Journal: bioRxiv

Article Title: TAX1BP1 recruitment reactivates autophagy of Tau aggregates through ULK1 and TBK1

doi: 10.64898/2026.02.12.705532

Figure Lengend Snippet: A) Schematic overview of the pelleting assay. B) Immunoblotting for autophagy factors after Tau aggregation and the pelleting assay. C) Quantification of the signal in the pellet fraction in comparison to the input samples. Data are presented as mean +/- s.d. (n = 3 biologically independent experiments). Multiple unpaired t-tests. D) Immunofluorescence images of Tau, p62, TAX1BP1, ATG13 and TBK1 p-S172 (scale bar = 20 µm). E) Pearson’s correlation (PCC) between Tau aggregates and p62, TAX1BP1, ATG13 and TBK1 p-S172. Data are presented as violin plots showing the distribution of individual data points. The solid line indicates the median; dashed lines represent the interquartile range. Each data point corresponds to the measured correlation within an individual cell. One-way ANOVA with Dunnett’s multiple comparisons test. F) Immunofluorescence images for p62 and p62 p-S403 in cells expressing Tau-mRuby after Tau seeding (scale bar = 20 µm). G) Pearson’s correlation (PCC) between Tau aggregates and p62 or p62 p-S403 in I. Data are presented as violin plots showing the distribution of individual data points. The solid line indicates the median; dashed lines represent the interquartile range. Each data point corresponds to measured correlation within an individual cell. H) Schematic summary of the results. *P < 0.05, **P < 0.005, ****P < 0.0001. ns, not significant.

Article Snippet: The following primary antibodies and dilution were used in this study: rabbit anti-Tau (1:2500; MA5-42451, Invitrogen), mouse anti-p62 (1:1000; #610832, Clone 3/p62 lck ligand, BD Bioscience), mouse anti-NBR1 (1:1000; H00004077-M01, Clone 6B11, Abnova), rabbit anti-TAX1BP1 (1:1000; #5105, clone D1D5, CST), rabbit anti-FIP200 (1:1000; #12436, clone D10D11, CST), rabbit anti-TBK1 (1:1000; #3013, CST), rabbit anti-TBK1 pS172 (1:1000; #5483, clone D52C2, CST), rabbit anti-ATG13 (1:1000; #13468, clone E1Y9V, CST), rabbit anti-ULK1 (1:1000; #8054, clone D8H5, CST), mouse anti-LC3B (1:1000; #3868S, clone D11, CST), rabbit ATG13 p-S355 (1:1000; #46329, CST), rabbit anti ULK1 p-S317 (1:1000; #37762, CST), rabbit anti-ULK1 p-S757 (1:1000; #14202, CST), rabbit anti-ATG9 (1:1000; #13509, clone D4O9D, CST), rabbit anti-ATG16L1 (1:1000; #8089, clone D6D5, CST), rabbit anti-ATG14 (1:1000; #5504, CST), mouse anti-GAPDH (1:20000; #G8795, clone 71.1, Sigma) The secondary antibodies used in this study were horseradish peroxidase (HRP)-conjugated polyclonal goat anti-mouse (1:10000, #115-035-003, Jackson ImmunoResearch Labs) and HRP-conjugated polyclonal goat anti-rabbit (1:10000; #111-035-003, Jackson ImmunoResearch Labs).

Techniques: Western Blot, Comparison, Immunofluorescence, Expressing

Journal: bioRxiv

Article Title: TAX1BP1 recruitment reactivates autophagy of Tau aggregates through ULK1 and TBK1

