Journal: Inflammation Research

Article Title: Genetic depletion of the early autophagy protein ATG13 impairs mitochondrial energy metabolism, augments oxidative stress, induces the polarization of macrophages to the M1 inflammatory mode, and compromises myelin integrity in skeletal muscle

doi: 10.1007/s00011-025-02158-6

Figure Lengend Snippet: The ROS-mediated inhibition of Sirtuin-1, but not Sirtuin-2, may be responsible for inflammation. A A fluorescence-based (excitation: emission = 355/460 nm) enzyme assay of sirtuin-1 (SIRT1) in splenic Mφs. The assay was performed as instructed by the manufacturer (BPS Biosciences) using a cell lysate containing 1 µg of total protein. B Fluorescence-based sirtuin-2 assay performed according to the manufacturer’s protocol (Abcam) with 1 µg of protein derived from the cell lysate of Mφ cells. C IB analyses followed by D densitometric analyses of Sirt-1/2, acetylated p65, and β-actin in splenic Mφ cells from NTg and Tg +/-ATG13 mice ( n = 3 per group). Dual IF analyses of SIRT-1 (red) and 3-nitrotyrosine (3NT; green) in the spleens of 10- to 12-week-old E NTg and F Tg +/-ATG13 mice ( n = 6/group). G 3D visualization of a single cell (ImageJ) showing the interaction between SIRT1 and 3NT. H Quantification followed by scatter boxplot analysis of the total number of 3NT-ir puncta per cell in 19 cells/group. The unpaired t test indicates **** p < 0.0005 versus the control. I Tg splenic macrophages were transfected with siRNA against sirt1 (250 pmol; Cat # AM16708; Thermo Fisher Scientific, MA), treated with 0.5 mg/mL LPS for 2 h, and then immunostained with a Sirt1 antibody to determine the efficiency of the siRNA. Enzyme activity assay of J SIRT1 and K SIRT2 after 30 min of incubation with siRNA-transfected and non-transfected cell lysates (1 µg protein). L IF analysis of acetylated p65 in Tg splenic Mφ cells transfected with Sirt1 siRNA and then treated with 0.5 mg/mL LPS. The results are presented as the means ± SEMs of three different experiments

Article Snippet: For the SIRT1 activity assay, SIRT1 activity was measured in spleen lysates from transgenic and nontransgenic mice using the SIRT1 Activity Assay Kit (Fluorometric) (BPS Bioscience, Cat# 50081) following the manufacturer’s protocol.

Techniques: Inhibition, Fluorescence, Enzymatic Assay, Derivative Assay, Control, Transfection, Enzyme Activity Assay, Incubation