pi3k p110 α  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc pi3k p110 α
    <t>PI3K/AKT</t> signaling blocks the inhibitory effect of PPAR γ on hypoxic-induced HSCs activation. Activation of PI3K with 740 Y-P (25 μ g/ml) of HSCs exposed to hypoxia in the presence or absence of RSG (50 nM). (a) PPAR γ , α -SMA, and desmin expression of HSCs in experimental groups were analyzed by western blotting. (b) α -SMA and desmin expression of HSCs in experimental groups were detected by immunofluorescence using Confocal microscopy. Inhibition of PI3K with LY294002 (20 μ M) in HSCs exposed to hypoxia. (c) Phosphorylated AKT, total AKT, PPAR γ , α -SMA, and desmin expression of HSCs in experimental groups were measured by western blotting. (d) α -SMA and desmin expression of HSCs in experimental groups were detected by immunofluorescence using Confocal microscopy. For each assay, n = 3. ∗ P < 0.05, ∗∗ P < 0.01.
    Pi3k P110 α, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pi3k p110 α/product/Cell Signaling Technology Inc
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    1) Product Images from "PPAR γ Antagonizes Hypoxia-Induced Activation of Hepatic Stellate Cell through Cross Mediating PI3K/AKT and cGMP/PKG Signaling"

    Article Title: PPAR γ Antagonizes Hypoxia-Induced Activation of Hepatic Stellate Cell through Cross Mediating PI3K/AKT and cGMP/PKG Signaling

    Journal: PPAR Research

    doi: 10.1155/2018/6970407

    PI3K/AKT signaling blocks the inhibitory effect of PPAR γ on hypoxic-induced HSCs activation. Activation of PI3K with 740 Y-P (25 μ g/ml) of HSCs exposed to hypoxia in the presence or absence of RSG (50 nM). (a) PPAR γ , α -SMA, and desmin expression of HSCs in experimental groups were analyzed by western blotting. (b) α -SMA and desmin expression of HSCs in experimental groups were detected by immunofluorescence using Confocal microscopy. Inhibition of PI3K with LY294002 (20 μ M) in HSCs exposed to hypoxia. (c) Phosphorylated AKT, total AKT, PPAR γ , α -SMA, and desmin expression of HSCs in experimental groups were measured by western blotting. (d) α -SMA and desmin expression of HSCs in experimental groups were detected by immunofluorescence using Confocal microscopy. For each assay, n = 3. ∗ P < 0.05, ∗∗ P < 0.01.
    Figure Legend Snippet: PI3K/AKT signaling blocks the inhibitory effect of PPAR γ on hypoxic-induced HSCs activation. Activation of PI3K with 740 Y-P (25 μ g/ml) of HSCs exposed to hypoxia in the presence or absence of RSG (50 nM). (a) PPAR γ , α -SMA, and desmin expression of HSCs in experimental groups were analyzed by western blotting. (b) α -SMA and desmin expression of HSCs in experimental groups were detected by immunofluorescence using Confocal microscopy. Inhibition of PI3K with LY294002 (20 μ M) in HSCs exposed to hypoxia. (c) Phosphorylated AKT, total AKT, PPAR γ , α -SMA, and desmin expression of HSCs in experimental groups were measured by western blotting. (d) α -SMA and desmin expression of HSCs in experimental groups were detected by immunofluorescence using Confocal microscopy. For each assay, n = 3. ∗ P < 0.05, ∗∗ P < 0.01.

    Techniques Used: Activation Assay, Expressing, Western Blot, Immunofluorescence, Confocal Microscopy, Inhibition

    sGC/cGMP/PKG and PI3K/AKT signals synergistically act on PPAR γ to inhibit hypoxia-induced HSC activation. (a) HSCs were exposed to hypoxia in the presence of cGMP analog, 8-Br-cGMP (1 mM), or agonist, Rp-8-Br-cGMPs (20 μ M). PI3K p100 α , p100 β , phosphorylated AKT, total AKT, PPAR γ , α -SMA, and desmin expression of HSCs in experimental groups were detected by western blotting. HSCs were exposed to hypoxia in the presence of PDE5 agonist, zaprinast (10 μ M). (b) PDE5, PPAR γ , α -SMA, and desmin expression of HSCs in experimental groups were measured by western blotting. (c)-(d) cGMP and PKG activity were tested, respectively. (e) Possible signal pathway involved in this study. Hypoxia induces HSCs activation. However, PPAR γ inhibits this effect, which is triggered by sGC/cGMP/PKG signaling. Hypoxia promotes PI3K/AKT signaling and PDE5, which inhibit cGMP and PPAR γ , respectively. Finally, sGC/cGMP/PKG and PI3K/AKT signals synergistically act on PPAR γ to inhibit hypoxia-induced HSC activation. For each assay, n = 3. ∗ P < 0.05, ∗∗ P < 0.01.
    Figure Legend Snippet: sGC/cGMP/PKG and PI3K/AKT signals synergistically act on PPAR γ to inhibit hypoxia-induced HSC activation. (a) HSCs were exposed to hypoxia in the presence of cGMP analog, 8-Br-cGMP (1 mM), or agonist, Rp-8-Br-cGMPs (20 μ M). PI3K p100 α , p100 β , phosphorylated AKT, total AKT, PPAR γ , α -SMA, and desmin expression of HSCs in experimental groups were detected by western blotting. HSCs were exposed to hypoxia in the presence of PDE5 agonist, zaprinast (10 μ M). (b) PDE5, PPAR γ , α -SMA, and desmin expression of HSCs in experimental groups were measured by western blotting. (c)-(d) cGMP and PKG activity were tested, respectively. (e) Possible signal pathway involved in this study. Hypoxia induces HSCs activation. However, PPAR γ inhibits this effect, which is triggered by sGC/cGMP/PKG signaling. Hypoxia promotes PI3K/AKT signaling and PDE5, which inhibit cGMP and PPAR γ , respectively. Finally, sGC/cGMP/PKG and PI3K/AKT signals synergistically act on PPAR γ to inhibit hypoxia-induced HSC activation. For each assay, n = 3. ∗ P < 0.05, ∗∗ P < 0.01.

    Techniques Used: Activation Assay, Expressing, Western Blot, Activity Assay

    pi3k p110 α  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc pi3k p110 α
    <t>PI3K/AKT</t> signaling blocks the inhibitory effect of PPAR γ on hypoxic-induced HSCs activation. Activation of PI3K with 740 Y-P (25 μ g/ml) of HSCs exposed to hypoxia in the presence or absence of RSG (50 nM). (a) PPAR γ , α -SMA, and desmin expression of HSCs in experimental groups were analyzed by western blotting. (b) α -SMA and desmin expression of HSCs in experimental groups were detected by immunofluorescence using Confocal microscopy. Inhibition of PI3K with LY294002 (20 μ M) in HSCs exposed to hypoxia. (c) Phosphorylated AKT, total AKT, PPAR γ , α -SMA, and desmin expression of HSCs in experimental groups were measured by western blotting. (d) α -SMA and desmin expression of HSCs in experimental groups were detected by immunofluorescence using Confocal microscopy. For each assay, n = 3. ∗ P < 0.05, ∗∗ P < 0.01.
    Pi3k P110 α, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pi3k p110 α/product/Cell Signaling Technology Inc
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pi3k p110 α - by Bioz Stars, 2023-03
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    Images

    1) Product Images from "PPAR γ Antagonizes Hypoxia-Induced Activation of Hepatic Stellate Cell through Cross Mediating PI3K/AKT and cGMP/PKG Signaling"

    Article Title: PPAR γ Antagonizes Hypoxia-Induced Activation of Hepatic Stellate Cell through Cross Mediating PI3K/AKT and cGMP/PKG Signaling

    Journal: PPAR Research

    doi: 10.1155/2018/6970407

    PI3K/AKT signaling blocks the inhibitory effect of PPAR γ on hypoxic-induced HSCs activation. Activation of PI3K with 740 Y-P (25 μ g/ml) of HSCs exposed to hypoxia in the presence or absence of RSG (50 nM). (a) PPAR γ , α -SMA, and desmin expression of HSCs in experimental groups were analyzed by western blotting. (b) α -SMA and desmin expression of HSCs in experimental groups were detected by immunofluorescence using Confocal microscopy. Inhibition of PI3K with LY294002 (20 μ M) in HSCs exposed to hypoxia. (c) Phosphorylated AKT, total AKT, PPAR γ , α -SMA, and desmin expression of HSCs in experimental groups were measured by western blotting. (d) α -SMA and desmin expression of HSCs in experimental groups were detected by immunofluorescence using Confocal microscopy. For each assay, n = 3. ∗ P < 0.05, ∗∗ P < 0.01.
    Figure Legend Snippet: PI3K/AKT signaling blocks the inhibitory effect of PPAR γ on hypoxic-induced HSCs activation. Activation of PI3K with 740 Y-P (25 μ g/ml) of HSCs exposed to hypoxia in the presence or absence of RSG (50 nM). (a) PPAR γ , α -SMA, and desmin expression of HSCs in experimental groups were analyzed by western blotting. (b) α -SMA and desmin expression of HSCs in experimental groups were detected by immunofluorescence using Confocal microscopy. Inhibition of PI3K with LY294002 (20 μ M) in HSCs exposed to hypoxia. (c) Phosphorylated AKT, total AKT, PPAR γ , α -SMA, and desmin expression of HSCs in experimental groups were measured by western blotting. (d) α -SMA and desmin expression of HSCs in experimental groups were detected by immunofluorescence using Confocal microscopy. For each assay, n = 3. ∗ P < 0.05, ∗∗ P < 0.01.

    Techniques Used: Activation Assay, Expressing, Western Blot, Immunofluorescence, Confocal Microscopy, Inhibition

    sGC/cGMP/PKG and PI3K/AKT signals synergistically act on PPAR γ to inhibit hypoxia-induced HSC activation. (a) HSCs were exposed to hypoxia in the presence of cGMP analog, 8-Br-cGMP (1 mM), or agonist, Rp-8-Br-cGMPs (20 μ M). PI3K p100 α , p100 β , phosphorylated AKT, total AKT, PPAR γ , α -SMA, and desmin expression of HSCs in experimental groups were detected by western blotting. HSCs were exposed to hypoxia in the presence of PDE5 agonist, zaprinast (10 μ M). (b) PDE5, PPAR γ , α -SMA, and desmin expression of HSCs in experimental groups were measured by western blotting. (c)-(d) cGMP and PKG activity were tested, respectively. (e) Possible signal pathway involved in this study. Hypoxia induces HSCs activation. However, PPAR γ inhibits this effect, which is triggered by sGC/cGMP/PKG signaling. Hypoxia promotes PI3K/AKT signaling and PDE5, which inhibit cGMP and PPAR γ , respectively. Finally, sGC/cGMP/PKG and PI3K/AKT signals synergistically act on PPAR γ to inhibit hypoxia-induced HSC activation. For each assay, n = 3. ∗ P < 0.05, ∗∗ P < 0.01.
    Figure Legend Snippet: sGC/cGMP/PKG and PI3K/AKT signals synergistically act on PPAR γ to inhibit hypoxia-induced HSC activation. (a) HSCs were exposed to hypoxia in the presence of cGMP analog, 8-Br-cGMP (1 mM), or agonist, Rp-8-Br-cGMPs (20 μ M). PI3K p100 α , p100 β , phosphorylated AKT, total AKT, PPAR γ , α -SMA, and desmin expression of HSCs in experimental groups were detected by western blotting. HSCs were exposed to hypoxia in the presence of PDE5 agonist, zaprinast (10 μ M). (b) PDE5, PPAR γ , α -SMA, and desmin expression of HSCs in experimental groups were measured by western blotting. (c)-(d) cGMP and PKG activity were tested, respectively. (e) Possible signal pathway involved in this study. Hypoxia induces HSCs activation. However, PPAR γ inhibits this effect, which is triggered by sGC/cGMP/PKG signaling. Hypoxia promotes PI3K/AKT signaling and PDE5, which inhibit cGMP and PPAR γ , respectively. Finally, sGC/cGMP/PKG and PI3K/AKT signals synergistically act on PPAR γ to inhibit hypoxia-induced HSC activation. For each assay, n = 3. ∗ P < 0.05, ∗∗ P < 0.01.

    Techniques Used: Activation Assay, Expressing, Western Blot, Activity Assay

    pi3k p110  (Cell Signaling Technology Inc)


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    Structured Review

    Cell Signaling Technology Inc pi3k p110
    A. Estrogen-induced <t>PI3K</t> phosphotylation. B. Estrogen-induced AKT phosphotylation. MCF-10A cells were exposed to a carcinogenic regimen of E2 and its metabolites – 2-OH-E2 or 4-OH-E2 as described in the legend of . At the end of transformation process, cells were treated for additional 30 minutes with E2, 2-OH-E2 or 4-OH-E2, respectively. The cellular extracts from treated and controls cells were immuno-precipitated with PI3K or AKT specific monoclonal antibodies and followed by Western detection of PI3K or AKT phosphorylation using phosphor-threonine antibody. Results are expressed as mean fold change of three separate experiments. *P<0.05, significantly different from contol. **P<0.001 indicates significantly different from control.
    Pi3k P110, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 86 stars, based on 1 article reviews
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    86/100 stars

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    1) Product Images from "Reactive Oxygen Species via Redox Signaling to PI3K/AKT Pathway Contribute to the Malignant Growth of 4-Hydroxy Estradiol-Transformed Mammary Epithelial Cells"

    Article Title: Reactive Oxygen Species via Redox Signaling to PI3K/AKT Pathway Contribute to the Malignant Growth of 4-Hydroxy Estradiol-Transformed Mammary Epithelial Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0054206

    A. Estrogen-induced PI3K phosphotylation. B. Estrogen-induced AKT phosphotylation. MCF-10A cells were exposed to a carcinogenic regimen of E2 and its metabolites – 2-OH-E2 or 4-OH-E2 as described in the legend of . At the end of transformation process, cells were treated for additional 30 minutes with E2, 2-OH-E2 or 4-OH-E2, respectively. The cellular extracts from treated and controls cells were immuno-precipitated with PI3K or AKT specific monoclonal antibodies and followed by Western detection of PI3K or AKT phosphorylation using phosphor-threonine antibody. Results are expressed as mean fold change of three separate experiments. *P<0.05, significantly different from contol. **P<0.001 indicates significantly different from control.
    Figure Legend Snippet: A. Estrogen-induced PI3K phosphotylation. B. Estrogen-induced AKT phosphotylation. MCF-10A cells were exposed to a carcinogenic regimen of E2 and its metabolites – 2-OH-E2 or 4-OH-E2 as described in the legend of . At the end of transformation process, cells were treated for additional 30 minutes with E2, 2-OH-E2 or 4-OH-E2, respectively. The cellular extracts from treated and controls cells were immuno-precipitated with PI3K or AKT specific monoclonal antibodies and followed by Western detection of PI3K or AKT phosphorylation using phosphor-threonine antibody. Results are expressed as mean fold change of three separate experiments. *P<0.05, significantly different from contol. **P<0.001 indicates significantly different from control.

    Techniques Used: Transformation Assay, Western Blot

    A. Inhibition of 4-OH-E2-induced PI3K phosphotylation by overexpression of MnSOD and catalase. B. Inhibition of 4-OH-E2-induced AKT phosphotylation by overexpression of MnSOD and catalase. For investigating inhibition of 4-OH-E2-induced cell transformation by ROS modifiers, MCF-10A cells were transfected with 50 MOI adenovirus expressing catalase or MnSOD. Cells overexpressing catalase or MnSOD were exposed to a carcinogenic regimen of 4-OH-E2 as described in the legend of . At the end of transformation process, cells were treated for additional 30 minutes with 100 ng/ml 4-OHE2. The cellular extracts from treated and controls cells were immuno-precipitated with PI3K or AKT specific monoclonal antibodies and followed by Western detection of PI3K or AKT phosphorylation using phosphor-threonine antibody. Results are expressed as mean ± SD of three separate experiments with control set as 100%. *P<0.05 indicates significantly different from E2, (P<0.05). **P<0.05, significantly different from control.
    Figure Legend Snippet: A. Inhibition of 4-OH-E2-induced PI3K phosphotylation by overexpression of MnSOD and catalase. B. Inhibition of 4-OH-E2-induced AKT phosphotylation by overexpression of MnSOD and catalase. For investigating inhibition of 4-OH-E2-induced cell transformation by ROS modifiers, MCF-10A cells were transfected with 50 MOI adenovirus expressing catalase or MnSOD. Cells overexpressing catalase or MnSOD were exposed to a carcinogenic regimen of 4-OH-E2 as described in the legend of . At the end of transformation process, cells were treated for additional 30 minutes with 100 ng/ml 4-OHE2. The cellular extracts from treated and controls cells were immuno-precipitated with PI3K or AKT specific monoclonal antibodies and followed by Western detection of PI3K or AKT phosphorylation using phosphor-threonine antibody. Results are expressed as mean ± SD of three separate experiments with control set as 100%. *P<0.05 indicates significantly different from E2, (P<0.05). **P<0.05, significantly different from control.

    Techniques Used: Inhibition, Over Expression, Transformation Assay, Transfection, Expressing, Western Blot

    A. Inhibition of 4-OH-E2-induced PI3K phosphotylation by co-treatment with Ebselen and N-acetylcysteine (NAC). B. Inhibition of 4-OH-E2-induced AKT phosphotylation by by co-treatment with Ebselen and NAC. For investigating inhibition of 4-OH-E2-induced cell transformation by ROS modifiers, MCF-10A cells pretreated for 2 hrs with 40 uM Ebselen or 10 mM NAC and were exposed to a carcinogenic regimen of 4-OH-E2 as described in the legend of . At the end of transformation process, cells were treated for additional 30 minutes with 100 ng/ml 4-OHE2. The cellular extracts from treated and controls cells were immuno-precipitated with PI3K or AKT specific monoclonal antibodies and followed by Western detection of PI3K or AKT phosphorylation using phosphor-threonine antibody. Results are expressed as mean ± SD of three separate experiments with control set as 100%. *P<0.05 indicates significantly different from E2, (P<0.05). **P<0.05, significantly different from control.
    Figure Legend Snippet: A. Inhibition of 4-OH-E2-induced PI3K phosphotylation by co-treatment with Ebselen and N-acetylcysteine (NAC). B. Inhibition of 4-OH-E2-induced AKT phosphotylation by by co-treatment with Ebselen and NAC. For investigating inhibition of 4-OH-E2-induced cell transformation by ROS modifiers, MCF-10A cells pretreated for 2 hrs with 40 uM Ebselen or 10 mM NAC and were exposed to a carcinogenic regimen of 4-OH-E2 as described in the legend of . At the end of transformation process, cells were treated for additional 30 minutes with 100 ng/ml 4-OHE2. The cellular extracts from treated and controls cells were immuno-precipitated with PI3K or AKT specific monoclonal antibodies and followed by Western detection of PI3K or AKT phosphorylation using phosphor-threonine antibody. Results are expressed as mean ± SD of three separate experiments with control set as 100%. *P<0.05 indicates significantly different from E2, (P<0.05). **P<0.05, significantly different from control.