doi: 10.64898/2026.02.12.705532

Figure Lengend Snippet: A) Schematic overview of the domain structure of TAX1BP1 and the tethering of TAX1BP1 truncations. B) Representative fluorescence-activated cell sorting (FACS) plots of Tau flux measurements in cells expressing full-length TAX1BP1, TAX1BP1ΔSKICH or TAX1BP1ΔZnF. C) FACS-based measurement of Tau flux upon its aggregation and tethering of TAX1BP1 WT, TAX1BP1ΔSKICH and TAX1BP1ΔZnF. Data are presented as mean +/- s.d. (n = 3 biologically independent experiments). Two-way ANOVA with Dunnett’s multiple comparisons test. D) Schematic overview of the microscopy-based protein-protein interaction assay in E. E) Representative images of microscopy-based protein-protein interaction assays between the SKICH domain of TAX1BP1 on the beads and GFP-ULK1 complex, mCh-PI3K complex, mCh-WIPI2, GFP-TBK1 and mCh-NAP1 (scale bar = 80 µm). F) Schematic depiction of the formation of the initiation hub on the surface of the SKICH domain of TAX1BP1 together with a size comparison between the SKICH domain and the proteins involved in the formation of the complex. G) Immunofluorescence images of TAX1BP1, ATG13, ULK1, FIP200 and TBK1 p-S172 with and without addition of rapalog (scale bar = 20 µm). ****P < 0.0001. ns, not significant.

Article Snippet: The following primary antibodies and dilution were used in this study: rabbit anti-Tau (1:2500; MA5-42451, Invitrogen), mouse anti-p62 (1:1000; #610832, Clone 3/p62 lck ligand, BD Bioscience), mouse anti-NBR1 (1:1000; H00004077-M01, Clone 6B11, Abnova), rabbit anti-TAX1BP1 (1:1000; #5105, clone D1D5, CST), rabbit anti-FIP200 (1:1000; #12436, clone D10D11, CST), rabbit anti-TBK1 (1:1000; #3013, CST), rabbit anti-TBK1 pS172 (1:1000; #5483, clone D52C2, CST), rabbit anti-ATG13 (1:1000; #13468, clone E1Y9V, CST), rabbit anti-ULK1 (1:1000; #8054, clone D8H5, CST), mouse anti-LC3B (1:1000; #3868S, clone D11, CST), rabbit ATG13 p-S355 (1:1000; #46329, CST), rabbit anti ULK1 p-S317 (1:1000; #37762, CST), rabbit anti-ULK1 p-S757 (1:1000; #14202, CST), rabbit anti-ATG9 (1:1000; #13509, clone D4O9D, CST), rabbit anti-ATG16L1 (1:1000; #8089, clone D6D5, CST), rabbit anti-ATG14 (1:1000; #5504, CST), mouse anti-GAPDH (1:20000; #G8795, clone 71.1, Sigma) The secondary antibodies used in this study were horseradish peroxidase (HRP)-conjugated polyclonal goat anti-mouse (1:10000, #115-035-003, Jackson ImmunoResearch Labs) and HRP-conjugated polyclonal goat anti-rabbit (1:10000; #111-035-003, Jackson ImmunoResearch Labs).

Techniques: Fluorescence, FACS, Expressing, Microscopy, Protein Protein Interaction Assay, Comparison, Immunofluorescence

Journal: bioRxiv

Article Title: TAX1BP1 recruitment reactivates autophagy of Tau aggregates through ULK1 and TBK1

doi: 10.64898/2026.02.12.705532

Figure Lengend Snippet: A) Immunoblotting of phospho-ULK1, ATG13, TBK1 and p62 upon Tau aggregation and TAX1BP1 tethering. B) Quantification of relative increase of phosphorylation levels upon TAX1BP1 tethering. Phosphorylation signal was normalized to the total protein. To account for inter-experimental variability, values are expressed as a relative to the -Rapalog samples within each biological replicate. The data are presented as mean +/- s.d. (n = 3 biologically independent experiments). Two-way ANOVA with Sidak’s multiple comparisons test. *P < 0.05, **P < 0.005. ns, not significant.

