“Vector”, pSV2 puro-Flag) were treated with gremlin (25 ng/mL) for indicated time, expressions of listed protein and mRNA were tested by Western blot assay C. and RT-qPCR assay D. respectively. Above cells were also subjected to UV (20 mJ/cm 2 ) radiation, or plus gremlin (25 ng/mL, 30 min prior UV), cell viability E. and apoptosis F. were tested. * P

    Journal: Oncotarget

    Article Title: Gremlin inhibits UV-induced skin cell damages via activating VEGFR2-Nrf2 signaling

    doi: 10.18632/oncotarget.12454

    Figure Lengend Snippet: Activation of Nrf2 signaling, downstream of VEGFR2-Akt, is required for gremlin-induced cytoprotection against UV Primary skin keratinocytes were treated with gremlin (1-100 ng/mL) for 8 hours, mRNA expression of listed genes was tested by RT-qPCR assay A. Primary skin keratinocytes were pretreated with 1 μM of SU5416 or 10 μM of MK-2206 for 30 min, cells were then treated with gremlin (25 ng/mL) for 8 hours, HO1 mRNA expression was tested B. Primary skin keratinocytes expressing the Nrf2 shRNA (“shNrf2”), dominant negative Nrf2 (S40T, “dnNrf2”, Flag-tagged) or empty vector (“Vector”, pSV2 puro-Flag) were treated with gremlin (25 ng/mL) for indicated time, expressions of listed protein and mRNA were tested by Western blot assay C. and RT-qPCR assay D. respectively. Above cells were also subjected to UV (20 mJ/cm 2 ) radiation, or plus gremlin (25 ng/mL, 30 min prior UV), cell viability E. and apoptosis F. were tested. * P

    Article Snippet: VEGFR2 inhibitors SU5416, ZD6474 and Axitinib as well as the Akt specific inhibitor MK-2206 were obtained from Selleck (Nanjing, China).

    Techniques: Activation Assay, Expressing, Quantitative RT-PCR, shRNA, Dominant Negative Mutation, Plasmid Preparation, Western Blot

    Cell morphology by Coomassie blue staining (×400). A. Blank control group; B. TGF-β 1 group; C. TGF-β 1 inhibitor group (SB525334); D. AKT inhibitor group (MK2206-2HCL); E. Combined inhibitors group (SB525334+MK2206-2HCL).

    Journal: American Journal of Translational Research

    Article Title: TGF-β1 induces human aortic vascular smooth muscle cell phenotype switch through PI3K/AKT/ID2 signaling

    doi:

    Figure Lengend Snippet: Cell morphology by Coomassie blue staining (×400). A. Blank control group; B. TGF-β 1 group; C. TGF-β 1 inhibitor group (SB525334); D. AKT inhibitor group (MK2206-2HCL); E. Combined inhibitors group (SB525334+MK2206-2HCL).

    Article Snippet: In a Transwell plate, 800 µl of DMEM, TGF-β1 (5 ng/ml) (added to four wells), SB525334 (10 ng/ml), MK2206-2HCl (5 ng/ml), or Combined inhibitors solution were respectively added to each of eight wells.

    Techniques: Staining

    Src/Akt/Erk signaling was constitutively activated and functionally involved in anoikis resistance in ID8-P1 cells A. pSrc, pAkt, pErk levels in ID8-P0 and ID8-P1 cells under different culture conditions by Western blot analyses. B. Survival of ID8-P1 cells under suspended conditions treated with Src siRNA (100 µM) and selective inhibitors: PP2 (10 µM), MK2206 (1 µM), PD98059 (30 µM). C. Quantification of soft agar colony numbers of ID8-P1 cells treated with inhibitors. D. Number of floating ID8-P1 cells in mouse peritoneal cavities treated with the Src selective inhibitor PP2 (daily i.p. injection at a dose of 2 mg/kg, n=3). E–G . Ectopic expression of CA-Src in ID8-P0 increased anoikis resistance. E. pSrc levels in ID8-P0 and ID8-P0-CA-Src cells by Western blot analyses. F. Anoikis assays of ID8-P0 and ID8-P0-CA-Src cells in SF medium. G. Colony formation of ID8-P0 and ID8-P0-CA-Src cells in soft agar. H. Numbers of floating ID8-P0 and ID8-P0-CA-Src cells in mouse peritoneal cavities. * P

