Journal: International Journal of Biological Sciences

Article Title: Magnolia Bioactive Constituent 4-O-Methylhonokiol Prevents the Impairment of Cardiac Insulin Signaling and the Cardiac Pathogenesis in High-Fat Diet-Induced Obese Mice

doi: 10.7150/ijbs.12101

Figure Lengend Snippet: Effects of MH on cardiac glucose metabolism. Protein expression of p-Akt, p-Akt2, and HK II in ND-fed mice (A) and HFD-fed mice was measured by western blot analysis. Data are presented as means ± SD (n=5). a, p<0.05 vs. ND; b, p< 0.05 vs. HFD.

Article Snippet: AMP-activated protein kinase α (AMPKα), phospho-AMPKα (Thr172), phospho-Akt (Ser473, p -Akt), total Akt ( t -Akt), phospho-Akt2 (Ser474, p -Akt2), and total Akt2 ( t -Akt2) were all obtained from Cell Signaling Technology (Danvers, MA), and 3-nitrotyrosine (3-NT) was purchased from Millipore (Billerica, MA).

Techniques: Expressing, Western Blot

Journal: International Journal of Biological Sciences

Article Title: Magnolia Bioactive Constituent 4-O-Methylhonokiol Prevents the Impairment of Cardiac Insulin Signaling and the Cardiac Pathogenesis in High-Fat Diet-Induced Obese Mice

doi: 10.7150/ijbs.12101

Figure Lengend Snippet: Effects of MH on cardiac glucose metabolism after insulin treatment for 15 min. The data for insulin treatment (black column) are presented as the fold change relative to baseline (without insulin treatment, white column). Protein expression of p-Akt, p-Akt2, and HK II in ND-fed mice (A) and HFD-fed mice (B) was measured by western blot analysis. Data are presented as means ± SD (n=5). a, p<0.05 vs. ND+Insulin; b, p< 0.05 vs. HFD+Insulin; c, p<0.05 vs. HFD/BL153+Insulin.

Article Snippet: AMP-activated protein kinase α (AMPKα), phospho-AMPKα (Thr172), phospho-Akt (Ser473, p -Akt), total Akt ( t -Akt), phospho-Akt2 (Ser474, p -Akt2), and total Akt2 ( t -Akt2) were all obtained from Cell Signaling Technology (Danvers, MA), and 3-nitrotyrosine (3-NT) was purchased from Millipore (Billerica, MA).

Techniques: Expressing, Western Blot

Journal: International Journal of Biological Sciences

Article Title: Magnolia Bioactive Constituent 4-O-Methylhonokiol Prevents the Impairment of Cardiac Insulin Signaling and the Cardiac Pathogenesis in High-Fat Diet-Induced Obese Mice

doi: 10.7150/ijbs.12101

Figure Lengend Snippet: A proposed mechanism for the effects of MH on cardiac insulin resistance in obese mice. MH might upregulate Akt (mainly Akt2) activity to ameliorate obesity-impaired cardiac insulin signaling. On the other hand, MH might attenuate oxidative stress-induced insulin resistance by activating the Akt-Nrf2 pathway. Lastly, MH might inhibit expression of CD36, which decreases FA uptake, and preserve the FA oxidation pathway (AMPK-Sirt1-PGC-1α), subsequently reducing intracellular lipid accumulation and finally reversing insulin signaling.

Article Snippet: AMP-activated protein kinase α (AMPKα), phospho-AMPKα (Thr172), phospho-Akt (Ser473, p -Akt), total Akt ( t -Akt), phospho-Akt2 (Ser474, p -Akt2), and total Akt2 ( t -Akt2) were all obtained from Cell Signaling Technology (Danvers, MA), and 3-nitrotyrosine (3-NT) was purchased from Millipore (Billerica, MA).