Article Snippet: The following primary antibodies and dilution were used in this study: rabbit anti-Tau (1:2500; MA5-42451, Invitrogen), mouse anti-p62 (1:1000; #610832, Clone 3/p62 lck ligand, BD Bioscience), mouse anti-NBR1 (1:1000; H00004077-M01, Clone 6B11, Abnova), rabbit anti-TAX1BP1 (1:1000; #5105, clone D1D5, CST), rabbit anti-FIP200 (1:1000; #12436, clone D10D11, CST), rabbit anti-TBK1 (1:1000; #3013, CST), rabbit anti-TBK1 pS172 (1:1000; #5483, clone D52C2, CST), rabbit anti-ATG13 (1:1000; #13468, clone E1Y9V, CST), rabbit anti-ULK1 (1:1000; #8054, clone D8H5, CST), mouse anti-LC3B (1:1000; #3868S, clone D11, CST), rabbit ATG13 p-S355 (1:1000; #46329, CST), rabbit anti ULK1 p-S317 (1:1000; #37762, CST), rabbit anti-ULK1 p-S757 (1:1000; #14202, CST), rabbit anti-ATG9 (1:1000; #13509, clone D4O9D, CST), rabbit anti-ATG16L1 (1:1000; #8089, clone D6D5, CST), rabbit anti-ATG14 (1:1000; #5504, CST), mouse anti-GAPDH (1:20000; #G8795, clone 71.1, Sigma) The secondary antibodies used in this study were horseradish peroxidase (HRP)-conjugated polyclonal goat anti-mouse (1:10000, #115-035-003, Jackson ImmunoResearch Labs) and HRP-conjugated polyclonal goat anti-rabbit (1:10000; #111-035-003, Jackson ImmunoResearch Labs).

Techniques: Western Blot, Phospho-proteomics

Journal: bioRxiv

Article Title: TAX1BP1 recruitment reactivates autophagy of Tau aggregates through ULK1 and TBK1

doi: 10.64898/2026.02.12.705532

Figure Lengend Snippet: A) Schematic overview of tethering ATG13 to Tau aggregates using the FKBP-FRB-Rapalog system. B) Immunofluorescence images of Tau and ATG13 (scale bar = 20 µm). C) Immunoblotting of ATG13, ULK1, FIP200 and TBK1 with and without addition of rapalog. D) Quantification of the fraction of protein pelleted upon rapalog treatment in C. E) Immunofluorescence images of Tau and ULK1 or FIP200 with and without rapalog treatment (scale bar = 20 µm) F) Schematic overview of tethering TBK1 to Tau aggregates using the FKBP-FRB-Rapalog system. G) Immunofluorescence images of Tau and TBK1 with and without rapalog treatment (scale bar = 20 µm). H) Comparison of the Tau flux upon tethering TAX1BP1, ATG13 or TBK1 to Tau aggregates following rapalog treatment. Data are presented as mean +/- s.d. (n = 3 biologically independent experiments). Two-way ANOVA with Dunnett’s multiple comparisons test. I) AF3 prediction of the ternary complex formed by the SKICH domain of TAX1BP1, aa1360-1385 of FIP200 and aa33-75 of NAP1. J) Schematic overview of the ATG13 and TBK1 co-tethering experiment. K) Comparison of the Tau flux upon tethering of ATG13 only or ATG13 and TBK1 co-tethering to the Tau aggregates. Data are presented as mean +/- s.d. (n = 3 biologically independent experiments). Two-way ANOVA with Dunnett’s multiple comparisons test. *P < 0.05, **P < 0.005, ***P < 0.001, ****P < 0.0001. ns, not significant.

Article Snippet: The following primary antibodies and dilution were used in this study: rabbit anti-Tau (1:2500; MA5-42451, Invitrogen), mouse anti-p62 (1:1000; #610832, Clone 3/p62 lck ligand, BD Bioscience), mouse anti-NBR1 (1:1000; H00004077-M01, Clone 6B11, Abnova), rabbit anti-TAX1BP1 (1:1000; #5105, clone D1D5, CST), rabbit anti-FIP200 (1:1000; #12436, clone D10D11, CST), rabbit anti-TBK1 (1:1000; #3013, CST), rabbit anti-TBK1 pS172 (1:1000; #5483, clone D52C2, CST), rabbit anti-ATG13 (1:1000; #13468, clone E1Y9V, CST), rabbit anti-ULK1 (1:1000; #8054, clone D8H5, CST), mouse anti-LC3B (1:1000; #3868S, clone D11, CST), rabbit ATG13 p-S355 (1:1000; #46329, CST), rabbit anti ULK1 p-S317 (1:1000; #37762, CST), rabbit anti-ULK1 p-S757 (1:1000; #14202, CST), rabbit anti-ATG9 (1:1000; #13509, clone D4O9D, CST), rabbit anti-ATG16L1 (1:1000; #8089, clone D6D5, CST), rabbit anti-ATG14 (1:1000; #5504, CST), mouse anti-GAPDH (1:20000; #G8795, clone 71.1, Sigma) The secondary antibodies used in this study were horseradish peroxidase (HRP)-conjugated polyclonal goat anti-mouse (1:10000, #115-035-003, Jackson ImmunoResearch Labs) and HRP-conjugated polyclonal goat anti-rabbit (1:10000; #111-035-003, Jackson ImmunoResearch Labs).

