Journal: Heliyon

Article Title: Effects of chronic treatment with metformin on brain glucose hypometabolism and central insulin actions in transgenic mice with tauopathy

doi: 10.1016/j.heliyon.2024.e35752

Figure Lengend Snippet: Identification of primers used in the different gene expression assays.

Article Snippet: Anti IRS1 , Rabbit , BioRad.

Techniques: Gene Expression, TaqMan Assay

Journal: Heliyon

Article Title: Effects of chronic treatment with metformin on brain glucose hypometabolism and central insulin actions in transgenic mice with tauopathy

doi: 10.1016/j.heliyon.2024.e35752

Figure Lengend Snippet: Antibodies used for protein detection by Western Blot.

Article Snippet: Anti IRS1 , Rabbit , BioRad.

Techniques: Western Blot

Journal: Heliyon

Article Title: Effects of chronic treatment with metformin on brain glucose hypometabolism and central insulin actions in transgenic mice with tauopathy

doi: 10.1016/j.heliyon.2024.e35752

Figure Lengend Snippet: Effect of metformin on the expression of the total (A, B and C), phosphorylated (D, E and F) and active (G, H and I) forms of IR, IRS1 and PI3K, respectively, in the frontal cortex of Tau-VLW transgenic mice. 9-months-old Tau mice were treated daily with metformin for another 9 months and then the expression of the indicated proteins was measured by Western blot as described in Experimental procedures. Total and phosphorylated forms were normalized by β-actin (representative bands are showed in ). Phosphorylated forms normalized to the total forms are represented as active forms. Data are represented as means ± SEM (n = 4–5). Student's unpaired t -test was used to analyse significant differences. *p < 0.05, **p < 0.01. The full-length blot is provided in supplementary material as jpg and jpg.

Article Snippet: Anti IRS1 , Rabbit , BioRad.

Techniques: Expressing, Transgenic Assay, Western Blot

Journal: Cell Discovery

Article Title: Spatiotemporal regulation of insulin signaling by liquid–liquid phase separation

doi: 10.1038/s41421-022-00430-1

Figure Lengend Snippet: a , b LLPS scores of human proteome, signaling transduction proteome and insulin-related proteins. The predictor PSAP was used to calculate LLPS scores. The percentage of proteins with LLPS feature ( a ) and average store ( b ) were presented in the 3 groups. n = 17,817 (human proteome), 5520 (signaling transduction proteome), and 173 (insulin-related proteins). The data are given as percentage ( a ) and the means ± SEM ( b ). Statistical analyses were carried out using Fisher’s Exact test ( a ) and Mann–Whitney U test ( b ). * p < 0.05. c Time-series imaging of IRS1-GFP-CRY2 (optoIRS1) upon induction with blue light in Cos-7 cells. Scale bar, 10 μm. d Blue light induced formation of optoDroplets of insulin-related proteins in Cos-7 cells. Scale bar, 10 μm. e FRAP analysis of optoDroplets of insulin-related proteins in Cos-7 cells. The optoDroplets subjected to FRAP analysis were highlighted in insets. Scale bar, 10 μm.

Article Snippet: For co-staining of endogenous IRS1/PKBβ, fixed cells were first incubated with the PKBβ primary antibody (Atlas Antibodies, HPA064521) and fluorophore-conjugated secondary antibody, and then stained with the Alexa564-labeled IRS1 antibody.

Techniques: Transduction, MANN-WHITNEY, Imaging

Journal: Cell Discovery

Article Title: Spatiotemporal regulation of insulin signaling by liquid–liquid phase separation

doi: 10.1038/s41421-022-00430-1

Figure Lengend Snippet: a In vitro LLPS assay with 20 μM recombinant His-tagged proteins GFP, GSK3β-GFP, PRAS40-GFP, BAD-GFP, and P27-GFP with or without 3% PEG8000. Scale bar, 10 μm. b In vitro LLPS assay with recombinant His-tagged full-length IRS1-GFP with 3% PEG8000 ± NaCl. Scale bar, 10 μm. c Schematic illustration of domains and fragments of mouse IRS1. Eight tyrosine sites regulated by insulin receptor are shown in the diagram. d In vitro LLPS assay with recombinant His-tagged four fragments IRS1-GFP with 3% PEG8000. Scale bar, 10 μm. e Fusion of droplets of His-tagged IRS1 N243-S600 -GFP. Red arrows showed the two droplets that fused during the experimental period. Time Zero started at 90 s after the addition of PEG8000. Scale bar, 10 μm.