    Journal: Oncogene

    Article Title: Anoikis resistance is a critical feature of highly aggressive ovarian cancer cells

    doi: 10.1038/onc.2014.264

    Figure Lengend Snippet: Src/Akt/Erk signaling was constitutively activated and functionally involved in anoikis resistance in ID8-P1 cells A. pSrc, pAkt, pErk levels in ID8-P0 and ID8-P1 cells under different culture conditions by Western blot analyses. B. Survival of ID8-P1 cells under suspended conditions treated with Src siRNA (100 µM) and selective inhibitors: PP2 (10 µM), MK2206 (1 µM), PD98059 (30 µM). C. Quantification of soft agar colony numbers of ID8-P1 cells treated with inhibitors. D. Number of floating ID8-P1 cells in mouse peritoneal cavities treated with the Src selective inhibitor PP2 (daily i.p. injection at a dose of 2 mg/kg, n=3). E–G . Ectopic expression of CA-Src in ID8-P0 increased anoikis resistance. E. pSrc levels in ID8-P0 and ID8-P0-CA-Src cells by Western blot analyses. F. Anoikis assays of ID8-P0 and ID8-P0-CA-Src cells in SF medium. G. Colony formation of ID8-P0 and ID8-P0-CA-Src cells in soft agar. H. Numbers of floating ID8-P0 and ID8-P0-CA-Src cells in mouse peritoneal cavities. * P

    Article Snippet: Materials The following reagents were used: PP2 (Tocris bioscience, Minneapolis, MN), MK2206 (Biovision, Milpitas, CA), PD98059 (Enzo Life Sciences, Farmingdale, NY), Chloroquine (CQ) (Sigma, St. Louis, MO), Bafilomycin A1 (baf A1) (Sigma, St. Louis, MO), Src siRNA (Dharmacon, Pittsburgh PA), Rotenone (Sigma, St. Louis, MO), 2-deoxyglucose (Sigma, St. Louis, MO), sodium oxamate (Sigma, St. Louis, MO).

    Techniques: Western Blot, Injection, Expressing

    MK-2206 and WZB117 synergistically induce apoptosis in MCF-7 and MDA-MB-231 cells. (A) Annexin V-FITC/PI staining and flow cytometric analysis. Cells were treated with 6 or 12 µM of MK-2206 and 60 µM of WZB117 either alone or in combination for 48 h. Representative dot plots and quantitative data from three independent experiments showed that MK-2206 and WZB117 significantly induced apoptosis. (B) Western blot analysis of proteins involved in apoptosis in MCF-7 cells. Cells were treated with 6 µM of MK-2206 and 60 µM of WZB117 either alone or in combination for 24 or 48 h. (C) Western blot analysis of proteins involved in apoptosis in MDA-MB-231 cells. Cells were treated with 12 µM of MK-2206 and 60 µM of WZB117 either alone or in combination for 24 or 48 h. Quantitative data are presented as the mean ± SEM of three independent experiments. Cleaved PARP and caspase-3 are marked with arrowheads . * P