Techniques: Activity Assay, Expressing

Journal: International Journal of Biological Sciences

Article Title: Magnolia Bioactive Constituent 4-O-Methylhonokiol Prevents the Impairment of Cardiac Insulin Signaling and the Cardiac Pathogenesis in High-Fat Diet-Induced Obese Mice

doi: 10.7150/ijbs.12101

Figure Lengend Snippet: Effects of MH on cardiac glucose metabolism. Protein expression of p-Akt, p-Akt2, and HK II in ND-fed mice (A) and HFD-fed mice was measured by western blot analysis. Data are presented as means ± SD (n=5). a, p<0.05 vs. ND; b, p< 0.05 vs. HFD.

Article Snippet: AMP-activated protein kinase α (AMPKα), phospho-AMPKα (Thr172), phospho-Akt (Ser473, p -Akt), total Akt ( t -Akt), phospho-Akt2 (Ser474, p -Akt2), and total Akt2 ( t -Akt2) were all obtained from Cell Signaling Technology (Danvers, MA), and 3-nitrotyrosine (3-NT) was purchased from Millipore (Billerica, MA).

Techniques: Expressing, Western Blot

Journal: International Journal of Biological Sciences

Article Title: Magnolia Bioactive Constituent 4-O-Methylhonokiol Prevents the Impairment of Cardiac Insulin Signaling and the Cardiac Pathogenesis in High-Fat Diet-Induced Obese Mice

doi: 10.7150/ijbs.12101

Figure Lengend Snippet: Effects of MH on cardiac glucose metabolism after insulin treatment for 15 min. The data for insulin treatment (black column) are presented as the fold change relative to baseline (without insulin treatment, white column). Protein expression of p-Akt, p-Akt2, and HK II in ND-fed mice (A) and HFD-fed mice (B) was measured by western blot analysis. Data are presented as means ± SD (n=5). a, p<0.05 vs. ND+Insulin; b, p< 0.05 vs. HFD+Insulin; c, p<0.05 vs. HFD/BL153+Insulin.

Article Snippet: AMP-activated protein kinase α (AMPKα), phospho-AMPKα (Thr172), phospho-Akt (Ser473, p -Akt), total Akt ( t -Akt), phospho-Akt2 (Ser474, p -Akt2), and total Akt2 ( t -Akt2) were all obtained from Cell Signaling Technology (Danvers, MA), and 3-nitrotyrosine (3-NT) was purchased from Millipore (Billerica, MA).

Techniques: Expressing, Western Blot

Journal: International Journal of Biological Sciences

Article Title: Magnolia Bioactive Constituent 4-O-Methylhonokiol Prevents the Impairment of Cardiac Insulin Signaling and the Cardiac Pathogenesis in High-Fat Diet-Induced Obese Mice

doi: 10.7150/ijbs.12101

Figure Lengend Snippet: A proposed mechanism for the effects of MH on cardiac insulin resistance in obese mice. MH might upregulate Akt (mainly Akt2) activity to ameliorate obesity-impaired cardiac insulin signaling. On the other hand, MH might attenuate oxidative stress-induced insulin resistance by activating the Akt-Nrf2 pathway. Lastly, MH might inhibit expression of CD36, which decreases FA uptake, and preserve the FA oxidation pathway (AMPK-Sirt1-PGC-1α), subsequently reducing intracellular lipid accumulation and finally reversing insulin signaling.

Article Snippet: AMP-activated protein kinase α (AMPKα), phospho-AMPKα (Thr172), phospho-Akt (Ser473, p -Akt), total Akt ( t -Akt), phospho-Akt2 (Ser474, p -Akt2), and total Akt2 ( t -Akt2) were all obtained from Cell Signaling Technology (Danvers, MA), and 3-nitrotyrosine (3-NT) was purchased from Millipore (Billerica, MA).