    Techniques Used: Inhibition, Transformation Assay, Western Blot

    antibodies against pi3k p110 α  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc antibodies against pi3k p110 α
    The effect of TMZ on DNA damage and repair regulating factors and key molecules of the <t>PI3K</t> signaling pathway in GBM cells. (a) SF295, U87, and U251 cells were treated with different concentrations of TMZ (10, 20, 40, 80, 160, 320, 640, and 1280 μ M) for 72 h. Cell viability was determined by WST-8 assay. (b)–(f) SF295 and U87 cells were treated with TMZ (120 μ M and 180 μ M) for 48 and 72 h. (b) The cells were then collected for western blotting detection of the DNA SSB marker XRCC1 and the DSB marker γ -H2AX. (c) The cells were harvested for qRT-PCR analysis of BRCA1 and RAD51. (d) Western blot analysis of DNA damage repair proteins, including p-ATM, BRCA1, RAD51, DNA-PKcs, and Ku80. (e) qRT-PCR analysis of PIK3CA and PIK3CB. (f) Western blot analysis of key proteins in the PI3K signaling pathway, including <t>PI3K-p110</t> α , PI3K-p110 β , p-Akt, Akt, p-mTOR, and mTOR. For western blotting, β -actin was used as a loading control. For qRT-PCR, gene expression was normalized to 18 s rRNA. All data are presented as the mean ± SD ( n = 3). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001; ∗∗∗∗ P < 0.0001.
    Antibodies Against Pi3k P110 α, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "ZSTK474 Sensitizes Glioblastoma to Temozolomide by Blocking Homologous Recombination Repair"

    Article Title: ZSTK474 Sensitizes Glioblastoma to Temozolomide by Blocking Homologous Recombination Repair

    Journal: BioMed Research International

    doi: 10.1155/2022/8568528

    The effect of TMZ on DNA damage and repair regulating factors and key molecules of the PI3K signaling pathway in GBM cells. (a) SF295, U87, and U251 cells were treated with different concentrations of TMZ (10, 20, 40, 80, 160, 320, 640, and 1280 μ M) for 72 h. Cell viability was determined by WST-8 assay. (b)–(f) SF295 and U87 cells were treated with TMZ (120 μ M and 180 μ M) for 48 and 72 h. (b) The cells were then collected for western blotting detection of the DNA SSB marker XRCC1 and the DSB marker γ -H2AX. (c) The cells were harvested for qRT-PCR analysis of BRCA1 and RAD51. (d) Western blot analysis of DNA damage repair proteins, including p-ATM, BRCA1, RAD51, DNA-PKcs, and Ku80. (e) qRT-PCR analysis of PIK3CA and PIK3CB. (f) Western blot analysis of key proteins in the PI3K signaling pathway, including PI3K-p110 α , PI3K-p110 β , p-Akt, Akt, p-mTOR, and mTOR. For western blotting, β -actin was used as a loading control. For qRT-PCR, gene expression was normalized to 18 s rRNA. All data are presented as the mean ± SD ( n = 3). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001; ∗∗∗∗ P < 0.0001.
    Figure Legend Snippet: The effect of TMZ on DNA damage and repair regulating factors and key molecules of the PI3K signaling pathway in GBM cells. (a) SF295, U87, and U251 cells were treated with different concentrations of TMZ (10, 20, 40, 80, 160, 320, 640, and 1280 μ M) for 72 h. Cell viability was determined by WST-8 assay. (b)–(f) SF295 and U87 cells were treated with TMZ (120 μ M and 180 μ M) for 48 and 72 h. (b) The cells were then collected for western blotting detection of the DNA SSB marker XRCC1 and the DSB marker γ -H2AX. (c) The cells were harvested for qRT-PCR analysis of BRCA1 and RAD51. (d) Western blot analysis of DNA damage repair proteins, including p-ATM, BRCA1, RAD51, DNA-PKcs, and Ku80. (e) qRT-PCR analysis of PIK3CA and PIK3CB. (f) Western blot analysis of key proteins in the PI3K signaling pathway, including PI3K-p110 α , PI3K-p110 β , p-Akt, Akt, p-mTOR, and mTOR. For western blotting, β -actin was used as a loading control. For qRT-PCR, gene expression was normalized to 18 s rRNA. All data are presented as the mean ± SD ( n = 3). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001; ∗∗∗∗ P < 0.0001.

    Techniques Used: Western Blot, Marker, Quantitative RT-PCR, Expressing

    Effect of PI3K inhibitors and TMZ as single agents or in combination on the survival of GBM cells. (a) SF295, U87, and U251 cells were treated with various concentrations of ZSTK474 (0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, and 2 μ M) for 72 h. Cell viability was determined by WST-8 assay. (b) Three GBM cell lines were incubated with ZSTK474 and TMZ as single agents or in combination (20%, 40%, 60%, 80%, and 100% of 2IC 50ZSTK474 or IC 50ZSTK474 : IC 50TMZ ) for 72 h. The combination effects were analyzed using CalcuSyn software, and the resulting CI-Fa plots are shown. (c) SF295 and U87 cells were treated with BYL719/TGX221 and TMZ as single agents or in combination (20%, 40%, 60%, 80%, and 100% of IC 50BYL719/TGX221 : IC 50TMZ ) for 72 h. The combination effects were analyzed using CalcuSyn software, and the resulting CI-Fa plots are shown. All data are presented as the mean ± SD ( n = 3).
    Figure Legend Snippet: Effect of PI3K inhibitors and TMZ as single agents or in combination on the survival of GBM cells. (a) SF295, U87, and U251 cells were treated with various concentrations of ZSTK474 (0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, and 2 μ M) for 72 h. Cell viability was determined by WST-8 assay. (b) Three GBM cell lines were incubated with ZSTK474 and TMZ as single agents or in combination (20%, 40%, 60%, 80%, and 100% of 2IC 50ZSTK474 or IC 50ZSTK474 : IC 50TMZ ) for 72 h. The combination effects were analyzed using CalcuSyn software, and the resulting CI-Fa plots are shown. (c) SF295 and U87 cells were treated with BYL719/TGX221 and TMZ as single agents or in combination (20%, 40%, 60%, 80%, and 100% of IC 50BYL719/TGX221 : IC 50TMZ ) for 72 h. The combination effects were analyzed using CalcuSyn software, and the resulting CI-Fa plots are shown. All data are presented as the mean ± SD ( n = 3).

    Techniques Used: Incubation, Software

    Combination indexes (CI) of PI3K inhibitors and TMZ in GBM cells.
    Figure Legend Snippet: Combination indexes (CI) of PI3K inhibitors and TMZ in GBM cells.

    Techniques Used:

    The combined use of ZSTK474 and TMZ reduced HR repair efficiency. (a)–(d) SF295 and U87 cells were treated with ZSTK474 and TMZ as single agents or in combination for 48 h. (a) The formation of RAD51 foci was assessed using immunofluorescence. Cell nuclei were stained with DAPI. Scale bar, 20 μ m. Representative images are shown. (b) Quantification of the number of RAD51-positive SF295 and U87 cells. (c) The cells were collected for western blotting detection of NBS1, p-ATM, BRCA1, BRCA2, DNA-PKcs, and Ku80. (d) Western blotting analysis of PI3K-p110 β , p-Akt, p-mTOR, and c-Myc. All data are presented as the mean ± SD ( n = 3). ∗ P < 0.05; ∗∗ P < 0.01.
    Figure Legend Snippet: The combined use of ZSTK474 and TMZ reduced HR repair efficiency. (a)–(d) SF295 and U87 cells were treated with ZSTK474 and TMZ as single agents or in combination for 48 h. (a) The formation of RAD51 foci was assessed using immunofluorescence. Cell nuclei were stained with DAPI. Scale bar, 20 μ m. Representative images are shown. (b) Quantification of the number of RAD51-positive SF295 and U87 cells. (c) The cells were collected for western blotting detection of NBS1, p-ATM, BRCA1, BRCA2, DNA-PKcs, and Ku80. (d) Western blotting analysis of PI3K-p110 β , p-Akt, p-mTOR, and c-Myc. All data are presented as the mean ± SD ( n = 3). ∗ P < 0.05; ∗∗ P < 0.01.

    Techniques Used: Immunofluorescence, Staining, Western Blot

    pi3k p110 β  (Cell Signaling Technology Inc)


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    Structured Review

    Cell Signaling Technology Inc pi3k p110 β
    The effect of TMZ on DNA damage and repair regulating factors and key molecules of the <t>PI3K</t> signaling pathway in GBM cells. (a) SF295, U87, and U251 cells were treated with different concentrations of TMZ (10, 20, 40, 80, 160, 320, 640, and 1280 μ M) for 72 h. Cell viability was determined by WST-8 assay. (b)–(f) SF295 and U87 cells were treated with TMZ (120 μ M and 180 μ M) for 48 and 72 h. (b) The cells were then collected for western blotting detection of the DNA SSB marker XRCC1 and the DSB marker γ -H2AX. (c) The cells were harvested for qRT-PCR analysis of BRCA1 and RAD51. (d) Western blot analysis of DNA damage repair proteins, including p-ATM, BRCA1, RAD51, DNA-PKcs, and Ku80. (e) qRT-PCR analysis of PIK3CA and PIK3CB. (f) Western blot analysis of key proteins in the PI3K signaling pathway, including <t>PI3K-p110</t> α , PI3K-p110 β , p-Akt, Akt, p-mTOR, and mTOR. For western blotting, β -actin was used as a loading control. For qRT-PCR, gene expression was normalized to 18 s rRNA. All data are presented as the mean ± SD ( n = 3). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001; ∗∗∗∗ P < 0.0001.
    Pi3k P110 β, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "ZSTK474 Sensitizes Glioblastoma to Temozolomide by Blocking Homologous Recombination Repair"

    Article Title: ZSTK474 Sensitizes Glioblastoma to Temozolomide by Blocking Homologous Recombination Repair

    Journal: BioMed Research International

    doi: 10.1155/2022/8568528

    The effect of TMZ on DNA damage and repair regulating factors and key molecules of the PI3K signaling pathway in GBM cells. (a) SF295, U87, and U251 cells were treated with different concentrations of TMZ (10, 20, 40, 80, 160, 320, 640, and 1280 μ M) for 72 h. Cell viability was determined by WST-8 assay. (b)–(f) SF295 and U87 cells were treated with TMZ (120 μ M and 180 μ M) for 48 and 72 h. (b) The cells were then collected for western blotting detection of the DNA SSB marker XRCC1 and the DSB marker γ -H2AX. (c) The cells were harvested for qRT-PCR analysis of BRCA1 and RAD51. (d) Western blot analysis of DNA damage repair proteins, including p-ATM, BRCA1, RAD51, DNA-PKcs, and Ku80. (e) qRT-PCR analysis of PIK3CA and PIK3CB. (f) Western blot analysis of key proteins in the PI3K signaling pathway, including PI3K-p110 α , PI3K-p110 β , p-Akt, Akt, p-mTOR, and mTOR. For western blotting, β -actin was used as a loading control. For qRT-PCR, gene expression was normalized to 18 s rRNA. All data are presented as the mean ± SD ( n = 3). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001; ∗∗∗∗ P < 0.0001.
    Figure Legend Snippet: The effect of TMZ on DNA damage and repair regulating factors and key molecules of the PI3K signaling pathway in GBM cells. (a) SF295, U87, and U251 cells were treated with different concentrations of TMZ (10, 20, 40, 80, 160, 320, 640, and 1280 μ M) for 72 h. Cell viability was determined by WST-8 assay. (b)–(f) SF295 and U87 cells were treated with TMZ (120 μ M and 180 μ M) for 48 and 72 h. (b) The cells were then collected for western blotting detection of the DNA SSB marker XRCC1 and the DSB marker γ -H2AX. (c) The cells were harvested for qRT-PCR analysis of BRCA1 and RAD51. (d) Western blot analysis of DNA damage repair proteins, including p-ATM, BRCA1, RAD51, DNA-PKcs, and Ku80. (e) qRT-PCR analysis of PIK3CA and PIK3CB. (f) Western blot analysis of key proteins in the PI3K signaling pathway, including PI3K-p110 α , PI3K-p110 β , p-Akt, Akt, p-mTOR, and mTOR. For western blotting, β -actin was used as a loading control. For qRT-PCR, gene expression was normalized to 18 s rRNA. All data are presented as the mean ± SD ( n = 3). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001; ∗∗∗∗ P < 0.0001.

    Techniques Used: Western Blot, Marker, Quantitative RT-PCR, Expressing

    Effect of PI3K inhibitors and TMZ as single agents or in combination on the survival of GBM cells. (a) SF295, U87, and U251 cells were treated with various concentrations of ZSTK474 (0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, and 2 μ M) for 72 h. Cell viability was determined by WST-8 assay. (b) Three GBM cell lines were incubated with ZSTK474 and TMZ as single agents or in combination (20%, 40%, 60%, 80%, and 100% of 2IC 50ZSTK474 or IC 50ZSTK474 : IC 50TMZ ) for 72 h. The combination effects were analyzed using CalcuSyn software, and the resulting CI-Fa plots are shown. (c) SF295 and U87 cells were treated with BYL719/TGX221 and TMZ as single agents or in combination (20%, 40%, 60%, 80%, and 100% of IC 50BYL719/TGX221 : IC 50TMZ ) for 72 h. The combination effects were analyzed using CalcuSyn software, and the resulting CI-Fa plots are shown. All data are presented as the mean ± SD ( n = 3).
    Figure Legend Snippet: Effect of PI3K inhibitors and TMZ as single agents or in combination on the survival of GBM cells. (a) SF295, U87, and U251 cells were treated with various concentrations of ZSTK474 (0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, and 2 μ M) for 72 h. Cell viability was determined by WST-8 assay. (b) Three GBM cell lines were incubated with ZSTK474 and TMZ as single agents or in combination (20%, 40%, 60%, 80%, and 100% of 2IC 50ZSTK474 or IC 50ZSTK474 : IC 50TMZ ) for 72 h. The combination effects were analyzed using CalcuSyn software, and the resulting CI-Fa plots are shown. (c) SF295 and U87 cells were treated with BYL719/TGX221 and TMZ as single agents or in combination (20%, 40%, 60%, 80%, and 100% of IC 50BYL719/TGX221 : IC 50TMZ ) for 72 h. The combination effects were analyzed using CalcuSyn software, and the resulting CI-Fa plots are shown. All data are presented as the mean ± SD ( n = 3).

    Techniques Used: Incubation, Software

    Combination indexes (CI) of PI3K inhibitors and TMZ in GBM cells.
    Figure Legend Snippet: Combination indexes (CI) of PI3K inhibitors and TMZ in GBM cells.

    Techniques Used:

    The combined use of ZSTK474 and TMZ reduced HR repair efficiency. (a)–(d) SF295 and U87 cells were treated with ZSTK474 and TMZ as single agents or in combination for 48 h. (a) The formation of RAD51 foci was assessed using immunofluorescence. Cell nuclei were stained with DAPI. Scale bar, 20 μ m. Representative images are shown. (b) Quantification of the number of RAD51-positive SF295 and U87 cells. (c) The cells were collected for western blotting detection of NBS1, p-ATM, BRCA1, BRCA2, DNA-PKcs, and Ku80. (d) Western blotting analysis of PI3K-p110 β , p-Akt, p-mTOR, and c-Myc. All data are presented as the mean ± SD ( n = 3). ∗ P < 0.05; ∗∗ P < 0.01.
    Figure Legend Snippet: The combined use of ZSTK474 and TMZ reduced HR repair efficiency. (a)–(d) SF295 and U87 cells were treated with ZSTK474 and TMZ as single agents or in combination for 48 h. (a) The formation of RAD51 foci was assessed using immunofluorescence. Cell nuclei were stained with DAPI. Scale bar, 20 μ m. Representative images are shown. (b) Quantification of the number of RAD51-positive SF295 and U87 cells. (c) The cells were collected for western blotting detection of NBS1, p-ATM, BRCA1, BRCA2, DNA-PKcs, and Ku80. (d) Western blotting analysis of PI3K-p110 β , p-Akt, p-mTOR, and c-Myc. All data are presented as the mean ± SD ( n = 3). ∗ P < 0.05; ∗∗ P < 0.01.

    Techniques Used: Immunofluorescence, Staining, Western Blot

    pi3k p110 isoforms  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc pi3k p110 isoforms
    Selectivity and effective concentrations of inhibitors used in this study (reported in the following references: 11, 26, 31, 34, 38, 40).
    Pi3k P110 Isoforms, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Effects of Novel Isoform-Selective Phosphoinositide 3-Kinase Inhibitors on Natural Killer Cell Function"

    Article Title: Effects of Novel Isoform-Selective Phosphoinositide 3-Kinase Inhibitors on Natural Killer Cell Function

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0099486

    Selectivity and effective concentrations of inhibitors used in this study (reported in the following references: 11, 26, 31, 34, 38, 40).
    Figure Legend Snippet: Selectivity and effective concentrations of inhibitors used in this study (reported in the following references: 11, 26, 31, 34, 38, 40).

    Techniques Used: In Vitro

    ( A ) Immunoblots were prepared using equivalent protein amounts from lysates of purified NK cells, T cells, B cells or two solid tumor cell lines. Blots were probed with PI3K isoform-specific antibodies and for β-actin as a loading control. ( B ) RMA-S, YAC-1, and EL4 H60 cells were labeled with calcein AM and co-cultured with NK cells at the indicated E:T ratios in the presence of 1 µM indicated inhibitors (TGX-221 was 0.5 µM) for 2 h. ( C ) RMA-S and YAC-1 cells were labeled with calcein AM and co-cultured with NK cells at 10∶1 E:T ratio in the presence of 1 µM indicated inhibitors for 2 h. ( D ) NK cells were treated with a higher concentration of MLN1117 (2 µM) and incubated with calcein AM-labeled YAC-1 cells at 10∶1 E:T ratio. Percentage specific lysis was calculated from pentaplicate cultures as follows: ((mean experimental release - mean spontaneous release)/(mean maximum release -mean spontaneous release)) x 100. The values are presented as the means ± SEM from three independent experiments. * p <0.05 and ** p <0.01, as determined by the unpaired Student's t test using Sigma Plot 10 software and as compared to the vehicle control group.
    Figure Legend Snippet: ( A ) Immunoblots were prepared using equivalent protein amounts from lysates of purified NK cells, T cells, B cells or two solid tumor cell lines. Blots were probed with PI3K isoform-specific antibodies and for β-actin as a loading control. ( B ) RMA-S, YAC-1, and EL4 H60 cells were labeled with calcein AM and co-cultured with NK cells at the indicated E:T ratios in the presence of 1 µM indicated inhibitors (TGX-221 was 0.5 µM) for 2 h. ( C ) RMA-S and YAC-1 cells were labeled with calcein AM and co-cultured with NK cells at 10∶1 E:T ratio in the presence of 1 µM indicated inhibitors for 2 h. ( D ) NK cells were treated with a higher concentration of MLN1117 (2 µM) and incubated with calcein AM-labeled YAC-1 cells at 10∶1 E:T ratio. Percentage specific lysis was calculated from pentaplicate cultures as follows: ((mean experimental release - mean spontaneous release)/(mean maximum release -mean spontaneous release)) x 100. The values are presented as the means ± SEM from three independent experiments. * p <0.05 and ** p <0.01, as determined by the unpaired Student's t test using Sigma Plot 10 software and as compared to the vehicle control group.

    Techniques Used: Western Blot, Purification, Labeling, Cell Culture, Concentration Assay, Incubation, Lysis, Software

    Wild-type C57BL/6 mice were orally dosed with vehicle, the p110α selective inhibitor MLN1117 (60 mg/kg), or the pan-PI3K inhibitor GDC-0941 (70 mg/kg) for 7 days (n = 4). Spleen and bone marrow (BM) were isolated and analyzed for NK cell development by flow cytometry. ( A ) Splenocytes were stained with CD21 and CD23 for marginal zone (MZ) B cell population. Among the B220 + population, CD21 high CD23 low cells were gated for MZ B cell population. ( B, C ) Percentage of B/T cells and Th/Tc cells in spleen were determined using surface staining with anti-B220/anti-Thy1.2 mAbs and anti-CD4/anti-CD8 mAbs, respectively. ( D ) NK cells as specified by CD3 − NK1.1 + were gated and analyzed in single-cell suspension of spleen and BM, respectively. ( E ) Spleen- or BM-derived CD3 − NK1.1 + NK cells were analyzed for NK subset based on the expression level of CD11b and CD27. ( F ) Spleen- or BM-derived CD3 − NCR1 + NK cells were analyzed for maturation marker, Ly49A, LY49C/I, Ly49D, Ly49G2, and Ly49H. ( G ) Spleen- or BM-derived CD3 − NCR1 + NK cells were analyzed for the expression of CD122, NKG2D, and NKG2A/C/E. The data are expressed as the means ± SEM. Statistical analysis was performed with the unpaired Student's t test using Sigma Plot 10 software to compare the differences between vehicle and each inhibitor-treated group. *, p <0.05, **, p <0.01, and ***, p <0.001.
    Figure Legend Snippet: Wild-type C57BL/6 mice were orally dosed with vehicle, the p110α selective inhibitor MLN1117 (60 mg/kg), or the pan-PI3K inhibitor GDC-0941 (70 mg/kg) for 7 days (n = 4). Spleen and bone marrow (BM) were isolated and analyzed for NK cell development by flow cytometry. ( A ) Splenocytes were stained with CD21 and CD23 for marginal zone (MZ) B cell population. Among the B220 + population, CD21 high CD23 low cells were gated for MZ B cell population. ( B, C ) Percentage of B/T cells and Th/Tc cells in spleen were determined using surface staining with anti-B220/anti-Thy1.2 mAbs and anti-CD4/anti-CD8 mAbs, respectively. ( D ) NK cells as specified by CD3 − NK1.1 + were gated and analyzed in single-cell suspension of spleen and BM, respectively. ( E ) Spleen- or BM-derived CD3 − NK1.1 + NK cells were analyzed for NK subset based on the expression level of CD11b and CD27. ( F ) Spleen- or BM-derived CD3 − NCR1 + NK cells were analyzed for maturation marker, Ly49A, LY49C/I, Ly49D, Ly49G2, and Ly49H. ( G ) Spleen- or BM-derived CD3 − NCR1 + NK cells were analyzed for the expression of CD122, NKG2D, and NKG2A/C/E. The data are expressed as the means ± SEM. Statistical analysis was performed with the unpaired Student's t test using Sigma Plot 10 software to compare the differences between vehicle and each inhibitor-treated group. *, p <0.05, **, p <0.01, and ***, p <0.001.