    Journal: Frontiers in Pharmacology

    Article Title: The Combination of MK-2206 and WZB117 Exerts a Synergistic Cytotoxic Effect Against Breast Cancer Cells

    doi: 10.3389/fphar.2019.01311

    Figure Lengend Snippet: MK-2206 and WZB117 synergistically induce apoptosis in MCF-7 and MDA-MB-231 cells. (A) Annexin V-FITC/PI staining and flow cytometric analysis. Cells were treated with 6 or 12 µM of MK-2206 and 60 µM of WZB117 either alone or in combination for 48 h. Representative dot plots and quantitative data from three independent experiments showed that MK-2206 and WZB117 significantly induced apoptosis. (B) Western blot analysis of proteins involved in apoptosis in MCF-7 cells. Cells were treated with 6 µM of MK-2206 and 60 µM of WZB117 either alone or in combination for 24 or 48 h. (C) Western blot analysis of proteins involved in apoptosis in MDA-MB-231 cells. Cells were treated with 12 µM of MK-2206 and 60 µM of WZB117 either alone or in combination for 24 or 48 h. Quantitative data are presented as the mean ± SEM of three independent experiments. Cleaved PARP and caspase-3 are marked with arrowheads . * P

    Article Snippet: Chemicals MK-2206 (purity ≥98% by HPLC) was purchased from BioVision, (Mountain View, CA).

    Techniques: Multiple Displacement Amplification, Staining, Flow Cytometry, Western Blot

    MK-2206 and WZB117 synergistically induce ROS in breast cancer cells. (A) Quantitative data of MCF-7 cells with ROS production. Cells were treated with 3 µM MK-2206 and 30 µM WZB117 either alone or in combination along with 10 µM DCFH-DA for 30 min. (B) Quantitative data of MDA-MB-231 cells with ROS production. Cells were treated with 6 µM MK-2206 and 30 µM WZB117 either alone or in combination along with 10 µM DCFH-DA for 30 min. DCFH-DA is non-fluorescent and is hydrolyzed by intracellular esterases to 2,7-dichlorodihydrofluorescein, which is then oxidized to fluorescent 2,7-dichlorofluorescein (DCF) by ROS. DCF fluorescence was detected by flow cytometric analysis and 10,000 cells were counted. The percentage of cells with ROS production was indicated. Data are presented as the mean ± SEM of at least three independent experiments. *** P

    Journal: Frontiers in Pharmacology

    Article Title: The Combination of MK-2206 and WZB117 Exerts a Synergistic Cytotoxic Effect Against Breast Cancer Cells

    doi: 10.3389/fphar.2019.01311

    Figure Lengend Snippet: MK-2206 and WZB117 synergistically induce ROS in breast cancer cells. (A) Quantitative data of MCF-7 cells with ROS production. Cells were treated with 3 µM MK-2206 and 30 µM WZB117 either alone or in combination along with 10 µM DCFH-DA for 30 min. (B) Quantitative data of MDA-MB-231 cells with ROS production. Cells were treated with 6 µM MK-2206 and 30 µM WZB117 either alone or in combination along with 10 µM DCFH-DA for 30 min. DCFH-DA is non-fluorescent and is hydrolyzed by intracellular esterases to 2,7-dichlorodihydrofluorescein, which is then oxidized to fluorescent 2,7-dichlorofluorescein (DCF) by ROS. DCF fluorescence was detected by flow cytometric analysis and 10,000 cells were counted. The percentage of cells with ROS production was indicated. Data are presented as the mean ± SEM of at least three independent experiments. *** P

    Article Snippet: Chemicals MK-2206 (purity ≥98% by HPLC) was purchased from BioVision, (Mountain View, CA).