Techniques: Activity Assay, Expressing

Journal: Cell reports

Article Title: Methylene-bridge tryptophan fatty acylation regulates PI3K-AKT signaling and glucose uptake

doi: 10.1016/j.celrep.2022.110509

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: phospho-AKT2(S473) , Cell Signaling Technology , Cat# 4060; RRID: AB_2224726.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Kinase Assay, Software

Journal: Cell reports

Article Title: Methylene-bridge tryptophan fatty acylation regulates PI3K-AKT signaling and glucose uptake

doi: 10.1016/j.celrep.2022.110509

Figure Lengend Snippet: (A) DHA and EPA specifically promote membrane-targeting proteins to the membrane. HepG2 cells were treated with DHA or EPA for 1 h before their membrane fractions were obtained and detected for AKT2, PDK1, TRAF6, SIRT3, COX4, PGK1, GAPDH, and Na-K-ATPase by western blotting.

Article Snippet: phospho-AKT2(S473) , Cell Signaling Technology , Cat# 4060; RRID: AB_2224726.

Techniques: Western Blot

Journal: Cell reports

Article Title: Methylene-bridge tryptophan fatty acylation regulates PI3K-AKT signaling and glucose uptake

doi: 10.1016/j.celrep.2022.110509

Figure Lengend Snippet: (A) DHA and EPA activate PDK1 and AKT2. Phosphorylation levels of substrates of PDK1 and AKT2 were detected in 3T3-L1 cells treated with DHA and EPA.

Article Snippet: phospho-AKT2(S473) , Cell Signaling Technology , Cat# 4060; RRID: AB_2224726.

Techniques:

Journal: Cell reports

Article Title: Methylene-bridge tryptophan fatty acylation regulates PI3K-AKT signaling and glucose uptake

doi: 10.1016/j.celrep.2022.110509

Figure Lengend Snippet: (A) DHA acylation induced insulin resistance. Insulin tolerance tests were performed in DHA-treated Akt2−/− 129/C57BL6 mice and Akt2−/− 129/C57BL6 mice overexpressing AKT2WT or AKT2W414L (n = 6, mean ± SEM).

Article Snippet: phospho-AKT2(S473) , Cell Signaling Technology , Cat# 4060; RRID: AB_2224726.

Techniques:

Journal: Cell reports

Article Title: Methylene-bridge tryptophan fatty acylation regulates PI3K-AKT signaling and glucose uptake

doi: 10.1016/j.celrep.2022.110509

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: phospho-AKT2(S473) , Cell Signaling Technology , Cat# 4060; RRID: AB_2224726.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Kinase Assay, Software

Journal: Cell reports

Article Title: Methylene-bridge tryptophan fatty acylation regulates PI3K-AKT signaling and glucose uptake

doi: 10.1016/j.celrep.2022.110509

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: phospho-AKT2(T308) , Cell Signaling Technology , Cat# 13038; RRID: AB_2629447.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Kinase Assay, Software

Journal: Cell reports

Article Title: Methylene-bridge tryptophan fatty acylation regulates PI3K-AKT signaling and glucose uptake

doi: 10.1016/j.celrep.2022.110509

Figure Lengend Snippet: (A) DHA and EPA specifically promote membrane-targeting proteins to the membrane. HepG2 cells were treated with DHA or EPA for 1 h before their membrane fractions were obtained and detected for AKT2, PDK1, TRAF6, SIRT3, COX4, PGK1, GAPDH, and Na-K-ATPase by western blotting.

Article Snippet: phospho-AKT2(T308) , Cell Signaling Technology , Cat# 13038; RRID: AB_2629447.

Techniques: Western Blot

Journal: Cell reports

Article Title: Methylene-bridge tryptophan fatty acylation regulates PI3K-AKT signaling and glucose uptake

doi: 10.1016/j.celrep.2022.110509

Figure Lengend Snippet: (A) DHA and EPA activate PDK1 and AKT2. Phosphorylation levels of substrates of PDK1 and AKT2 were detected in 3T3-L1 cells treated with DHA and EPA.

