Journal: Experimental Diabetes Research

Article Title: Insulin Receptor Substrate 2 Expression and Involvement in Neuronal Insulin Resistance in Diabetic Neuropathy

doi: 10.1155/2011/212571

Figure Lengend Snippet: IRS isoform expression in murine lumbar DRG. IRS isoforms were examined using RT-PCR ((a) and (b)) and Western blot ((c) and (d)). (a) RT-PCR was performed on adult C57Bl/6 mouse lumbar DRG ( n = 3 mice) and comparisons were made among IRS1, IRS2, IRS3, and IRS4. GAPDH was used as the housekeeping gene. ΔCt values for IRS2 were significantly lower than any other isoform. (b) Analysis of IRS mRNA levels that were normalized to IRS1 mRNA levels revealed that IRS2 mRNA expression was 27-fold higher than IRS1 in the DRG. *denotes P < .05 n = 3 mice. ((c) and (d)) Representative Western blots of IRS1 and IRS2 protein in mouse lumbar DRG. Equal amounts of protein (20 μ g) were loaded for each lane and samples were separated on 4–15% tris-glycine gel. Both IRS1 (c) and IRS2 (d) proteins were readily detectable. All mice were 8-week-old nondiabetic C57Bl/6 males.

Article Snippet: Primary antibodies were used at the following dilutions and incubations: total IRS2 (Millipore) 1 : 400 overnight at 4°C, mouse monoclonal Peripherin (Millipore) 1 : 2000 overnight at 4°C, and mouse monoclonal Neurofilament 200 (Sigma) 1 : 2000 overnight at 4°C.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Experimental Diabetes Research

Article Title: Insulin Receptor Substrate 2 Expression and Involvement in Neuronal Insulin Resistance in Diabetic Neuropathy

doi: 10.1155/2011/212571

Figure Lengend Snippet: IRS2 protein expression pattern in murine lumbar DRG. Fluorescence immunohistochemistry was used to examine IRS2 expression in adult C57Bl/6 mouse lumbar DRG. ((a) and (d)) Photomicrographs of IRS2 immunoreactivity in DRG neurons. IRS2 was expressed in most neurons of the DRG in mice. ((b) and (e)) Photomicrographs of the same sections stained with antibodies to NF-200 (b), which labels large, myelinated neurons in the DRG or peripherin (e), which labels unmyelinated small DRG neurons. ((c) and (f)) Merged images of IRS2 and NF-200 (c) labeling illustrate that many IRS2-positive neurons also express NF-200, suggesting that many IRS2-positive neurons are large myelinated neurons. Similarly, merged images of IRS2 and peripherin (f) labeling illustrate that many IRS2-positive neurons coexpress peripherin, suggesting that many IRS2-positive neurons are small unmyelinated neurons. Scale bar = 100 μ m.

Article Snippet: Primary antibodies were used at the following dilutions and incubations: total IRS2 (Millipore) 1 : 400 overnight at 4°C, mouse monoclonal Peripherin (Millipore) 1 : 2000 overnight at 4°C, and mouse monoclonal Neurofilament 200 (Sigma) 1 : 2000 overnight at 4°C.

Techniques: Expressing, Fluorescence, Immunohistochemistry, Staining, Labeling

Journal: Experimental Diabetes Research

Article Title: Insulin Receptor Substrate 2 Expression and Involvement in Neuronal Insulin Resistance in Diabetic Neuropathy

doi: 10.1155/2011/212571

Figure Lengend Snippet: pSer(731)IRS2 is elevated in DRG neurons from type 1 and type 2 diabetic mice. Protein was harvested from adult mouse DRG culture from diabetic ob/ob and nondiabetic mice (a), freshly isolated DRG from STZ-injected diabetic and nondiabetic C57Bl/6 mice (b), and from DRG neurons grown in hyperglycemic and control conditions (c). Western blots were performed using antibodies that recognized phosphorylated ser731 resides on IRS2, and levels of Ser(731)IRS2 were normalized to total IRS2. (a) Comparisons of pSer(731)IRS2 levels in nondiabetic and diabetic ob/ob mice revealed a significant increase in pSer(731)IRS2 levels in diabetic mice. *denotes P < .05 versus nondiabetics. n = 6 for nondiabetic mice and n = 7 for diabetic mice. (b) Diabetes was induced in 8-week-old C57Bl/6 male mice with STZ, and diabetes was allowed to progress for 6 weeks. Similar to ob/ob diabetic mice, pSer(731)IRS2 levels were significantly elevated in STZ-injected diabetic mice. *denotes P < .05 versus nondiabetics. n = 5 for nondiabetic mice and n = 8 for diabetic mice. (c) DRG neurons from nondiabetic animals were grown in 10 mM (control) and 25 mM (hyperglycemic) glucose concentrations. There was no significant change in IRS2 serine phosphorylation levels between groups. n = 6 for 10 mM glucose and n = 7 for 25 mM glucose.