    Techniques: Multiple Displacement Amplification, Fluorescence, Flow Cytometry

    WZB117 may enhance the growth inhibitory effect of MK-2206 via inhibition of GLUT1 in MCF-7 and MDA-MB-231 cells. (A) WZB117 inhibits glucose uptake in MCF-7 and MDA-MB-231 cells. Cells were incubated with 2-NBDG in the presence of DMSO, 6 µM MK-2206 and/or 60 µM WZB117 (MCF-7) or 12 µM MK-2206 and/or 60 µM WZB117 (MDA-MB-231) for 1.5 h and harvested for flow cytometric analysis of 2-NBDG fluorescence, and relative 2-NBDG uptake was calculated. Data are presented as the mean ± SEM of three independent experiments. (B) Knockdown of GLUT1 enhances the growth inhibitory effect of MK-2206 in MCF-7 and MDA-MB-231 cells. Cells were transfected with control siRNA (siCTL) or GLUT1 siRNA (siGLUT1) and then treated with MK-2206 (6 µM in MCF-7 cells or 12 µM in MDA-MB-231 cells) for 72 h, followed by the MTT assay to assess cell viability. Data are presented as the mean ± SEM of three independent experiments. (C) Western blot analysis of GLUT1 in MCF-7 cells after siRNA transfection and MK-2206 treatment. MCF-7 cells were transfected with control siRNA (siCTL) or GLUT1 siRNA (siGLUT1), allowed to recover for 48 h, and then treated with 6 µM MK-2206 for 48 h before harvested for Western blot analysis. *** P

    Journal: Frontiers in Pharmacology

    Article Title: The Combination of MK-2206 and WZB117 Exerts a Synergistic Cytotoxic Effect Against Breast Cancer Cells

    doi: 10.3389/fphar.2019.01311

    Figure Lengend Snippet: WZB117 may enhance the growth inhibitory effect of MK-2206 via inhibition of GLUT1 in MCF-7 and MDA-MB-231 cells. (A) WZB117 inhibits glucose uptake in MCF-7 and MDA-MB-231 cells. Cells were incubated with 2-NBDG in the presence of DMSO, 6 µM MK-2206 and/or 60 µM WZB117 (MCF-7) or 12 µM MK-2206 and/or 60 µM WZB117 (MDA-MB-231) for 1.5 h and harvested for flow cytometric analysis of 2-NBDG fluorescence, and relative 2-NBDG uptake was calculated. Data are presented as the mean ± SEM of three independent experiments. (B) Knockdown of GLUT1 enhances the growth inhibitory effect of MK-2206 in MCF-7 and MDA-MB-231 cells. Cells were transfected with control siRNA (siCTL) or GLUT1 siRNA (siGLUT1) and then treated with MK-2206 (6 µM in MCF-7 cells or 12 µM in MDA-MB-231 cells) for 72 h, followed by the MTT assay to assess cell viability. Data are presented as the mean ± SEM of three independent experiments. (C) Western blot analysis of GLUT1 in MCF-7 cells after siRNA transfection and MK-2206 treatment. MCF-7 cells were transfected with control siRNA (siCTL) or GLUT1 siRNA (siGLUT1), allowed to recover for 48 h, and then treated with 6 µM MK-2206 for 48 h before harvested for Western blot analysis. *** P

    Article Snippet: Chemicals MK-2206 (purity ≥98% by HPLC) was purchased from BioVision, (Mountain View, CA).

    Techniques: Inhibition, Multiple Displacement Amplification, Incubation, Flow Cytometry, Fluorescence, Transfection, MTT Assay, Western Blot

    MK-2206 and WZB117 affect proteins involved in DNA damage signaling and repair, determined by Western blot analysis, and induce DNA damage, determined by the comet assay. (A) MCF-7 cells. (B) MDA-MB-231 cells. MCF-7 cells were treated with 6 µM MK-2206 and 60 µM WZB117 and MDA-MB-231 cells were treated with 12 µM MK-2206 and 60 µM WZB117, either alone or in combination, for 24 or 48 h. Cells were then harvested for Western blot analysis. Quantitative data are presented as the mean ± SEM of three independent experiments. (C) Comet images. (D) Quantitative data of comet assay. MCF-7 cells were treated with 6 µM MK-2206 and/or 60 µM WZB117 for 1 h and MDA-MB-231 cells were treated with 12 µM MK-2206 and/or 60 µM WZB117 for 6 h and subjected to alkaline comet assay. Data are presented as the mean ± SEM of three independent experiments. *P