Article Snippet: phospho-AKT2(T308) , Cell Signaling Technology , Cat# 13038; RRID: AB_2629447.

Techniques:

Journal: Cell reports

Article Title: Methylene-bridge tryptophan fatty acylation regulates PI3K-AKT signaling and glucose uptake

doi: 10.1016/j.celrep.2022.110509

Figure Lengend Snippet: (A) DHA acylation induced insulin resistance. Insulin tolerance tests were performed in DHA-treated Akt2−/− 129/C57BL6 mice and Akt2−/− 129/C57BL6 mice overexpressing AKT2WT or AKT2W414L (n = 6, mean ± SEM).

Article Snippet: phospho-AKT2(T308) , Cell Signaling Technology , Cat# 13038; RRID: AB_2629447.

Techniques:

Journal: Cell reports

Article Title: Methylene-bridge tryptophan fatty acylation regulates PI3K-AKT signaling and glucose uptake

doi: 10.1016/j.celrep.2022.110509

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: phospho-AKT2(T308) , Cell Signaling Technology , Cat# 13038; RRID: AB_2629447.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Kinase Assay, Software

Journal: Nutrients

Article Title: Cinnamaldehyde and Curcumin Prime Akt2 for Insulin-Stimulated Activation

doi: 10.3390/nu14163301

Figure Lengend Snippet: Composition of Akt isoforms in primary human subcutaneous preadipocytes. ( A – D ) Electropherograms of capillary isoelectric focusing (cIEF) immunoassays of ( A ) Akt1, ( B ) Akt2, ( C ) Akt3, and ( D ) pan-Akt or all Akt isoforms. The letter p represents phosphorylation of Akt isoforms. Peaks on electropherograms are color-coded with Akt1 (blue), Akt2 (green), and Akt3 (red). ( E ) Percentage of Akt1 (blue), Akt2 (green), and Akt3 (red) as a function of all Akt isoforms. ( F – H ) Electropherograms of cIEF immunoassays of ( F ) pAkt (Thr308), ( G ) pAkt (Thr450), and ( H ) pAkt (S473). The dashed line highlights the distribution of pAkt (Thr450) as a function of pI values.

Article Snippet: Primary antibodies used for this study were: pan-Akt (cat. no. 8312, Santa Cruz Biotech, Dallas, TX, USA), Akt 1 (cat. no. 2938, cat. no. 2938, Cell Signaling Technology (CST), Danvers, MA, USA), Akt2 (cat. no. cs3063, CST), Akt3 (cat. no. 8018, CST), p-Akt (S473) (cat. no. 9271, CST), p-Akt1 (S473) (cat. no. 9018, CST), p-Akt2 (S474) (cat. no. cs8599, CST), p-Akt (S124) (cat. no. PA5-38251, Thermo Fisher Scientific, Waltham, MA, USA), p-Akt (S246) (cat. no. PA5-38352, Thermo Fisher Scientific, Waltham, MA, USA), p-Akt (T308) (cat. no. cs2965, CST), p-Akt (T450) (cat. no. cs9267, CST), p-Akt (Y475) (p-Akt1 (Y474) or p-Akt2 (Y475), cat. no. PA5-38351, Thermo Fisher Scientific, Waltham, MA, USA), HSP70 (cat. no. cs4872, CST), and β-actin (cat. no. MAB8929, R&D Systems, Minneapolis, MN, USA).