    Techniques Used: Isolation, Flow Cytometry, Staining, Derivative Assay, Expressing, Marker, Software

    pi3k p110  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc pi3k p110
    (A) SLAMF3 expression was confirmed by Western blot analysis in SLAMF3-over-expressing Huh-7 and mock cells at 48 h post-transfection. ERK1/2, JNK, p38, AKT and mTOR proteins were detected as controls. The activation levels of MAPK ERK1/2 (Thr 202/Tyr 204), JNK (Thr 183/Tyr 185), p38 (Thr 180/Tyr 183), <t>PI3K</t> <t>(p85/p110</t> Thr 467/Tyr 199), AKT (Ser 473, Thr 308) and mTOR (Ser 2448, Ser 2481) are represented. One of three representative experiments is shown here. (B) The results of an active caspase assay based on cell-permeable fluorochrome inhibitor of caspases (FLICA). Caspase activity was evaluated in HCC cells (Huh-7 and HepG2 lines) at the indicated times after the ectopic introduction of SLAMF3 vector. Caspase activity is shown in the SLAMF3 neg subpopulation (in white) and the SLAMF3 pos (overlaid in grey) subpopulation from one representative of two independent experiments.
    Pi3k P110, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Identification of SLAMF3 (CD229) as an Inhibitor of Hepatocellular Carcinoma Cell Proliferation and Tumour Progression"

    Article Title: Identification of SLAMF3 (CD229) as an Inhibitor of Hepatocellular Carcinoma Cell Proliferation and Tumour Progression

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0082918

    (A) SLAMF3 expression was confirmed by Western blot analysis in SLAMF3-over-expressing Huh-7 and mock cells at 48 h post-transfection. ERK1/2, JNK, p38, AKT and mTOR proteins were detected as controls. The activation levels of MAPK ERK1/2 (Thr 202/Tyr 204), JNK (Thr 183/Tyr 185), p38 (Thr 180/Tyr 183), PI3K (p85/p110 Thr 467/Tyr 199), AKT (Ser 473, Thr 308) and mTOR (Ser 2448, Ser 2481) are represented. One of three representative experiments is shown here. (B) The results of an active caspase assay based on cell-permeable fluorochrome inhibitor of caspases (FLICA). Caspase activity was evaluated in HCC cells (Huh-7 and HepG2 lines) at the indicated times after the ectopic introduction of SLAMF3 vector. Caspase activity is shown in the SLAMF3 neg subpopulation (in white) and the SLAMF3 pos (overlaid in grey) subpopulation from one representative of two independent experiments.
    Figure Legend Snippet: (A) SLAMF3 expression was confirmed by Western blot analysis in SLAMF3-over-expressing Huh-7 and mock cells at 48 h post-transfection. ERK1/2, JNK, p38, AKT and mTOR proteins were detected as controls. The activation levels of MAPK ERK1/2 (Thr 202/Tyr 204), JNK (Thr 183/Tyr 185), p38 (Thr 180/Tyr 183), PI3K (p85/p110 Thr 467/Tyr 199), AKT (Ser 473, Thr 308) and mTOR (Ser 2448, Ser 2481) are represented. One of three representative experiments is shown here. (B) The results of an active caspase assay based on cell-permeable fluorochrome inhibitor of caspases (FLICA). Caspase activity was evaluated in HCC cells (Huh-7 and HepG2 lines) at the indicated times after the ectopic introduction of SLAMF3 vector. Caspase activity is shown in the SLAMF3 neg subpopulation (in white) and the SLAMF3 pos (overlaid in grey) subpopulation from one representative of two independent experiments.

    Techniques Used: Expressing, Western Blot, Transfection, Activation Assay, Caspase Assay, Activity Assay, Plasmid Preparation

    pi3k p110 α 4249 antibodies  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc pi3k p110 α 4249 antibodies
    S1P-induced COX-2 expression is mediated through <t>PI3K/Akt</t> in HCFs. (a) Cells were pretreated without or with LY294002 (0.3, 3, and 30 μ M) for 1 h and then incubated with 15 μ M S1P for 8 h. The levels of COX-2 and GAPDH used as an internal control were analyzed by Western blot. (b) Cells were transfected without or with COX-2 promoter-luciferase reporter gene, pretreated with LY294002 (30 μ M) for 1 h, and then incubated with 15 μ M S1P for 4 h (mRNA level) or 1 h (promoter activity). The COX-2 mRNA and promoter activity were analyzed by real-time PCR (open bar) and promoter assay (gray bar). (c) Cells were transfected with scrambled, <t>p110</t> (left panel), or Akt (right panel) siRNA for 24 h and then exposed to 15 μ M S1P for 8 h. The levels of COX-2, GAPDH, p110, and Akt proteins were analyzed by Western blot. (d) Cells were pretreated with or without 30 μ M LY294002, 10 μ M MMP2/9 inhibitor, 10 μ g/ml CRM197, or 10 μ M AG1478 for 1 h and then incubated with 15 μ M S1P for 0.5, 1, 3, 5, and 10 min. The levels of phospho-Akt and GAPDH were determined by Western blot. (e) Cells were pretreated without or with 30 μ M LY294002 for 1 h and then incubated with 15 μ M S1P for 0, 3, 5, 10, 15, and 30 min. The levels of phospho-p44/p42, phospho-p38, phospho-JNK1/2, and GAPDH were determined by Western blot. Data are expressed as the mean ± SEM of three individual experiments ( n = 3). # P < 0.05, as compared with the control or pretreatment with inhibitor indicated in the figure.
    Pi3k P110 α 4249 Antibodies, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Sphingosine 1-Phosphate-Upregulated COX-2/PGE 2 System Contributes to Human Cardiac Fibroblast Apoptosis: Involvement of MMP-9-Dependent Transactivation of EGFR Cascade"

    Article Title: Sphingosine 1-Phosphate-Upregulated COX-2/PGE 2 System Contributes to Human Cardiac Fibroblast Apoptosis: Involvement of MMP-9-Dependent Transactivation of EGFR Cascade

    Journal: Oxidative Medicine and Cellular Longevity

    doi: 10.1155/2022/7664290

    S1P-induced COX-2 expression is mediated through PI3K/Akt in HCFs. (a) Cells were pretreated without or with LY294002 (0.3, 3, and 30 μ M) for 1 h and then incubated with 15 μ M S1P for 8 h. The levels of COX-2 and GAPDH used as an internal control were analyzed by Western blot. (b) Cells were transfected without or with COX-2 promoter-luciferase reporter gene, pretreated with LY294002 (30 μ M) for 1 h, and then incubated with 15 μ M S1P for 4 h (mRNA level) or 1 h (promoter activity). The COX-2 mRNA and promoter activity were analyzed by real-time PCR (open bar) and promoter assay (gray bar). (c) Cells were transfected with scrambled, p110 (left panel), or Akt (right panel) siRNA for 24 h and then exposed to 15 μ M S1P for 8 h. The levels of COX-2, GAPDH, p110, and Akt proteins were analyzed by Western blot. (d) Cells were pretreated with or without 30 μ M LY294002, 10 μ M MMP2/9 inhibitor, 10 μ g/ml CRM197, or 10 μ M AG1478 for 1 h and then incubated with 15 μ M S1P for 0.5, 1, 3, 5, and 10 min. The levels of phospho-Akt and GAPDH were determined by Western blot. (e) Cells were pretreated without or with 30 μ M LY294002 for 1 h and then incubated with 15 μ M S1P for 0, 3, 5, 10, 15, and 30 min. The levels of phospho-p44/p42, phospho-p38, phospho-JNK1/2, and GAPDH were determined by Western blot. Data are expressed as the mean ± SEM of three individual experiments ( n = 3). # P < 0.05, as compared with the control or pretreatment with inhibitor indicated in the figure.
    Figure Legend Snippet: S1P-induced COX-2 expression is mediated through PI3K/Akt in HCFs. (a) Cells were pretreated without or with LY294002 (0.3, 3, and 30 μ M) for 1 h and then incubated with 15 μ M S1P for 8 h. The levels of COX-2 and GAPDH used as an internal control were analyzed by Western blot. (b) Cells were transfected without or with COX-2 promoter-luciferase reporter gene, pretreated with LY294002 (30 μ M) for 1 h, and then incubated with 15 μ M S1P for 4 h (mRNA level) or 1 h (promoter activity). The COX-2 mRNA and promoter activity were analyzed by real-time PCR (open bar) and promoter assay (gray bar). (c) Cells were transfected with scrambled, p110 (left panel), or Akt (right panel) siRNA for 24 h and then exposed to 15 μ M S1P for 8 h. The levels of COX-2, GAPDH, p110, and Akt proteins were analyzed by Western blot. (d) Cells were pretreated with or without 30 μ M LY294002, 10 μ M MMP2/9 inhibitor, 10 μ g/ml CRM197, or 10 μ M AG1478 for 1 h and then incubated with 15 μ M S1P for 0.5, 1, 3, 5, and 10 min. The levels of phospho-Akt and GAPDH were determined by Western blot. (e) Cells were pretreated without or with 30 μ M LY294002 for 1 h and then incubated with 15 μ M S1P for 0, 3, 5, 10, 15, and 30 min. The levels of phospho-p44/p42, phospho-p38, phospho-JNK1/2, and GAPDH were determined by Western blot. Data are expressed as the mean ± SEM of three individual experiments ( n = 3). # P < 0.05, as compared with the control or pretreatment with inhibitor indicated in the figure.

    Techniques Used: Expressing, Incubation, Western Blot, Transfection, Luciferase, Activity Assay, Real-time Polymerase Chain Reaction, Promoter Assay

    Schematic signaling pathways are involved in S1P-induced COX-2/PGE 2 expression associated with activation of caspase-3 leading to apoptosis in HCFs. S1P could be upregulated mediated through estrogen/estrogen receptor-stimulated sphingosine kinase-1 (SphK1) activity , binding with S1PR1/3 coupled to G q or G i protein results in MMP9-mediated HB-EGF cleavage leading to activation of EGFR/PI3K/Akt/MAPKs (i.e., p42/p44 MAPK, p38 MAPK, and JNK1/2)-dependent AP-1 c-Jun. The COX-2 transcription and PGE 2 generation are dependently regulated by an S1PR1/3-mediated EGFR transactivation to activate AP-1 binding activity. These signaling pathways contribute to the activation of AP-1 required for COX-2 expression and PGE 2 generation in HCFs.
    Figure Legend Snippet: Schematic signaling pathways are involved in S1P-induced COX-2/PGE 2 expression associated with activation of caspase-3 leading to apoptosis in HCFs. S1P could be upregulated mediated through estrogen/estrogen receptor-stimulated sphingosine kinase-1 (SphK1) activity , binding with S1PR1/3 coupled to G q or G i protein results in MMP9-mediated HB-EGF cleavage leading to activation of EGFR/PI3K/Akt/MAPKs (i.e., p42/p44 MAPK, p38 MAPK, and JNK1/2)-dependent AP-1 c-Jun. The COX-2 transcription and PGE 2 generation are dependently regulated by an S1PR1/3-mediated EGFR transactivation to activate AP-1 binding activity. These signaling pathways contribute to the activation of AP-1 required for COX-2 expression and PGE 2 generation in HCFs.

    Techniques Used: Expressing, Activation Assay, Activity Assay, Binding Assay

    pi3k p110  (Cell Signaling Technology Inc)


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    Structured Review

    Cell Signaling Technology Inc pi3k p110
    <t>PI3K/AKT</t> and JNK/c-Jun pathway facilitated by Quisinostat were required for cell cycle arrest and cell apoptosis. (A) HCCLM3 and SMMC-7721 cells were treated with quisinostat (12.5, 25.0 and 50.0nM) for 48 h. Western blot analysis was used to evaluate expressions of <t>PI3K-p110,</t> PI3K-p85, phosphorylation of AKT 473 , JNK, phosphorylation of JNK and c-Jun. Data, mean ± SD (n = 3); *, P < 0.05; **, P < 0.01 compared with DMSO group. (B) Cells were preincubated for 5 h with or without MK2206 2HCL(5μM), followed by culturing with 25nM quisinostat for 48 h, then conducting the cell cycle analysis and (C) Expression levels of phosphorylation of AKT 473 and p21 proteins were detected by Western Blotting. Data were shown as mean ± SD. n = 3; *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with the DMSO group. (D) Cells were pretreated with or without 40μM Z-VAD-FMK for 2 h, followed by culturing with 25nM quisinostat for 48 h to conduct apoptosis analysis, (E) cell proliferation analysis and (F) Expression levels of cle-Caspase9, cle-Caspase3, PARP, and cle-PARP proteins were detected by Western Blotting. Data were shown as mean ± SD. n = 3; *P <0.05; **P < 0.01; ***P < 0.001 compared with Quisinostat group. (G) Cells were pretreated with SP600125 (6μM) for 5 h followed by culturing with 25nM quisinostat for 48 h to conduct apoptosis analysis, (H) cell proliferation analysis and (I) Expression levels of phosphorylation of JNK, cle-Caspase3 and cle-PARP proteins were detected by Western Blotting. Data were shown as mean ± SD. n = 3; *P <0.05; **P < 0.01; ***P < 0.001 compared with Quisinostat group.
    Pi3k P110, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "The HDAC Inhibitor Quisinostat (JNJ-26481585) Supresses Hepatocellular Carcinoma alone and Synergistically in Combination with Sorafenib by G0/G1 phase arrest and Apoptosis induction"

    Article Title: The HDAC Inhibitor Quisinostat (JNJ-26481585) Supresses Hepatocellular Carcinoma alone and Synergistically in Combination with Sorafenib by G0/G1 phase arrest and Apoptosis induction

    Journal: International Journal of Biological Sciences

    doi: 10.7150/ijbs.27661

    PI3K/AKT and JNK/c-Jun pathway facilitated by Quisinostat were required for cell cycle arrest and cell apoptosis. (A) HCCLM3 and SMMC-7721 cells were treated with quisinostat (12.5, 25.0 and 50.0nM) for 48 h. Western blot analysis was used to evaluate expressions of PI3K-p110, PI3K-p85, phosphorylation of AKT 473 , JNK, phosphorylation of JNK and c-Jun. Data, mean ± SD (n = 3); *, P < 0.05; **, P < 0.01 compared with DMSO group. (B) Cells were preincubated for 5 h with or without MK2206 2HCL(5μM), followed by culturing with 25nM quisinostat for 48 h, then conducting the cell cycle analysis and (C) Expression levels of phosphorylation of AKT 473 and p21 proteins were detected by Western Blotting. Data were shown as mean ± SD. n = 3; *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with the DMSO group. (D) Cells were pretreated with or without 40μM Z-VAD-FMK for 2 h, followed by culturing with 25nM quisinostat for 48 h to conduct apoptosis analysis, (E) cell proliferation analysis and (F) Expression levels of cle-Caspase9, cle-Caspase3, PARP, and cle-PARP proteins were detected by Western Blotting. Data were shown as mean ± SD. n = 3; *P <0.05; **P < 0.01; ***P < 0.001 compared with Quisinostat group. (G) Cells were pretreated with SP600125 (6μM) for 5 h followed by culturing with 25nM quisinostat for 48 h to conduct apoptosis analysis, (H) cell proliferation analysis and (I) Expression levels of phosphorylation of JNK, cle-Caspase3 and cle-PARP proteins were detected by Western Blotting. Data were shown as mean ± SD. n = 3; *P <0.05; **P < 0.01; ***P < 0.001 compared with Quisinostat group.
    Figure Legend Snippet: PI3K/AKT and JNK/c-Jun pathway facilitated by Quisinostat were required for cell cycle arrest and cell apoptosis. (A) HCCLM3 and SMMC-7721 cells were treated with quisinostat (12.5, 25.0 and 50.0nM) for 48 h. Western blot analysis was used to evaluate expressions of PI3K-p110, PI3K-p85, phosphorylation of AKT 473 , JNK, phosphorylation of JNK and c-Jun. Data, mean ± SD (n = 3); *, P < 0.05; **, P < 0.01 compared with DMSO group. (B) Cells were preincubated for 5 h with or without MK2206 2HCL(5μM), followed by culturing with 25nM quisinostat for 48 h, then conducting the cell cycle analysis and (C) Expression levels of phosphorylation of AKT 473 and p21 proteins were detected by Western Blotting. Data were shown as mean ± SD. n = 3; *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with the DMSO group. (D) Cells were pretreated with or without 40μM Z-VAD-FMK for 2 h, followed by culturing with 25nM quisinostat for 48 h to conduct apoptosis analysis, (E) cell proliferation analysis and (F) Expression levels of cle-Caspase9, cle-Caspase3, PARP, and cle-PARP proteins were detected by Western Blotting. Data were shown as mean ± SD. n = 3; *P <0.05; **P < 0.01; ***P < 0.001 compared with Quisinostat group. (G) Cells were pretreated with SP600125 (6μM) for 5 h followed by culturing with 25nM quisinostat for 48 h to conduct apoptosis analysis, (H) cell proliferation analysis and (I) Expression levels of phosphorylation of JNK, cle-Caspase3 and cle-PARP proteins were detected by Western Blotting. Data were shown as mean ± SD. n = 3; *P <0.05; **P < 0.01; ***P < 0.001 compared with Quisinostat group.

    Techniques Used: Western Blot, Cell Cycle Assay, Expressing

    pi3k p110  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc pi3k p110
    TDRG1 is significantly upregulated in testicular seminoma tissues than in normal testicular tissues. Notes: ( A ) RT-PCR showed that the relative gene expression level of TDRG1 is significantly higher in testicular seminoma tissues than in normal testicular tissues. ( B i) Representative Western blotting images that are associated with TDRG1 and GAPDH protein expression in both testicular seminoma and normal testicular tissues. ( B ii) Western blotting showed that the relative protein expression level of TDRG1 is significantly higher in testicular seminoma tissues than in normal testicular tissues. RT-PCR and Western blotting were performed to detect the expression levels of TDRG1 in both testicular seminoma tissues (n=33) and normal testicular tissues (n=33). ( C i) Immunohistochemical staining pictures of both testicular seminoma and normal testicular tissue samples with anti- TDRG1 , <t>anti-p-PI3K/p110,</t> and anti-p-Akt antibodies. ( C ii) The protein expression levels of TDRG1 , p-PI3K/p110, and p-Akt are significantly upregulated in testicular seminoma tissues compared to normal testicular tissues. Protein expression levels of TDRG1 , p-PI3K/p110, and p-Akt were detected by an immunohistochemistry assay. The tissue microarray chips TE802 and TE2081 contained 80 points (from 80 testicular seminoma tissue samples) and 138 points (from 46 testicular seminoma tissue samples, three points per sample), respectively. TEN601 had 60 points (from 30 normal testicular tissue samples, two points per sample). Bars representing cancer are averages of data from 218 points (80+138) in two tissue microarray chips. Bars representing the normal tissue samples are the average of data from 60 points in a tissue microarray chip. * P <0.05 vs control group. Cancer: testicular seminoma; normal: normal testicular tissues. Abbreviations: TDRG1 , testis development-related gene 1; RT-PCR, reverse transcription polymerase chain reaction; PI3K, phosphoinositide-3 kinase; GADPH, glyceral-dehyde-3-phosphate dehydrogenase.
    Pi3k P110, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "TDRG1 functions in testicular seminoma are dependent on the PI3K/Akt/mTOR signaling pathway"

    Article Title: TDRG1 functions in testicular seminoma are dependent on the PI3K/Akt/mTOR signaling pathway