    Journal: Frontiers in Pharmacology

    Article Title: The Combination of MK-2206 and WZB117 Exerts a Synergistic Cytotoxic Effect Against Breast Cancer Cells

    doi: 10.3389/fphar.2019.01311

    Figure Lengend Snippet: MK-2206 and WZB117 affect proteins involved in DNA damage signaling and repair, determined by Western blot analysis, and induce DNA damage, determined by the comet assay. (A) MCF-7 cells. (B) MDA-MB-231 cells. MCF-7 cells were treated with 6 µM MK-2206 and 60 µM WZB117 and MDA-MB-231 cells were treated with 12 µM MK-2206 and 60 µM WZB117, either alone or in combination, for 24 or 48 h. Cells were then harvested for Western blot analysis. Quantitative data are presented as the mean ± SEM of three independent experiments. (C) Comet images. (D) Quantitative data of comet assay. MCF-7 cells were treated with 6 µM MK-2206 and/or 60 µM WZB117 for 1 h and MDA-MB-231 cells were treated with 12 µM MK-2206 and/or 60 µM WZB117 for 6 h and subjected to alkaline comet assay. Data are presented as the mean ± SEM of three independent experiments. *P

    Article Snippet: Chemicals MK-2206 (purity ≥98% by HPLC) was purchased from BioVision, (Mountain View, CA).

    Techniques: Western Blot, Single Cell Gel Electrophoresis, Multiple Displacement Amplification, Alkaline Single Cell Gel Electrophoresis

    The effect of MK-2206 and WZB117 on proteins involved in the Akt/mTOR signaling pathway. (A) MCF-7 cells. (B) MDA-MB-231 cells. MCF-7 cells were treated with 6 µM MK-2206 and 60 µM WZB117, and MDA-MB-231 cells were treated with 12 µM MK-2206 and 60µM WZB117, either alone or in combination, for 24 or 48 h. Cells were then harvested for Western blot analysis. Quantitative data are presented as the mean ± SEM of three independent experiments. *P

    Journal: Frontiers in Pharmacology

    Article Title: The Combination of MK-2206 and WZB117 Exerts a Synergistic Cytotoxic Effect Against Breast Cancer Cells

    doi: 10.3389/fphar.2019.01311

    Figure Lengend Snippet: The effect of MK-2206 and WZB117 on proteins involved in the Akt/mTOR signaling pathway. (A) MCF-7 cells. (B) MDA-MB-231 cells. MCF-7 cells were treated with 6 µM MK-2206 and 60 µM WZB117, and MDA-MB-231 cells were treated with 12 µM MK-2206 and 60µM WZB117, either alone or in combination, for 24 or 48 h. Cells were then harvested for Western blot analysis. Quantitative data are presented as the mean ± SEM of three independent experiments. *P

    Article Snippet: Chemicals MK-2206 (purity ≥98% by HPLC) was purchased from BioVision, (Mountain View, CA).

    Techniques: Multiple Displacement Amplification, Western Blot

    MK-2206 and WZB117 induce DNA damage and may compromise DNA repair. (A) Images of MCF-7 cells stained with Rad51 ( green ) and γ-H2AX ( red ) 24 h after drug treatment. The nucleus was counterstained with DAPI ( blue ). Scale bar , 20 µm. (B) Quantitative data of Rad51-positive and γ-H2AX-positive MCF-7 cells. (C) Quantitative data of Rad51-positive and γ-H2AX-positive MDA-MB-231 cells. MCF-7 cells were treated with 6 µM MK-2206 and/or 60 µM WZB117 and MDA-MB-231 cells were treated with 12 µM MK-2206 and/or 60 µM WZB117 for 24 h. Rad51-positive cells were defined as those with five or more Rad51 nuclear foci. Data are presented as the mean ± SEM of three independent experiments. * P

