Journal: Autophagy

Article Title: Chronic enteritis triggered by diet westernization is driven by epithelial ATG16L1-mediated autophagy

doi: 10.1080/15548627.2025.2600906

Figure Lengend Snippet: Atg16l1 is required for PUFA-induced TLR2 activity in IECs. (A) Relative AP-1 activation in siCtrl or siATG16L1 mTLR2 reporter cell line after stimulation with vehicle, oxidized phosphatidylcholine (oxPAPC) or the lipid peroxidation by-product 4-hydroxynonenal (4-HNE) for 24 h ( n = 8). (B) Enrichment plots showing significant downregulation of MAPK signaling pathway in AA and SDA stimulated IECs. siGpx4 siAtg16l1 IECs were compared to siGpx4 IECs ( n = 4). Red and blue color bar: this bar corresponds to the ranked list of genes. * red: indicates genes in the MAPK signaling pathway that are upregulated in siGpx4 siAtg16l1 +AA/+SDA compared to siGpx4 +AA/+SDA. Blue: indicates genes in the MAPK signaling pathway that are downregulated in siGpx4 siAtg16l1 +AA/+SDA compared to siGpx4 +AA/+SDA. The intensity of the color reflects the degree of upregulation or downregulation. (C) A representative immunoblot of (phospho-)MAPK/JNK and (phospho-) JUN/c-Jun in siCtrl, siGpx4 , siAtg16l1 and siGpx4 siAtg16l1 IECs after ω-6 PUFA (AA) or ω-3 PUFA (SDA) stimulation for 24 h. ( n ≥4). GAPDH served as the loading control. (D) Enrichment plots showing significant downregulation of the protein processing at the endoplasmic reticulum pathway in AA and SDA stimulated IECs. siGpx4 siAtg16l1 IECs were compared to siGpx4 IECs ( n = 4). Red and blue color bar: this bar corresponds to the ranked list of genes. * red: indicates genes in the protein processing at the endoplasmic reticulum pathway that are upregulated in siGpx4 siAtg16l1 +AA/+SDA compared to siGpx4 +AA/+SDA. Blue: indicates genes in the protein processing at the endoplasmic reticulum pathway that are downregulated in siGpx4 siAtg16l1 +AA/+SDA compared to siGpx4 +AA/+SDA. The intensity of the color reflects the degree of upregulation or downregulation. (E) a representative immunoblot of (phospho-)ERN1/IRE1α and HSPA5/GRP78 in siCtrl, siGpx4 , siAtg16l1 and siGpx4 siAtg16l1 IECs after ω-6 PUFA (AA) or ω-3 PUFA (SDA) stimulation for 24 h. ( n ≥4). GAPDH served as the loading control. (F, G) A representative immunoblot (F) and quantification by densitometry (G) of epithelial HSPA5/GRP78 relative to GAPDH of WT, Gpx4 ±IEC , atg16l1 -/-IEC and Gpx4 ±IEC ; atg16l1 -/-IEC mice after three months exposure to a PUFA-enriched western diet ( n = 5). Each dot represents one experimental animal. GAPDH served as the loading control in F. * p < 0.05 , **** p < 0.0001 .

Article Snippet: The following reagents were used: arachidonic acid (AA, 20 μM; Sigma, A3611), stearidonic acid (SDA, 50 μM; Sigma, SMB00291), rapamycin (Rapa, 500 nM; Enzo, A275-0005), bafilomycin A 1 (100 nM; InvivoGen, tlrl-baf1), chloroquine (50 μM; Sigma, C6628), oxPAPC (100 μg/ml; Avanti Polar Lipids, 870604P), 4-HNE (100 μM; Sigma, 393,204), TNF (50 ng ml −1 ; Peprotech, 315-01A), cycloheximide (CHX, 10 mg ml −1 ; Sigma, 239,765), Pam2CSK4 (0,1 μg ml −1 ; Invivogen, tlrl-pm2s-1), celecoxib (CEB, 0.1 μM, 1 μM, 10 μM; Tocris, 3786/10).

