Journal: Journal of Diabetes Research

Article Title: Caveolin-1 Is Essential for the Improvement of Insulin Sensitivity through AKT Activation during Glargine Treatment on Diabetic Mice

doi: 10.1155/2021/9943344

Figure Lengend Snippet: The expression of Caveolin-1, p-AKT/AKT, and PI3K in adipose tissue of NC, type 2 diabetic mice, and insulin-treated T2DM mice (a). The band intensity of Caveolin-1, p-AKT/AKT, and PI3K was quantified as shown in (b–d), respectively (data presented as mean ± SEM. ∗ P < 0.05 vs. NC mice. # P < 0.05 compared with T2DM mice, n = 5).

Article Snippet: The primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA), which included Caveolin-1-Ab (Cat. #3267), phosphorylated (p)-Akt (Ser473)-Ab (Cat. #4060, 1 : 2000), Akt-Ab (Cat. #4691), PI3K-Ab (Cat. #4255), and β -actin (Cat. #8457).

Techniques: Expressing

Journal: Journal of Diabetes Research

Article Title: Caveolin-1 Is Essential for the Improvement of Insulin Sensitivity through AKT Activation during Glargine Treatment on Diabetic Mice

doi: 10.1155/2021/9943344

Figure Lengend Snippet: The expression of Caveolin-1, p-AKT/AKT, and PI3K among five different groups (a). The band intensity of Caveolin-1, p-AKT/AKT, and PI3K was quantified as shown in (b–d), respectively (data are expressed as mean ± SEM. ∗ P < 0.05 compared with the insulin group. # P < 0.05 compared with the Ctrl-shRNA group; a represents P < 0.05 compared with the NC group, and b represents P < 0.05 compared with the T2DM group, n = 5).

Article Snippet: The primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA), which included Caveolin-1-Ab (Cat. #3267), phosphorylated (p)-Akt (Ser473)-Ab (Cat. #4060, 1 : 2000), Akt-Ab (Cat. #4691), PI3K-Ab (Cat. #4255), and β -actin (Cat. #8457).

Techniques: Expressing, shRNA

Journal: Theranostics

Article Title: Inhibition of acylglycerol kinase sensitizes DLBCL to venetoclax via upregulation of FOXO1-mediated BCL-2 expression

doi: 10.7150/thno.72786

Figure Lengend Snippet: AGK inhibits BCL-2 expression by phosphorylation PTEN and subsequent AKT-FOXO1 signaling. A. The levels of AGK and BCL-2 were determined in SU-DHL2, OCI-LY1, TMD8, SU-DHL4, SU-DHL6, SU-DHL10, and SU-DHL16 DLBCL cell lines. B. Spearman correlation analysis of AGK and BCL-2 expression in DLBCL cell lines. C. AGK and BCL-2 expression was measured with immunoblotting and BCL-2 expression was quantified in shRNA control and shRNA AGK SU-DHL4 cells from three independent experiments. D. AGK and BCL-2 expression was measured in control and AGK overexpression SU-DHL4 cells, and BCL-2 expression was quantified from three independent experiments. E. The amounts of p-PTEN and PTEN in SU-DHL2, OCI-LY1, TMD8, SU-DHL4, SU-DHL6, SU-DHL10, and SU-DHL16 DLBCL cell lines were determined by immunoblotting. F. Spearman correlation analysis of AGK and PTEN phosphorylation levels in DLBCL cell lines. G-H. Phosphorylation levels of PTEN, AKT and FOXO1 were measured and quantified in control and AGK knockdown SU-DHL4 cells from three independent experiments. I-J The levels of FOXO1 and p-FOXO1 were measured in cytoplasmic and nuclear fractions (I) and the band intensities were quantified (J). K. The binding of FOXO1 to BCL-2 promoter in SU-DHL4 was determined by chromatin immunoprecipitation analysis. Data were expressed as mean ± SEM from three independent experiments (A-J) or two independent experiments with triplicates (K), and analyzed for statistical significance by t -test. (* P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: After blocking with 5% milk for 2 h, the blots were probed with the anti-AGK (Abcam, ab137616), anti-BCL-2 (Abclonal, A19693), anti-PTEN (CST, #9188), anti-pPTEN (CST, #9551), anti-AKT (CST, #4691), anti-pAKT(T308) (CST, #2965), anti-pAKT (S473) (CST, #4060), anti-FOXO1 (CST, #2880), anti-pFOXO1 (CST, #9464), anti-β-Actin (CST, #4970) and anti-GAPDH (Servicebio, GB11002) antibodies overnight at 4 °C.

Techniques: Expressing, Western Blot, shRNA, Over Expression, Binding Assay, Chromatin Immunoprecipitation

Journal: Theranostics

Article Title: Inhibition of acylglycerol kinase sensitizes DLBCL to venetoclax via upregulation of FOXO1-mediated BCL-2 expression