    Journal: OncoTargets and therapy

    doi: 10.2147/OTT.S97294

    TDRG1 is significantly upregulated in testicular seminoma tissues than in normal testicular tissues. Notes: ( A ) RT-PCR showed that the relative gene expression level of TDRG1 is significantly higher in testicular seminoma tissues than in normal testicular tissues. ( B i) Representative Western blotting images that are associated with TDRG1 and GAPDH protein expression in both testicular seminoma and normal testicular tissues. ( B ii) Western blotting showed that the relative protein expression level of TDRG1 is significantly higher in testicular seminoma tissues than in normal testicular tissues. RT-PCR and Western blotting were performed to detect the expression levels of TDRG1 in both testicular seminoma tissues (n=33) and normal testicular tissues (n=33). ( C i) Immunohistochemical staining pictures of both testicular seminoma and normal testicular tissue samples with anti- TDRG1 , anti-p-PI3K/p110, and anti-p-Akt antibodies. ( C ii) The protein expression levels of TDRG1 , p-PI3K/p110, and p-Akt are significantly upregulated in testicular seminoma tissues compared to normal testicular tissues. Protein expression levels of TDRG1 , p-PI3K/p110, and p-Akt were detected by an immunohistochemistry assay. The tissue microarray chips TE802 and TE2081 contained 80 points (from 80 testicular seminoma tissue samples) and 138 points (from 46 testicular seminoma tissue samples, three points per sample), respectively. TEN601 had 60 points (from 30 normal testicular tissue samples, two points per sample). Bars representing cancer are averages of data from 218 points (80+138) in two tissue microarray chips. Bars representing the normal tissue samples are the average of data from 60 points in a tissue microarray chip. * P <0.05 vs control group. Cancer: testicular seminoma; normal: normal testicular tissues. Abbreviations: TDRG1 , testis development-related gene 1; RT-PCR, reverse transcription polymerase chain reaction; PI3K, phosphoinositide-3 kinase; GADPH, glyceral-dehyde-3-phosphate dehydrogenase.
    Figure Legend Snippet: TDRG1 is significantly upregulated in testicular seminoma tissues than in normal testicular tissues. Notes: ( A ) RT-PCR showed that the relative gene expression level of TDRG1 is significantly higher in testicular seminoma tissues than in normal testicular tissues. ( B i) Representative Western blotting images that are associated with TDRG1 and GAPDH protein expression in both testicular seminoma and normal testicular tissues. ( B ii) Western blotting showed that the relative protein expression level of TDRG1 is significantly higher in testicular seminoma tissues than in normal testicular tissues. RT-PCR and Western blotting were performed to detect the expression levels of TDRG1 in both testicular seminoma tissues (n=33) and normal testicular tissues (n=33). ( C i) Immunohistochemical staining pictures of both testicular seminoma and normal testicular tissue samples with anti- TDRG1 , anti-p-PI3K/p110, and anti-p-Akt antibodies. ( C ii) The protein expression levels of TDRG1 , p-PI3K/p110, and p-Akt are significantly upregulated in testicular seminoma tissues compared to normal testicular tissues. Protein expression levels of TDRG1 , p-PI3K/p110, and p-Akt were detected by an immunohistochemistry assay. The tissue microarray chips TE802 and TE2081 contained 80 points (from 80 testicular seminoma tissue samples) and 138 points (from 46 testicular seminoma tissue samples, three points per sample), respectively. TEN601 had 60 points (from 30 normal testicular tissue samples, two points per sample). Bars representing cancer are averages of data from 218 points (80+138) in two tissue microarray chips. Bars representing the normal tissue samples are the average of data from 60 points in a tissue microarray chip. * P <0.05 vs control group. Cancer: testicular seminoma; normal: normal testicular tissues. Abbreviations: TDRG1 , testis development-related gene 1; RT-PCR, reverse transcription polymerase chain reaction; PI3K, phosphoinositide-3 kinase; GADPH, glyceral-dehyde-3-phosphate dehydrogenase.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Immunohistochemical staining, Staining, Immunohistochemistry, Microarray

    Relative TDRG1 expression in both gene and protein levels of seminoma TCam-2 cells after different treatments. Notes: ( A ) Relative gene expression level of TDRG1 in seminoma TCam-2 cells was remarkably upregulated and downregulated after the gene was knocked out and overexpressed, respectively, on the basis of RT-PCR (n=5). ( B ) Relative protein expression level of TDRG1 in seminoma TCam-2 cells was remarkably upregulated and downregulated after the gene was knocked out and overexpressed, respectively, but was unaffected after treatment with LY294002 or insulin-like growth factor-1 on the basis of Western blotting. * P <0.05 and ** P <0.01 vs control group. Vector: enhanced green fluorescent protein plasmid-C1 vector. Abbreviations: TDRG1 , testis development-related gene 1; RT-PCR, reverse transcription polymerase chain reaction; PI3K, phosphoinositide-3 kinase; TDRG1 (−), the knockout of TDRG1 ; TDRG1 (+), the overexpression of TDRG1 ; PI3K (−), the inhibition of PI3K by LY294002; PI3K (+), the activation of PI3K by insulin-like growth factor-1.
    Figure Legend Snippet: Relative TDRG1 expression in both gene and protein levels of seminoma TCam-2 cells after different treatments. Notes: ( A ) Relative gene expression level of TDRG1 in seminoma TCam-2 cells was remarkably upregulated and downregulated after the gene was knocked out and overexpressed, respectively, on the basis of RT-PCR (n=5). ( B ) Relative protein expression level of TDRG1 in seminoma TCam-2 cells was remarkably upregulated and downregulated after the gene was knocked out and overexpressed, respectively, but was unaffected after treatment with LY294002 or insulin-like growth factor-1 on the basis of Western blotting. * P <0.05 and ** P <0.01 vs control group. Vector: enhanced green fluorescent protein plasmid-C1 vector. Abbreviations: TDRG1 , testis development-related gene 1; RT-PCR, reverse transcription polymerase chain reaction; PI3K, phosphoinositide-3 kinase; TDRG1 (−), the knockout of TDRG1 ; TDRG1 (+), the overexpression of TDRG1 ; PI3K (−), the inhibition of PI3K by LY294002; PI3K (+), the activation of PI3K by insulin-like growth factor-1.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Plasmid Preparation, Knock-Out, Over Expression, Inhibition, Activation Assay

    The effect of TDRG1 - and PI3K-mediated signaling on the cell proliferation rate, cell proliferation index, and apoptosis rate. Notes: ( A ) Cells with both TDRG1 knockout and PI3K inhibition exhibited decreased cell proliferation. ( B i and B ii) Cells with both TDRG1 knockout and PI3K inhibition exhibited decreased cell proliferation index. ( C i and C ii) Cells with both TDRG1 knockout and PI3K inhibition exhibited increased apoptosis rate. Seminoma TCam-2 cells were subjected to different treatments: the TDRG1 knockout, TDRG1 overexpression, PI3K inhibition (LY294002 administration), or PI3K activation (IGF-1 administration). The cell proliferation rate was assessed using MTT kits at different time points after the treatments, and cell proliferation index and apoptosis rate were evaluated using flow cytometry at 72 h. The cell proliferation index was obtained by calculating the percentage of cells in the S, G2, and M phases. * P <0.05 and ** P <0.01 vs control group. Vector: enhanced green fluorescent protein plasmid-C1 vector. Abbreviations: TDRG1 , testis development-related gene 1; PI3K, phosphoinositide-3 kinase; IGF-1, insulin-like growth factor-1; MTT, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide; h, hours; TDRG1 (−), the knockout of TDRG1 ; TDRG1 (+), the overexpression of TDRG1 ; PI3K (−), the inhibition of PI3K by LY294002; PI3K (+), the activation of PI3K by IGF-1; FL2-A, fluorescence 2 area; FITC, fluorescein isothiocyanate.
    Figure Legend Snippet: The effect of TDRG1 - and PI3K-mediated signaling on the cell proliferation rate, cell proliferation index, and apoptosis rate. Notes: ( A ) Cells with both TDRG1 knockout and PI3K inhibition exhibited decreased cell proliferation. ( B i and B ii) Cells with both TDRG1 knockout and PI3K inhibition exhibited decreased cell proliferation index. ( C i and C ii) Cells with both TDRG1 knockout and PI3K inhibition exhibited increased apoptosis rate. Seminoma TCam-2 cells were subjected to different treatments: the TDRG1 knockout, TDRG1 overexpression, PI3K inhibition (LY294002 administration), or PI3K activation (IGF-1 administration). The cell proliferation rate was assessed using MTT kits at different time points after the treatments, and cell proliferation index and apoptosis rate were evaluated using flow cytometry at 72 h. The cell proliferation index was obtained by calculating the percentage of cells in the S, G2, and M phases. * P <0.05 and ** P <0.01 vs control group. Vector: enhanced green fluorescent protein plasmid-C1 vector. Abbreviations: TDRG1 , testis development-related gene 1; PI3K, phosphoinositide-3 kinase; IGF-1, insulin-like growth factor-1; MTT, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide; h, hours; TDRG1 (−), the knockout of TDRG1 ; TDRG1 (+), the overexpression of TDRG1 ; PI3K (−), the inhibition of PI3K by LY294002; PI3K (+), the activation of PI3K by IGF-1; FL2-A, fluorescence 2 area; FITC, fluorescein isothiocyanate.

    Techniques Used: Knock-Out, Inhibition, Over Expression, Activation Assay, Flow Cytometry, Plasmid Preparation, Fluorescence

    The effect of TDRG1 - and PI3K-mediated signaling on the cell adhesive capacity and cell invasion. Notes: ( A ) The cell adhesive capacity was increased in the cells with both TDRG1 knockout and PI3K inhibition and decreased in the cells with both TDRG1 overexpression and PI3K activation. ( B i) Pictures of cell passing through transwells. ( B ii) The cell invasion was increased in the cells with both TDRG1 knockout and PI3K inhibition and decreased in the cells with both TDRG1 overexpression and PI3K activation. Seminoma TCam-2 cells were subjected to different treatments: the TDRG1 knockout, TDRG1 overexpression, PI3K inhibition (LY294002 administration), or PI3K activation (IGF-1 administration). * P <0.05 and ** P <0.01 vs control group. Vector: enhanced green fluorescent protein plasmid-C1 vector. Abbreviations: TDRG1 , testis development-related gene 1; PI3K, phosphoinositide-3 kinase; IGF-1, insulin-like growth factor-1; TDRG1 (−), the knockout of TDRG1 ; TDRG1 (+), the overexpression of TDRG1 ; PI3K (−), the inhibition of PI3K by LY294002; PI3K (+), the activation of PI3K by IGF-1.
    Figure Legend Snippet: The effect of TDRG1 - and PI3K-mediated signaling on the cell adhesive capacity and cell invasion. Notes: ( A ) The cell adhesive capacity was increased in the cells with both TDRG1 knockout and PI3K inhibition and decreased in the cells with both TDRG1 overexpression and PI3K activation. ( B i) Pictures of cell passing through transwells. ( B ii) The cell invasion was increased in the cells with both TDRG1 knockout and PI3K inhibition and decreased in the cells with both TDRG1 overexpression and PI3K activation. Seminoma TCam-2 cells were subjected to different treatments: the TDRG1 knockout, TDRG1 overexpression, PI3K inhibition (LY294002 administration), or PI3K activation (IGF-1 administration). * P <0.05 and ** P <0.01 vs control group. Vector: enhanced green fluorescent protein plasmid-C1 vector. Abbreviations: TDRG1 , testis development-related gene 1; PI3K, phosphoinositide-3 kinase; IGF-1, insulin-like growth factor-1; TDRG1 (−), the knockout of TDRG1 ; TDRG1 (+), the overexpression of TDRG1 ; PI3K (−), the inhibition of PI3K by LY294002; PI3K (+), the activation of PI3K by IGF-1.

    Techniques Used: Knock-Out, Inhibition, Over Expression, Activation Assay, Plasmid Preparation

    Expression levels of various proteins in seminoma TCam-2 cells after different treatments based on a Western blot assay. Notes: Seminoma TCam-2 cells were subjected to different treatments: the TDRG1 knockout, TDRG1 overexpression, PI3K inhibition (LY294002 administration), or PI3K activation (IGF-1 administration). Inhibiting TDRG1 causes a dramatic reduction in the protein expression levels of p-PI3K/p85, p-PI3K/p110, p-Akt, and p-mTOR (S2448) but not PI3K/p85, PI3K/p110, mTOR, p-mTOR (S2481), and p-mTOR (T2446). In contrast, overexpression of TDRG1 promoted the expression of p-PI3K/p85, p-Akt, and p-mTOR (S2448). The treatment with LY294002 inhibited the expression of PI3K/p110, p-PI3K/p110, p-Akt, and p-mTOR (S2448), while pharmacological stimulation with IGF-1 caused a marked elevation in the phosphorylation levels of PI3K/p110, Akt, and mTOR (S2448). * P <0.05 and ** P <0.01 vs control group. Vector: enhanced green fluorescent protein plasmid-C1 vector. Abbreviations: TDRG1 , testis development-related gene 1; PI3K, phosphoinositide-3 kinase; IGF-1, insulin-like growth factor-1; mTOR, mammalian target of rapamycin; TDRG1 (−), the knockout of TDRG1 ; TDRG1 (+), the overexpression of TDRG1 ; PI3K (−), the inhibition of PI3K by LY294002; PI3K (+), the activation of PI3K by IGF-1; GADPH, glyceraldehyde-3-phosphate dehydrogenase.
    Figure Legend Snippet: Expression levels of various proteins in seminoma TCam-2 cells after different treatments based on a Western blot assay. Notes: Seminoma TCam-2 cells were subjected to different treatments: the TDRG1 knockout, TDRG1 overexpression, PI3K inhibition (LY294002 administration), or PI3K activation (IGF-1 administration). Inhibiting TDRG1 causes a dramatic reduction in the protein expression levels of p-PI3K/p85, p-PI3K/p110, p-Akt, and p-mTOR (S2448) but not PI3K/p85, PI3K/p110, mTOR, p-mTOR (S2481), and p-mTOR (T2446). In contrast, overexpression of TDRG1 promoted the expression of p-PI3K/p85, p-Akt, and p-mTOR (S2448). The treatment with LY294002 inhibited the expression of PI3K/p110, p-PI3K/p110, p-Akt, and p-mTOR (S2448), while pharmacological stimulation with IGF-1 caused a marked elevation in the phosphorylation levels of PI3K/p110, Akt, and mTOR (S2448). * P <0.05 and ** P <0.01 vs control group. Vector: enhanced green fluorescent protein plasmid-C1 vector. Abbreviations: TDRG1 , testis development-related gene 1; PI3K, phosphoinositide-3 kinase; IGF-1, insulin-like growth factor-1; mTOR, mammalian target of rapamycin; TDRG1 (−), the knockout of TDRG1 ; TDRG1 (+), the overexpression of TDRG1 ; PI3K (−), the inhibition of PI3K by LY294002; PI3K (+), the activation of PI3K by IGF-1; GADPH, glyceraldehyde-3-phosphate dehydrogenase.

    Techniques Used: Expressing, Western Blot, Knock-Out, Over Expression, Inhibition, Activation Assay, Plasmid Preparation

    p pi3k p110  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc p pi3k p110
    TDRG1 is significantly upregulated in testicular seminoma tissues than in normal testicular tissues. Notes: ( A ) RT-PCR showed that the relative gene expression level of TDRG1 is significantly higher in testicular seminoma tissues than in normal testicular tissues. ( B i) Representative Western blotting images that are associated with TDRG1 and GAPDH protein expression in both testicular seminoma and normal testicular tissues. ( B ii) Western blotting showed that the relative protein expression level of TDRG1 is significantly higher in testicular seminoma tissues than in normal testicular tissues. RT-PCR and Western blotting were performed to detect the expression levels of TDRG1 in both testicular seminoma tissues (n=33) and normal testicular tissues (n=33). ( C i) Immunohistochemical staining pictures of both testicular seminoma and normal testicular tissue samples with anti- TDRG1 , <t>anti-p-PI3K/p110,</t> and anti-p-Akt antibodies. ( C ii) The protein expression levels of TDRG1 , p-PI3K/p110, and p-Akt are significantly upregulated in testicular seminoma tissues compared to normal testicular tissues. Protein expression levels of TDRG1 , p-PI3K/p110, and p-Akt were detected by an immunohistochemistry assay. The tissue microarray chips TE802 and TE2081 contained 80 points (from 80 testicular seminoma tissue samples) and 138 points (from 46 testicular seminoma tissue samples, three points per sample), respectively. TEN601 had 60 points (from 30 normal testicular tissue samples, two points per sample). Bars representing cancer are averages of data from 218 points (80+138) in two tissue microarray chips. Bars representing the normal tissue samples are the average of data from 60 points in a tissue microarray chip. * P <0.05 vs control group. Cancer: testicular seminoma; normal: normal testicular tissues. Abbreviations: TDRG1 , testis development-related gene 1; RT-PCR, reverse transcription polymerase chain reaction; PI3K, phosphoinositide-3 kinase; GADPH, glyceral-dehyde-3-phosphate dehydrogenase.
    P Pi3k P110, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/p pi3k p110/product/Cell Signaling Technology Inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    p pi3k p110 - by Bioz Stars, 2023-03
    86/100 stars

    Images

    1) Product Images from "TDRG1 functions in testicular seminoma are dependent on the PI3K/Akt/mTOR signaling pathway"

    Article Title: TDRG1 functions in testicular seminoma are dependent on the PI3K/Akt/mTOR signaling pathway

    Journal: OncoTargets and therapy

    doi: 10.2147/OTT.S97294

    TDRG1 is significantly upregulated in testicular seminoma tissues than in normal testicular tissues. Notes: ( A ) RT-PCR showed that the relative gene expression level of TDRG1 is significantly higher in testicular seminoma tissues than in normal testicular tissues. ( B i) Representative Western blotting images that are associated with TDRG1 and GAPDH protein expression in both testicular seminoma and normal testicular tissues. ( B ii) Western blotting showed that the relative protein expression level of TDRG1 is significantly higher in testicular seminoma tissues than in normal testicular tissues. RT-PCR and Western blotting were performed to detect the expression levels of TDRG1 in both testicular seminoma tissues (n=33) and normal testicular tissues (n=33). ( C i) Immunohistochemical staining pictures of both testicular seminoma and normal testicular tissue samples with anti- TDRG1 , anti-p-PI3K/p110, and anti-p-Akt antibodies. ( C ii) The protein expression levels of TDRG1 , p-PI3K/p110, and p-Akt are significantly upregulated in testicular seminoma tissues compared to normal testicular tissues. Protein expression levels of TDRG1 , p-PI3K/p110, and p-Akt were detected by an immunohistochemistry assay. The tissue microarray chips TE802 and TE2081 contained 80 points (from 80 testicular seminoma tissue samples) and 138 points (from 46 testicular seminoma tissue samples, three points per sample), respectively. TEN601 had 60 points (from 30 normal testicular tissue samples, two points per sample). Bars representing cancer are averages of data from 218 points (80+138) in two tissue microarray chips. Bars representing the normal tissue samples are the average of data from 60 points in a tissue microarray chip. * P <0.05 vs control group. Cancer: testicular seminoma; normal: normal testicular tissues. Abbreviations: TDRG1 , testis development-related gene 1; RT-PCR, reverse transcription polymerase chain reaction; PI3K, phosphoinositide-3 kinase; GADPH, glyceral-dehyde-3-phosphate dehydrogenase.
    Figure Legend Snippet: TDRG1 is significantly upregulated in testicular seminoma tissues than in normal testicular tissues. Notes: ( A ) RT-PCR showed that the relative gene expression level of TDRG1 is significantly higher in testicular seminoma tissues than in normal testicular tissues. ( B i) Representative Western blotting images that are associated with TDRG1 and GAPDH protein expression in both testicular seminoma and normal testicular tissues. ( B ii) Western blotting showed that the relative protein expression level of TDRG1 is significantly higher in testicular seminoma tissues than in normal testicular tissues. RT-PCR and Western blotting were performed to detect the expression levels of TDRG1 in both testicular seminoma tissues (n=33) and normal testicular tissues (n=33). ( C i) Immunohistochemical staining pictures of both testicular seminoma and normal testicular tissue samples with anti- TDRG1 , anti-p-PI3K/p110, and anti-p-Akt antibodies. ( C ii) The protein expression levels of TDRG1 , p-PI3K/p110, and p-Akt are significantly upregulated in testicular seminoma tissues compared to normal testicular tissues. Protein expression levels of TDRG1 , p-PI3K/p110, and p-Akt were detected by an immunohistochemistry assay. The tissue microarray chips TE802 and TE2081 contained 80 points (from 80 testicular seminoma tissue samples) and 138 points (from 46 testicular seminoma tissue samples, three points per sample), respectively. TEN601 had 60 points (from 30 normal testicular tissue samples, two points per sample). Bars representing cancer are averages of data from 218 points (80+138) in two tissue microarray chips. Bars representing the normal tissue samples are the average of data from 60 points in a tissue microarray chip. * P <0.05 vs control group. Cancer: testicular seminoma; normal: normal testicular tissues. Abbreviations: TDRG1 , testis development-related gene 1; RT-PCR, reverse transcription polymerase chain reaction; PI3K, phosphoinositide-3 kinase; GADPH, glyceral-dehyde-3-phosphate dehydrogenase.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Immunohistochemical staining, Staining, Immunohistochemistry, Microarray