Techniques: Immunofluorescence, Western Blot, Comparison

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: Generation of Tg +/-ATG13 mice and evaluation of ATG13 expression in the spleen. A A schematic strategy for the generation of Tg +/-ATG13 mice. B Genotype data generated with different primer sets (as mentioned in the Methods section) for identifying the LacZ repressor Tg-ATG13 (~ 500 bp = 450 bp) and Tg +/-ATG13 (~ 800 bp = 797 bp) mice. Nontransgenic (NTg) mice did not show any band at ~ 500 or ~ 800 bp. C Splenic tissue from 10-week-old male NTg and Tg +/-ATG13 mice ( n = 5/group) was paraffin-embedded, cut at 5 μm thickness, and double immunostained for ATG13 and the activated Mφ marker IBA1. D The parametric Pearson correlation was performed to monitor the relationship between IBA1 and ATG13 expression in 100 dual-stained cells. A linear regression plot was generated in GraphPad Prism 10, which indicated that the slope (Pearson coefficient = r = -0.4420) was significantly nonzero, with F 1,98 =23.80 and ** p < 0.001. E Immunoblot analyses of ATG13, ATG101, and the loading control β-actin are shown . The relative densities of F ATG13 and G ATG101 were calculated as the ratio of the target band intensity to the respective actin band density and are plotted as histograms. The Mann‒Whitney U test was performed to test the significance of the difference in the means between groups; *** p < 0.001 vs. ATG13 of NTg. NS = nonsignificant. The results were confirmed after three independent experiments were performed for n = 5 animals/group

Article Snippet: ATG13 , 10181-2-AP , Protein tech , IB (1:500), IF (1:100).

Techniques: Expressing, Generated, Marker, Staining, Western Blot, Control

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: Autophagy impairment in the splenic Mφs of Tg +/-ATG13 mice. A Dual IF analysis of LC3 (rabbit anti-LC3; Cat#; ProteinTech; dilution 1:100) and CD11b (mouse anti-CD11b; Cat#; Invitrogen; dilution 1:100) in 5 μm thick paraffin-embedded sections of 10–12-week-old nontransgenic and hemizygous atg13 knockout mice ( n = 6/group). ( Inset ) Magnified dual IF images of (a) NTg and (b) Tg +/-ATG13 . Dual flow cytometry of PE-labeled LC3 and FITC-labeled CD11b in purified Mφs isolated from 10- to 12-week-old B NTg and C Tg +/- ATG13 mice (male). The total number of gated events was 20,000/group. The cells under the enclosed area represent a distinct population of CD11b-ir cells, which are also LC3-positive. D Histogram analyses to quantify the CD11b + LC3 + cells among the NTg and Tg +/-ATG13 Mφs. E Immunoblot analyses of LC3II revealed two distinct bands corresponding to autophagy-active LC3BII (lower) and autophagy-inactive LC3BI (upper). IB analyses of WDFY3 (~ 100 kD) and p62 (> 60 kD) were also performed in splenic Mφs of NTg and Tg +/-ATG13 mice. Beta-actin immunoblotting was performed as a loading control. F The relative densitometric analysis results were plotted after normalization to the respective β-actin bands. The white and gray bars represent the LC3B band densities of the NTg and Tg +/-ATG13 mice, respectively. (An unpaired t test was performed to test the significance of the difference in the means between groups; * p < 0.05 vs. the LC3BI of Tg +/-ATG13 . NS = not significant. G DAB immunostaining of splenic sections of WDFY3 (rabbit; cat# Invitrogen, dilution 1:100) ( n = 5/group) counterstained with hematoxylin. (inset) Enclosed zones are magnified (a = NTg, b = Tg). H WDFY3 + cells were counted in 5 different images from 5 mice/group following the quantification per sq.mm. of sections (unpaired t test; *** p < 0.005 = 0.002844221). I & J Representative flow cytometry analyses of WDFY3 (PE-tagged) and CD11b (FITC-tagged) cells (10000 gated events). K Histogram of the number of gated cells in the circled population. L The scatter bar graph shows the counts of CD11b + WDFY3 + cells in six analyses per group (unpaired t test shows **** p < 0.0005). The results are presented as the mean ± SD of three experiments

Article Snippet: ATG13 , 10181-2-AP , Protein tech , IB (1:500), IF (1:100).

Techniques: Knock-Out, Flow Cytometry, Labeling, Purification, Isolation, Western Blot, Control, Immunostaining

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: Polarization of M1Mφ cells in the spleens of Tg+/-ATG13 mice. A Dual IF analysis of CD40 (rabbit anti-CD40; Cat#; ProteinTech; dilution 1:100) and IBA1 (mouse anti-IBA1; Cat#; Invitrogen; dilution 1:100) in 5 μm thick paraffin-embedded spleen sections from 10- to 12-week-old male NTg and Tg+/-ATG13 mice (n=6/group). B Quantification of CD40-ir cells in the 50 μm radius of blood vessels in the red pulp zone was performed. An unpaired t test was performed to verify the significance of the difference in the means between groups, and the results are shown as ****p<0.0005. The results were confirmed after counting 7 independent images per group. C IB followed by D β-actin-normalized densitometric analyses; ***p<0.005 (unpaired t test) between groups. Dual flow cytometry of APC-labeled CD40 and FITC-labeled CD11b in purified Mφs isolated from 10- to 12-week-old E NTg and F Tg+/- ATG13 mice (n=6 male). G The quantification analysis of CD11b+ CD40+ population was performed followed by measuring the significance by unpaired t-test (***p<0.005) . The total number of gated events was 20,000/group. H & I Similarly, dual flow cytometry analysis of CD86 (PE-tagged; dilution 1:100) and CD11b (FITC-tagged; dilution 1:100) followed by J quantification (n=7 analysis/group) was performed (****p<0.0005; unpaired t test). K & L Dual flow cytometry analysis of CD163 (APC-tagged; dilution 1:100) and CD11b (FITC-tagged; dilution 1:100) followed by M quantification (n=7 analysis; ****p<0.0005 by unpaired t test) was performed on purified Mφ cells. The results are presented as the mean ± SD of three experiments

Article Snippet: ATG13 , 10181-2-AP , Protein tech , IB (1:500), IF (1:100).

Techniques: Flow Cytometry, Labeling, Purification, Isolation

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: Functional characterization of M1Mφ cells in the spleens of Tg +/−ATG13 mice. Dual IF analysis of IBA1 (mouse anti-IBA1; Cat#; Invitrogen; dilution 1:100) with M1Mφ functional markers, including A iNOS (rabbit anti-CD40; Cat#; ProteinTech; dilution 1:100), B serine 468phospho (S468P) NFκB subunit p65, and C acetylated p65 in paraffin-embedded spleen sections of 10- to 12-week-old NTg and Tg +/−ATG13 mice ( n = 6/group). D Quantification analyses of iNOS (pink bars), S468Pp65 (blue bars), and acetylated p65 (green bars) in n = 6 independent images. Unpaired t tests were used to test the significance of the mean results; *** p < 0.005 and **** p < 0.0005 versus NTg. E 3D surface plots (ImageJ software) were drawn to visualize the fluorescence intensities and the distribution of iNOS, S468Pp65, and acep65-ir cells in the red pulp regions. The red pulp regions were separated from the white pulp regions (arrowhead) by a dotted white line. A distinctively lower distribution of immunoreactive cells was observed in the white pulp regions. The results are presented as the mean ± SD of three experiments

Article Snippet: ATG13 , 10181-2-AP , Protein tech , IB (1:500), IF (1:100).

Techniques: Functional Assay, Software, Fluorescence

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: Functional characterization of the M2Mφ phenotype in splenic Mφ cells from Tg +/−ATG13 mice. A Dual IF analysis of the M2Mφ functional marker arginase-1 (APC-tagged) and the M2Mφ surface marker CD163 (FITC-tagged). The enclosed subsets show CD163 + arginase 1 + cells among the purified splenic Mφs. Total gated events = 10,000. B Scatter histogram displaying the number of CD163 + Arginase 1 + cells in n = 6 analyses/group. An unpaired t test was used to determine the significance of the difference in the means between groups, *** p < 0.005. C Paraffin-embedded splenic tissue sections of NTg and Tg +/- ATG13 were immunostained with IBA1 (green) and Arginase-1(red). Nuclei were stained with DAPI. D Phagocytosis assay of live Mφ cells by immunocytochemistry with pHrodo™ green conjugated BioParticles™ (Cat#P35381; ThermoFisher; 10 6 cells in 10,000 Mφ cells). E Quantification of phagocytic cells in n = 6 images/group followed by an unpaired t test to verify the significance of the difference; *** p < 0.005. F Dual flow cytometry of CD206 (APC-tagged) and Bioparticles™ (pHrodogreen-tagged) followed by G quantification of Bioparticles™ (%)-conjugated CD206+ cells ( n = 6 analyses). An unpaired t test was used to verify the significance of the mean, and the results are indicated by * p < 0.05. Total gated events = 20,000. H Dual flow cytometry of CD163 (APC-tagged) and LysoTracker™ (red-DND99-tagged detected in PE filter; ThermoFisher; Cat# pHrodogreen-tagged) followed by I quantification of LysoTracker™ (%)-conjugated CD163 + ve cells ( n = 6 analyses). An unpaired t test was used to verify the significance of the mean, and the results are indicated by * p < 0.05. Total gate = 50,000. P62 IF analysis in J NTg and K Tg Mφs treated with 100 nM Bafilomycin A1 for 2 h under control and serum-starved condition (24 h). L Numbers and M size for p62-ir puncta were measured in 12 independent cells per group by ImageJ software, displayed in histogram analyses. The significance of mean was calculated by Kruskal-Wallis test, resulting in * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.0001. The results are presented as the mean ± SD of three experiments

Article Snippet: ATG13 , 10181-2-AP , Protein tech , IB (1:500), IF (1:100).

Techniques: Functional Assay, Marker, Purification, Staining, Phagocytosis Assay, Immunocytochemistry, Flow Cytometry, Control, Software

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: Production of ROS and evaluation of metabolic events responsible for ROS production in Mφ cells from Tg +/−ATG13 mice. FC analysis with the ROS sensor H2DCFDA (FITC-filter) in A NTg and B Tg Mφ cells. The enclosed ellipsoids are subsets of the ROS-producing population in purified splenic Mφs. Total gated events = 10,000. C Scatter histogram displaying the number of DCFDA + cells in n =7 analyses/group. An unpaired t test was used to determine the significance of the difference in the means between groups, with ** p < 0.01. D The MitoSOX™ green kinetic assay was done to measure superoxide radicals in NTg and Tg +/−ATG13 Mφs (Gray = Tg; Black = NTg). E Fluorescence microscopy was done to visualize superoxide production after 6 h of starvation alone or with ULK-1 inhibitor SBI-0206965 (50 & 100 nM) or ULK-1 agonist BL-918 (100 nM). F The Seahorse metabolic assay of mitochondrial oxidative phosphorylation (OXPHOS) was evaluated with XF96 equipment (Agilent) by measuring the oxygen consumption rate (OCR) followed by visualization and analysis with Agilent Wave™ software. Approximately 200,000 cells were treated with OLM = Oligomycin (2 µg/mL); FCCP = Fluoro-carbonyl cyanide phenylhydrazone (2 µM), ROT = rotenone, and AA = antimycin A. G ATP utilization and H reserve capacity indicative of the capacity of performing OXPHOS under stress was evaluated by scatter histogram analyses. **** p < 0.0005 versus NTg based on an unpaired t test. I Glycolysis was evaluated by measuring the extracellular acidification rate (ECAR) in a Seahorse XF96 system and subsequent analysis via Wave TM software. GLU = glucose, 2DG = 2 Deoxy-glucose. Scatter histogram analyses of J basal glycolysis and K glycolytic capacity. An unpaired t test revealed **** p < 0.0005 versus NTg. L NTg and Tg +/−ATG13 Mφs were serum-starved for 24 h followed by measuring lactate production in supernatants. An unpaired t-test displayed ** p < 0.005 vs. NTg. M LDH release assay was measured by CyQUANT™ LDH assay (ThermoFisher) kit (paired t test ** p < 0.01). The results are presented as the mean ± SD of three experiments

Article Snippet: ATG13 , 10181-2-AP , Protein tech , IB (1:500), IF (1:100).

Techniques: Purification, Kinetic Assay, Fluorescence, Microscopy, Metabolic Assay, Phospho-proteomics, Software, Lactate Dehydrogenase Assay, CyQUANT Assay

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: ATG13 , 10181-2-AP , Protein tech , IB (1:500), IF (1:100).

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

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: ATG13 ablation mediates perivascular infiltration of M1Mφ cells and may compromise myelin integrity. A Dual IF analysis of the M1Mφ marker CD40 (green; dil 1:100) and the blood vessel endothelium marker laminin alpha-5 (LAMA5; red; 1:100). B Scatter histogram analyses of CD40-ir cells at a 10 μm radius from the outer wall of the blood vessel. The unpaired t test represents the significance of the difference in the mean between two groups, with **** p < 0.0005: n = 13 vessels/group. C Dual IF analysis of the M2Mφ markers CD206 (green) and LAMA5 (red). D Scatter histogram analyses of CD206-ir cells at a 10 μm radius from the outer wall of the blood vessel. NS = not significant according to the count ( n = 13 vessels/group). E LFB staining of myelin (lighter blue) in muscle-serving nerve bundles counterstained with cresyl violet (deep blue) in the biceps femoris muscle of NTg and Tg +/−ATG13 mice ( n = 5/group). F 3D surface plot (ImageJ) showing axonal fibers (cyan circles labeled with red arrowheads) with decreased myelin integrity. The results are presented as the mean ± SEM of three different experiments

Article Snippet: ATG13 , 10181-2-AP , Protein tech , IB (1:500), IF (1:100).

Techniques: Marker, Staining, Labeling

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 post-treadmill exacerbation of mitochondrial energy metabolism in Tg +/−ATG13 mice. A Mitochondrial OCR of purified splenic Mφs from the NTg without treadmill exercise (BSL = baseline) and after 4 h post-Treadmill exercise (PTM) ( n = 3/group). B Mitochondrial OCR of splenic Mφs from Tg +/−ATG13 mice before and 4 h after treadmill exercise ( n = 3 per group). C Spare or reserve capacity, indicative of the ability of mitochondria to restore OXPHOS after stress, was measured in all four groups. Black bars = NTg without a treadmill, white bars = NTg after 4 h of treadmill exercise, dark gray bars = Tg without a treadmill, and lighter gray bars = Tg after 4 h of treadmill exercise. Two-way ANOVA (effectors: genotype and stressor) was adopted, followed by multiple comparison analyses to monitor the significance of the difference in the means between groups. **** p < 0.0005, * p < 0.05, and ns = not significant. Glycolysis was measured in enriched Mφs and subsequently compared between D NTg without a treadmill and 4 h PTM, as well as between E Tg +/−ATG13 without a treadmill and 4 h PTM ( n = 6 per group). F The glycolytic capacity, which is indicative of restorative ability after stress, was measured in all four groups. Two-way ANOVA was performed to test the significance of differences in means between groups. **** p < 0.0005, and ns = not significant. G LDH assay was performed in cell lysate as directed in CyQUANT™ LDH assay kit (ThermoFisher). Two-way ANOVAfollowed by multiple comparison analyses revealed * p < 0.05, ** p < 0.01 and ns = not significant versus respective controls. The results are presented as the mean ± SEM of three different experiments

Article Snippet: ATG13 , 10181-2-AP , Protein tech , IB (1:500), IF (1:100).

Techniques: Purification, Comparison, Lactate Dehydrogenase Assay, CyQUANT Assay

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: Muscle fatigue in Tg+/−ATG13 mice after treadmill exercise. A Representative trackplots indicating the gross movement of both NTg (left) and Tg+/−ATG13 (right) mice (10–12 weeks old) on the day before (-1 day), immediately after (0 day), and 2 days after treadmill exercise (4–14 rpm for 15 min). B Average speed and C mobility time were calculated at different time points during treadmill exercise. Two-way ANOVA followed by multiple comparison analysis (GraphPad Prism) was used to analyze the significance of differences in the means between groups. ****p < 0.0005 and *p < 0.05. D EMG recordings followed by the summation of frequencies were plotted as a heatmap. The all-limb grip strength measurements were performed E before and F one day after single-session treadmill (14 rpm /15 mins) exercise in a digital meter as described under method section. The recording was performed over 5 min in n = 6 NTg and Tg+/−ATG13 mice. The results are presented as the mean ± SEM of three different experiments

Article Snippet: ATG13 , 10181-2-AP , Protein tech , IB (1:500), IF (1:100).