Techniques: Activity Assay, Activation Assay, Western Blot, Control

Journal: Nature Immunology

Article Title: Delaying pyroptosis with an AI-screened gasdermin D pore blocker mitigates inflammatory response

doi: 10.1038/s41590-025-02280-x

Figure Lengend Snippet: a , Representative images of CHMP4–GFP puncta (arrowheads; left) and percentage of CHMP4 speckle + (top right) or Annexin V + (bottom right) cells in BMDMs incubated with PBS, 15 µM SK56, 2 mM EDTA or EDTA + SK56 at 2 h after the addition of 1 μg ml −1 LPS + 10 μM nigericin; scale bar, 50 µm; n = 5 repeats. b , Immunoblots (top) and quantification (bottom) showing GSDMD-NT in the supernatant from THP-1 cells treated with PBS, 30 µM DSF, 15 µM SK56 or 15 µM SK56 at 120 min after LPS + nigericin treatment; n = 3 repeats. c , Representative images (left) and percentage of GSDMD-NT–BFP/cytomembrane-CellMask Orange + (right) cells in calcein-AM-labeled (green) mouse wild-type BMDCs incubated with 2 μg ml −1 pyroptotic cytomembrane fragments from mouse wild-type BMDMs transfected with a GSDMD-casp–BFP construct and incubated with LPS + nigericin (PCF BFP ), 20 µM SK56 (PCF BFP + SK56) or 20 µM SK56 scrambled (synthetic SK56 scrambled peptide; PCF BFP + SK56 scrambled ), pyroptotic cytomembrane fragments from mouse Gsdmd −/− BMDMs incubated with LPS + nigericin and PBS (PCF Gsdmd −/− ), cytomembranes from wild-type BMDMs incubated with PBS (NCF) or pyroptotic cytomembrane fragments from mouse BMDMs incubated with LPS + nigericin and 10 µg ml −1 BFP (PCF + BFP), 10 µg ml −1 GSDMD-NT–BFP or 10 µg ml −1 BFP for 2 h; green, calcein-AM + BMDCs; red, CellMask Orange + NCF, PCF or PCF Gsdmd −/− ; blue, GSDMD-NT–BFP, BFP or PCF BFP . The white arrow indicates cytomembrane outside BMDCs, and the red arrow indicates phagocytosed cytomembrane; scale bar, 25 μm; n = 3 repeats. d , ELISA of secreted (left) and cell-associated (right) IL-1β from wild-type BMDCs treated with PBS, 10 μg ml −1 BFP, 1 μg ml −1 GSDMD-NT, 20 µM SK56, SK56 + GSDMD-NT, 120 µM oxPAPC, SK56 + oxPAPC or 2 μg ml −1 pyroptotic cytomembrane fragments from mouse wild-type or Gsdmd −/− BMDMs as in c (NCF, PCF, PCF + SK56, PCF Gsdmd −/− , PCF Gsdmd −/− + SK56), treated or not treated with Pam3 for 12 h; n = 4 repeats. All data are shown as mean ± s.d., and P values were determined by two-tailed Student’s t -test; NS, not significant ( P > 0.05).

Article Snippet: Following treatment, the cells were washed three times with PBS and subsequently stimulated according to their respective experimental groups with oxPAPC (120 μM; tlrl-oxp1, InvivoGen), GSDMD-NT (1 μg ml −1 ; P9442, FineTest), NFC (2 μg ml −1 ), PCF (2 μg ml −1 ), BFP (10 μg ml −1 ; P08114, Solarbio), SK56 (20 μM) and scrambled SK56 (SK56 scrambled ).

Techniques: Incubation, Western Blot, Labeling, Transfection, Construct, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Scientific Reports

Article Title: Phage-mediated TLR2 signaling attenuates intracellular Mycobacterium abscessus survival in macrophages

doi: 10.1038/s41598-025-07320-y

Figure Lengend Snippet: Impact of TLR2/TLR4 signaling activation on MAB infection. ( A ) The intracellular growth dynamics of MAB in TLR2 and TLR4-stimulated THP-1 cells before and after bacterial infection. The bacterial survival rates were assessed through viable CFU counts at 1 h, 24 h, and 72 h after MAB infection. The data is presented as the mean ± SD of three independent experiments, each conducted in triplicate. * p < 0.05, ** p < 0.01 denote statistical significance between the TLR-treated groups and the MAB infection alone group at the corresponding time points. Following the 2 h MAB infection of THP-1 monolayers, either ( B ) TLR2/4 inhibitor oxPAPC, ( C ) TLR2 inhibitor TL2-C29 or ( D ) TLR4 inhibitor CLI-095 from InvivoGen was added to THP-1 cell monolayers for 30 min and then exposed to phages at 10:1 ratio. Intracellular MAB growth was evaluated at day 3 post-infection where the control group did not receive antagonist treatment. * p < 0.05 and ** p < 0.01, denote statistical significance between the TLR-treated and non-treated corresponding groups. The data is presented as the mean ± SD of three independent experiments each performed in three technical replicates.

Article Snippet: Initially, we employed the oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine from InvivoGen (oxPAPC), a modified low-density lipoprotein of mammalian cell membrane, which acts as a dual inhibitor for TLR2 and TLR4 signaling.

Techniques: Activation Assay, Infection, Control