    Relative TDRG1 expression in both gene and protein levels of seminoma TCam-2 cells after different treatments. Notes: ( A ) Relative gene expression level of TDRG1 in seminoma TCam-2 cells was remarkably upregulated and downregulated after the gene was knocked out and overexpressed, respectively, on the basis of RT-PCR (n=5). ( B ) Relative protein expression level of TDRG1 in seminoma TCam-2 cells was remarkably upregulated and downregulated after the gene was knocked out and overexpressed, respectively, but was unaffected after treatment with LY294002 or insulin-like growth factor-1 on the basis of Western blotting. * P <0.05 and ** P <0.01 vs control group. Vector: enhanced green fluorescent protein plasmid-C1 vector. Abbreviations: TDRG1 , testis development-related gene 1; RT-PCR, reverse transcription polymerase chain reaction; PI3K, phosphoinositide-3 kinase; TDRG1 (−), the knockout of TDRG1 ; TDRG1 (+), the overexpression of TDRG1 ; PI3K (−), the inhibition of PI3K by LY294002; PI3K (+), the activation of PI3K by insulin-like growth factor-1.
    Figure Legend Snippet: Relative TDRG1 expression in both gene and protein levels of seminoma TCam-2 cells after different treatments. Notes: ( A ) Relative gene expression level of TDRG1 in seminoma TCam-2 cells was remarkably upregulated and downregulated after the gene was knocked out and overexpressed, respectively, on the basis of RT-PCR (n=5). ( B ) Relative protein expression level of TDRG1 in seminoma TCam-2 cells was remarkably upregulated and downregulated after the gene was knocked out and overexpressed, respectively, but was unaffected after treatment with LY294002 or insulin-like growth factor-1 on the basis of Western blotting. * P <0.05 and ** P <0.01 vs control group. Vector: enhanced green fluorescent protein plasmid-C1 vector. Abbreviations: TDRG1 , testis development-related gene 1; RT-PCR, reverse transcription polymerase chain reaction; PI3K, phosphoinositide-3 kinase; TDRG1 (−), the knockout of TDRG1 ; TDRG1 (+), the overexpression of TDRG1 ; PI3K (−), the inhibition of PI3K by LY294002; PI3K (+), the activation of PI3K by insulin-like growth factor-1.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Plasmid Preparation, Knock-Out, Over Expression, Inhibition, Activation Assay

    The effect of TDRG1 - and PI3K-mediated signaling on the cell proliferation rate, cell proliferation index, and apoptosis rate. Notes: ( A ) Cells with both TDRG1 knockout and PI3K inhibition exhibited decreased cell proliferation. ( B i and B ii) Cells with both TDRG1 knockout and PI3K inhibition exhibited decreased cell proliferation index. ( C i and C ii) Cells with both TDRG1 knockout and PI3K inhibition exhibited increased apoptosis rate. Seminoma TCam-2 cells were subjected to different treatments: the TDRG1 knockout, TDRG1 overexpression, PI3K inhibition (LY294002 administration), or PI3K activation (IGF-1 administration). The cell proliferation rate was assessed using MTT kits at different time points after the treatments, and cell proliferation index and apoptosis rate were evaluated using flow cytometry at 72 h. The cell proliferation index was obtained by calculating the percentage of cells in the S, G2, and M phases. * P <0.05 and ** P <0.01 vs control group. Vector: enhanced green fluorescent protein plasmid-C1 vector. Abbreviations: TDRG1 , testis development-related gene 1; PI3K, phosphoinositide-3 kinase; IGF-1, insulin-like growth factor-1; MTT, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide; h, hours; TDRG1 (−), the knockout of TDRG1 ; TDRG1 (+), the overexpression of TDRG1 ; PI3K (−), the inhibition of PI3K by LY294002; PI3K (+), the activation of PI3K by IGF-1; FL2-A, fluorescence 2 area; FITC, fluorescein isothiocyanate.
    Figure Legend Snippet: The effect of TDRG1 - and PI3K-mediated signaling on the cell proliferation rate, cell proliferation index, and apoptosis rate. Notes: ( A ) Cells with both TDRG1 knockout and PI3K inhibition exhibited decreased cell proliferation. ( B i and B ii) Cells with both TDRG1 knockout and PI3K inhibition exhibited decreased cell proliferation index. ( C i and C ii) Cells with both TDRG1 knockout and PI3K inhibition exhibited increased apoptosis rate. Seminoma TCam-2 cells were subjected to different treatments: the TDRG1 knockout, TDRG1 overexpression, PI3K inhibition (LY294002 administration), or PI3K activation (IGF-1 administration). The cell proliferation rate was assessed using MTT kits at different time points after the treatments, and cell proliferation index and apoptosis rate were evaluated using flow cytometry at 72 h. The cell proliferation index was obtained by calculating the percentage of cells in the S, G2, and M phases. * P <0.05 and ** P <0.01 vs control group. Vector: enhanced green fluorescent protein plasmid-C1 vector. Abbreviations: TDRG1 , testis development-related gene 1; PI3K, phosphoinositide-3 kinase; IGF-1, insulin-like growth factor-1; MTT, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide; h, hours; TDRG1 (−), the knockout of TDRG1 ; TDRG1 (+), the overexpression of TDRG1 ; PI3K (−), the inhibition of PI3K by LY294002; PI3K (+), the activation of PI3K by IGF-1; FL2-A, fluorescence 2 area; FITC, fluorescein isothiocyanate.

    Techniques Used: Knock-Out, Inhibition, Over Expression, Activation Assay, Flow Cytometry, Plasmid Preparation, Fluorescence

    The effect of TDRG1 - and PI3K-mediated signaling on the cell adhesive capacity and cell invasion. Notes: ( A ) The cell adhesive capacity was increased in the cells with both TDRG1 knockout and PI3K inhibition and decreased in the cells with both TDRG1 overexpression and PI3K activation. ( B i) Pictures of cell passing through transwells. ( B ii) The cell invasion was increased in the cells with both TDRG1 knockout and PI3K inhibition and decreased in the cells with both TDRG1 overexpression and PI3K activation. Seminoma TCam-2 cells were subjected to different treatments: the TDRG1 knockout, TDRG1 overexpression, PI3K inhibition (LY294002 administration), or PI3K activation (IGF-1 administration). * P <0.05 and ** P <0.01 vs control group. Vector: enhanced green fluorescent protein plasmid-C1 vector. Abbreviations: TDRG1 , testis development-related gene 1; PI3K, phosphoinositide-3 kinase; IGF-1, insulin-like growth factor-1; TDRG1 (−), the knockout of TDRG1 ; TDRG1 (+), the overexpression of TDRG1 ; PI3K (−), the inhibition of PI3K by LY294002; PI3K (+), the activation of PI3K by IGF-1.
    Figure Legend Snippet: The effect of TDRG1 - and PI3K-mediated signaling on the cell adhesive capacity and cell invasion. Notes: ( A ) The cell adhesive capacity was increased in the cells with both TDRG1 knockout and PI3K inhibition and decreased in the cells with both TDRG1 overexpression and PI3K activation. ( B i) Pictures of cell passing through transwells. ( B ii) The cell invasion was increased in the cells with both TDRG1 knockout and PI3K inhibition and decreased in the cells with both TDRG1 overexpression and PI3K activation. Seminoma TCam-2 cells were subjected to different treatments: the TDRG1 knockout, TDRG1 overexpression, PI3K inhibition (LY294002 administration), or PI3K activation (IGF-1 administration). * P <0.05 and ** P <0.01 vs control group. Vector: enhanced green fluorescent protein plasmid-C1 vector. Abbreviations: TDRG1 , testis development-related gene 1; PI3K, phosphoinositide-3 kinase; IGF-1, insulin-like growth factor-1; TDRG1 (−), the knockout of TDRG1 ; TDRG1 (+), the overexpression of TDRG1 ; PI3K (−), the inhibition of PI3K by LY294002; PI3K (+), the activation of PI3K by IGF-1.

    Techniques Used: Knock-Out, Inhibition, Over Expression, Activation Assay, Plasmid Preparation

    Expression levels of various proteins in seminoma TCam-2 cells after different treatments based on a Western blot assay. Notes: Seminoma TCam-2 cells were subjected to different treatments: the TDRG1 knockout, TDRG1 overexpression, PI3K inhibition (LY294002 administration), or PI3K activation (IGF-1 administration). Inhibiting TDRG1 causes a dramatic reduction in the protein expression levels of p-PI3K/p85, p-PI3K/p110, p-Akt, and p-mTOR (S2448) but not PI3K/p85, PI3K/p110, mTOR, p-mTOR (S2481), and p-mTOR (T2446). In contrast, overexpression of TDRG1 promoted the expression of p-PI3K/p85, p-Akt, and p-mTOR (S2448). The treatment with LY294002 inhibited the expression of PI3K/p110, p-PI3K/p110, p-Akt, and p-mTOR (S2448), while pharmacological stimulation with IGF-1 caused a marked elevation in the phosphorylation levels of PI3K/p110, Akt, and mTOR (S2448). * P <0.05 and ** P <0.01 vs control group. Vector: enhanced green fluorescent protein plasmid-C1 vector. Abbreviations: TDRG1 , testis development-related gene 1; PI3K, phosphoinositide-3 kinase; IGF-1, insulin-like growth factor-1; mTOR, mammalian target of rapamycin; TDRG1 (−), the knockout of TDRG1 ; TDRG1 (+), the overexpression of TDRG1 ; PI3K (−), the inhibition of PI3K by LY294002; PI3K (+), the activation of PI3K by IGF-1; GADPH, glyceraldehyde-3-phosphate dehydrogenase.
    Figure Legend Snippet: Expression levels of various proteins in seminoma TCam-2 cells after different treatments based on a Western blot assay. Notes: Seminoma TCam-2 cells were subjected to different treatments: the TDRG1 knockout, TDRG1 overexpression, PI3K inhibition (LY294002 administration), or PI3K activation (IGF-1 administration). Inhibiting TDRG1 causes a dramatic reduction in the protein expression levels of p-PI3K/p85, p-PI3K/p110, p-Akt, and p-mTOR (S2448) but not PI3K/p85, PI3K/p110, mTOR, p-mTOR (S2481), and p-mTOR (T2446). In contrast, overexpression of TDRG1 promoted the expression of p-PI3K/p85, p-Akt, and p-mTOR (S2448). The treatment with LY294002 inhibited the expression of PI3K/p110, p-PI3K/p110, p-Akt, and p-mTOR (S2448), while pharmacological stimulation with IGF-1 caused a marked elevation in the phosphorylation levels of PI3K/p110, Akt, and mTOR (S2448). * P <0.05 and ** P <0.01 vs control group. Vector: enhanced green fluorescent protein plasmid-C1 vector. Abbreviations: TDRG1 , testis development-related gene 1; PI3K, phosphoinositide-3 kinase; IGF-1, insulin-like growth factor-1; mTOR, mammalian target of rapamycin; TDRG1 (−), the knockout of TDRG1 ; TDRG1 (+), the overexpression of TDRG1 ; PI3K (−), the inhibition of PI3K by LY294002; PI3K (+), the activation of PI3K by IGF-1; GADPH, glyceraldehyde-3-phosphate dehydrogenase.

    Techniques Used: Expressing, Western Blot, Knock-Out, Over Expression, Inhibition, Activation Assay, Plasmid Preparation

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    <t>PI3K/AKT</t> signaling blocks the inhibitory effect of PPAR γ on hypoxic-induced HSCs activation. Activation of PI3K with 740 Y-P (25 μ g/ml) of HSCs exposed to hypoxia in the presence or absence of RSG (50 nM). (a) PPAR γ , α -SMA, and desmin expression of HSCs in experimental groups were analyzed by western blotting. (b) α -SMA and desmin expression of HSCs in experimental groups were detected by immunofluorescence using Confocal microscopy. Inhibition of PI3K with LY294002 (20 μ M) in HSCs exposed to hypoxia. (c) Phosphorylated AKT, total AKT, PPAR γ , α -SMA, and desmin expression of HSCs in experimental groups were measured by western blotting. (d) α -SMA and desmin expression of HSCs in experimental groups were detected by immunofluorescence using Confocal microscopy. For each assay, n = 3. ∗ P < 0.05, ∗∗ P < 0.01.
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    A. Estrogen-induced <t>PI3K</t> phosphotylation. B. Estrogen-induced AKT phosphotylation. MCF-10A cells were exposed to a carcinogenic regimen of E2 and its metabolites – 2-OH-E2 or 4-OH-E2 as described in the legend of . At the end of transformation process, cells were treated for additional 30 minutes with E2, 2-OH-E2 or 4-OH-E2, respectively. The cellular extracts from treated and controls cells were immuno-precipitated with PI3K or AKT specific monoclonal antibodies and followed by Western detection of PI3K or AKT phosphorylation using phosphor-threonine antibody. Results are expressed as mean fold change of three separate experiments. *P<0.05, significantly different from contol. **P<0.001 indicates significantly different from control.
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    antibodies against pi3k p110 α  (Cell Signaling Technology Inc)
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    The effect of TMZ on DNA damage and repair regulating factors and key molecules of the <t>PI3K</t> signaling pathway in GBM cells. (a) SF295, U87, and U251 cells were treated with different concentrations of TMZ (10, 20, 40, 80, 160, 320, 640, and 1280 μ M) for 72 h. Cell viability was determined by WST-8 assay. (b)–(f) SF295 and U87 cells were treated with TMZ (120 μ M and 180 μ M) for 48 and 72 h. (b) The cells were then collected for western blotting detection of the DNA SSB marker XRCC1 and the DSB marker γ -H2AX. (c) The cells were harvested for qRT-PCR analysis of BRCA1 and RAD51. (d) Western blot analysis of DNA damage repair proteins, including p-ATM, BRCA1, RAD51, DNA-PKcs, and Ku80. (e) qRT-PCR analysis of PIK3CA and PIK3CB. (f) Western blot analysis of key proteins in the PI3K signaling pathway, including <t>PI3K-p110</t> α , PI3K-p110 β , p-Akt, Akt, p-mTOR, and mTOR. For western blotting, β -actin was used as a loading control. For qRT-PCR, gene expression was normalized to 18 s rRNA. All data are presented as the mean ± SD ( n = 3). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001; ∗∗∗∗ P < 0.0001.
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    The effect of TMZ on DNA damage and repair regulating factors and key molecules of the <t>PI3K</t> signaling pathway in GBM cells. (a) SF295, U87, and U251 cells were treated with different concentrations of TMZ (10, 20, 40, 80, 160, 320, 640, and 1280 μ M) for 72 h. Cell viability was determined by WST-8 assay. (b)–(f) SF295 and U87 cells were treated with TMZ (120 μ M and 180 μ M) for 48 and 72 h. (b) The cells were then collected for western blotting detection of the DNA SSB marker XRCC1 and the DSB marker γ -H2AX. (c) The cells were harvested for qRT-PCR analysis of BRCA1 and RAD51. (d) Western blot analysis of DNA damage repair proteins, including p-ATM, BRCA1, RAD51, DNA-PKcs, and Ku80. (e) qRT-PCR analysis of PIK3CA and PIK3CB. (f) Western blot analysis of key proteins in the PI3K signaling pathway, including <t>PI3K-p110</t> α , PI3K-p110 β , p-Akt, Akt, p-mTOR, and mTOR. For western blotting, β -actin was used as a loading control. For qRT-PCR, gene expression was normalized to 18 s rRNA. All data are presented as the mean ± SD ( n = 3). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001; ∗∗∗∗ P < 0.0001.
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    Cell Signaling Technology Inc pi3k p110 isoforms
    Selectivity and effective concentrations of inhibitors used in this study (reported in the following references: 11, 26, 31, 34, 38, 40).
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    S1P-induced COX-2 expression is mediated through <t>PI3K/Akt</t> in HCFs. (a) Cells were pretreated without or with LY294002 (0.3, 3, and 30 μ M) for 1 h and then incubated with 15 μ M S1P for 8 h. The levels of COX-2 and GAPDH used as an internal control were analyzed by Western blot. (b) Cells were transfected without or with COX-2 promoter-luciferase reporter gene, pretreated with LY294002 (30 μ M) for 1 h, and then incubated with 15 μ M S1P for 4 h (mRNA level) or 1 h (promoter activity). The COX-2 mRNA and promoter activity were analyzed by real-time PCR (open bar) and promoter assay (gray bar). (c) Cells were transfected with scrambled, <t>p110</t> (left panel), or Akt (right panel) siRNA for 24 h and then exposed to 15 μ M S1P for 8 h. The levels of COX-2, GAPDH, p110, and Akt proteins were analyzed by Western blot. (d) Cells were pretreated with or without 30 μ M LY294002, 10 μ M MMP2/9 inhibitor, 10 μ g/ml CRM197, or 10 μ M AG1478 for 1 h and then incubated with 15 μ M S1P for 0.5, 1, 3, 5, and 10 min. The levels of phospho-Akt and GAPDH were determined by Western blot. (e) Cells were pretreated without or with 30 μ M LY294002 for 1 h and then incubated with 15 μ M S1P for 0, 3, 5, 10, 15, and 30 min. The levels of phospho-p44/p42, phospho-p38, phospho-JNK1/2, and GAPDH were determined by Western blot. Data are expressed as the mean ± SEM of three individual experiments ( n = 3). # P < 0.05, as compared with the control or pretreatment with inhibitor indicated in the figure.
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    TDRG1 is significantly upregulated in testicular seminoma tissues than in normal testicular tissues. Notes: ( A ) RT-PCR showed that the relative gene expression level of TDRG1 is significantly higher in testicular seminoma tissues than in normal testicular tissues. ( B i) Representative Western blotting images that are associated with TDRG1 and GAPDH protein expression in both testicular seminoma and normal testicular tissues. ( B ii) Western blotting showed that the relative protein expression level of TDRG1 is significantly higher in testicular seminoma tissues than in normal testicular tissues. RT-PCR and Western blotting were performed to detect the expression levels of TDRG1 in both testicular seminoma tissues (n=33) and normal testicular tissues (n=33). ( C i) Immunohistochemical staining pictures of both testicular seminoma and normal testicular tissue samples with anti- TDRG1 , <t>anti-p-PI3K/p110,</t> and anti-p-Akt antibodies. ( C ii) The protein expression levels of TDRG1 , p-PI3K/p110, and p-Akt are significantly upregulated in testicular seminoma tissues compared to normal testicular tissues. Protein expression levels of TDRG1 , p-PI3K/p110, and p-Akt were detected by an immunohistochemistry assay. The tissue microarray chips TE802 and TE2081 contained 80 points (from 80 testicular seminoma tissue samples) and 138 points (from 46 testicular seminoma tissue samples, three points per sample), respectively. TEN601 had 60 points (from 30 normal testicular tissue samples, two points per sample). Bars representing cancer are averages of data from 218 points (80+138) in two tissue microarray chips. Bars representing the normal tissue samples are the average of data from 60 points in a tissue microarray chip. * P <0.05 vs control group. Cancer: testicular seminoma; normal: normal testicular tissues. Abbreviations: TDRG1 , testis development-related gene 1; RT-PCR, reverse transcription polymerase chain reaction; PI3K, phosphoinositide-3 kinase; GADPH, glyceral-dehyde-3-phosphate dehydrogenase.
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    PI3K/AKT signaling blocks the inhibitory effect of PPAR γ on hypoxic-induced HSCs activation. Activation of PI3K with 740 Y-P (25 μ g/ml) of HSCs exposed to hypoxia in the presence or absence of RSG (50 nM). (a) PPAR γ , α -SMA, and desmin expression of HSCs in experimental groups were analyzed by western blotting. (b) α -SMA and desmin expression of HSCs in experimental groups were detected by immunofluorescence using Confocal microscopy. Inhibition of PI3K with LY294002 (20 μ M) in HSCs exposed to hypoxia. (c) Phosphorylated AKT, total AKT, PPAR γ , α -SMA, and desmin expression of HSCs in experimental groups were measured by western blotting. (d) α -SMA and desmin expression of HSCs in experimental groups were detected by immunofluorescence using Confocal microscopy. For each assay, n = 3. ∗ P < 0.05, ∗∗ P < 0.01.

    Journal: PPAR Research

    Article Title: PPAR γ Antagonizes Hypoxia-Induced Activation of Hepatic Stellate Cell through Cross Mediating PI3K/AKT and cGMP/PKG Signaling

    doi: 10.1155/2018/6970407

    Figure Lengend Snippet: PI3K/AKT signaling blocks the inhibitory effect of PPAR γ on hypoxic-induced HSCs activation. Activation of PI3K with 740 Y-P (25 μ g/ml) of HSCs exposed to hypoxia in the presence or absence of RSG (50 nM). (a) PPAR γ , α -SMA, and desmin expression of HSCs in experimental groups were analyzed by western blotting. (b) α -SMA and desmin expression of HSCs in experimental groups were detected by immunofluorescence using Confocal microscopy. Inhibition of PI3K with LY294002 (20 μ M) in HSCs exposed to hypoxia. (c) Phosphorylated AKT, total AKT, PPAR γ , α -SMA, and desmin expression of HSCs in experimental groups were measured by western blotting. (d) α -SMA and desmin expression of HSCs in experimental groups were detected by immunofluorescence using Confocal microscopy. For each assay, n = 3. ∗ P < 0.05, ∗∗ P < 0.01.

    Article Snippet: Primary antibodies used in this study are as follows: PPAR γ (ab209350) (1 : 1,500), α -SMA (ab5694), desmin (ab15200), AKT (ab8805), Phospho-AKT (ab81283), PI3K p110 β (ab151549), PDE5 (ab14672) (1 : 1,000; ABcam, USA), and PI3K p110 α (#4249) (1 : 1,200; Cell Signaling Technology, USA).