    Journal: Frontiers in Pharmacology

    Article Title: The Combination of MK-2206 and WZB117 Exerts a Synergistic Cytotoxic Effect Against Breast Cancer Cells

    doi: 10.3389/fphar.2019.01311

    Figure Lengend Snippet: MK-2206 and WZB117 induce DNA damage and may compromise DNA repair. (A) Images of MCF-7 cells stained with Rad51 ( green ) and γ-H2AX ( red ) 24 h after drug treatment. The nucleus was counterstained with DAPI ( blue ). Scale bar , 20 µm. (B) Quantitative data of Rad51-positive and γ-H2AX-positive MCF-7 cells. (C) Quantitative data of Rad51-positive and γ-H2AX-positive MDA-MB-231 cells. MCF-7 cells were treated with 6 µM MK-2206 and/or 60 µM WZB117 and MDA-MB-231 cells were treated with 12 µM MK-2206 and/or 60 µM WZB117 for 24 h. Rad51-positive cells were defined as those with five or more Rad51 nuclear foci. Data are presented as the mean ± SEM of three independent experiments. * P

    Article Snippet: Chemicals MK-2206 (purity ≥98% by HPLC) was purchased from BioVision, (Mountain View, CA).

    Techniques: Staining, Multiple Displacement Amplification

    MK-2206 and WZB117 synergistically inhibit the growth of MCF-7 and MDA-MB-231 breast cancer cells. (A) Dose–response curves of cells treated with MK-2206 and WZB117 either alone or in combination for 48 h. Cell viability was measured by the MTT assay. (B) MK-2206 and WZB117 inhibit colony formation. A set of colony formation images of MDA-MB-231 cells are illustrated in the upper panels and quantitative results of colony formation are shown in the lower panels . MCF-7 cells were treated with 0.5 µM MK-2206 and/or 5 µM WZB117, and MDA-MB-231 cells were treated with 6 µM MK-2206 and/or 30 µM WZB117 for 24 h, then cultured in drug-free culture medium for 7 days (MCF-7) or 9 days (MDA-MB-231), and colonies were then scored. CTL , the DMSO vehicle control; MK , MK-2206; WZB , WZB117, MK+WZB , the combination of MK-2206 and WZB117. Data are presented as the mean ± SEM of three independent experiments. ** P

    Journal: Frontiers in Pharmacology

    Article Title: The Combination of MK-2206 and WZB117 Exerts a Synergistic Cytotoxic Effect Against Breast Cancer Cells

    doi: 10.3389/fphar.2019.01311

    Figure Lengend Snippet: MK-2206 and WZB117 synergistically inhibit the growth of MCF-7 and MDA-MB-231 breast cancer cells. (A) Dose–response curves of cells treated with MK-2206 and WZB117 either alone or in combination for 48 h. Cell viability was measured by the MTT assay. (B) MK-2206 and WZB117 inhibit colony formation. A set of colony formation images of MDA-MB-231 cells are illustrated in the upper panels and quantitative results of colony formation are shown in the lower panels . MCF-7 cells were treated with 0.5 µM MK-2206 and/or 5 µM WZB117, and MDA-MB-231 cells were treated with 6 µM MK-2206 and/or 30 µM WZB117 for 24 h, then cultured in drug-free culture medium for 7 days (MCF-7) or 9 days (MDA-MB-231), and colonies were then scored. CTL , the DMSO vehicle control; MK , MK-2206; WZB , WZB117, MK+WZB , the combination of MK-2206 and WZB117. Data are presented as the mean ± SEM of three independent experiments. ** P

    Article Snippet: Chemicals MK-2206 (purity ≥98% by HPLC) was purchased from BioVision, (Mountain View, CA).

