Journal: International Journal of Molecular Sciences

Article Title: Dominant Role of PI3K p110α over p110β in Insulin and β-Adrenergic Receptor Signalling

doi: 10.3390/ijms222312813

Figure Lengend Snippet: p110α is the principal PI3K isoform engaged in insulin signalling in brown adipocytes. Brown pre-adipocytes or differentiated adipocytes were treated with A66 (1 μM, blue graph bars) or TGX221 (0.5 μM, red graph bars) or a combination of both inhibitors (purple graph bars) followed by stimulation with 100 nM of insulin for 15 min at 37 °C. ( A ) Levels of Akt (T308) phosphorylation were determined by immunoblot analysis. Phosphorylation levels were normalised to total Akt levels detected in a second blot performed in parallel using the same lysates. ( B ) Ribosomal protein S6 (rpS6) (S240/244) phosphorylation was also detected on the same immunoblots. ( C ) The levels of expression of p110α and p110β in pre-adipocytes and differentiated adipocytes used in the signalling experiments were also determined by immunoblot analysis. p110α and p110β signal intensities were normalised to vinculin (used as a loading control). ( D ) Akt (T308) phosphorylation in p110α-deficient (p110α DEL ) brown adipocytes treated and stimulated as above. Representative immunoblots and bar graphs with data from three or four ( n = 3–4) independent experiments are shown. Data are presented as mean ± SEM. Statistical analysis was performed by one-way ANOVA with Dunnett’s multiple comparisons test between insulin-stimulated vehicle-treated and insulin-stimulated inhibitor-treated samples ( A , B , D ) or by unpaired two-tailed t -test ( C ). Comparisons between pre-adipocytes and differentiated adipocytes in ( B ) were performed by two-way ANOVA with Sidak’s multiple comparisons test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: PI3K p110α-selective inhibitor A66 (cat. no. 5595) was from Tocris/Bio-Techne (Minneapolis, MN, USA), PI3K p110β-selective inhibitor TGX221 (item no. 10007349), EPAC1 inhibitor CE3F4 (item no. 17767), and PKA inhibitor (PKI) fragment (2-22)-amide (item no. 17486) were from Cayman Chemical (Ann Arbor, MI, USA).

Techniques: Western Blot, Expressing, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Dominant Role of PI3K p110α over p110β in Insulin and β-Adrenergic Receptor Signalling

doi: 10.3390/ijms222312813

Figure Lengend Snippet: p110α is the principal PI3K isoform engaged in insulin signalling in 3T3-L1 adipocytes and Hepa 1.6 cells. 3T3-L1 pre-adipocytes or differentiated adipocytes or murine Hepa 1.6 hepatocytes were treated with A66 (1 μM, blue graph bars) or TGX221 (0.5 μM, red graph bars) or a combination of both inhibitors (purple graph bars) followed by stimulation with 100 nM of insulin for 15 min at 37 °C. ( A ) Levels of Akt (T308) phosphorylation in 3T3-L1 pre-adipocytes and adipocytes were determined by immunoblot analysis. Phosphorylation levels were normalised to total Akt levels detected in a second blot performed in parallel using the same lysates. ( B ) Ribosomal protein S6 (rpS6) phosphorylation (S240/244) was also detected in the same immunoblots. ( C ) The levels of expression of p110α and p110β in pre-adipocytes and differentiated 3T3-L1 adipocytes used for the signalling experiments were also determined by immunoblot analysis. ( D ) Akt (T308) and rpS6 (S240/244) phosphorylation in Hepa 1-6 murine hepatoma cells treated and stimulated as above. Representative immunoblots and bar graphs with pooled data from three or four ( n = 3–4) independent experiments are shown. Data are presented as mean ± SEM. ( E ) Insulin-stimulated glucose uptake in 3T3-L1 adipocytes treated with inhibitors as above. Data from three ( n = 3) independent experiments are shown. Statistical analysis was performed by one-way ANOVA with Dunnett’s multiple comparisons test between insulin-stimulated vehicle-treated and insulin-stimulated inhibitor-treated samples ( A , B , D , E ) or by unpaired two-tailed t -test ( C ). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: PI3K p110α-selective inhibitor A66 (cat. no. 5595) was from Tocris/Bio-Techne (Minneapolis, MN, USA), PI3K p110β-selective inhibitor TGX221 (item no. 10007349), EPAC1 inhibitor CE3F4 (item no. 17767), and PKA inhibitor (PKI) fragment (2-22)-amide (item no. 17486) were from Cayman Chemical (Ann Arbor, MI, USA).