Article Snippet: Primary antibodies were used at the following dilutions and incubations: total IRS2 (Millipore) 1 : 400 overnight at 4°C, mouse monoclonal Peripherin (Millipore) 1 : 2000 overnight at 4°C, and mouse monoclonal Neurofilament 200 (Sigma) 1 : 2000 overnight at 4°C.

Techniques: Isolation, Injection, Western Blot

Journal: Experimental Diabetes Research

Article Title: Insulin Receptor Substrate 2 Expression and Involvement in Neuronal Insulin Resistance in Diabetic Neuropathy

doi: 10.1155/2011/212571

Figure Lengend Snippet: Total IRS2 and IR protein levels in mouse lumbar DRG. Protein was harvested from adult mouse DRG culture from diabetic ob/ob and nondiabetic mice. Western blots were performed using antibodies that recognized total IRS2 (a) or IR β subunit levels (b). In both cases protein levels were normalized to actin. (a) Total IRS2 levels were slightly decreased in diabetic ob/ob mice although this trend was not statistically significant ( P > .05). n = 7 for nondiabetic mice and n = 7 for diabetic mice. (b) IR β subunit protein levels were not statistically different between diabetic and nondiabetic mice ( P > .05). n = 5 for nondiabetic mice and n = 6 for diabetic mice.

Article Snippet: Primary antibodies were used at the following dilutions and incubations: total IRS2 (Millipore) 1 : 400 overnight at 4°C, mouse monoclonal Peripherin (Millipore) 1 : 2000 overnight at 4°C, and mouse monoclonal Neurofilament 200 (Sigma) 1 : 2000 overnight at 4°C.

Techniques: Western Blot

Journal: International Journal of Biological Sciences

Article Title: CircFAT1 Promotes Lung Adenocarcinoma Progression by Sequestering miR-7 from Repressing IRS2-ERK-mediated CCND1 Expression

doi: 10.7150/ijbs.70889

Figure Lengend Snippet: IRS2 is a direct target of miR-7, and circFAT1 regulates LUAD cells proliferation via miR-7/IRS2/p-ERK1/2/CCND1 pathway. (A) Potential target genes of miR-7 predicted by 5 different algorithms. The numbers of genes predicted by individual tools uniquely or different combinations were indicated. (B) Venn diagram showing the numbers of miR-7 target genes predicted by all five tools and downregulated by both miR-7 mimic and shcircFAT1. (C) Schematic illustration of two predicted miR-7-binding sites (sites 1588 and 2307) in IRS2 3'UTR and the sequences of the sites (3'UTR-WT) and their mutant (3'UTR-Mut) that cloned into luciferase reporter. MiR-7 sequence was included to show matched sequences (Red). The complementary sequences were used in mutants (Green). (D) The relative luciferase activities in 293 T cells after transfected with IRS2 3'UTR-WT or IRS2 3'UTR-Mut reporters and miR-7 mimic or mimic control to show site 2307 was functional. (E) Knockdown efficiencies of shIRS2 tested in A549 and PC9 cells as detected by RT-qPCR. (F) CCK8 assay showing growth curves of A549 and PC9 cells infected with shIRS2 or shNC. (G) Relative IRS2 mRNA expression level in A549 and PC9 cells transduced with shcircFAT1 as detected by RT-qPCR to show downregulation of IRS2 by shcircFAT1. (H) Relative mRNA expression level of IRS2 in A549 and PC9 cells transfected with miR-7 as detected by RT-qPCR, showing downregulation of IRS2 by miR-7. (I and J, Left) Western blot assay showing that shcircFAT1 (I) and miR-7 (J) downregulated IRS2, p-ERK1/2, CCND1, but not ERK1/2 in A549 and PC9 cells. (Right) Quantification of western blot signals. (K) Expression level of IRS2 in 34 paired LUAD clinical samples tested by RT-qPCR. (L-N) RT-qPCR assay showing downregulation of CCND1 mRNA in A549 and PC9 cells by shcircFAT1(L), miR-7 (M) and shIRS2 (N). (O, left) Western blot analysis to show that shIRS2 downregulated p-ERK1/2 and CCND1, but not ERK1/2 protein levels in A549 and PC9 cells. (Right) Quantification of western blot signals. Data are presented as mean ± SD; * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant; ctr, miR-7 mimic control; Inh, miR-7 inhibitor; Inh ctr, miR-7 inhibitor control.

Article Snippet: The primary antibodies used were anti-IRS2 (1:100, Proteintech, China), anti-CCND1 (1:100, Servicebio, China), anti-Ki67 (1:500, Proteintech, China).