    Techniques: Multiple Displacement Amplification, MTT Assay, Cell Culture, CTL Assay

    The MK-2206 and WZB117 combination affects DNA repair via HR and NHEJ. (A) HR and NHEJ assays in MCF-7 cells. (B) HR and NHEJ assays in MDA-MB-231 cells. For drug treatment, MCF-7 cells were treated with 6 µM MK-2206 and/or 60 µM WZB117 and MDA-MB-231 cells were treated with 12 µM MK-2206 and/or 60 µM WZB117 for 24 h. Data are presented as the mean ± SEM of three independent experiments, except NHEJ assay in MCF-7 cells ( N = 2).*P

    Journal: Frontiers in Pharmacology

    Article Title: The Combination of MK-2206 and WZB117 Exerts a Synergistic Cytotoxic Effect Against Breast Cancer Cells

    doi: 10.3389/fphar.2019.01311

    Figure Lengend Snippet: The MK-2206 and WZB117 combination affects DNA repair via HR and NHEJ. (A) HR and NHEJ assays in MCF-7 cells. (B) HR and NHEJ assays in MDA-MB-231 cells. For drug treatment, MCF-7 cells were treated with 6 µM MK-2206 and/or 60 µM WZB117 and MDA-MB-231 cells were treated with 12 µM MK-2206 and/or 60 µM WZB117 for 24 h. Data are presented as the mean ± SEM of three independent experiments, except NHEJ assay in MCF-7 cells ( N = 2).*P

    Article Snippet: Chemicals MK-2206 (purity ≥98% by HPLC) was purchased from BioVision, (Mountain View, CA).

    Techniques: Non-Homologous End Joining, Multiple Displacement Amplification

    Exosomal miR-205 regulates the PTEN-AKT signalling pathway in HUVECs. ( A ) Interference efficiency was measured by western blotting. ( B ) Western blotting analysis of PTEN, p-AKT, AKT, and α-tubulin in HUVECs incubated with NC-Exos, miR-205-Exos, miR-205 inhibitor, NC and mimic groups. ( C ) Western blotting was used to detect PTEN, p-AKT and AKT expression in HUVECs after treatment with miR-205-Exos, miR-205-Exos+miR-205 inhibitor, NC, miR-205 mimic and miR-205 mimic+PTEN. ( D, E ) Effects of miR-205 inhibitor, NC, mimic, PTEN+inhibitor, PTEN, PTEN+mimic on HUVEC tube formation ability and migration were assessed by tube formation and Transwell migration assays. ( F, G ) Effects of miR-205 inhibitor, NC, mimic, siPTEN+inhibitor, siPTEN, siPTEN+mimic on HUVEC tube formation ability and migration were examined by tube formation and transwell migration assays. The scale bar in (D, F) represents 100 µm. The scale bar in (E, G) represents 50 µm. ( H ) Effects of GDC-0941 and MK-2206 on tube formation and migration abilities induced by exosomal miR-205 in HUVECs. The scale bar in the upper panel represents 100 µm. The scale bar in the bottom panel represents 50 µm. ( I ) Western blot analysis shows the effects of GDC-0941 and MK-2206. All experiments were repeated three times, and the results are presented as the mean ± SEM. Statistical significance was determined by a two-tailed, unpaired Student's t test. ** P

    Journal: Theranostics

    Article Title: Ovarian cancer cell-secreted exosomal miR-205 promotes metastasis by inducing angiogenesis

    doi: 10.7150/thno.37455

    Figure Lengend Snippet: Exosomal miR-205 regulates the PTEN-AKT signalling pathway in HUVECs. ( A ) Interference efficiency was measured by western blotting. ( B ) Western blotting analysis of PTEN, p-AKT, AKT, and α-tubulin in HUVECs incubated with NC-Exos, miR-205-Exos, miR-205 inhibitor, NC and mimic groups. ( C ) Western blotting was used to detect PTEN, p-AKT and AKT expression in HUVECs after treatment with miR-205-Exos, miR-205-Exos+miR-205 inhibitor, NC, miR-205 mimic and miR-205 mimic+PTEN. ( D, E ) Effects of miR-205 inhibitor, NC, mimic, PTEN+inhibitor, PTEN, PTEN+mimic on HUVEC tube formation ability and migration were assessed by tube formation and Transwell migration assays. ( F, G ) Effects of miR-205 inhibitor, NC, mimic, siPTEN+inhibitor, siPTEN, siPTEN+mimic on HUVEC tube formation ability and migration were examined by tube formation and transwell migration assays. The scale bar in (D, F) represents 100 µm. The scale bar in (E, G) represents 50 µm. ( H ) Effects of GDC-0941 and MK-2206 on tube formation and migration abilities induced by exosomal miR-205 in HUVECs. The scale bar in the upper panel represents 100 µm. The scale bar in the bottom panel represents 50 µm. ( I ) Western blot analysis shows the effects of GDC-0941 and MK-2206. All experiments were repeated three times, and the results are presented as the mean ± SEM. Statistical significance was determined by a two-tailed, unpaired Student's t test. ** P