Techniques:

Journal: Nutrients

Article Title: Cinnamaldehyde and Curcumin Prime Akt2 for Insulin-Stimulated Activation

doi: 10.3390/nu14163301

Figure Lengend Snippet: Insulin induces changes to the electropherograms of Akt2 and Akt3. Electropherograms of cIEF immunoassays of ( A ) Akt1, ( B ) Akt2, ( C ) Akt3, ( D ) p-Akt (Thr308), ( E ) p-Akt (Thr450), and ( F ) p-Akt (Ser473) following treatment with insulin for 30 min. Arrows point to new peaks that appeared after treatment with insulin. The dashed line highlights the distribution of p-Akt (Thr450) as a function of pI values. ( G ) Capillary Western (CW) immunoassays of p-Akt (Thr308), p-Akt (Thr450), and p-Akt (Ser473) before and after treatment with insulin. Pan-Akt and β-actin served as the loading controls. ( H ) Relative expression level of p-Akt (Thr308), p-Akt (Thr450), and p-Akt (Ser473) before (solid black) and after (textured) treatment with insulin. Error bars indicate standard deviations across six repeated measurements using CW immunoassays per experimental condition. Asterisks indicate a statistical significance of p ≤ 0.01 versus untreated control.

Article Snippet: Primary antibodies used for this study were: pan-Akt (cat. no. 8312, Santa Cruz Biotech, Dallas, TX, USA), Akt 1 (cat. no. 2938, cat. no. 2938, Cell Signaling Technology (CST), Danvers, MA, USA), Akt2 (cat. no. cs3063, CST), Akt3 (cat. no. 8018, CST), p-Akt (S473) (cat. no. 9271, CST), p-Akt1 (S473) (cat. no. 9018, CST), p-Akt2 (S474) (cat. no. cs8599, CST), p-Akt (S124) (cat. no. PA5-38251, Thermo Fisher Scientific, Waltham, MA, USA), p-Akt (S246) (cat. no. PA5-38352, Thermo Fisher Scientific, Waltham, MA, USA), p-Akt (T308) (cat. no. cs2965, CST), p-Akt (T450) (cat. no. cs9267, CST), p-Akt (Y475) (p-Akt1 (Y474) or p-Akt2 (Y475), cat. no. PA5-38351, Thermo Fisher Scientific, Waltham, MA, USA), HSP70 (cat. no. cs4872, CST), and β-actin (cat. no. MAB8929, R&D Systems, Minneapolis, MN, USA).

Techniques: Western Blot, Expressing

Journal: Nutrients

Article Title: Cinnamaldehyde and Curcumin Prime Akt2 for Insulin-Stimulated Activation

doi: 10.3390/nu14163301

Figure Lengend Snippet: Cinnamaldehyde and curcumin induce changes to Akt2 posttranslational modification. ( A – E ) Distribution of Akt2 as a function of isoelectric points in ( A ) control untreated preadipocytes, or ( B – E ) preadipocytes treated with ( B ) insulin, ( C ) cinnamaldehyde, ( D ) curcumin, or ( E ) combined cinnamaldehyde and curcumin. ( F ) Distribution of p-Akt2 (S474) as a function of isoelectric points in untreated control preadipocytes (top electropherogram), or preadipocytes treated with insulin (second electropherogram), cinnamaldehyde (third electropherogram), curcumin (fourth electropherogram), and combined cinnamaldehyde and curcumin (bottom electropherogram). Arrows point to the appearance of new peaks following treatment versus untreated control.

Article Snippet: Primary antibodies used for this study were: pan-Akt (cat. no. 8312, Santa Cruz Biotech, Dallas, TX, USA), Akt 1 (cat. no. 2938, cat. no. 2938, Cell Signaling Technology (CST), Danvers, MA, USA), Akt2 (cat. no. cs3063, CST), Akt3 (cat. no. 8018, CST), p-Akt (S473) (cat. no. 9271, CST), p-Akt1 (S473) (cat. no. 9018, CST), p-Akt2 (S474) (cat. no. cs8599, CST), p-Akt (S124) (cat. no. PA5-38251, Thermo Fisher Scientific, Waltham, MA, USA), p-Akt (S246) (cat. no. PA5-38352, Thermo Fisher Scientific, Waltham, MA, USA), p-Akt (T308) (cat. no. cs2965, CST), p-Akt (T450) (cat. no. cs9267, CST), p-Akt (Y475) (p-Akt1 (Y474) or p-Akt2 (Y475), cat. no. PA5-38351, Thermo Fisher Scientific, Waltham, MA, USA), HSP70 (cat. no. cs4872, CST), and β-actin (cat. no. MAB8929, R&D Systems, Minneapolis, MN, USA).