    Techniques: Activation Assay, Expressing, Western Blot, Immunofluorescence, Confocal Microscopy, Inhibition

    sGC/cGMP/PKG and PI3K/AKT signals synergistically act on PPAR γ to inhibit hypoxia-induced HSC activation. (a) HSCs were exposed to hypoxia in the presence of cGMP analog, 8-Br-cGMP (1 mM), or agonist, Rp-8-Br-cGMPs (20 μ M). PI3K p100 α , p100 β , phosphorylated AKT, total AKT, PPAR γ , α -SMA, and desmin expression of HSCs in experimental groups were detected by western blotting. HSCs were exposed to hypoxia in the presence of PDE5 agonist, zaprinast (10 μ M). (b) PDE5, PPAR γ , α -SMA, and desmin expression of HSCs in experimental groups were measured by western blotting. (c)-(d) cGMP and PKG activity were tested, respectively. (e) Possible signal pathway involved in this study. Hypoxia induces HSCs activation. However, PPAR γ inhibits this effect, which is triggered by sGC/cGMP/PKG signaling. Hypoxia promotes PI3K/AKT signaling and PDE5, which inhibit cGMP and PPAR γ , respectively. Finally, sGC/cGMP/PKG and PI3K/AKT signals synergistically act on PPAR γ to inhibit hypoxia-induced HSC activation. For each assay, n = 3. ∗ P < 0.05, ∗∗ P < 0.01.

    Journal: PPAR Research

    Article Title: PPAR γ Antagonizes Hypoxia-Induced Activation of Hepatic Stellate Cell through Cross Mediating PI3K/AKT and cGMP/PKG Signaling

    doi: 10.1155/2018/6970407

    Figure Lengend Snippet: sGC/cGMP/PKG and PI3K/AKT signals synergistically act on PPAR γ to inhibit hypoxia-induced HSC activation. (a) HSCs were exposed to hypoxia in the presence of cGMP analog, 8-Br-cGMP (1 mM), or agonist, Rp-8-Br-cGMPs (20 μ M). PI3K p100 α , p100 β , phosphorylated AKT, total AKT, PPAR γ , α -SMA, and desmin expression of HSCs in experimental groups were detected by western blotting. HSCs were exposed to hypoxia in the presence of PDE5 agonist, zaprinast (10 μ M). (b) PDE5, PPAR γ , α -SMA, and desmin expression of HSCs in experimental groups were measured by western blotting. (c)-(d) cGMP and PKG activity were tested, respectively. (e) Possible signal pathway involved in this study. Hypoxia induces HSCs activation. However, PPAR γ inhibits this effect, which is triggered by sGC/cGMP/PKG signaling. Hypoxia promotes PI3K/AKT signaling and PDE5, which inhibit cGMP and PPAR γ , respectively. Finally, sGC/cGMP/PKG and PI3K/AKT signals synergistically act on PPAR γ to inhibit hypoxia-induced HSC activation. For each assay, n = 3. ∗ P < 0.05, ∗∗ P < 0.01.

    Article Snippet: Primary antibodies used in this study are as follows: PPAR γ (ab209350) (1 : 1,500), α -SMA (ab5694), desmin (ab15200), AKT (ab8805), Phospho-AKT (ab81283), PI3K p110 β (ab151549), PDE5 (ab14672) (1 : 1,000; ABcam, USA), and PI3K p110 α (#4249) (1 : 1,200; Cell Signaling Technology, USA).

    Techniques: Activation Assay, Expressing, Western Blot, Activity Assay

    A. Estrogen-induced PI3K phosphotylation. B. Estrogen-induced AKT phosphotylation. MCF-10A cells were exposed to a carcinogenic regimen of E2 and its metabolites – 2-OH-E2 or 4-OH-E2 as described in the legend of . At the end of transformation process, cells were treated for additional 30 minutes with E2, 2-OH-E2 or 4-OH-E2, respectively. The cellular extracts from treated and controls cells were immuno-precipitated with PI3K or AKT specific monoclonal antibodies and followed by Western detection of PI3K or AKT phosphorylation using phosphor-threonine antibody. Results are expressed as mean fold change of three separate experiments. *P<0.05, significantly different from contol. **P<0.001 indicates significantly different from control.

    Journal: PLoS ONE

    Article Title: Reactive Oxygen Species via Redox Signaling to PI3K/AKT Pathway Contribute to the Malignant Growth of 4-Hydroxy Estradiol-Transformed Mammary Epithelial Cells

    doi: 10.1371/journal.pone.0054206

    Figure Lengend Snippet: A. Estrogen-induced PI3K phosphotylation. B. Estrogen-induced AKT phosphotylation. MCF-10A cells were exposed to a carcinogenic regimen of E2 and its metabolites – 2-OH-E2 or 4-OH-E2 as described in the legend of . At the end of transformation process, cells were treated for additional 30 minutes with E2, 2-OH-E2 or 4-OH-E2, respectively. The cellular extracts from treated and controls cells were immuno-precipitated with PI3K or AKT specific monoclonal antibodies and followed by Western detection of PI3K or AKT phosphorylation using phosphor-threonine antibody. Results are expressed as mean fold change of three separate experiments. *P<0.05, significantly different from contol. **P<0.001 indicates significantly different from control.

    Article Snippet: All antibodies; PI3K (p110), phospho PI3K (p85), phospho-AKT (ser 473) and total AKT antibodies were purchased from Cell Signaling Technology Inc. (Boston, MA).

    Techniques: Transformation Assay, Western Blot

    A. Inhibition of 4-OH-E2-induced PI3K phosphotylation by overexpression of MnSOD and catalase. B. Inhibition of 4-OH-E2-induced AKT phosphotylation by overexpression of MnSOD and catalase. For investigating inhibition of 4-OH-E2-induced cell transformation by ROS modifiers, MCF-10A cells were transfected with 50 MOI adenovirus expressing catalase or MnSOD. Cells overexpressing catalase or MnSOD were exposed to a carcinogenic regimen of 4-OH-E2 as described in the legend of . At the end of transformation process, cells were treated for additional 30 minutes with 100 ng/ml 4-OHE2. The cellular extracts from treated and controls cells were immuno-precipitated with PI3K or AKT specific monoclonal antibodies and followed by Western detection of PI3K or AKT phosphorylation using phosphor-threonine antibody. Results are expressed as mean ± SD of three separate experiments with control set as 100%. *P<0.05 indicates significantly different from E2, (P<0.05). **P<0.05, significantly different from control.

    Journal: PLoS ONE

    Article Title: Reactive Oxygen Species via Redox Signaling to PI3K/AKT Pathway Contribute to the Malignant Growth of 4-Hydroxy Estradiol-Transformed Mammary Epithelial Cells

    doi: 10.1371/journal.pone.0054206

    Figure Lengend Snippet: A. Inhibition of 4-OH-E2-induced PI3K phosphotylation by overexpression of MnSOD and catalase. B. Inhibition of 4-OH-E2-induced AKT phosphotylation by overexpression of MnSOD and catalase. For investigating inhibition of 4-OH-E2-induced cell transformation by ROS modifiers, MCF-10A cells were transfected with 50 MOI adenovirus expressing catalase or MnSOD. Cells overexpressing catalase or MnSOD were exposed to a carcinogenic regimen of 4-OH-E2 as described in the legend of . At the end of transformation process, cells were treated for additional 30 minutes with 100 ng/ml 4-OHE2. The cellular extracts from treated and controls cells were immuno-precipitated with PI3K or AKT specific monoclonal antibodies and followed by Western detection of PI3K or AKT phosphorylation using phosphor-threonine antibody. Results are expressed as mean ± SD of three separate experiments with control set as 100%. *P<0.05 indicates significantly different from E2, (P<0.05). **P<0.05, significantly different from control.

    Article Snippet: All antibodies; PI3K (p110), phospho PI3K (p85), phospho-AKT (ser 473) and total AKT antibodies were purchased from Cell Signaling Technology Inc. (Boston, MA).

    Techniques: Inhibition, Over Expression, Transformation Assay, Transfection, Expressing, Western Blot

    A. Inhibition of 4-OH-E2-induced PI3K phosphotylation by co-treatment with Ebselen and N-acetylcysteine (NAC). B. Inhibition of 4-OH-E2-induced AKT phosphotylation by by co-treatment with Ebselen and NAC. For investigating inhibition of 4-OH-E2-induced cell transformation by ROS modifiers, MCF-10A cells pretreated for 2 hrs with 40 uM Ebselen or 10 mM NAC and were exposed to a carcinogenic regimen of 4-OH-E2 as described in the legend of . At the end of transformation process, cells were treated for additional 30 minutes with 100 ng/ml 4-OHE2. The cellular extracts from treated and controls cells were immuno-precipitated with PI3K or AKT specific monoclonal antibodies and followed by Western detection of PI3K or AKT phosphorylation using phosphor-threonine antibody. Results are expressed as mean ± SD of three separate experiments with control set as 100%. *P<0.05 indicates significantly different from E2, (P<0.05). **P<0.05, significantly different from control.

    Journal: PLoS ONE

    Article Title: Reactive Oxygen Species via Redox Signaling to PI3K/AKT Pathway Contribute to the Malignant Growth of 4-Hydroxy Estradiol-Transformed Mammary Epithelial Cells

    doi: 10.1371/journal.pone.0054206

    Figure Lengend Snippet: A. Inhibition of 4-OH-E2-induced PI3K phosphotylation by co-treatment with Ebselen and N-acetylcysteine (NAC). B. Inhibition of 4-OH-E2-induced AKT phosphotylation by by co-treatment with Ebselen and NAC. For investigating inhibition of 4-OH-E2-induced cell transformation by ROS modifiers, MCF-10A cells pretreated for 2 hrs with 40 uM Ebselen or 10 mM NAC and were exposed to a carcinogenic regimen of 4-OH-E2 as described in the legend of . At the end of transformation process, cells were treated for additional 30 minutes with 100 ng/ml 4-OHE2. The cellular extracts from treated and controls cells were immuno-precipitated with PI3K or AKT specific monoclonal antibodies and followed by Western detection of PI3K or AKT phosphorylation using phosphor-threonine antibody. Results are expressed as mean ± SD of three separate experiments with control set as 100%. *P<0.05 indicates significantly different from E2, (P<0.05). **P<0.05, significantly different from control.

    Article Snippet: All antibodies; PI3K (p110), phospho PI3K (p85), phospho-AKT (ser 473) and total AKT antibodies were purchased from Cell Signaling Technology Inc. (Boston, MA).

    Techniques: Inhibition, Transformation Assay, Western Blot

    The effect of TMZ on DNA damage and repair regulating factors and key molecules of the PI3K signaling pathway in GBM cells. (a) SF295, U87, and U251 cells were treated with different concentrations of TMZ (10, 20, 40, 80, 160, 320, 640, and 1280 μ M) for 72 h. Cell viability was determined by WST-8 assay. (b)–(f) SF295 and U87 cells were treated with TMZ (120 μ M and 180 μ M) for 48 and 72 h. (b) The cells were then collected for western blotting detection of the DNA SSB marker XRCC1 and the DSB marker γ -H2AX. (c) The cells were harvested for qRT-PCR analysis of BRCA1 and RAD51. (d) Western blot analysis of DNA damage repair proteins, including p-ATM, BRCA1, RAD51, DNA-PKcs, and Ku80. (e) qRT-PCR analysis of PIK3CA and PIK3CB. (f) Western blot analysis of key proteins in the PI3K signaling pathway, including PI3K-p110 α , PI3K-p110 β , p-Akt, Akt, p-mTOR, and mTOR. For western blotting, β -actin was used as a loading control. For qRT-PCR, gene expression was normalized to 18 s rRNA. All data are presented as the mean ± SD ( n = 3). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001; ∗∗∗∗ P < 0.0001.

    Journal: BioMed Research International

    Article Title: ZSTK474 Sensitizes Glioblastoma to Temozolomide by Blocking Homologous Recombination Repair

    doi: 10.1155/2022/8568528

    Figure Lengend Snippet: The effect of TMZ on DNA damage and repair regulating factors and key molecules of the PI3K signaling pathway in GBM cells. (a) SF295, U87, and U251 cells were treated with different concentrations of TMZ (10, 20, 40, 80, 160, 320, 640, and 1280 μ M) for 72 h. Cell viability was determined by WST-8 assay. (b)–(f) SF295 and U87 cells were treated with TMZ (120 μ M and 180 μ M) for 48 and 72 h. (b) The cells were then collected for western blotting detection of the DNA SSB marker XRCC1 and the DSB marker γ -H2AX. (c) The cells were harvested for qRT-PCR analysis of BRCA1 and RAD51. (d) Western blot analysis of DNA damage repair proteins, including p-ATM, BRCA1, RAD51, DNA-PKcs, and Ku80. (e) qRT-PCR analysis of PIK3CA and PIK3CB. (f) Western blot analysis of key proteins in the PI3K signaling pathway, including PI3K-p110 α , PI3K-p110 β , p-Akt, Akt, p-mTOR, and mTOR. For western blotting, β -actin was used as a loading control. For qRT-PCR, gene expression was normalized to 18 s rRNA. All data are presented as the mean ± SD ( n = 3). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001; ∗∗∗∗ P < 0.0001.

    Article Snippet: Antibodies against PI3K-p110 α (#4255), PI3K-p110 β (#3011), Akt (#9272), phospho-Akt (Ser473) (#4060), mTOR (#2983), phospho-mTOR (Ser2448) (#5536), c-Myc (#18583), β -actin (#4970), NBS1 (#14956), phospho-ATM (Ser1981) (#13050), BRCA1 (#9010), BRCA2 (#10741), RAD51 (#8875), DNA-PKcs (#38168), Ku80 (#2753), Caspase 3 (#9662), and PARP (#9532) were purchased from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Western Blot, Marker, Quantitative RT-PCR, Expressing

    Effect of PI3K inhibitors and TMZ as single agents or in combination on the survival of GBM cells. (a) SF295, U87, and U251 cells were treated with various concentrations of ZSTK474 (0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, and 2 μ M) for 72 h. Cell viability was determined by WST-8 assay. (b) Three GBM cell lines were incubated with ZSTK474 and TMZ as single agents or in combination (20%, 40%, 60%, 80%, and 100% of 2IC 50ZSTK474 or IC 50ZSTK474 : IC 50TMZ ) for 72 h. The combination effects were analyzed using CalcuSyn software, and the resulting CI-Fa plots are shown. (c) SF295 and U87 cells were treated with BYL719/TGX221 and TMZ as single agents or in combination (20%, 40%, 60%, 80%, and 100% of IC 50BYL719/TGX221 : IC 50TMZ ) for 72 h. The combination effects were analyzed using CalcuSyn software, and the resulting CI-Fa plots are shown. All data are presented as the mean ± SD ( n = 3).

    Journal: BioMed Research International

    Article Title: ZSTK474 Sensitizes Glioblastoma to Temozolomide by Blocking Homologous Recombination Repair

    doi: 10.1155/2022/8568528

    Figure Lengend Snippet: Effect of PI3K inhibitors and TMZ as single agents or in combination on the survival of GBM cells. (a) SF295, U87, and U251 cells were treated with various concentrations of ZSTK474 (0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, and 2 μ M) for 72 h. Cell viability was determined by WST-8 assay. (b) Three GBM cell lines were incubated with ZSTK474 and TMZ as single agents or in combination (20%, 40%, 60%, 80%, and 100% of 2IC 50ZSTK474 or IC 50ZSTK474 : IC 50TMZ ) for 72 h. The combination effects were analyzed using CalcuSyn software, and the resulting CI-Fa plots are shown. (c) SF295 and U87 cells were treated with BYL719/TGX221 and TMZ as single agents or in combination (20%, 40%, 60%, 80%, and 100% of IC 50BYL719/TGX221 : IC 50TMZ ) for 72 h. The combination effects were analyzed using CalcuSyn software, and the resulting CI-Fa plots are shown. All data are presented as the mean ± SD ( n = 3).

    Article Snippet: Antibodies against PI3K-p110 α (#4255), PI3K-p110 β (#3011), Akt (#9272), phospho-Akt (Ser473) (#4060), mTOR (#2983), phospho-mTOR (Ser2448) (#5536), c-Myc (#18583), β -actin (#4970), NBS1 (#14956), phospho-ATM (Ser1981) (#13050), BRCA1 (#9010), BRCA2 (#10741), RAD51 (#8875), DNA-PKcs (#38168), Ku80 (#2753), Caspase 3 (#9662), and PARP (#9532) were purchased from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Incubation, Software

    Combination indexes (CI) of PI3K inhibitors and TMZ in GBM cells.

    Journal: BioMed Research International

    Article Title: ZSTK474 Sensitizes Glioblastoma to Temozolomide by Blocking Homologous Recombination Repair

    doi: 10.1155/2022/8568528

    Figure Lengend Snippet: Combination indexes (CI) of PI3K inhibitors and TMZ in GBM cells.

    Article Snippet: Antibodies against PI3K-p110 α (#4255), PI3K-p110 β (#3011), Akt (#9272), phospho-Akt (Ser473) (#4060), mTOR (#2983), phospho-mTOR (Ser2448) (#5536), c-Myc (#18583), β -actin (#4970), NBS1 (#14956), phospho-ATM (Ser1981) (#13050), BRCA1 (#9010), BRCA2 (#10741), RAD51 (#8875), DNA-PKcs (#38168), Ku80 (#2753), Caspase 3 (#9662), and PARP (#9532) were purchased from Cell Signaling Technology (Danvers, MA, USA).

    Techniques:

    The combined use of ZSTK474 and TMZ reduced HR repair efficiency. (a)–(d) SF295 and U87 cells were treated with ZSTK474 and TMZ as single agents or in combination for 48 h. (a) The formation of RAD51 foci was assessed using immunofluorescence. Cell nuclei were stained with DAPI. Scale bar, 20 μ m. Representative images are shown. (b) Quantification of the number of RAD51-positive SF295 and U87 cells. (c) The cells were collected for western blotting detection of NBS1, p-ATM, BRCA1, BRCA2, DNA-PKcs, and Ku80. (d) Western blotting analysis of PI3K-p110 β , p-Akt, p-mTOR, and c-Myc. All data are presented as the mean ± SD ( n = 3). ∗ P < 0.05; ∗∗ P < 0.01.

    Journal: BioMed Research International

    Article Title: ZSTK474 Sensitizes Glioblastoma to Temozolomide by Blocking Homologous Recombination Repair

    doi: 10.1155/2022/8568528

    Figure Lengend Snippet: The combined use of ZSTK474 and TMZ reduced HR repair efficiency. (a)–(d) SF295 and U87 cells were treated with ZSTK474 and TMZ as single agents or in combination for 48 h. (a) The formation of RAD51 foci was assessed using immunofluorescence. Cell nuclei were stained with DAPI. Scale bar, 20 μ m. Representative images are shown. (b) Quantification of the number of RAD51-positive SF295 and U87 cells. (c) The cells were collected for western blotting detection of NBS1, p-ATM, BRCA1, BRCA2, DNA-PKcs, and Ku80. (d) Western blotting analysis of PI3K-p110 β , p-Akt, p-mTOR, and c-Myc. All data are presented as the mean ± SD ( n = 3). ∗ P < 0.05; ∗∗ P < 0.01.

    Article Snippet: Antibodies against PI3K-p110 α (#4255), PI3K-p110 β (#3011), Akt (#9272), phospho-Akt (Ser473) (#4060), mTOR (#2983), phospho-mTOR (Ser2448) (#5536), c-Myc (#18583), β -actin (#4970), NBS1 (#14956), phospho-ATM (Ser1981) (#13050), BRCA1 (#9010), BRCA2 (#10741), RAD51 (#8875), DNA-PKcs (#38168), Ku80 (#2753), Caspase 3 (#9662), and PARP (#9532) were purchased from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Immunofluorescence, Staining, Western Blot

    The effect of TMZ on DNA damage and repair regulating factors and key molecules of the PI3K signaling pathway in GBM cells. (a) SF295, U87, and U251 cells were treated with different concentrations of TMZ (10, 20, 40, 80, 160, 320, 640, and 1280 μ M) for 72 h. Cell viability was determined by WST-8 assay. (b)–(f) SF295 and U87 cells were treated with TMZ (120 μ M and 180 μ M) for 48 and 72 h. (b) The cells were then collected for western blotting detection of the DNA SSB marker XRCC1 and the DSB marker γ -H2AX. (c) The cells were harvested for qRT-PCR analysis of BRCA1 and RAD51. (d) Western blot analysis of DNA damage repair proteins, including p-ATM, BRCA1, RAD51, DNA-PKcs, and Ku80. (e) qRT-PCR analysis of PIK3CA and PIK3CB. (f) Western blot analysis of key proteins in the PI3K signaling pathway, including PI3K-p110 α , PI3K-p110 β , p-Akt, Akt, p-mTOR, and mTOR. For western blotting, β -actin was used as a loading control. For qRT-PCR, gene expression was normalized to 18 s rRNA. All data are presented as the mean ± SD ( n = 3). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001; ∗∗∗∗ P < 0.0001.