Techniques: Western Blot, Expressing, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Dominant Role of PI3K p110α over p110β in Insulin and β-Adrenergic Receptor Signalling

doi: 10.3390/ijms222312813

Figure Lengend Snippet: p110α maximal activation by insulin requires interaction with Ras. Wild-type (non-striped graph bars) and mutant (striped graph bars) p110α-RBD MEFs were treated with A66 (1 μM, blue graph bars) or TGX221 (0.5 μM, red graph bars) or a combination of both inhibitors (purple graph bars) followed by stimulation with 100 nM of insulin or with 15.6 nM of EGF (green graph bars) for 15 min at 37 °C. ( A ) Levels of Akt (T308) phosphorylation were determined by immunoblot analysis. Phosphorylation levels were normalised to vinculin used as a loading control. rpS6 (S240/244) phosphorylation ( B ) and ERK1/2 (T202/Y204) phosphorylation ( C ) were also detected in the same immunoblots. Representative immunoblots and corresponding bar graphs with pooled data from five ( n = 5) independent experiments are shown. Data are presented as mean ± SEM. Statistical analysis was performed by two-way ANOVA with Dunnett’s multiple comparisons test between insulin-stimulated vehicle-treated and insulin-stimulated inhibitor-treated samples of the same genotype or Sidak’s multiple comparisons test between samples of different genotypes. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: PI3K p110α-selective inhibitor A66 (cat. no. 5595) was from Tocris/Bio-Techne (Minneapolis, MN, USA), PI3K p110β-selective inhibitor TGX221 (item no. 10007349), EPAC1 inhibitor CE3F4 (item no. 17767), and PKA inhibitor (PKI) fragment (2-22)-amide (item no. 17486) were from Cayman Chemical (Ann Arbor, MI, USA).

Techniques: Activation Assay, Mutagenesis, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Dominant Role of PI3K p110α over p110β in Insulin and β-Adrenergic Receptor Signalling

doi: 10.3390/ijms222312813

Figure Lengend Snippet: p110α is the principal PI3K isoform engaged in β-adrenergic signalling in adipocytes. Brown pre-adipocytes ( A ) or differentiated adipocytes ( B ) or differentiated 3T3-L1 adipocytes ( C ) were treated with A66 (1 μM, blue graph bars) or TGX221 (0.5 μM, red graph bars) or a combination of both inhibitors (purple graph bars) followed by stimulation with 1 μM of norepinephrine (NE, striped graph bars) for 15 min at 37 °C. Levels of Akt T308 phosphorylation were determined by immunoblot analysis. Phosphorylation levels were normalised to total Akt levels detected in a second blot performed in parallel using the same lysates. Representative immunoblots and corresponding bar graphs with pooled data from three or four ( n = 3–4) independent experiments are shown. Data are presented as mean ± SEM. Statistical analysis was performed by one-way ANOVA with Dunnett’s multiple comparisons test between vehicle-treated NE-stimulated and all other samples. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: PI3K p110α-selective inhibitor A66 (cat. no. 5595) was from Tocris/Bio-Techne (Minneapolis, MN, USA), PI3K p110β-selective inhibitor TGX221 (item no. 10007349), EPAC1 inhibitor CE3F4 (item no. 17767), and PKA inhibitor (PKI) fragment (2-22)-amide (item no. 17486) were from Cayman Chemical (Ann Arbor, MI, USA).

Techniques: Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Dominant Role of PI3K p110α over p110β in Insulin and β-Adrenergic Receptor Signalling

doi: 10.3390/ijms222312813

Figure Lengend Snippet: Activation of Akt downstream of β-AR is mediated by EPAC1. ( A ) Brown pre-adipocytes were treated with the EPAC1 inhibitor CE3F4 (20 μM, dark green graph bars) or the PKA inhibitor fragment (6-22) amide (PKI, 5 μM, yellow graph bars) or a combination of both inhibitors (bright green graph bar) followed by stimulation with 1 μM of norepinephrine (NE, striped graph bars) for 15 min at 37 °C. Levels of Akt T308 phosphorylation were determined by immunoblot analysis. Phosphorylation levels were normalised to total Akt levels detected in a second blot performed in parallel using the same lysates. A representative immunoblot and a bar graph with data from four ( n = 4) independent experiments are shown. ( B ) Dose response of NE-stimulated Akt phosphorylation to CE3F4. Data are presented as mean ± SEM. Statistical analysis was performed by one-way ANOVA with Sidak’s multiple comparisons test between pre-determined pairs of treatments ( A ) or by repeated measures one-way ANOVA with Dunnett’s multiple comparisons test ( B ). * p < 0.05; ** p < 0.01; *** p < 0.001. ( C ) Schematic representation of p110α-dependent and Akt-mediated activation of PDE upon β-AR stimulation. According to the model, PI3K p110α and Akt are activated in the β-AR/cAMP pathway through EPAC1 and its target Rap1, as part of a feedback loop that downregulates cAMP levels through Akt-mediated PDE activation.