Techniques: Binding Assay, Mutagenesis, Clone Assay, Luciferase, Sequencing, Transfection, Functional Assay, Quantitative RT-PCR, CCK-8 Assay, Infection, Expressing, Transduction, Western Blot

Journal: International Journal of Biological Sciences

Article Title: CircFAT1 Promotes Lung Adenocarcinoma Progression by Sequestering miR-7 from Repressing IRS2-ERK-mediated CCND1 Expression

doi: 10.7150/ijbs.70889

Figure Lengend Snippet: CircFAT1 promotes A549 LUAD cell tumorigenesis in vivo . (A and B) Xenograft assay showing A549 cells with circFAT1 knockdown (A) or overexpression (B) by lentiviruses inhibited or promoted tumor formation, respectively (n=5). (C and D) Volume of the xenograft tumors under circFAT1 knockdown (C) and overexpression (D) conditions. The volumes of tumors were estimated by measuring sizes every other day. (E) Weight of harvested xenograft tumors derived from A549 cells with circFAT1-knockdown (Upper) or overexpression (Lower). (F) Immunofluorescent staining of xenograft tumors showing that the protein levels of IRS2, CCND1 and Ki67 were decreased in circFAT1-knocked-down tumor tissues and increased in circFAT1-overexpressed tissue. Scale bar, 100 µm. Data are presented as mean ± SD; * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The primary antibodies used were anti-IRS2 (1:100, Proteintech, China), anti-CCND1 (1:100, Servicebio, China), anti-Ki67 (1:500, Proteintech, China).

Techniques: In Vivo, Xenograft Assay, Over Expression, Derivative Assay, Staining

Journal: International Journal of Biological Sciences

Article Title: CircFAT1 Promotes Lung Adenocarcinoma Progression by Sequestering miR-7 from Repressing IRS2-ERK-mediated CCND1 Expression

doi: 10.7150/ijbs.70889

Figure Lengend Snippet: Working model of circFAT1 action. (Left) CircFAT1 sequence-specifically sequester miR-7, resulting in an increase of IRS2 mRNA and protein, which in turn enhance ERK1/2 phosphorylation and CCND1 production, consequently promoting tumor cell growth. (Right) CircFAT1 enhances the effectiveness of the chemotherapeutic drug DDP.

Article Snippet: The primary antibodies used were anti-IRS2 (1:100, Proteintech, China), anti-CCND1 (1:100, Servicebio, China), anti-Ki67 (1:500, Proteintech, China).

Techniques: Sequencing

Journal: PLoS ONE

Article Title: KLF15 Is a Molecular Link between Endoplasmic Reticulum Stress and Insulin Resistance

doi: 10.1371/journal.pone.0077851

Figure Lengend Snippet: A . Inflammatory marker expression . Total RNA was extracted from hepatocytes isolated from chow-fed 4-month-old male WT and KLF15 -/- mice (top) or from liver tissues isolated from WT and KLF15 -/- male mice that received HFD (60% kcal from fat) for 8 weeks starting at age 2 months, were fasted for 5h, i.p. injected with 10mU/g insulin and sacrificed 10 minutes post-injection (n=3; bottom). First strand cDNA was subjected to QPCR using primers against TNFα and MCP-1 . B . Activation of hepatic JNK, AKT and markers of energy availability . Western analysis of protein lysates prepared from (left) hepatocytes that were isolated from chow-fed 3.5-month-old male WT and KLF15 -/- mice and harvested 10 minutes after addition of saline or 100nM insulin, or from (right) liver tissues described in (A). C . Proximal insulin signaling pathway activity . Western analysis of IRS-1 and IRS-2 immunoprecipitated from protein lysates of liver tissues from WT and KLF15 -/- male mice that received HFD (60% kcal from fat) for 8 weeks starting at age 2 months, were fasted for 5h, i.p. injected with saline or 10mU/g insulin and sacrificed 10 minutes post-injection (n=3). D . Effect of KLF15 overexpression on mTORC1 activity . Hepatocytes were isolated from standard chow-fed 2.5-month-old female WT mice and infected with EV or KLF15 -containing adenovirus. Protein lysates were subjected to immunoblotting with the antibodies shown. E . Effect of KLF15 deficiency on insulin and amino acid-mediated activation of mTORC1 . Hepatocytes were isolated from 3.5 month-old male WT and KLF15 -/- mice. Following an overnight incubation in serum-free Williams E medium (without L-glutamine), cells were harvested after a 45-minute treatment with PBS or (top) 10mM L-glutamine, (middle) 10mM L-leucine or after a 10-minute treatment with PBS or 100nM insulin (bottom). Protein lysates were subjected to immunoblotting with the antibodies shown. primary hepatocyte experiments were performed twice in triplicate; each lane (in B, D and E) indicates a technical replicate. For liver blots, each lane represents one mouse. Statistical analysis was performed using Student’s t-test for unpaired samples. Values = mean ± SEM. *p<0.05; **p<0.01.

Article Snippet: Proteins resolved by SDS-PAGE were transferred to a nitrocellulose membrane and immunoblotting was performed using antibodies against total IRS-1, phosphorylated tyrosine (PY99) (Santa Cruz Biotechnology) and phosphorylated IRS-1(Ser307), total IRS-2 (Cell Signaling).

Techniques: Marker, Expressing, Isolation, Injection, Activation Assay, Western Blot, Activity Assay, Immunoprecipitation, Over Expression, Infection, Incubation