Article Snippet: For co-staining of endogenous IRS1/PKBβ, fixed cells were first incubated with the PKBβ primary antibody (Atlas Antibodies, HPA064521) and fluorophore-conjugated secondary antibody, and then stained with the Alexa564-labeled IRS1 antibody.

Techniques: In Vitro, Recombinant

Journal: Cell Discovery

Article Title: Spatiotemporal regulation of insulin signaling by liquid–liquid phase separation

doi: 10.1038/s41421-022-00430-1

Figure Lengend Snippet: a , b Concentration-dependent formation of IRS1-GFP condensates in U2OS cells. Various amounts of IRS1-GFP plasmid were transfected into cells (1× IRS1-GFP indicates 0.4 μg plasmid DNA per well in a 12-well plate) to achieve increased expression levels of IRS1-GFP protein in U2OS cells. The formation of IRS1-GFP condensates was detected via fluorescence confocal microscope ( a ), and the expression levels of IRS1-GFP and endogenous IRS1 were determined via immunoblotting ( b ). Scale bar, 10 μm. c , d Formation of condensates of IRS1-GFP or IRS1 8Y/F -GFP or IRS1 G972R -GFP in HEK293T cells. The 8 tyrosine residues whose phosphorylation is dependent on IR were mutated to non-phosphorylatable phenylalanine residues on IRS1 8Y/F -GFP. c Representative images. d Quantification of IRS1-GFP condensates per cell. n = 113–156. Statistical analyses were carried out via one-way ANOVA. n.s., not significant. Scale bar, 10 μm. e , f FRAP analysis of wild-type or mutant IRS1-GFP condensates in U2OS cells. e Quantification of FRAP analyses. f Representative images. Scale bar, 10 μm. The condensates subjected to FRAP were indicated with white arrows. The ROI intensity was normalized to the value before photo-bleaching and given as the means ± SEM. n = 34–43. Statistical analyses were carried out via one-way ANOVA. * p < 0.05 (IRS1 G972R -GFP vs. WT IRS1-GFP, IRS1 G972R -GFP vs IRS1 8Y/F -GFP). g The material exchange between IRS1-GFP condensates and cytoplasm was examined by FRAP analysis in U2OS cells. The whole condensate of IRS1-GFP was photo-bleached and monitored subsequently. Scale bar, 10 μm. h Effects of 1,6-hexanediol on IRS1-GFP condensates. 1,6-hexanediol (5%) was added to U2OS cells, and IRS1-GFP condensates were monitored subsequently. Scale bar, 10 μm.

Article Snippet: For co-staining of endogenous IRS1/PKBβ, fixed cells were first incubated with the PKBβ primary antibody (Atlas Antibodies, HPA064521) and fluorophore-conjugated secondary antibody, and then stained with the Alexa564-labeled IRS1 antibody.

Techniques: Concentration Assay, Plasmid Preparation, Transfection, Expressing, Fluorescence, Microscopy, Western Blot, Mutagenesis

Journal: Cell Discovery

Article Title: Spatiotemporal regulation of insulin signaling by liquid–liquid phase separation