Article Snippet: PI3K p110α-selective inhibitor A66 (cat. no. 5595) was from Tocris/Bio-Techne (Minneapolis, MN, USA), PI3K p110β-selective inhibitor TGX221 (item no. 10007349), EPAC1 inhibitor CE3F4 (item no. 17767), and PKA inhibitor (PKI) fragment (2-22)-amide (item no. 17486) were from Cayman Chemical (Ann Arbor, MI, USA).

Techniques: Activation Assay, Western Blot

Journal: Translational Oncology

Article Title: Effect of transmembrane protein 100 on prostate cancer progression by regulating SCNN1D through the FAK/PI3K/AKT pathway

doi: 10.1016/j.tranon.2022.101578

Figure Lengend Snippet: Overexpression of TMEM100 inhibits the cell cycle and apoptosis of PCa cells and regulate FAK/PI3K/AKT signaling pathway. (A) Effects of TMEM100 overexpression on cell cycle by flow cytometry in DU145 and PC3 cells. (B) Effects of TMEM100 overexpression on cell apoptosis by flow cytometry in DU145 and PC3 cells. (C) Changes in the apoptotic and cell cycle-related proteins were detected by Western blot in DU145 and PC3 cells. (D) The analysis results of enrichment of KEEG pathway. (E) Changes in the FAK/PI3K/AKT signaling pathway proteins were detected by Western blot in DU145 and PC3 cells. (F) The qualification of apoptotic and cell cycle-related proteins by densitometry (G) The qualification of FAK/PI3K/AKT signaling pathway proteins by densitometry. * P <0.05 VS control group.

Article Snippet: The membranes were blocked with 5% skim milk for 1 h at 25 °C and incubated for 12 h at 4 °C with primary antibodies: anti-TMEM100 (cat.no.A16653; 1:1000; ABclonal), anti-E-cadherin (cat.no.20874–1-AP; 1:5000; Proteintech), anti-N-cadherin (cat.no.ab76011; 1:10000; Abcam), anti-MMP-9 (cat.no.ab76003; 1:2000; Abcam), anti-vimentin (cat.no.10366–1-AP; 1:2000; Proteintech), anti-Bax (cat.no.50599–2; 1:5000; Proteintech), anti-Bcl-2 (cat.no.265931; 1:1000; Proteintech), anti-Caspase-3 (cat.no.19677–1; 1:100; Proteintech), anti-CyclinD1 (cat.no.60186–1; 1:5000; Proteintech), anti-p-FAK (cat.no.8556; 1:1000; CST), anti-FAK (cat.no.71433; 1:1000; CST), anti-p-PI3K (cat.no.17366; 1:1000; CST), anti-PI3K (cat.no.T40064; 1:1000; Abmart), anti-p-AKT (cat.no.4060; 1:1000; CST), anti-AKT (cat.no.60203; 1:5000; Proteintech), anti-Beta actin (cat.no.20536; 1:1000; Proteintech).

Techniques: Over Expression, Flow Cytometry, Western Blot

Journal: Translational Oncology

Article Title: Effect of transmembrane protein 100 on prostate cancer progression by regulating SCNN1D through the FAK/PI3K/AKT pathway

doi: 10.1016/j.tranon.2022.101578

Figure Lengend Snippet: TMEM100 inhibits migration, invasion and EMT by regulating SCNN1D through FAK/PI3K/AKT signaling pathway. (A-B) The depletion of SCNN1D increases migration and invasion ability in TMEM100-overexpression DU145 and PC3 cells. (C) The depletion of SCNN1D rescue the function that TMEM100 overexpression hindered the protein level of p-FAK, p-PI3K and p-AKT. (D) TMEM100 affected cell apoptosis, cell cycle and EMT via regulating SCNN1D by western blot analysis. * P <0.05 VS control group, # P <0.05 VS TMEM100 overexpression group.

Article Snippet: The membranes were blocked with 5% skim milk for 1 h at 25 °C and incubated for 12 h at 4 °C with primary antibodies: anti-TMEM100 (cat.no.A16653; 1:1000; ABclonal), anti-E-cadherin (cat.no.20874–1-AP; 1:5000; Proteintech), anti-N-cadherin (cat.no.ab76011; 1:10000; Abcam), anti-MMP-9 (cat.no.ab76003; 1:2000; Abcam), anti-vimentin (cat.no.10366–1-AP; 1:2000; Proteintech), anti-Bax (cat.no.50599–2; 1:5000; Proteintech), anti-Bcl-2 (cat.no.265931; 1:1000; Proteintech), anti-Caspase-3 (cat.no.19677–1; 1:100; Proteintech), anti-CyclinD1 (cat.no.60186–1; 1:5000; Proteintech), anti-p-FAK (cat.no.8556; 1:1000; CST), anti-FAK (cat.no.71433; 1:1000; CST), anti-p-PI3K (cat.no.17366; 1:1000; CST), anti-PI3K (cat.no.T40064; 1:1000; Abmart), anti-p-AKT (cat.no.4060; 1:1000; CST), anti-AKT (cat.no.60203; 1:5000; Proteintech), anti-Beta actin (cat.no.20536; 1:1000; Proteintech).