Techniques: Modification

Journal: Nutrients

Article Title: Cinnamaldehyde and Curcumin Prime Akt2 for Insulin-Stimulated Activation

doi: 10.3390/nu14163301

Figure Lengend Snippet: Cinnamaldehyde and curcumin enhance insulin-stimulated activation of Akt2. ( A – D ) Distribution of Akt2 as a function of isoelectric points in preadipocytes treated with ( A ) insulin alone, ( B ) insulin and cinnamaldehyde, ( C ) insulin and curcumin, or ( D ) insulin, cinnamaldehyde and curcumin. ( E ) Distribution of p-Akt2 (S474) as a function of isoelectric points in preadipocytes treated with insulin alone (top electropherogram), insulin and cinnamaldehyde (second electropherogram), insulin and curcumin (third electropherogram), or insulin, cinnamaldehyde, and curcumin (bottom electropherogram). ( F ) Relative abundance of p-Akt2 (S474) as a function of treatment condition. Error bars are standard deviations across nine repeated measurements. Single asterisk (*) indicates p -value ≤ 0.01 versus treatment with insulin alone. Double asterisk (**) indicates p -value ≤ 0.01 versus treatment with insulin and cinnamaldehyde or insulin and curcumin.

Article Snippet: Primary antibodies used for this study were: pan-Akt (cat. no. 8312, Santa Cruz Biotech, Dallas, TX, USA), Akt 1 (cat. no. 2938, cat. no. 2938, Cell Signaling Technology (CST), Danvers, MA, USA), Akt2 (cat. no. cs3063, CST), Akt3 (cat. no. 8018, CST), p-Akt (S473) (cat. no. 9271, CST), p-Akt1 (S473) (cat. no. 9018, CST), p-Akt2 (S474) (cat. no. cs8599, CST), p-Akt (S124) (cat. no. PA5-38251, Thermo Fisher Scientific, Waltham, MA, USA), p-Akt (S246) (cat. no. PA5-38352, Thermo Fisher Scientific, Waltham, MA, USA), p-Akt (T308) (cat. no. cs2965, CST), p-Akt (T450) (cat. no. cs9267, CST), p-Akt (Y475) (p-Akt1 (Y474) or p-Akt2 (Y475), cat. no. PA5-38351, Thermo Fisher Scientific, Waltham, MA, USA), HSP70 (cat. no. cs4872, CST), and β-actin (cat. no. MAB8929, R&D Systems, Minneapolis, MN, USA).

Techniques: Activation Assay

Journal: Nutrients

Article Title: Cinnamaldehyde and Curcumin Prime Akt2 for Insulin-Stimulated Activation

doi: 10.3390/nu14163301

Figure Lengend Snippet: Cinnamaldehyde and curcumin increase pan-Akt phosphorylation. ( A – C ) Distribution of p-Akt (T450) as a function of isoelectric points in ( A ) untreated control preadipocytes or ( B , C ) preadipocytes treated with ( B ) cinnamaldehyde or ( C ) curcumin. The dashed line highlights the distribution trend. ( D – F ) Distribution of ( D ) p-Akt (S124), ( E ) p-Akt (S246), and ( F ) p-Akt (Y475) in untreated control preadipocytes (top electropherogram), or preadipocytes treated with cinnamaldehyde (middle electropherogram) or curcumin (bottom electropherogram).