doi: 10.1038/s41421-022-00430-1

Figure Lengend Snippet: a Dynamics of IRS1-GFP condensates in U2OS cells upon insulin stimulation. Cells transfected with IRS1-GFP were stimulated with insulin (100 nM) for indicated time. Insets show the formation of IRS1-GFP condensates. Scale bar, 10 μm. b , c Formation of endogenous IRS1 condensates in L6 muscle cells upon insulin stimulation. b Representative images. c Fitted curves of cell distribution in terms of IRS1 condensate (puncta) number per cell. The values were fitted into curves using polynomials. n = 81–96. Scale bar, 5 μm. d , e Formation of endogenous IRS1 condensates in U2OS cells upon insulin stimulation. d Fitted curves of cell distribution in terms of IRS1 condensate (puncta) number per cell. The values were fitted into curves using polynomials. n = 102–119. e Representative images. Scale bar, 10 μm. f , g Effects of palmitate (PA) treatment on insulin-induced formation of IRS1 condensates in L6 muscle cells. Cells were treated with or without palmitate (400 μM) for 24 h before stimulated with or without insulin (100 nM). f Representative images. Scale bar, 10 μm. g Quantification of IRS1 puncta per cell. n = 50. The data are given as the means ± SEM. Statistical analyses were carried out via two-way ANOVA. * p < 0.05, and *** p < 0.001. h – j Effects of the PKC inhibitor Gö6983 on PKB phosphorylation and IRS1 puncta formation in L6 muscle cells. Cells were treated with or without palmitate for 24 h before stimulated with or without insulin in the presence or absence of Gö6983 (1 μM). h Representative images of IRS1 puncta. Scale bar, 10 μm. i Quantification of IRS1 puncta per cell. n = 55-62. j Immunoblotting analysis of PKB phosphorylation. The data are given as the means ± SEM. Statistical analyses were carried out via two-way ANOVA. *** p < 0.001. n.s., not significant.

Article Snippet: For co-staining of endogenous IRS1/PKBβ, fixed cells were first incubated with the PKBβ primary antibody (Atlas Antibodies, HPA064521) and fluorophore-conjugated secondary antibody, and then stained with the Alexa564-labeled IRS1 antibody.

Techniques: Transfection, Western Blot

Journal: Cell Discovery

Article Title: Spatiotemporal regulation of insulin signaling by liquid–liquid phase separation

doi: 10.1038/s41421-022-00430-1

Figure Lengend Snippet: a Cellular localization of mCherry-p85 with co-expressed GFP in U2OS cells. Scale bar, 10 μm. b , c Cellular localization of IRS1-GFP co-expressed with mCherry-p85 ( b ) and mCherry-p110 ( c ) in response to insulin stimulation in U2OS cells. Scale bar, 10 μm. Quantification of colocalization was shown in Supplementary Fig. . d – f Cellular localization of IRS1-GFP co-expressed with PIP2 sensor ( d ), mCherry-PDK1 ( e ), and mCherry-SIN1 ( f ) in response to insulin stimulation in HEK293T cells. Scale bar, 10 μm. Quantification of colocalization was shown in Supplementary Fig. . g Cellular localization of IRS1-GFP co-expressed with PIP3 sensor in response to insulin stimulation. IRS1-GFP was expressed at low and high levels in U2OS cells. Scale bar, 10 μm. Quantification of colocalization was shown in Supplementary Fig. . h Cellular localization of IRS1-GFP co-expressed with PKBβ-mCherry in response to insulin stimulation. IRS1-GFP was expressed at low and high levels in Cos-7 cells. Arrows indicated PKBβ-mCherry that was co-localized with IRS1 condensates upon insulin stimulation. Scale bar, 10 μm. Quantification of colocalization was shown in Supplementary Fig. .

Article Snippet: For co-staining of endogenous IRS1/PKBβ, fixed cells were first incubated with the PKBβ primary antibody (Atlas Antibodies, HPA064521) and fluorophore-conjugated secondary antibody, and then stained with the Alexa564-labeled IRS1 antibody.

Techniques:

Journal: Cell Discovery

Article Title: Spatiotemporal regulation of insulin signaling by liquid–liquid phase separation

doi: 10.1038/s41421-022-00430-1

Figure Lengend Snippet: a Time-series imaging of IRS1-GFP and PIP3 sensor in U2OS cells upon insulin stimulation. The merged images of IRS1-GFP and PIP3 sensor were enlarged and shown in the upper left corners. Scale bar, 10 μm. b Cellular localization of endogenous IRS1 and p85 in U2OS cells in response to insulin stimulation. The IRS1 puncta co-localized with p85 were indicated with white arrows. The p85 antibody was labeled with Alexa564. Scale bar, 10 μm. c Cellular localization of endogenous IRS1 and PIP2 sensor in U2OS cells in response to insulin stimulation. The IRS1 puncta co-localized with PIP2 sensor were indicated with white arrows. Scale bar, 10 μm. d Cellular localization of endogenous IRS1 and PIP3 sensor in U2OS cells in response to insulin stimulation. The IRS1 puncta co-localized with PIP3 sensor were indicated with white arrows. Scale bar, 10 μm. e Cellular localization of endogenous IRS1 and PKBβ in U2OS cells in response to insulin stimulation. The IRS1 puncta co-localized with PKBβ were indicated with white arrows. The IRS1 antibody was labeled with Alexa564. Scale bar, 10 μm.