Techniques: Migration, Over Expression, Western Blot

Journal: Translational Oncology

Article Title: Effect of transmembrane protein 100 on prostate cancer progression by regulating SCNN1D through the FAK/PI3K/AKT pathway

doi: 10.1016/j.tranon.2022.101578

Figure Lengend Snippet: TMEM100 inhibited prostate cancer progression by regulating the FAK/PI3K/AKT signaling pathway via SCNN1D.

Article Snippet: The membranes were blocked with 5% skim milk for 1 h at 25 °C and incubated for 12 h at 4 °C with primary antibodies: anti-TMEM100 (cat.no.A16653; 1:1000; ABclonal), anti-E-cadherin (cat.no.20874–1-AP; 1:5000; Proteintech), anti-N-cadherin (cat.no.ab76011; 1:10000; Abcam), anti-MMP-9 (cat.no.ab76003; 1:2000; Abcam), anti-vimentin (cat.no.10366–1-AP; 1:2000; Proteintech), anti-Bax (cat.no.50599–2; 1:5000; Proteintech), anti-Bcl-2 (cat.no.265931; 1:1000; Proteintech), anti-Caspase-3 (cat.no.19677–1; 1:100; Proteintech), anti-CyclinD1 (cat.no.60186–1; 1:5000; Proteintech), anti-p-FAK (cat.no.8556; 1:1000; CST), anti-FAK (cat.no.71433; 1:1000; CST), anti-p-PI3K (cat.no.17366; 1:1000; CST), anti-PI3K (cat.no.T40064; 1:1000; Abmart), anti-p-AKT (cat.no.4060; 1:1000; CST), anti-AKT (cat.no.60203; 1:5000; Proteintech), anti-Beta actin (cat.no.20536; 1:1000; Proteintech).

Techniques:

Journal: Oncology Reports

Article Title: Exploration of anti‑osteosarcoma activity of asiatic acid based on network pharmacology and in vitro experiments

doi: 10.3892/or.2023.8692

Figure Lengend Snippet: Asiatic acid inhibits PI3K/AKT and activates ROS/MAPK pathways in osteosarcoma cells. Osteosarcoma cells were treated with asiatic acid for 24 h. (A) Protein levels of p-PI3K, PI3K, p-AKT, and AKT as measured by western blot analyses. (B) Intracellular ROS content by flow cytometry. (C) Protein levels of p-ERK1/2, ERK1/2, p-p38, p38, p-JNK, and JNK by western blot analyses. *P<0.05 vs. control group. ROS, reactive oxygen species; p-, phosphorylated.

Article Snippet: The voltage dependent anion channel 1 (VDAC1; cat. no. ab14734), p-AKT (cat. no. ab192623), AKT (cat. no. ab179463), PI3K (cat. no. ab191606), LC3 (cat. no. ab48394) and p62 (cat. no. ab56416) antibodies were obtained from Abcam.

Techniques: Western Blot, Flow Cytometry

Journal: Thoracic Cancer

Article Title: miR ‐1301‐3p promotes invasion and migration and EMT progression in esophageal cancer by downregulating NBL1 expression

doi: 10.1111/1759-7714.15093

Figure Lengend Snippet: MiR‐1301‐3p/NBL1 axis affects esophageal cancer (ESCA) cell invasion, migration and epithelial‐mesenchymal transition (EMT) via the PI3K/AKT signaling pathway. (a) Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis based on the results of single‐gene differential analysis of NBL1. (b) Gene set enrichment analysis (GSEA) analysis comparing the NBL1 high‐expression group (red) to the low‐expression group (blue) of esophageal cancer tissues. (c, d) The levels of phosphorylated and unphosphorylated PI3K and AKT were detected by Western blot. (e) Mechanism diagram.

Article Snippet: The following primary antibodies were used: rabbit anti‐human E‐cadherin (1:1000, Cat# 3195S, CST), rabbit anti‐human vimentin (1:1000, Cat# 5741S, CST), rabbit glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) (1:10000, Cat# 3683S, CST), rabbit anti‐human NBL1 (1:500, Cat#FNab05566, FineTest), rabbit anti‐human Phospho‐PI3K (1:500, Cat#ab182651, Abcam), rabbit anti‐human PI3K(1:1000, Cat#4249, CST), rabbit anti‐human Phospho‐AKT (1:2000, Cat#4060, CST), rabbit anti‐human AKT (1:1000, Cat#4691, CST).

Techniques: Migration, Expressing, Western Blot