    Article Snippet: GDC-0941 (200 nM) and MK-2206 (200 nM) were purchased from APExBIO.

    Techniques: Western Blot, Incubation, Expressing, Migration, Two Tailed Test

    CXCL16 induction in L. donovani -infected macrophages is AKT/mTOR dependent but TLR independent. (A and B) WT, MyD88 KO, and UNC93B KO BMDM cultures were inoculated with L. donovani , treated with 250 ng/ml LPG, 100 ng/ml LPS, or 250 ng/ml poly(I:C), or left uninfected/untreated for 24 h. CXCL16 protein expression was assessed by Western blotting. β-Actin was used as a loading control. Parasite infection was monitored by probing for Leishmania IMPDH. Induction of COX-2 served as a surrogate for disrupted TLR signaling in MyD88 KO cells. (C) BMDM cultures were inoculated with L. donovani WT, lpg1 -KO, or lpg1 -KO+ LPG1 for 24 h or left uninfected, and the activity of AKT/mTOR signaling was monitored by Western blotting using phospho-specific and total antibodies against AKT and rpS6, respectively. CXCL16 expression and efficacy of infection were examined as for panel A. (D) BMDM cultures were pretreated with 2 μM MK-2206, 200 nM Torin-1, or an equal volume of dimethyl sulfoxide (DMSO) (vehicle) for 1 h and then infected with L. donovani promastigotes for 24 h. The phosphorylation status and total levels of AKT and rpS6 were assessed as for panel C. Results are representative of at least two independent biological replicates.

    Journal: Infection and Immunity

    Article Title: Leishmania donovani Lipophosphoglycan Increases Macrophage-Dependent Chemotaxis of CXCR6-Expressing Cells via CXCL16 Induction

    doi: 10.1128/IAI.00064-19

    Figure Lengend Snippet: CXCL16 induction in L. donovani -infected macrophages is AKT/mTOR dependent but TLR independent. (A and B) WT, MyD88 KO, and UNC93B KO BMDM cultures were inoculated with L. donovani , treated with 250 ng/ml LPG, 100 ng/ml LPS, or 250 ng/ml poly(I:C), or left uninfected/untreated for 24 h. CXCL16 protein expression was assessed by Western blotting. β-Actin was used as a loading control. Parasite infection was monitored by probing for Leishmania IMPDH. Induction of COX-2 served as a surrogate for disrupted TLR signaling in MyD88 KO cells. (C) BMDM cultures were inoculated with L. donovani WT, lpg1 -KO, or lpg1 -KO+ LPG1 for 24 h or left uninfected, and the activity of AKT/mTOR signaling was monitored by Western blotting using phospho-specific and total antibodies against AKT and rpS6, respectively. CXCL16 expression and efficacy of infection were examined as for panel A. (D) BMDM cultures were pretreated with 2 μM MK-2206, 200 nM Torin-1, or an equal volume of dimethyl sulfoxide (DMSO) (vehicle) for 1 h and then infected with L. donovani promastigotes for 24 h. The phosphorylation status and total levels of AKT and rpS6 were assessed as for panel C. Results are representative of at least two independent biological replicates.

    Article Snippet: Torin-1 and MK-2206 were acquired from Cayman Chemical.

    Techniques: Infection, Expressing, Western Blot, Activity Assay