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, Phospho-proteomics, 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 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: Scientific Reports

Article Title: Passive repetitive stretching is associated with greater muscle mass and cross-sectional area in the sarcopenic muscle

doi: 10.1038/s41598-021-94709-0

Figure Lengend Snippet: Real-time PCR gene expression of Akt, p70S6K, 4E-BP1, and muscle-specific E3 ubiquitin ligases MAFbx and MuRF1. US unstretched, St stretched. Values are expressed as mean ± SEM. *P < 0.05, n = 8/group.

Article Snippet: After blocking with a blocking buffer (Boster Biological Technology, Pleasanton, CA, USA) for 1.5 h at room temperature, the membrane was incubated with primary antibodies (diluted in TBS-T) against p-p70S6K (Thr 389) (1:1,000; A0533; Assay Biotechnology Company, Sunnyvale, CA, USA) and p70S6K (1:1,000; CSB-PA003686; CUSABIO, Houston, TX, USA), p-4E-BP1 (Thr37/46) (1:1,000; #2855; Cell Signaling technology, Danvers, MA, USA) and 4E-BP1 (1:1000; CSB-PA007994; CUSABIO), p-AKT (S473) (1:1000; CSB-PA000466; CUSABIO), AKT (1:1000; CSB-PA008132; CUSABIO), GAPDH (1:1000; CSB-PA004666; CUSABIO) for 2 h at room temperature.

Techniques: Real-time Polymerase Chain Reaction, Gene Expression, Ubiquitin Proteomics

Journal: Scientific Reports

Article Title: Passive repetitive stretching is associated with greater muscle mass and cross-sectional area in the sarcopenic muscle

doi: 10.1038/s41598-021-94709-0

Figure Lengend Snippet: The phosphorylation levels of Akt, p70S6K, and 4E-BP1 measured with Western blotting. Representative western blot images and quantification of phosphorylation levels of ( A ) Akt, ( B ) p70S6K, and ( C ) 4E-BP1. US unstretched, St stretched. Values are expressed as mean ± SEM. *P < 0.05, n = 7/group.

Article Snippet: After blocking with a blocking buffer (Boster Biological Technology, Pleasanton, CA, USA) for 1.5 h at room temperature, the membrane was incubated with primary antibodies (diluted in TBS-T) against p-p70S6K (Thr 389) (1:1,000; A0533; Assay Biotechnology Company, Sunnyvale, CA, USA) and p70S6K (1:1,000; CSB-PA003686; CUSABIO, Houston, TX, USA), p-4E-BP1 (Thr37/46) (1:1,000; #2855; Cell Signaling technology, Danvers, MA, USA) and 4E-BP1 (1:1000; CSB-PA007994; CUSABIO), p-AKT (S473) (1:1000; CSB-PA000466; CUSABIO), AKT (1:1000; CSB-PA008132; CUSABIO), GAPDH (1:1000; CSB-PA004666; CUSABIO) for 2 h at room temperature.

Techniques: Phospho-proteomics, Western Blot

Journal: Scientific Reports

Article Title: Passive repetitive stretching is associated with greater muscle mass and cross-sectional area in the sarcopenic muscle

doi: 10.1038/s41598-021-94709-0

Figure Lengend Snippet: Sequences of real-time PCR primers used.

Article Snippet: After blocking with a blocking buffer (Boster Biological Technology, Pleasanton, CA, USA) for 1.5 h at room temperature, the membrane was incubated with primary antibodies (diluted in TBS-T) against p-p70S6K (Thr 389) (1:1,000; A0533; Assay Biotechnology Company, Sunnyvale, CA, USA) and p70S6K (1:1,000; CSB-PA003686; CUSABIO, Houston, TX, USA), p-4E-BP1 (Thr37/46) (1:1,000; #2855; Cell Signaling technology, Danvers, MA, USA) and 4E-BP1 (1:1000; CSB-PA007994; CUSABIO), p-AKT (S473) (1:1000; CSB-PA000466; CUSABIO), AKT (1:1000; CSB-PA008132; CUSABIO), GAPDH (1:1000; CSB-PA004666; CUSABIO) for 2 h at room temperature.

Techniques: Real-time Polymerase Chain Reaction