Journal: The Journal of Biological Chemistry

Article Title: Xenophagy of invasive bacteria is differentially activated and modulated via a TLR-TRAF6-Beclin1 axis in echinoderms

doi: 10.1016/j.jbc.2022.101667

Figure Lengend Snippet: L PS V. splendidus and LPS E. coli mediate the K63-linked ubiquitination of Aj TRAF6 based on Aj TLR3 and Aj Toll cascades, which interacts with Aj Beclin1 to regulate xenophagy in A. japonicus coelomocyte. Sea cucumber primary coelomocyte were stimulated with LPS V. splendidus ( A ) or LPS E. coli ( B ) for the indicated times (0 and 12 h) following Aj TLR3 ( C ), Aj Toll ( D ), and Aj TRAF6 ( E and F ) silencing for 24 h. G , LC3 fluorescence intensity detection after interference of Aj TRAF6 followed by LPS V. splendidus or LPS E. coli exposure for 12 h. H , LC3 fluorescence intensity detection after interference of Aj ULK followed by LPS V. splendidus or LPS E. coli exposure for 12 h. Western blotting analysis of immunoprecipitated (IP) Aj TRAF6 samples was performed to determine the presence of K63-linked ubiquitin (Ub K63). The membranes in ( A ) and ( B ) were stripped and analyzed for interaction with Aj Beclin-1. Whole-cell lysates were analyzed by western blotting as indicated. The protein band density was calculated using ImageJ software. The heavy chain of the TRAF6 antibody was detected by the secondary antibody and was labeled Ig band. The data, which are presented as the means ± SDs ( n = 3) relative to the 0 h (control) in Fig. S2 . For LC3 positive signal, the cells were fixed and stained with anti-LC3 at the indicated time points (0 and 12 h). After nuclear staining with DAPI, green signal represents autophagosomes was visualized under a confocal microscope and statistically analyzed; scale bar = 5 μm. The relative LC3 positivity in 1000 cells from each indicated sample was determined. The data are presented as the means ± SDs ( n = 3) relative to the control, are shown in bar graphs ( lower panel in G and H ). The asterisks indicate significant differences compared with the control group: ∗ p < 0.05 and ∗∗ p < 0.01 ( t test).

Article Snippet: In addition, antibodies against Ub-K63 (T56579S), β-actin (M20011S), TRAF6 (T55175S), Beclin1 (T55092S) were purchased from Abmart.

Techniques: Ubiquitin Proteomics, Fluorescence, Western Blot, Immunoprecipitation, Software, Labeling, Control, Staining, Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Xenophagy of invasive bacteria is differentially activated and modulated via a TLR-TRAF6-Beclin1 axis in echinoderms

doi: 10.1016/j.jbc.2022.101667

Figure Lengend Snippet: LPS V. splendidus and LPS E. coli differentially induce A. japonicus coelomocyte xenophagy by Aj A20. Sea cucumber primary coelomocyte were stimulated with LPS V. splendidus ( A ) or LPS E. coli ( B ) for the indicated times (0 and 12 h) following Aj TLR3 ( C ), Aj Toll ( D ), and Aj TRAF6 ( E and F ) silencing for 24 h. Western blotting analysis of immunoprecipitated (IP) Aj Beclin1 samples was performed to determine the presence of K63-linked ubiquitin (Ub K63). Whole-cell lysates were analyzed by western blotting as indicated. The protein band density was calculated using ImageJ software. The heavy chain of the Beclin1 antibody was detected by the secondary antibody and was labeled Ig band. The data are presented as the means ± SDs ( n = 3) relative to the 0 h (control) in Fig. S3 . The V indicate significant differences compared with the control group: ∗ p < 0.05 and ∗∗ p < 0.01 ( t test).

Article Snippet: In addition, antibodies against Ub-K63 (T56579S), β-actin (M20011S), TRAF6 (T55175S), Beclin1 (T55092S) were purchased from Abmart.

Techniques: Western Blot, Immunoprecipitation, Ubiquitin Proteomics, Software, Labeling, Control

Journal: The Journal of Biological Chemistry

Article Title: Xenophagy of invasive bacteria is differentially activated and modulated via a TLR-TRAF6-Beclin1 axis in echinoderms

doi: 10.1016/j.jbc.2022.101667

Figure Lengend Snippet: Differential TLR-mediated xenophagy in A. japonicus after V. splendidus and E. coli infection. The exposure of coelomocyte to LPS V. splendidus and LPS E. coli were mediated by Aj TLR3 and Aj Toll, respectively. Activated Aj TLR3 and Aj Toll both promoted Aj TRAF6 ubiquitination, which further increased the K63-linked ubiquitination of Aj Beclin1 and triggered the formation of autophagosomes. Inconsistently, the engagement of Aj TLR3 also triggered a signaling pathway that led to the expression of Aj A20. The increased abundance of Aj A20 might limit Aj TLR3-induced autophagy through the deubiquitination of Aj Beclin1. The precise mechanism of A20 and deubiquitination should be verified in future studies. The yellow solid line indicates E. coli -induced autophagy based on the Aj Toll signaling pathway; the green solid and imaginary lines indicate V. splendidus -induced autophagy based on the Aj TLR3 signaling pathway; the red solid line indicates the common xenophagy processes in both V. splendidus - and E. coli -induced autophagy.

Article Snippet: In addition, antibodies against Ub-K63 (T56579S), β-actin (M20011S), TRAF6 (T55175S), Beclin1 (T55092S) were purchased from Abmart.

Techniques: Infection, Ubiquitin Proteomics, Expressing