    Journal: BioMed Research International

    Article Title: ZSTK474 Sensitizes Glioblastoma to Temozolomide by Blocking Homologous Recombination Repair

    doi: 10.1155/2022/8568528

    Figure Lengend Snippet: The effect of TMZ on DNA damage and repair regulating factors and key molecules of the PI3K signaling pathway in GBM cells. (a) SF295, U87, and U251 cells were treated with different concentrations of TMZ (10, 20, 40, 80, 160, 320, 640, and 1280 μ M) for 72 h. Cell viability was determined by WST-8 assay. (b)–(f) SF295 and U87 cells were treated with TMZ (120 μ M and 180 μ M) for 48 and 72 h. (b) The cells were then collected for western blotting detection of the DNA SSB marker XRCC1 and the DSB marker γ -H2AX. (c) The cells were harvested for qRT-PCR analysis of BRCA1 and RAD51. (d) Western blot analysis of DNA damage repair proteins, including p-ATM, BRCA1, RAD51, DNA-PKcs, and Ku80. (e) qRT-PCR analysis of PIK3CA and PIK3CB. (f) Western blot analysis of key proteins in the PI3K signaling pathway, including PI3K-p110 α , PI3K-p110 β , p-Akt, Akt, p-mTOR, and mTOR. For western blotting, β -actin was used as a loading control. For qRT-PCR, gene expression was normalized to 18 s rRNA. All data are presented as the mean ± SD ( n = 3). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001; ∗∗∗∗ P < 0.0001.

    Article Snippet: Antibodies against PI3K-p110 α (#4255), PI3K-p110 β (#3011), Akt (#9272), phospho-Akt (Ser473) (#4060), mTOR (#2983), phospho-mTOR (Ser2448) (#5536), c-Myc (#18583), β -actin (#4970), NBS1 (#14956), phospho-ATM (Ser1981) (#13050), BRCA1 (#9010), BRCA2 (#10741), RAD51 (#8875), DNA-PKcs (#38168), Ku80 (#2753), Caspase 3 (#9662), and PARP (#9532) were purchased from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Western Blot, Marker, Quantitative RT-PCR, Expressing

    Effect of PI3K inhibitors and TMZ as single agents or in combination on the survival of GBM cells. (a) SF295, U87, and U251 cells were treated with various concentrations of ZSTK474 (0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, and 2 μ M) for 72 h. Cell viability was determined by WST-8 assay. (b) Three GBM cell lines were incubated with ZSTK474 and TMZ as single agents or in combination (20%, 40%, 60%, 80%, and 100% of 2IC 50ZSTK474 or IC 50ZSTK474 : IC 50TMZ ) for 72 h. The combination effects were analyzed using CalcuSyn software, and the resulting CI-Fa plots are shown. (c) SF295 and U87 cells were treated with BYL719/TGX221 and TMZ as single agents or in combination (20%, 40%, 60%, 80%, and 100% of IC 50BYL719/TGX221 : IC 50TMZ ) for 72 h. The combination effects were analyzed using CalcuSyn software, and the resulting CI-Fa plots are shown. All data are presented as the mean ± SD ( n = 3).

    Journal: BioMed Research International

    Article Title: ZSTK474 Sensitizes Glioblastoma to Temozolomide by Blocking Homologous Recombination Repair

    doi: 10.1155/2022/8568528

    Figure Lengend Snippet: Effect of PI3K inhibitors and TMZ as single agents or in combination on the survival of GBM cells. (a) SF295, U87, and U251 cells were treated with various concentrations of ZSTK474 (0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, and 2 μ M) for 72 h. Cell viability was determined by WST-8 assay. (b) Three GBM cell lines were incubated with ZSTK474 and TMZ as single agents or in combination (20%, 40%, 60%, 80%, and 100% of 2IC 50ZSTK474 or IC 50ZSTK474 : IC 50TMZ ) for 72 h. The combination effects were analyzed using CalcuSyn software, and the resulting CI-Fa plots are shown. (c) SF295 and U87 cells were treated with BYL719/TGX221 and TMZ as single agents or in combination (20%, 40%, 60%, 80%, and 100% of IC 50BYL719/TGX221 : IC 50TMZ ) for 72 h. The combination effects were analyzed using CalcuSyn software, and the resulting CI-Fa plots are shown. All data are presented as the mean ± SD ( n = 3).

    Article Snippet: Antibodies against PI3K-p110 α (#4255), PI3K-p110 β (#3011), Akt (#9272), phospho-Akt (Ser473) (#4060), mTOR (#2983), phospho-mTOR (Ser2448) (#5536), c-Myc (#18583), β -actin (#4970), NBS1 (#14956), phospho-ATM (Ser1981) (#13050), BRCA1 (#9010), BRCA2 (#10741), RAD51 (#8875), DNA-PKcs (#38168), Ku80 (#2753), Caspase 3 (#9662), and PARP (#9532) were purchased from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Incubation, Software

    Combination indexes (CI) of PI3K inhibitors and TMZ in GBM cells.

    Journal: BioMed Research International

    Article Title: ZSTK474 Sensitizes Glioblastoma to Temozolomide by Blocking Homologous Recombination Repair

    doi: 10.1155/2022/8568528

    Figure Lengend Snippet: Combination indexes (CI) of PI3K inhibitors and TMZ in GBM cells.

    Article Snippet: Antibodies against PI3K-p110 α (#4255), PI3K-p110 β (#3011), Akt (#9272), phospho-Akt (Ser473) (#4060), mTOR (#2983), phospho-mTOR (Ser2448) (#5536), c-Myc (#18583), β -actin (#4970), NBS1 (#14956), phospho-ATM (Ser1981) (#13050), BRCA1 (#9010), BRCA2 (#10741), RAD51 (#8875), DNA-PKcs (#38168), Ku80 (#2753), Caspase 3 (#9662), and PARP (#9532) were purchased from Cell Signaling Technology (Danvers, MA, USA).

    Techniques:

    The combined use of ZSTK474 and TMZ reduced HR repair efficiency. (a)–(d) SF295 and U87 cells were treated with ZSTK474 and TMZ as single agents or in combination for 48 h. (a) The formation of RAD51 foci was assessed using immunofluorescence. Cell nuclei were stained with DAPI. Scale bar, 20 μ m. Representative images are shown. (b) Quantification of the number of RAD51-positive SF295 and U87 cells. (c) The cells were collected for western blotting detection of NBS1, p-ATM, BRCA1, BRCA2, DNA-PKcs, and Ku80. (d) Western blotting analysis of PI3K-p110 β , p-Akt, p-mTOR, and c-Myc. All data are presented as the mean ± SD ( n = 3). ∗ P < 0.05; ∗∗ P < 0.01.

    Journal: BioMed Research International

    Article Title: ZSTK474 Sensitizes Glioblastoma to Temozolomide by Blocking Homologous Recombination Repair

    doi: 10.1155/2022/8568528

    Figure Lengend Snippet: The combined use of ZSTK474 and TMZ reduced HR repair efficiency. (a)–(d) SF295 and U87 cells were treated with ZSTK474 and TMZ as single agents or in combination for 48 h. (a) The formation of RAD51 foci was assessed using immunofluorescence. Cell nuclei were stained with DAPI. Scale bar, 20 μ m. Representative images are shown. (b) Quantification of the number of RAD51-positive SF295 and U87 cells. (c) The cells were collected for western blotting detection of NBS1, p-ATM, BRCA1, BRCA2, DNA-PKcs, and Ku80. (d) Western blotting analysis of PI3K-p110 β , p-Akt, p-mTOR, and c-Myc. All data are presented as the mean ± SD ( n = 3). ∗ P < 0.05; ∗∗ P < 0.01.

    Article Snippet: Antibodies against PI3K-p110 α (#4255), PI3K-p110 β (#3011), Akt (#9272), phospho-Akt (Ser473) (#4060), mTOR (#2983), phospho-mTOR (Ser2448) (#5536), c-Myc (#18583), β -actin (#4970), NBS1 (#14956), phospho-ATM (Ser1981) (#13050), BRCA1 (#9010), BRCA2 (#10741), RAD51 (#8875), DNA-PKcs (#38168), Ku80 (#2753), Caspase 3 (#9662), and PARP (#9532) were purchased from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Immunofluorescence, Staining, Western Blot

    Selectivity and effective concentrations of inhibitors used in this study (reported in the following references: 11, 26, 31, 34, 38, 40).

    Journal: PLoS ONE

    Article Title: Effects of Novel Isoform-Selective Phosphoinositide 3-Kinase Inhibitors on Natural Killer Cell Function

    doi: 10.1371/journal.pone.0099486

    Figure Lengend Snippet: Selectivity and effective concentrations of inhibitors used in this study (reported in the following references: 11, 26, 31, 34, 38, 40).

    Article Snippet: Proteins were transferred to nitrocellulose and probed with the following antibodies to the different PI3K p110 isoforms: p110α (C73F8), p110β (C33D4), p110γ (D55D5) (Cell Signaling Technology), p110δ(Abcam, Cambridge, MA) and Actin (Sigma Aldrich).

    Techniques: In Vitro

    ( A ) Immunoblots were prepared using equivalent protein amounts from lysates of purified NK cells, T cells, B cells or two solid tumor cell lines. Blots were probed with PI3K isoform-specific antibodies and for β-actin as a loading control. ( B ) RMA-S, YAC-1, and EL4 H60 cells were labeled with calcein AM and co-cultured with NK cells at the indicated E:T ratios in the presence of 1 µM indicated inhibitors (TGX-221 was 0.5 µM) for 2 h. ( C ) RMA-S and YAC-1 cells were labeled with calcein AM and co-cultured with NK cells at 10∶1 E:T ratio in the presence of 1 µM indicated inhibitors for 2 h. ( D ) NK cells were treated with a higher concentration of MLN1117 (2 µM) and incubated with calcein AM-labeled YAC-1 cells at 10∶1 E:T ratio. Percentage specific lysis was calculated from pentaplicate cultures as follows: ((mean experimental release - mean spontaneous release)/(mean maximum release -mean spontaneous release)) x 100. The values are presented as the means ± SEM from three independent experiments. * p <0.05 and ** p <0.01, as determined by the unpaired Student's t test using Sigma Plot 10 software and as compared to the vehicle control group.

    Journal: PLoS ONE

    Article Title: Effects of Novel Isoform-Selective Phosphoinositide 3-Kinase Inhibitors on Natural Killer Cell Function

    doi: 10.1371/journal.pone.0099486

    Figure Lengend Snippet: ( A ) Immunoblots were prepared using equivalent protein amounts from lysates of purified NK cells, T cells, B cells or two solid tumor cell lines. Blots were probed with PI3K isoform-specific antibodies and for β-actin as a loading control. ( B ) RMA-S, YAC-1, and EL4 H60 cells were labeled with calcein AM and co-cultured with NK cells at the indicated E:T ratios in the presence of 1 µM indicated inhibitors (TGX-221 was 0.5 µM) for 2 h. ( C ) RMA-S and YAC-1 cells were labeled with calcein AM and co-cultured with NK cells at 10∶1 E:T ratio in the presence of 1 µM indicated inhibitors for 2 h. ( D ) NK cells were treated with a higher concentration of MLN1117 (2 µM) and incubated with calcein AM-labeled YAC-1 cells at 10∶1 E:T ratio. Percentage specific lysis was calculated from pentaplicate cultures as follows: ((mean experimental release - mean spontaneous release)/(mean maximum release -mean spontaneous release)) x 100. The values are presented as the means ± SEM from three independent experiments. * p <0.05 and ** p <0.01, as determined by the unpaired Student's t test using Sigma Plot 10 software and as compared to the vehicle control group.

    Article Snippet: Proteins were transferred to nitrocellulose and probed with the following antibodies to the different PI3K p110 isoforms: p110α (C73F8), p110β (C33D4), p110γ (D55D5) (Cell Signaling Technology), p110δ(Abcam, Cambridge, MA) and Actin (Sigma Aldrich).

    Techniques: Western Blot, Purification, Labeling, Cell Culture, Concentration Assay, Incubation, Lysis, Software

    Wild-type C57BL/6 mice were orally dosed with vehicle, the p110α selective inhibitor MLN1117 (60 mg/kg), or the pan-PI3K inhibitor GDC-0941 (70 mg/kg) for 7 days (n = 4). Spleen and bone marrow (BM) were isolated and analyzed for NK cell development by flow cytometry. ( A ) Splenocytes were stained with CD21 and CD23 for marginal zone (MZ) B cell population. Among the B220 + population, CD21 high CD23 low cells were gated for MZ B cell population. ( B, C ) Percentage of B/T cells and Th/Tc cells in spleen were determined using surface staining with anti-B220/anti-Thy1.2 mAbs and anti-CD4/anti-CD8 mAbs, respectively. ( D ) NK cells as specified by CD3 − NK1.1 + were gated and analyzed in single-cell suspension of spleen and BM, respectively. ( E ) Spleen- or BM-derived CD3 − NK1.1 + NK cells were analyzed for NK subset based on the expression level of CD11b and CD27. ( F ) Spleen- or BM-derived CD3 − NCR1 + NK cells were analyzed for maturation marker, Ly49A, LY49C/I, Ly49D, Ly49G2, and Ly49H. ( G ) Spleen- or BM-derived CD3 − NCR1 + NK cells were analyzed for the expression of CD122, NKG2D, and NKG2A/C/E. The data are expressed as the means ± SEM. Statistical analysis was performed with the unpaired Student's t test using Sigma Plot 10 software to compare the differences between vehicle and each inhibitor-treated group. *, p <0.05, **, p <0.01, and ***, p <0.001.

    Journal: PLoS ONE

    Article Title: Effects of Novel Isoform-Selective Phosphoinositide 3-Kinase Inhibitors on Natural Killer Cell Function

    doi: 10.1371/journal.pone.0099486

    Figure Lengend Snippet: Wild-type C57BL/6 mice were orally dosed with vehicle, the p110α selective inhibitor MLN1117 (60 mg/kg), or the pan-PI3K inhibitor GDC-0941 (70 mg/kg) for 7 days (n = 4). Spleen and bone marrow (BM) were isolated and analyzed for NK cell development by flow cytometry. ( A ) Splenocytes were stained with CD21 and CD23 for marginal zone (MZ) B cell population. Among the B220 + population, CD21 high CD23 low cells were gated for MZ B cell population. ( B, C ) Percentage of B/T cells and Th/Tc cells in spleen were determined using surface staining with anti-B220/anti-Thy1.2 mAbs and anti-CD4/anti-CD8 mAbs, respectively. ( D ) NK cells as specified by CD3 − NK1.1 + were gated and analyzed in single-cell suspension of spleen and BM, respectively. ( E ) Spleen- or BM-derived CD3 − NK1.1 + NK cells were analyzed for NK subset based on the expression level of CD11b and CD27. ( F ) Spleen- or BM-derived CD3 − NCR1 + NK cells were analyzed for maturation marker, Ly49A, LY49C/I, Ly49D, Ly49G2, and Ly49H. ( G ) Spleen- or BM-derived CD3 − NCR1 + NK cells were analyzed for the expression of CD122, NKG2D, and NKG2A/C/E. The data are expressed as the means ± SEM. Statistical analysis was performed with the unpaired Student's t test using Sigma Plot 10 software to compare the differences between vehicle and each inhibitor-treated group. *, p <0.05, **, p <0.01, and ***, p <0.001.

    Article Snippet: Proteins were transferred to nitrocellulose and probed with the following antibodies to the different PI3K p110 isoforms: p110α (C73F8), p110β (C33D4), p110γ (D55D5) (Cell Signaling Technology), p110δ(Abcam, Cambridge, MA) and Actin (Sigma Aldrich).

    Techniques: Isolation, Flow Cytometry, Staining, Derivative Assay, Expressing, Marker, Software

    S1P-induced COX-2 expression is mediated through PI3K/Akt in HCFs. (a) Cells were pretreated without or with LY294002 (0.3, 3, and 30 μ M) for 1 h and then incubated with 15 μ M S1P for 8 h. The levels of COX-2 and GAPDH used as an internal control were analyzed by Western blot. (b) Cells were transfected without or with COX-2 promoter-luciferase reporter gene, pretreated with LY294002 (30 μ M) for 1 h, and then incubated with 15 μ M S1P for 4 h (mRNA level) or 1 h (promoter activity). The COX-2 mRNA and promoter activity were analyzed by real-time PCR (open bar) and promoter assay (gray bar). (c) Cells were transfected with scrambled, p110 (left panel), or Akt (right panel) siRNA for 24 h and then exposed to 15 μ M S1P for 8 h. The levels of COX-2, GAPDH, p110, and Akt proteins were analyzed by Western blot. (d) Cells were pretreated with or without 30 μ M LY294002, 10 μ M MMP2/9 inhibitor, 10 μ g/ml CRM197, or 10 μ M AG1478 for 1 h and then incubated with 15 μ M S1P for 0.5, 1, 3, 5, and 10 min. The levels of phospho-Akt and GAPDH were determined by Western blot. (e) Cells were pretreated without or with 30 μ M LY294002 for 1 h and then incubated with 15 μ M S1P for 0, 3, 5, 10, 15, and 30 min. The levels of phospho-p44/p42, phospho-p38, phospho-JNK1/2, and GAPDH were determined by Western blot. Data are expressed as the mean ± SEM of three individual experiments ( n = 3). # P < 0.05, as compared with the control or pretreatment with inhibitor indicated in the figure.

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Sphingosine 1-Phosphate-Upregulated COX-2/PGE 2 System Contributes to Human Cardiac Fibroblast Apoptosis: Involvement of MMP-9-Dependent Transactivation of EGFR Cascade

    doi: 10.1155/2022/7664290

    Figure Lengend Snippet: S1P-induced COX-2 expression is mediated through PI3K/Akt in HCFs. (a) Cells were pretreated without or with LY294002 (0.3, 3, and 30 μ M) for 1 h and then incubated with 15 μ M S1P for 8 h. The levels of COX-2 and GAPDH used as an internal control were analyzed by Western blot. (b) Cells were transfected without or with COX-2 promoter-luciferase reporter gene, pretreated with LY294002 (30 μ M) for 1 h, and then incubated with 15 μ M S1P for 4 h (mRNA level) or 1 h (promoter activity). The COX-2 mRNA and promoter activity were analyzed by real-time PCR (open bar) and promoter assay (gray bar). (c) Cells were transfected with scrambled, p110 (left panel), or Akt (right panel) siRNA for 24 h and then exposed to 15 μ M S1P for 8 h. The levels of COX-2, GAPDH, p110, and Akt proteins were analyzed by Western blot. (d) Cells were pretreated with or without 30 μ M LY294002, 10 μ M MMP2/9 inhibitor, 10 μ g/ml CRM197, or 10 μ M AG1478 for 1 h and then incubated with 15 μ M S1P for 0.5, 1, 3, 5, and 10 min. The levels of phospho-Akt and GAPDH were determined by Western blot. (e) Cells were pretreated without or with 30 μ M LY294002 for 1 h and then incubated with 15 μ M S1P for 0, 3, 5, 10, 15, and 30 min. The levels of phospho-p44/p42, phospho-p38, phospho-JNK1/2, and GAPDH were determined by Western blot. Data are expressed as the mean ± SEM of three individual experiments ( n = 3). # P < 0.05, as compared with the control or pretreatment with inhibitor indicated in the figure.

    Article Snippet: Phospho-JNK1/2 (#4668, Thr 183 /Tyr 185 ), phospho-p38 MAPK (#9211, Thr 180 /Tyr 182 ), phospho-p42/p44 MAPK (#9101, Thr 202 /Tyr 204 ), phospho-EGFR (#4407, Tyr 1173 ), phospho-Akt (#9271, Ser 473 ), c-Jun (#9165), and PI3K p110 α (#4249) antibodies were from Cell Signaling (Danvers, MA, USA).

    Techniques: Expressing, Incubation, Western Blot, Transfection, Luciferase, Activity Assay, Real-time Polymerase Chain Reaction, Promoter Assay

    Schematic signaling pathways are involved in S1P-induced COX-2/PGE 2 expression associated with activation of caspase-3 leading to apoptosis in HCFs. S1P could be upregulated mediated through estrogen/estrogen receptor-stimulated sphingosine kinase-1 (SphK1) activity , binding with S1PR1/3 coupled to G q or G i protein results in MMP9-mediated HB-EGF cleavage leading to activation of EGFR/PI3K/Akt/MAPKs (i.e., p42/p44 MAPK, p38 MAPK, and JNK1/2)-dependent AP-1 c-Jun. The COX-2 transcription and PGE 2 generation are dependently regulated by an S1PR1/3-mediated EGFR transactivation to activate AP-1 binding activity. These signaling pathways contribute to the activation of AP-1 required for COX-2 expression and PGE 2 generation in HCFs.