Article Snippet: Primary antibodies used for this study were: pan-Akt (cat. no. 8312, Santa Cruz Biotech, Dallas, TX, USA), Akt 1 (cat. no. 2938, cat. no. 2938, Cell Signaling Technology (CST), Danvers, MA, USA), Akt2 (cat. no. cs3063, CST), Akt3 (cat. no. 8018, CST), p-Akt (S473) (cat. no. 9271, CST), p-Akt1 (S473) (cat. no. 9018, CST), p-Akt2 (S474) (cat. no. cs8599, CST), p-Akt (S124) (cat. no. PA5-38251, Thermo Fisher Scientific, Waltham, MA, USA), p-Akt (S246) (cat. no. PA5-38352, Thermo Fisher Scientific, Waltham, MA, USA), p-Akt (T308) (cat. no. cs2965, CST), p-Akt (T450) (cat. no. cs9267, CST), p-Akt (Y475) (p-Akt1 (Y474) or p-Akt2 (Y475), cat. no. PA5-38351, Thermo Fisher Scientific, Waltham, MA, USA), HSP70 (cat. no. cs4872, CST), and β-actin (cat. no. MAB8929, R&D Systems, Minneapolis, MN, USA).

Techniques:

Journal: Nutrients

Article Title: Cinnamaldehyde and Curcumin Prime Akt2 for Insulin-Stimulated Activation

doi: 10.3390/nu14163301

Figure Lengend Snippet: Dual inhibition of PP2A and PTP1B activates Akt2. ( A – C ) Distribution of Akt2 as a function of isoelectric points in control untreated preadipocytes (top electropherograms) or in preadipocytes treated with ( A ) okadaic acid, ( B ) PTP1Bi, or ( C ) combined okadaic acid and PTP1B (bottom electropherograms). Arrows point to new peaks or peaks that experience elevated abundance in treated preadipocytes versus untreated control preadipocytes. ( D ) Distribution of p-Akt2 (S474) as a function of isoelectric point in control untreated preadipocytes (top electropherogram), or preadipocytes treated with okadaic acid (second electropherogram), PTP1Bi (third electropherogram), or combined okadaic acid and PTP1Bi (bottom electropherogram).

Article Snippet: Primary antibodies used for this study were: pan-Akt (cat. no. 8312, Santa Cruz Biotech, Dallas, TX, USA), Akt 1 (cat. no. 2938, cat. no. 2938, Cell Signaling Technology (CST), Danvers, MA, USA), Akt2 (cat. no. cs3063, CST), Akt3 (cat. no. 8018, CST), p-Akt (S473) (cat. no. 9271, CST), p-Akt1 (S473) (cat. no. 9018, CST), p-Akt2 (S474) (cat. no. cs8599, CST), p-Akt (S124) (cat. no. PA5-38251, Thermo Fisher Scientific, Waltham, MA, USA), p-Akt (S246) (cat. no. PA5-38352, Thermo Fisher Scientific, Waltham, MA, USA), p-Akt (T308) (cat. no. cs2965, CST), p-Akt (T450) (cat. no. cs9267, CST), p-Akt (Y475) (p-Akt1 (Y474) or p-Akt2 (Y475), cat. no. PA5-38351, Thermo Fisher Scientific, Waltham, MA, USA), HSP70 (cat. no. cs4872, CST), and β-actin (cat. no. MAB8929, R&D Systems, Minneapolis, MN, USA).

Techniques: Inhibition

Journal: Science Advances

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

doi: 10.1126/sciadv.abn7298

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

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

Techniques: Functional Assay

Journal: Science Advances

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

doi: 10.1126/sciadv.abn7298

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

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

Techniques: Translocation Assay, Two Tailed Test

Journal: Science Advances

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

doi: 10.1126/sciadv.abn7298

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

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

Techniques:

Journal: Science Advances

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

doi: 10.1126/sciadv.abn7298

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

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

Techniques: Functional Assay

Journal: Science Advances

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

doi: 10.1126/sciadv.abn7298

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

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

Techniques: Translocation Assay, Two Tailed Test

Journal: Science Advances

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

doi: 10.1126/sciadv.abn7298

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

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

Techniques: