Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Exosomes derived from fibroblasts in DFUs delay wound healing by delivering miR-93-5p to target macrophage ATG16L1.

doi: 10.1016/j.bbadis.2024.167640

Figure Lengend Snippet: Fig. 3. miR-93-5p affects macrophage autophagy function by affecting Atg16L1. (A) Nine mRNAs were identifed based based on TargetScan, miRDB, miRTarBase and Human Autophage Database. (B) Schematic representation of the wild-type and mutant-type binding site between the 3′UTR of ATG16L1 and miR-93-5p. (C) Relative luciferase activity of 3′UTR-ATG16L1-luc constructs in HEK293T cells after transfection of miR-93-5p mimics/NC; n = 3 per group. (D) The protein levels of ATG16L1 in bone marrow-derived macrophages transfected with miR-93-5p NC/mimics or PBS (Control) by Western blot. (E) The protein levels of ATG16L1 in skin tissues from normal or diabetic wound (DFUs) by Western blot. (F) Immunofluorescence staining for ATG16L1 (red) and F4/80 (green) in skin tissues from normal or diabetic wound (DFUs); scale bar, 100 μm. (G) The protein levels of LC3b and P62 in BMDMs transfected with miR-93-5p NC/mimics or PBS by Western blot, immunofluorescence staining for LC3b (green) in bone marrow-derived macrophages transfected with miR-93-5p NC/mimics or PBS; scale bar, 50 μm. (H) The protein levels of ATG16L1, LC3 and P62 in fibroblast-derived exosomes treated bone marrow-derived macrophages transfected with miR-93-5p inhibitor NC/in hibitor or PBS (Control) by Western blot. Data are expressed as mean ± SD, ns P > 0.05, **P < 0.01 (unpaired t-test or ANOVA). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Next, sections were incubated overnight at 4 ◦C with primary antibodies against F4/80 (Servicebio, GB11027), Atg16L1 (CST, #8089), LC3B (Proteintech, 81004-1-RR), α-SMA (Proteintech,14395-1AP) and Collagen Type I (Proteintech, 14695-1-AP).

Techniques: Mutagenesis, Binding Assay, Luciferase, Activity Assay, Construct, Transfection, Derivative Assay, Control, Western Blot, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Autophagy limits inflammatory gene expression through targeting of nuclear p65/RelA by LC3 and p62 for lysosomal degradation

doi: 10.1101/2022.11.02.514846

Figure Lengend Snippet: a ) Western blot (WB) analysis of cytoplasmic and nuclear fractions for the autophagy marker LC3 and NF-κB subunit p65 (low exposure, high exposure) in untreated cells (Ut), after 90 min irradiation (IR 20 Gy) or 1 hour TNFα (10 ng) treatment in monocytic leukemia cells (THP1). PARP1 and LDHA are used as loading controls for nuclear and cytoplasmic fractions respectively. LC3-I, LC3-II, non-lipidated and lipidated forms of LC3. Data are representative of n=3 independent experiment. b ) U2-OS osteosarcoma cells stably expressing GFP-LC3 were used in a GFP-LC3 puncta formation assay and analyzed in untreated cells, 1 hour after irradiation (IR 20 Gy) or TNFα treatment. Earle’s Balanced Salt Solution (EBSS) was used as autophagy positive control for starvation treatment. Images are representative fluorescence confocal microscopic photographs of n=3 independent experiments (scale bars: 20 µm). c ) HEK293T cells were pre-treated with Bafilomycin A1 (200 nM) or DMSO (vehicle) for 2 hours and analyzed before (Ctr: control) or 1 hour after irradiation (IR 20 Gy) by WB. Rapamycin (Rapa) and starvation (EBSS) treatments were used as positive controls for autophagy induction. All data are representative of n=3 independent experiments. d ) Proximity Ligation Assay (PLA) was used to demonstrate binding between LC3 and p65 in untreated conditions or after TNFα (10 ng) stimulation or irradiation (IR 20 Gy) treatments using A549 adenocarcinoma cells. Cells stained only with anti-p65 primary antibodies were used as a technical negative control (Control). Each red spot represents a single interaction. Nuclei were stained with DAPI. Scale bars: 100 µm and 20 µm (insets). e ) Quantification of corrected total cell fluorescence (CTCF) of nuclear p65:LC3 PLA foci from (d). Data are means ± SEM of three independent experiments. Statistical analyses were performed by one-way ANOVA followed by Bonferroni’s multiple comparison test (*p < 0.05) using GraphPad Prism 8. f-h ) HEK293T cell lysates were added to beads with immobilized GST fusion LC3-like modifiers (GST, GST-LC3A, GST-LC3B, GST-LC3C, GST-GABARAP, GST-GABARAPL1, GST-GABARAPL2, GST-Ub, GST-4XUb (f) or the LC3 like modifiers lacking the unique N-terminus (dN) (g) and GST-LC3BF52A-V53A (h)), followed by WB using an antibody against endogenous p65. Ponceau stain gels (bottom) represent the amount of GST beads constructs used for the GST-pulldown assays. All data are representative of n≥3 independent experiments.

Article Snippet: Primary antibodies dilution was done in Duolink® Antibody Diluent (LC3B, Novus Biological NB100-2220, NF-κB p65 (F6), Santa Cruz sc-8008 and SQSTM1/p62 Enzolife BML-PW9860 diluted at 1:100) and incubated overnight at 4°C.

Techniques: Western Blot, Marker, Irradiation, Stable Transfection, Expressing, Tube Formation Assay, Positive Control, Fluorescence, Control, Proximity Ligation Assay, Binding Assay, Staining, Negative Control, Comparison, Construct

Journal: bioRxiv

Article Title: Autophagy limits inflammatory gene expression through targeting of nuclear p65/RelA by LC3 and p62 for lysosomal degradation

doi: 10.1101/2022.11.02.514846

Figure Lengend Snippet: a ) Full-length myc-p65 and truncation mutants have been transfected into HEK293T cells. Cell extracts were added to beads with immobilized GST-LC3B and immunoblotted for myc. LC3B was able to bind only to full-length myc-p65 and to the truncation mutants including residues 193 to 306. Ponceau stain gels (bottom) represent the amount of GST beads constructs used for the GST-pulldown assays. All data are representative of n≥3 independent experiments. b ) Schematic representation of myc-p65 full length (FL) and truncation mutants indicating which bind to LC3. c ) HEK293T cell lysates were added to GST-LC3B in presence or absence of NEM (N-ethylmaleimide). Overexpressed myc-p65 binds to LC3B only in the presence of NEM. All data are representative of n=3 independent experiments. d ) HEK293T cells were transfected with empty GFP vector (control), WT, or ΔUBA deleted mutant of GFP-tagged p62. After 24 hours, cell lysates were immunoprecipitated with GFP-Trap beads and immunoblotted for GFP fusion proteins and p65. ( e ) PLA between p65 and p62 (red spots) was performed in U2-OS cells stably expressing GFP-LC3 (green spots). Top panels are representative images of cells treated only with p65 antibody (top left panel), or cells after transfection with a mixture of two siRNAs targeting p62 (top right panel; see Fig. S3b for p62 siRNA knockdown efficiency), which were used as negative controls. Bottom panels are representative images of PLA performed in untreated, TNFα (10 ng) stimulation or irradiation (20 Gy) conditions. Each red spot represents a single interaction of p62 and p65 proteins. Yellow arrowheads point to p65-p62 interacting proteins (PLA red signals) that co-localize with LC3 puncta structures (autophagosome in green). Nuclei were stained with DAPI. Scale bars 10 µm. ( F ) Pearson’s Correlation Coefficients for the colocalization of PLA signals (p65:p62) with GFP-LC3 from (e) quantified using JACoP plugin of ImageJ . Data are means ± SEM of three independent experiments (n≥5 cells for each condition). Statistical analyses were performed by two-way ANOVA followed by Bonferroni’s multiple comparison test (*p<0.05; **p < 0.01; n.s.: not significant) using GraphPad Prism 8.

Article Snippet: Primary antibodies dilution was done in Duolink® Antibody Diluent (LC3B, Novus Biological NB100-2220, NF-κB p65 (F6), Santa Cruz sc-8008 and SQSTM1/p62 Enzolife BML-PW9860 diluted at 1:100) and incubated overnight at 4°C.

Techniques: Transfection, Staining, Construct, Plasmid Preparation, Control, Mutagenesis, Immunoprecipitation, Stable Transfection, Expressing, Knockdown, Irradiation, Comparison

Journal: bioRxiv

Article Title: Autophagy limits inflammatory gene expression through targeting of nuclear p65/RelA by LC3 and p62 for lysosomal degradation

doi: 10.1101/2022.11.02.514846

Figure Lengend Snippet: a ) Immunofluorescence staining for p65 (magenta) and LAMP1 or LC3 (green) using U2-OS cells pre-treated 30 minutes with bafilomycin A1 (200 nM) or DMSO (control) in untreated or TNFα (10 ng) stimulated conditions. White arrowheads indicate colocalization of p65 in the lysosomes (LAMP1) or autophagosome (LC3) compartments. DAPI was used for the staining of nuclei. Z-stack images shown are representative fluorescence confocal microscopic photographs of n=3 independent experiments (scale bars 20 µm). b and c ) Cycloheximide-chase assay performed in HCT116 transfected with myc-p65 24 hours before treatment. Cells were washed once after 15 minutes of TNFα stimulation and cycloheximide was added. The time course was performed for a total of 6 hours as shown in the graph and analyzed by western blot ( b ) at the times indicated using an antibody against myc. Vinculin is used as loading control and quantification of the protein abundance was done using ImageJ ( c ). Data are representative of n=3 independent experiments. d ) HEK293T cells were transfected with GFP empty vector (control) or GFP-LC3. After 24 hours, cells were treated with TNFα (10 ng) and cell lysates were collected at the time points indicated. Immunoprecipitation was performed with GFP-Trap beads and samples immunoblotted for GFP fusion protein and p65. ( e ) HCT116 were transfected with myc-p65 plasmid 24 hours before the 15 minutes TNFα treatment. Cells were washed once with PBS and fresh medium was added for 6 hours (T 2 , left blot) or 24 hours (T 3 , right blot) with or without MG132 (10 µM) or chloroquine (CQ, 60 µM). On top is a schematic representation of the experimental setup used. Western blot analysis was performed using antibodies against myc and β-actin as loading control. Antibodies against NIK and LC3-I/II were used as controls for MG132 and CQ inhibitors respectively. All data are representative of n=3 independent experiments.

Article Snippet: Primary antibodies dilution was done in Duolink® Antibody Diluent (LC3B, Novus Biological NB100-2220, NF-κB p65 (F6), Santa Cruz sc-8008 and SQSTM1/p62 Enzolife BML-PW9860 diluted at 1:100) and incubated overnight at 4°C.

Techniques: Immunofluorescence, Staining, Control, Fluorescence, Transfection, Western Blot, Quantitative Proteomics, Plasmid Preparation, Immunoprecipitation

Journal: bioRxiv

Article Title: Autophagy limits inflammatory gene expression through targeting of nuclear p65/RelA by LC3 and p62 for lysosomal degradation

doi: 10.1101/2022.11.02.514846

Figure Lengend Snippet: a, b ) Nuclear extracts from WT and ATG16L1 KO RAW 264.7 cells were collected at the time points indicated after LPS (10µg) treatment and analyzed by WB (a) for p65. PARP1 is used as nuclear loading control. Quantifications of the protein abundance (b) were measured using ImageJ and statistical analysis was performed by Two-way ANOVA followed by Bonferroni’s multiple comparison test (**p<0.01, ****p<0.0001) using GraphPad Prism 8 (n=3). c ) WT and ATG16L1 KO RAW 264.7 cells were treated with LPS for the times indicated and whole cell extracts were analyzed by EMSA for NF-κB activity (top panel) or immunoblotted (WB) with the indicated antibodies (bottom panel). Data are representative of n=3 independent experiments. d ) Cytoplasmic and nuclear fractions from THP1 cells were extracted at 0 or 90 min after LPS (10 µg) treatment for immunoprecipitation (IP) with anti-p65 antibody, followed by WB analysis with anti-p65 or anti-p62 antibodies. LDHA and PARP1 antibodies were used as loading controls for cytoplasmic and nuclear fractions, respectively. IgG was used for IP control. e ) Immunofluorescence staining for p65 (red) and p62 (green) using THP1 cells pre-treated with leptomycin B (LMB 40 nM) for 2-hours followed by LPS (10 µg) treatment for the time indicated. Yellow arrowheads indicate colocalization between p65 and p62 in the nuclei stained with DAPI. Z-stack images shown are representative fluorescence confocal microscopic photographs of n=3 independent experiments (scale bars 10 µm).

Article Snippet: Primary antibodies dilution was done in Duolink® Antibody Diluent (LC3B, Novus Biological NB100-2220, NF-κB p65 (F6), Santa Cruz sc-8008 and SQSTM1/p62 Enzolife BML-PW9860 diluted at 1:100) and incubated overnight at 4°C.

Techniques: Control, Quantitative Proteomics, Comparison, Activity Assay, Immunoprecipitation, Immunofluorescence, Staining, Fluorescence

Journal: bioRxiv

Article Title: Development of the ULK1-Recruiting Chimeras (ULKRECs) to enable proximity-induced and ULK1-dependent degradation of mitochondria

doi: 10.1101/2024.04.15.589474

Figure Lengend Snippet: ULKRECs rely on ULK1 for the redirection of autophagy components to mitochondria and subsequent mitophagic degradation (A) Western blot of mouse embryonic fibroblasts (MEF) WT and MEF ULK1/2 -/- treated with 1 μM ULKRECs, 5 μM CCCP or both and probed for ULK1, Mfn2 and Actin – quantification of the relative intensity of Mfn2 shown in (B), normalised to Actin. Representative immunofluorescence of MEF WT (C) and MEF ULK1/2 -/- (D) treated with 5 μM CCCP, 1 μM ULKRECs or both and immunostained for Alexa 488 ATP5a and Alexa 647 LC3B. Quantification of the colocalised LC3 to ATP5a spot area shown in WT vs ULK1/2 -/- treated MEF cells shown in (E). Data shown are mean ± SD, n = 3, *p<0.05, **p<0.01, ***p<0.001, Two-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: Plates were incubated overnight at 4°C with 1:400 LC3B (D11) XP Rabbit mAb Alexa Fluor 647 Conjugate (Cell Signalling Technologies, #65299) and 1:400 ATP5a (Cell Signalling Technologies, #18023) lightning-linked to Alexa Fluor 488.

Techniques: Western Blot, Immunofluorescence