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Sphingosine 1-Phosphate-Upregulated COX-2/PGE 2 System Contributes to Human Cardiac Fibroblast Apoptosis: Involvement of MMP-9-Dependent Transactivation of EGFR Cascade

    doi: 10.1155/2022/7664290

    Figure Lengend Snippet: Schematic signaling pathways are involved in S1P-induced COX-2/PGE 2 expression associated with activation of caspase-3 leading to apoptosis in HCFs. S1P could be upregulated mediated through estrogen/estrogen receptor-stimulated sphingosine kinase-1 (SphK1) activity , binding with S1PR1/3 coupled to G q or G i protein results in MMP9-mediated HB-EGF cleavage leading to activation of EGFR/PI3K/Akt/MAPKs (i.e., p42/p44 MAPK, p38 MAPK, and JNK1/2)-dependent AP-1 c-Jun. The COX-2 transcription and PGE 2 generation are dependently regulated by an S1PR1/3-mediated EGFR transactivation to activate AP-1 binding activity. These signaling pathways contribute to the activation of AP-1 required for COX-2 expression and PGE 2 generation in HCFs.

    Article Snippet: Phospho-JNK1/2 (#4668, Thr 183 /Tyr 185 ), phospho-p38 MAPK (#9211, Thr 180 /Tyr 182 ), phospho-p42/p44 MAPK (#9101, Thr 202 /Tyr 204 ), phospho-EGFR (#4407, Tyr 1173 ), phospho-Akt (#9271, Ser 473 ), c-Jun (#9165), and PI3K p110 α (#4249) antibodies were from Cell Signaling (Danvers, MA, USA).

    Techniques: Expressing, Activation Assay, Activity Assay, Binding Assay

    TDRG1 is significantly upregulated in testicular seminoma tissues than in normal testicular tissues. Notes: ( A ) RT-PCR showed that the relative gene expression level of TDRG1 is significantly higher in testicular seminoma tissues than in normal testicular tissues. ( B i) Representative Western blotting images that are associated with TDRG1 and GAPDH protein expression in both testicular seminoma and normal testicular tissues. ( B ii) Western blotting showed that the relative protein expression level of TDRG1 is significantly higher in testicular seminoma tissues than in normal testicular tissues. RT-PCR and Western blotting were performed to detect the expression levels of TDRG1 in both testicular seminoma tissues (n=33) and normal testicular tissues (n=33). ( C i) Immunohistochemical staining pictures of both testicular seminoma and normal testicular tissue samples with anti- TDRG1 , anti-p-PI3K/p110, and anti-p-Akt antibodies. ( C ii) The protein expression levels of TDRG1 , p-PI3K/p110, and p-Akt are significantly upregulated in testicular seminoma tissues compared to normal testicular tissues. Protein expression levels of TDRG1 , p-PI3K/p110, and p-Akt were detected by an immunohistochemistry assay. The tissue microarray chips TE802 and TE2081 contained 80 points (from 80 testicular seminoma tissue samples) and 138 points (from 46 testicular seminoma tissue samples, three points per sample), respectively. TEN601 had 60 points (from 30 normal testicular tissue samples, two points per sample). Bars representing cancer are averages of data from 218 points (80+138) in two tissue microarray chips. Bars representing the normal tissue samples are the average of data from 60 points in a tissue microarray chip. * P <0.05 vs control group. Cancer: testicular seminoma; normal: normal testicular tissues. Abbreviations: TDRG1 , testis development-related gene 1; RT-PCR, reverse transcription polymerase chain reaction; PI3K, phosphoinositide-3 kinase; GADPH, glyceral-dehyde-3-phosphate dehydrogenase.

    Journal: OncoTargets and therapy

    Article Title: TDRG1 functions in testicular seminoma are dependent on the PI3K/Akt/mTOR signaling pathway

    doi: 10.2147/OTT.S97294

    Figure Lengend Snippet: TDRG1 is significantly upregulated in testicular seminoma tissues than in normal testicular tissues. Notes: ( A ) RT-PCR showed that the relative gene expression level of TDRG1 is significantly higher in testicular seminoma tissues than in normal testicular tissues. ( B i) Representative Western blotting images that are associated with TDRG1 and GAPDH protein expression in both testicular seminoma and normal testicular tissues. ( B ii) Western blotting showed that the relative protein expression level of TDRG1 is significantly higher in testicular seminoma tissues than in normal testicular tissues. RT-PCR and Western blotting were performed to detect the expression levels of TDRG1 in both testicular seminoma tissues (n=33) and normal testicular tissues (n=33). ( C i) Immunohistochemical staining pictures of both testicular seminoma and normal testicular tissue samples with anti- TDRG1 , anti-p-PI3K/p110, and anti-p-Akt antibodies. ( C ii) The protein expression levels of TDRG1 , p-PI3K/p110, and p-Akt are significantly upregulated in testicular seminoma tissues compared to normal testicular tissues. Protein expression levels of TDRG1 , p-PI3K/p110, and p-Akt were detected by an immunohistochemistry assay. The tissue microarray chips TE802 and TE2081 contained 80 points (from 80 testicular seminoma tissue samples) and 138 points (from 46 testicular seminoma tissue samples, three points per sample), respectively. TEN601 had 60 points (from 30 normal testicular tissue samples, two points per sample). Bars representing cancer are averages of data from 218 points (80+138) in two tissue microarray chips. Bars representing the normal tissue samples are the average of data from 60 points in a tissue microarray chip. * P <0.05 vs control group. Cancer: testicular seminoma; normal: normal testicular tissues. Abbreviations: TDRG1 , testis development-related gene 1; RT-PCR, reverse transcription polymerase chain reaction; PI3K, phosphoinositide-3 kinase; GADPH, glyceral-dehyde-3-phosphate dehydrogenase.

    Article Snippet: Antibodies against PI3K/p85 (1:8,000), phospho-PI3K/p85 (p-PI3K/p85, 1:8,000), p-Akt (1:4,000), and p-mTOR (T2446, 1:4,000) were obtained from Abcam Biotechnology (Cambridge, UK); mTOR (1:4,000), p-mTOR (Ser2448, 1:4,000), and p-mTOR (Ser2481, 1:4,000) antibodies were purchased from Cell Signaling Technology (Boston, MA, USA); PI3K/p110 (1:4,000) and p-PI3K/p110 (1:4,000) antibodies were obtained from Boaoshen Biotechnology (Beijing, People’s Republic of China).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Immunohistochemical staining, Staining, Immunohistochemistry, Microarray

    Relative TDRG1 expression in both gene and protein levels of seminoma TCam-2 cells after different treatments. Notes: ( A ) Relative gene expression level of TDRG1 in seminoma TCam-2 cells was remarkably upregulated and downregulated after the gene was knocked out and overexpressed, respectively, on the basis of RT-PCR (n=5). ( B ) Relative protein expression level of TDRG1 in seminoma TCam-2 cells was remarkably upregulated and downregulated after the gene was knocked out and overexpressed, respectively, but was unaffected after treatment with LY294002 or insulin-like growth factor-1 on the basis of Western blotting. * P <0.05 and ** P <0.01 vs control group. Vector: enhanced green fluorescent protein plasmid-C1 vector. Abbreviations: TDRG1 , testis development-related gene 1; RT-PCR, reverse transcription polymerase chain reaction; PI3K, phosphoinositide-3 kinase; TDRG1 (−), the knockout of TDRG1 ; TDRG1 (+), the overexpression of TDRG1 ; PI3K (−), the inhibition of PI3K by LY294002; PI3K (+), the activation of PI3K by insulin-like growth factor-1.

    Journal: OncoTargets and therapy

    Article Title: TDRG1 functions in testicular seminoma are dependent on the PI3K/Akt/mTOR signaling pathway

    doi: 10.2147/OTT.S97294

    Figure Lengend Snippet: Relative TDRG1 expression in both gene and protein levels of seminoma TCam-2 cells after different treatments. Notes: ( A ) Relative gene expression level of TDRG1 in seminoma TCam-2 cells was remarkably upregulated and downregulated after the gene was knocked out and overexpressed, respectively, on the basis of RT-PCR (n=5). ( B ) Relative protein expression level of TDRG1 in seminoma TCam-2 cells was remarkably upregulated and downregulated after the gene was knocked out and overexpressed, respectively, but was unaffected after treatment with LY294002 or insulin-like growth factor-1 on the basis of Western blotting. * P <0.05 and ** P <0.01 vs control group. Vector: enhanced green fluorescent protein plasmid-C1 vector. Abbreviations: TDRG1 , testis development-related gene 1; RT-PCR, reverse transcription polymerase chain reaction; PI3K, phosphoinositide-3 kinase; TDRG1 (−), the knockout of TDRG1 ; TDRG1 (+), the overexpression of TDRG1 ; PI3K (−), the inhibition of PI3K by LY294002; PI3K (+), the activation of PI3K by insulin-like growth factor-1.

    Article Snippet: Antibodies against PI3K/p85 (1:8,000), phospho-PI3K/p85 (p-PI3K/p85, 1:8,000), p-Akt (1:4,000), and p-mTOR (T2446, 1:4,000) were obtained from Abcam Biotechnology (Cambridge, UK); mTOR (1:4,000), p-mTOR (Ser2448, 1:4,000), and p-mTOR (Ser2481, 1:4,000) antibodies were purchased from Cell Signaling Technology (Boston, MA, USA); PI3K/p110 (1:4,000) and p-PI3K/p110 (1:4,000) antibodies were obtained from Boaoshen Biotechnology (Beijing, People’s Republic of China).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Plasmid Preparation, Knock-Out, Over Expression, Inhibition, Activation Assay

    The effect of TDRG1 - and PI3K-mediated signaling on the cell proliferation rate, cell proliferation index, and apoptosis rate. Notes: ( A ) Cells with both TDRG1 knockout and PI3K inhibition exhibited decreased cell proliferation. ( B i and B ii) Cells with both TDRG1 knockout and PI3K inhibition exhibited decreased cell proliferation index. ( C i and C ii) Cells with both TDRG1 knockout and PI3K inhibition exhibited increased apoptosis rate. Seminoma TCam-2 cells were subjected to different treatments: the TDRG1 knockout, TDRG1 overexpression, PI3K inhibition (LY294002 administration), or PI3K activation (IGF-1 administration). The cell proliferation rate was assessed using MTT kits at different time points after the treatments, and cell proliferation index and apoptosis rate were evaluated using flow cytometry at 72 h. The cell proliferation index was obtained by calculating the percentage of cells in the S, G2, and M phases. * P <0.05 and ** P <0.01 vs control group. Vector: enhanced green fluorescent protein plasmid-C1 vector. Abbreviations: TDRG1 , testis development-related gene 1; PI3K, phosphoinositide-3 kinase; IGF-1, insulin-like growth factor-1; MTT, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide; h, hours; TDRG1 (−), the knockout of TDRG1 ; TDRG1 (+), the overexpression of TDRG1 ; PI3K (−), the inhibition of PI3K by LY294002; PI3K (+), the activation of PI3K by IGF-1; FL2-A, fluorescence 2 area; FITC, fluorescein isothiocyanate.

    Journal: OncoTargets and therapy

    Article Title: TDRG1 functions in testicular seminoma are dependent on the PI3K/Akt/mTOR signaling pathway

    doi: 10.2147/OTT.S97294

    Figure Lengend Snippet: The effect of TDRG1 - and PI3K-mediated signaling on the cell proliferation rate, cell proliferation index, and apoptosis rate. Notes: ( A ) Cells with both TDRG1 knockout and PI3K inhibition exhibited decreased cell proliferation. ( B i and B ii) Cells with both TDRG1 knockout and PI3K inhibition exhibited decreased cell proliferation index. ( C i and C ii) Cells with both TDRG1 knockout and PI3K inhibition exhibited increased apoptosis rate. Seminoma TCam-2 cells were subjected to different treatments: the TDRG1 knockout, TDRG1 overexpression, PI3K inhibition (LY294002 administration), or PI3K activation (IGF-1 administration). The cell proliferation rate was assessed using MTT kits at different time points after the treatments, and cell proliferation index and apoptosis rate were evaluated using flow cytometry at 72 h. The cell proliferation index was obtained by calculating the percentage of cells in the S, G2, and M phases. * P <0.05 and ** P <0.01 vs control group. Vector: enhanced green fluorescent protein plasmid-C1 vector. Abbreviations: TDRG1 , testis development-related gene 1; PI3K, phosphoinositide-3 kinase; IGF-1, insulin-like growth factor-1; MTT, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide; h, hours; TDRG1 (−), the knockout of TDRG1 ; TDRG1 (+), the overexpression of TDRG1 ; PI3K (−), the inhibition of PI3K by LY294002; PI3K (+), the activation of PI3K by IGF-1; FL2-A, fluorescence 2 area; FITC, fluorescein isothiocyanate.

    Article Snippet: Antibodies against PI3K/p85 (1:8,000), phospho-PI3K/p85 (p-PI3K/p85, 1:8,000), p-Akt (1:4,000), and p-mTOR (T2446, 1:4,000) were obtained from Abcam Biotechnology (Cambridge, UK); mTOR (1:4,000), p-mTOR (Ser2448, 1:4,000), and p-mTOR (Ser2481, 1:4,000) antibodies were purchased from Cell Signaling Technology (Boston, MA, USA); PI3K/p110 (1:4,000) and p-PI3K/p110 (1:4,000) antibodies were obtained from Boaoshen Biotechnology (Beijing, People’s Republic of China).

    Techniques: Knock-Out, Inhibition, Over Expression, Activation Assay, Flow Cytometry, Plasmid Preparation, Fluorescence

    The effect of TDRG1 - and PI3K-mediated signaling on the cell adhesive capacity and cell invasion. Notes: ( A ) The cell adhesive capacity was increased in the cells with both TDRG1 knockout and PI3K inhibition and decreased in the cells with both TDRG1 overexpression and PI3K activation. ( B i) Pictures of cell passing through transwells. ( B ii) The cell invasion was increased in the cells with both TDRG1 knockout and PI3K inhibition and decreased in the cells with both TDRG1 overexpression and PI3K activation. Seminoma TCam-2 cells were subjected to different treatments: the TDRG1 knockout, TDRG1 overexpression, PI3K inhibition (LY294002 administration), or PI3K activation (IGF-1 administration). * P <0.05 and ** P <0.01 vs control group. Vector: enhanced green fluorescent protein plasmid-C1 vector. Abbreviations: TDRG1 , testis development-related gene 1; PI3K, phosphoinositide-3 kinase; IGF-1, insulin-like growth factor-1; TDRG1 (−), the knockout of TDRG1 ; TDRG1 (+), the overexpression of TDRG1 ; PI3K (−), the inhibition of PI3K by LY294002; PI3K (+), the activation of PI3K by IGF-1.

    Journal: OncoTargets and therapy

    Article Title: TDRG1 functions in testicular seminoma are dependent on the PI3K/Akt/mTOR signaling pathway

    doi: 10.2147/OTT.S97294

    Figure Lengend Snippet: The effect of TDRG1 - and PI3K-mediated signaling on the cell adhesive capacity and cell invasion. Notes: ( A ) The cell adhesive capacity was increased in the cells with both TDRG1 knockout and PI3K inhibition and decreased in the cells with both TDRG1 overexpression and PI3K activation. ( B i) Pictures of cell passing through transwells. ( B ii) The cell invasion was increased in the cells with both TDRG1 knockout and PI3K inhibition and decreased in the cells with both TDRG1 overexpression and PI3K activation. Seminoma TCam-2 cells were subjected to different treatments: the TDRG1 knockout, TDRG1 overexpression, PI3K inhibition (LY294002 administration), or PI3K activation (IGF-1 administration). * P <0.05 and ** P <0.01 vs control group. Vector: enhanced green fluorescent protein plasmid-C1 vector. Abbreviations: TDRG1 , testis development-related gene 1; PI3K, phosphoinositide-3 kinase; IGF-1, insulin-like growth factor-1; TDRG1 (−), the knockout of TDRG1 ; TDRG1 (+), the overexpression of TDRG1 ; PI3K (−), the inhibition of PI3K by LY294002; PI3K (+), the activation of PI3K by IGF-1.

    Article Snippet: Antibodies against PI3K/p85 (1:8,000), phospho-PI3K/p85 (p-PI3K/p85, 1:8,000), p-Akt (1:4,000), and p-mTOR (T2446, 1:4,000) were obtained from Abcam Biotechnology (Cambridge, UK); mTOR (1:4,000), p-mTOR (Ser2448, 1:4,000), and p-mTOR (Ser2481, 1:4,000) antibodies were purchased from Cell Signaling Technology (Boston, MA, USA); PI3K/p110 (1:4,000) and p-PI3K/p110 (1:4,000) antibodies were obtained from Boaoshen Biotechnology (Beijing, People’s Republic of China).

    Techniques: Knock-Out, Inhibition, Over Expression, Activation Assay, Plasmid Preparation

    Expression levels of various proteins in seminoma TCam-2 cells after different treatments based on a Western blot assay. Notes: Seminoma TCam-2 cells were subjected to different treatments: the TDRG1 knockout, TDRG1 overexpression, PI3K inhibition (LY294002 administration), or PI3K activation (IGF-1 administration). Inhibiting TDRG1 causes a dramatic reduction in the protein expression levels of p-PI3K/p85, p-PI3K/p110, p-Akt, and p-mTOR (S2448) but not PI3K/p85, PI3K/p110, mTOR, p-mTOR (S2481), and p-mTOR (T2446). In contrast, overexpression of TDRG1 promoted the expression of p-PI3K/p85, p-Akt, and p-mTOR (S2448). The treatment with LY294002 inhibited the expression of PI3K/p110, p-PI3K/p110, p-Akt, and p-mTOR (S2448), while pharmacological stimulation with IGF-1 caused a marked elevation in the phosphorylation levels of PI3K/p110, Akt, and mTOR (S2448). * P <0.05 and ** P <0.01 vs control group. Vector: enhanced green fluorescent protein plasmid-C1 vector. Abbreviations: TDRG1 , testis development-related gene 1; PI3K, phosphoinositide-3 kinase; IGF-1, insulin-like growth factor-1; mTOR, mammalian target of rapamycin; TDRG1 (−), the knockout of TDRG1 ; TDRG1 (+), the overexpression of TDRG1 ; PI3K (−), the inhibition of PI3K by LY294002; PI3K (+), the activation of PI3K by IGF-1; GADPH, glyceraldehyde-3-phosphate dehydrogenase.

    Journal: OncoTargets and therapy

    Article Title: TDRG1 functions in testicular seminoma are dependent on the PI3K/Akt/mTOR signaling pathway

    doi: 10.2147/OTT.S97294

    Figure Lengend Snippet: Expression levels of various proteins in seminoma TCam-2 cells after different treatments based on a Western blot assay. Notes: Seminoma TCam-2 cells were subjected to different treatments: the TDRG1 knockout, TDRG1 overexpression, PI3K inhibition (LY294002 administration), or PI3K activation (IGF-1 administration). Inhibiting TDRG1 causes a dramatic reduction in the protein expression levels of p-PI3K/p85, p-PI3K/p110, p-Akt, and p-mTOR (S2448) but not PI3K/p85, PI3K/p110, mTOR, p-mTOR (S2481), and p-mTOR (T2446). In contrast, overexpression of TDRG1 promoted the expression of p-PI3K/p85, p-Akt, and p-mTOR (S2448). The treatment with LY294002 inhibited the expression of PI3K/p110, p-PI3K/p110, p-Akt, and p-mTOR (S2448), while pharmacological stimulation with IGF-1 caused a marked elevation in the phosphorylation levels of PI3K/p110, Akt, and mTOR (S2448). * P <0.05 and ** P <0.01 vs control group. Vector: enhanced green fluorescent protein plasmid-C1 vector. Abbreviations: TDRG1 , testis development-related gene 1; PI3K, phosphoinositide-3 kinase; IGF-1, insulin-like growth factor-1; mTOR, mammalian target of rapamycin; TDRG1 (−), the knockout of TDRG1 ; TDRG1 (+), the overexpression of TDRG1 ; PI3K (−), the inhibition of PI3K by LY294002; PI3K (+), the activation of PI3K by IGF-1; GADPH, glyceraldehyde-3-phosphate dehydrogenase.

    Article Snippet: Antibodies against PI3K/p85 (1:8,000), phospho-PI3K/p85 (p-PI3K/p85, 1:8,000), p-Akt (1:4,000), and p-mTOR (T2446, 1:4,000) were obtained from Abcam Biotechnology (Cambridge, UK); mTOR (1:4,000), p-mTOR (Ser2448, 1:4,000), and p-mTOR (Ser2481, 1:4,000) antibodies were purchased from Cell Signaling Technology (Boston, MA, USA); PI3K/p110 (1:4,000) and p-PI3K/p110 (1:4,000) antibodies were obtained from Boaoshen Biotechnology (Beijing, People’s Republic of China).

    Techniques: Expressing, Western Blot, Knock-Out, Over Expression, Inhibition, Activation Assay, Plasmid Preparation