Article Snippet: For co-staining of endogenous IRS1/PKBβ, fixed cells were first incubated with the PKBβ primary antibody (Atlas Antibodies, HPA064521) and fluorophore-conjugated secondary antibody, and then stained with the Alexa564-labeled IRS1 antibody.

Techniques: Imaging, Labeling

Journal: Cell Discovery

Article Title: Spatiotemporal regulation of insulin signaling by liquid–liquid phase separation

doi: 10.1038/s41421-022-00430-1

Figure Lengend Snippet: a Cellular localization of IRS1 8Y/F -GFP co-expressed with mCherry-p85, mCherry-PDK1, mCherry-SIN1 and PIP2 sensor in HEK293T cells. Scale bar, 10 μm. b Cellular localization of IRS1 8Y/F -GFP co-expressed with PIP3 sensor in response to insulin stimulation in HEK293T cells. Scale bar, 10 μm. c Cellular localization of IRS1Δ(PH-PTB)-GFP co-expressed with mCherry-p85, mCherry-PDK1, mCherry-SIN1, and PIP2 sensor in HEK293T cells. Scale bar, 10 μm. d Cellular localization of IRS1Δ(PH-PTB)-GFP co-expressed with PIP3 sensor in response to insulin stimulation in HEK293T cells. Scale bar, 10 μm.

Article Snippet: For co-staining of endogenous IRS1/PKBβ, fixed cells were first incubated with the PKBβ primary antibody (Atlas Antibodies, HPA064521) and fluorophore-conjugated secondary antibody, and then stained with the Alexa564-labeled IRS1 antibody.

Techniques:

Journal: Cell Discovery

Article Title: Spatiotemporal regulation of insulin signaling by liquid–liquid phase separation

doi: 10.1038/s41421-022-00430-1

Figure Lengend Snippet: A schematic illustration of IRS1 condensates serving as intracellular signal hubs to execute insulin signal transduction. IRS1 condensates form through LLPS in concentration- or insulin-dependent manners. At the basal state, multiple components including PI(4,5)P2, PI3K, PDK1, and SIN1 are present in IRS1 condensates. Upon insulin stimulation, more components such as PIP3 and PKB appear in IRS1 condensates to mediate insulin signal transduction.

Article Snippet: For co-staining of endogenous IRS1/PKBβ, fixed cells were first incubated with the PKBβ primary antibody (Atlas Antibodies, HPA064521) and fluorophore-conjugated secondary antibody, and then stained with the Alexa564-labeled IRS1 antibody.

Techniques: Transduction, Concentration Assay

Journal: Frontiers in Pharmacology

Article Title: Edgeworthia gardneri (Wall.) Meisn. Water Extract Ameliorates Palmitate Induced Insulin Resistance by Regulating IRS1/GSK3β/FoxO1 Signaling Pathway in Human HepG2 Hepatocytes

doi: 10.3389/fphar.2019.01666

Figure Lengend Snippet: WEE affected the molecular components of IRS1/GSK-3β/FoxO1 pathways in PA treated HepG2 cells. HepG2 cells were incubated with normal glucose (5.5 mM) or high glucose (30 mM) plus PA (0.3 mM) in the absence or presence of WEE (200 and 300 μg/ml) for 24 h and followed by being stimulated with insulin (100 nM) for 20 min. Then, the phosphorylation and total levels of IRβ, IRS-1, GSK3β, CREB, c-Jun, FoxO1, Akt, p38, JNK, and ERK were detected by Western blotting. Bar graphs show the relative expression of indicated proteins. All proteins were normalized for β-actin levels. Data presented in bar charts are mean ± SEM values from three independent experiments. Groups are significantly different from the control group at *p < 0.05, **p < 0.01. The groups are significantly different from the IR group at # p < 0.05 and ## p < 0.01 determined by Dunnett’s multiple comparisons test.

Article Snippet: Phospho-IRS1 (Tyr 612, catalog no. PAB12628) was purchased from Abnova (Taiwan, China).

Techniques: Incubation, Western Blot, Expressing, Control