Techniques: Comparison

Journal: Translational Cancer Research

Article Title: PHGDH knockdown activates autophagic flux to suppress migration and invasion of gastric cancer cells

doi: 10.21037/tcr-2025-1817

Figure Lengend Snippet: Expression analysis of autophagy-related proteins and pathway profiling after PHGDH knockdown in gastric cancer cells. (A) KEGG pathway enrichment analysis based on proteomic data from PHGDH-knockdown cells. (B) Immunoblotting was performed to quantify LC3 conversion and P62 degradation after transient PHGDH knockdown. (C) Western blot detection of LC3, P62, mTOR, and p-mTOR protein levels following PHGDH knockdown and treatment with or without 3-MA (12.5 µM). (D) Western blot detection of LC3, P62, mTOR, and p-mTOR protein levels after PHGDH knockdown and treatment with or without CQ (12.5 µM). (E) Fluorescence signals of autophagosomes and autophagic flux were observed using an mRFP-GFP-LC3 dual-labeled adenoviral system following PHGDH knockdown. (F) Western blot assessment of autophagy initiation-related proteins ULK1, p-ULK1, FIP200, ATG13, and ATG101 post-PHGDH knockdown. All Western blot experiments were performed in triplicate (n=3) and yielded consistent results. KEGG, Kyoto Encyclopedia of Genes and Genomes; NC, negative control; PHGDH, phosphoglycerate dehydrogenase.

Article Snippet: Antibodies against p62 (8025), mTOR (2983), phospho-mTOR (Ser2448, 5536), ULK1 (8504), HIF-1α (14179), Atg13 (13273), FIP200 (12436), Atg101 (13492), phospho-ULK1 (Ser757, 14202), phospho-ULK1 (Ser555, 5869), and GAPDH (2118) were all supplied by Cell Signaling Technology (CST, Danvers, Massachusetts, USA).

Techniques: Expressing, Knockdown, Western Blot, Fluorescence, Labeling, Negative Control

Journal: Translational Cancer Research

Article Title: PHGDH knockdown activates autophagic flux to suppress migration and invasion of gastric cancer cells

doi: 10.21037/tcr-2025-1817

Figure Lengend Snippet: Expression analysis of autophagy-related proteins and pathway profiling after PHGDH knockdown in gastric cancer cells. (A) KEGG pathway enrichment analysis based on proteomic data from PHGDH-knockdown cells. (B) Immunoblotting was performed to quantify LC3 conversion and P62 degradation after transient PHGDH knockdown. (C) Western blot detection of LC3, P62, mTOR, and p-mTOR protein levels following PHGDH knockdown and treatment with or without 3-MA (12.5 µM). (D) Western blot detection of LC3, P62, mTOR, and p-mTOR protein levels after PHGDH knockdown and treatment with or without CQ (12.5 µM). (E) Fluorescence signals of autophagosomes and autophagic flux were observed using an mRFP-GFP-LC3 dual-labeled adenoviral system following PHGDH knockdown. (F) Western blot assessment of autophagy initiation-related proteins ULK1, p-ULK1, FIP200, ATG13, and ATG101 post-PHGDH knockdown. All Western blot experiments were performed in triplicate (n=3) and yielded consistent results. KEGG, Kyoto Encyclopedia of Genes and Genomes; NC, negative control; PHGDH, phosphoglycerate dehydrogenase.

Article Snippet: Antibodies against p62 (8025), mTOR (2983), phospho-mTOR (Ser2448, 5536), ULK1 (8504), HIF-1α (14179), Atg13 (13273), FIP200 (12436), Atg101 (13492), phospho-ULK1 (Ser757, 14202), phospho-ULK1 (Ser555, 5869), and GAPDH (2118) were all supplied by Cell Signaling Technology (CST, Danvers, Massachusetts, USA).

Techniques: Expressing, Knockdown, Western Blot, Fluorescence, Labeling, Negative Control