doi: 10.7150/thno.72786

Figure Lengend Snippet: Knockdown of AGK augments venetoclax efficacy in vivo . A. Schematic diagram of xenograft tumor model and venetoclax treatment strategy. B-E. Tumor growth curves of mice receiving shRNA control, shRNA AGK#1 cells with or without venetoclax treatment (n = 6, each group). F. Tumor weights of shRNA control and shRNA AGK#1 groups with or without venetoclax treatment (n = 6, each group). G-H. H&E staining, cleaved-caspase3 and TUNEL staining of shRNA control and shRNA AGK groups with or without venetoclax treatment, and the percentages of positive area of cleaved-caspase3 and TUNEL were quantified. I-J. The levels of AGK, BCL-2, p-FOXO1, FOXO1, p-AKT and AKT were measured (I) and quantified (J) in the tumor tissues isolated from shRNA control and shRNA AGK groups (n = 6, each group). K-L. FOXO1 staining of shRNA control and shRNA AGK group tumor tissues (K) and the percentages of FOXO1 expression in cytoplasmic and nuclear were quantified (L). Scale bar, 100 μm or 25 μm. Data were expressed as mean ± SEM and analyzed for statistical significance by One-way ANOVA (F, H, L) or t -test (J) (* P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: After blocking with 5% milk for 2 h, the blots were probed with the anti-AGK (Abcam, ab137616), anti-BCL-2 (Abclonal, A19693), anti-PTEN (CST, #9188), anti-pPTEN (CST, #9551), anti-AKT (CST, #4691), anti-pAKT(T308) (CST, #2965), anti-pAKT (S473) (CST, #4060), anti-FOXO1 (CST, #2880), anti-pFOXO1 (CST, #9464), anti-β-Actin (CST, #4970) and anti-GAPDH (Servicebio, GB11002) antibodies overnight at 4 °C.

Techniques: In Vivo, shRNA, Staining, TUNEL Assay, Isolation, Expressing

Journal: Theranostics

Article Title: Inhibition of acylglycerol kinase sensitizes DLBCL to venetoclax via upregulation of FOXO1-mediated BCL-2 expression

doi: 10.7150/thno.72786

Figure Lengend Snippet: The expression of AGK is inversely associated with BCL-2 expression in DLBCL patients. A. H&E staining, BCL-2, AGK and FOXO1 staining of BCL-2 low and BCL-2 high DLBCL tissue samples. n = 33, scale bar, 100 μm or 25 μm. Black arrow: cytoplasmic FOXO1; Red arrow: nuclear FOXO1. B. Quantitation of the percentages of AGK positive cell in BCL-2 low (n = 17) and BCL-2 high (n = 16) DLBCL paraffin sections. C. Quantitation of the ratios of nuclear and cytoplasmic FOXO1 in BCL-2 low (n = 15) and BCL-2 high (n = 14) DLBCL paraffin sections. D. Spearman correlation analysis of the ratios of nuclear versus cytoplasmic FOXO1 with the expression of AGK. E. Schematic figure of the regulation of AGK on venetoclax sensitivity. In DLBCL cells, AGK phosphorylates and inactivates PTEN, leading to enhanced phosphorylation of AKT and FOXO1 and reduced FOXO1 nuclear translocation and its target gene BCL-2 expression. AGK knockdown could reverse the effect of AGK-PTEN-FOXO1-BCL-2 axis, upregulate the expression of BCL-2 and increase the sensibility to venetoclax-induced apoptosis. Ibrutinib inhibits the expression of AGK in DLBCL cells. Data were expressed as mean ± SEM and analyzed for statistical significance by t test (B, D). (** P < 0.01; *** P < 0.001; ns, not significant).

Article Snippet: After blocking with 5% milk for 2 h, the blots were probed with the anti-AGK (Abcam, ab137616), anti-BCL-2 (Abclonal, A19693), anti-PTEN (CST, #9188), anti-pPTEN (CST, #9551), anti-AKT (CST, #4691), anti-pAKT(T308) (CST, #2965), anti-pAKT (S473) (CST, #4060), anti-FOXO1 (CST, #2880), anti-pFOXO1 (CST, #9464), anti-β-Actin (CST, #4970) and anti-GAPDH (Servicebio, GB11002) antibodies overnight at 4 °C.

Techniques: Expressing, Staining, Quantitation Assay, Translocation Assay

Journal: Theranostics

Article Title: Aligned chitosan nanofiber hydrogel grafted with peptides mimicking bioactive brain-derived neurotrophic factor and vascular endothelial growth factor repair long-distance sciatic nerve defects in rats

doi: 10.7150/thno.36272

Figure Lengend Snippet: ACG-RGI/KLT regulated Schwann cells in vitro . (A) Images of cultured Schwann cells in the ACG, ACG-RGI/KLT, and control groups. Schwann cells were stained with anti-S100 (red) and DAPI (blue). (B) The expression levels of several markers reflecting the proliferation, adhesion, and secretory function of cells were examined by Western blotting (WB). (C-I) Statistical analysis of brain-derived neurotrophic factor (BNGF), nerve growth factor (NGF), glial cell-derived neurotrophic factor (GDNF), vascular endothelial growth factor (VEGF), neural cell adhesion molecule 1 (NCAM1), proliferating cell nuclear antigen (PCNA), and phosphorylated protein kinase B (p-AKT) protein levels, respectively. (K-N) qRT-PCR results indicated the relative expression levels of BNGF, NGF, GDNF, VEGF, and NCAM1. Data are presented as means ± standard error of the mean, n = 3 for each group. ** p <0.01, * p <0.05.

Article Snippet: WB was performed using antibodies to VEGF (ab32152), nerve growth factor (NGF; ab6199), BDNF (ab108319), glial cell-derived neurotrophic factor (GDNF; ab18956), neural cell adhesion molecule 1 (NCAM1; ab9018), and growth associated protein 43 (GAP43; ab12274) purchased from Abcam, and to proliferating cell nuclear antigen (PCNA; #13110), protein kinase B (AKT; #4691), phosphorylated protein kinase B (p-AKT; #4060), CD31 (#77699), and GAPDH (#51332) purchased from Cell Signaling Technology.

Techniques: In Vitro, Cell Culture, Staining, Expressing, Western Blot, Derivative Assay, Quantitative RT-PCR