Journal: The EMBO Journal

Article Title: Hepatic ASPG-mediated lysophosphatidylinositol catabolism impairs insulin signal transduction

doi: 10.1038/s44318-025-00525-x

Figure Lengend Snippet: ( A ) Relative mRNA levels of Sepp1 in livers (left, n = 5 female) and SELENOP protein levels in serum (right, WT n = 8, LKO n = 6, female) from the WT and Aspg LKO mice that were fed a HFD for 20 weeks. ( B ) Adenoviruses expressing Aspg (Adv-ASPG) or the vector control (Adv-GFP) infected the primary hepatocytes followed by with or without palmitate (200 μM) treating the cells for 24 h. Then the mRNA levels of Aspg and Sepp1 were determined by quantitative real-time PCR ( n = 3 each group). ( C ) The SELENOP protein levels in the culture medium of cells in ( B ) ( n = 3 each group). ( D ) Conditional medium collected from primary hepatocytes overexpressing Adv-ASPG after palmitic acid treatment for 24 h was applied to 3T3L1 adipocytes for 48 h. Western blot analysis of the phosphorylation and total protein levels of INSR and AKT in 3T3L1 adipocytes ( n = 3). ( E , F ) Lentiviral Foxo1 shRNA infected mouse primary hepatocytes followed by treatment with palmitate (36 h), insulin (24 h). Sepp1 mRNA ( E ) and SELENOP protein levels ( F ) were measured ( n = 5 each group). ( G ) Relative mRNA levels of Sepp1 in the primary hepatocytes isolated from the WT and Aspg LKO mice. The cells were treated with or without AKTi (2 μM) for 2 h before the assay ( n = 3 each group). ( H ) Relative mRNA levels of Sepp1 in the mouse primary hepatocytes. The cells were treated with palmitate (36 h), insulin (24 h), LPI (12 h) and AKTi (2 h) as indicated following by a quantitative RT-PCR analysis ( n = 3). ( I , J ) Mouse primary hepatocytes were treated by PA (36 h) and ML-193 (6 h) prior to the Sepp1 mRNA ( I ) and SELENOP protein assay ( J ). n = 5 each group. For all: Data are represented as mean ± SEM. Statistical analysis was performed by unpaired two-tailed Student’s t test for ( A ), by two-way ANOVA followed by Tukey’s test for ( B , C , G ), by one-way ANOVA followed by Tukey’s test for ( E , F , H – J ). .

Article Snippet: In order to ascertain the impact of LPI on primary hepatocytes, hepatocytes were treated for 12 h with 5 μM of LPI (Avanti Polar Lipids, #850091 P) and/or 10 μM ML-193 (MCE, #HY-110125) for 6 h. To observe the regulation on Sepp1 expression, primary hepatocytes were treated with 10 nM insulin for 24 h and 5 μM LPI for 12 h with or without 2 μM AKTi (MCE, # HY-10355) for 2 h.

Techniques: Expressing, Plasmid Preparation, Control, Infection, Real-time Polymerase Chain Reaction, Western Blot, Phospho-proteomics, shRNA, Isolation, Quantitative RT-PCR, Two Tailed Test

Journal: Molecular & Cellular Proteomics : MCP

Article Title: mTORC2 Regulates Non-homologous End Joining Through Modulating the Temporal Dynamics of 53BP1

doi: 10.1016/j.mcpro.2025.101035

Figure Lengend Snippet: mTORC2 regulated NHEJ through downstream kinase Akt. A , Rictor, Akt, phosphorylated Akt (S473) from Scr and ACHN-shRictor cell lysate were immunoblotted using the corresponding antibodies. β-actin was used as the loading control. B , the effect of Akt inhibitor (Akt VIII) on Akt phosphorylation (S473) was determined by Western blotting. C , ACHN cells were untreated or irradiated with 2.5 Gy IR followed by incubation with 10 μM Akt VIII. After incubated for the indicated time periods, cells were fixed, permeabilized, and stained for 53BP1 and cell nuclei. Insets represent enlarged images of 53BP1 foci in single cell nuclei. Scale bar, 20 μm. D , the numbers of 53BP1 foci per cell with indicated time points were quantified using the Image J software. The quantitative data are expressed as means ± SD from 100 randomly picked cells on each slide (∗∗∗ p < 0.001, by two-sided unpaired Student’s t test). E , the levels of 53BP1 phosphorylation in ACHN cells incubated with 10 μM Akt VIII were examined by immunoprecipitation followed by western blotting with anti-PXS∗P antibody.

Article Snippet: The second-generation mTOR inhibitor AZD2014, Akt VIII and rapamycin were purchased from MedChemExpress.

Techniques: Control, Phospho-proteomics, Western Blot, Irradiation, Incubation, Staining, Software, Immunoprecipitation