Journal: Advanced Science

Article Title: Granzyme B PET Imaging Reveals Lgmn + Macrophage‐Mediated Immune Evasion and Guides Immunotherapy in EGFR‐TKI‐Resistant NSCLC

doi: 10.1002/advs.77837

Figure Lengend Snippet: Lgmn recruits Chip to ubiquitinate Eno1 to regulate the dysfunction of CD8 + T cells. (A) The expression of glycolysis‐related enzymes in CD8 + T cells treated with Lgmn fl/fl /EVs and Lgmn cko /EVs from proteomics analysis by LC‐MS/MS. (B) Cell lysates were immunoprecipitated using anti‐Lgmn antibodies, and Eno1 expression was detected by Western blotting. (C, D) Eno1 expression in CD8 + T cells treated with Lgmn fl/fl /EVs and Lgmn cko /EVs was detected by qRT‐PCR (C) and Western blotting (D). (E, F) Detection of Eno1 expression in CD8 + T cells overexpressing Lgmn by immunoblotting (E) and qRT‐PCR (F). (G, H) Detection of Eno1 protein stability in CD8 + T cells overexpressing Lgmn (G) or treated with EVs (H) by immunoblotting. (I) Detect Eno1 expression in CD8 + T cells overexpressing Lgmn or treated with MG132 using Western blotting. (J) Detect Eno1 expression in CD8 + T cells overexpressing Lgmn or treated with CQ using Western blotting. (K) Detect Eno1 ubiquitination in CD8 + T cells overexpressing Lgmn using Western blotting. (L) Cell lysates were immunoprecipitated using anti‐Lgmn or anti‐Eno1 antibodies, and the proteins interacting with Lgmn and Eno1 were detected by LC‐MS. (M) Co‐IP assays were conducted to observe the interaction between Lgmn, Chip, and Eno1. (N) Chip was knocked down, and Eno1 ubiquitination in CD8 + T cells was detected by Western blotting. (O) Chip was knocked down, and Lgmn was overexpressed; Eno1 expression and ubiquitination in CD8 + T cells were detected by Western blotting. (P) Cell lysates were immunoprecipitated using anti‐Eno1 antibodies, and Chip expression was detected by Western blotting. (Q, R) Chip was knocked down, and Lgmn was overexpressed; glucose uptake in CD8 + T cells was detected (Q), and TIM‐3 + and PD‐1 + CD8 + T cells were detected by flow cytometry (R). (S) Illustration of Lgmn enhancing the interaction between Chip and Eno1, and subsequently modulating CD8 + T cell exhaustion through metabolic reprogramming.

Article Snippet: Primary antibodies used are as follows: anti‐β‐actin (#4970, Cell Signaling Technology, 1:2000), anti‐Lgmn (A23776, ABclonal, 1:1000), anti‐Eno1 (A11448, ABclonal, 1:1000), anti‐Chip (#2080, Cell Signaling Technology, 1:1000), anti‐HA (AE105, ABclonal, 1:20000), anti‐FLAG (AE005, ABclonal, 1:10000), anti‐Ubiquitin (A19686, ABclonal, 1:1000).

Techniques: Expressing, Liquid Chromatography with Mass Spectroscopy, Immunoprecipitation, Western Blot, Quantitative RT-PCR, Ubiquitin Proteomics, Co-Immunoprecipitation Assay, Flow Cytometry

Journal: MedComm

Article Title: Global Lactylome Reveals Lactylation‐Dependent Mechanisms Underlying CXC Motif Chemokine Ligand 12 Expression in Pulmonary Endothelium During Acute Respiratory Distress Syndrome

doi: 10.1002/mco2.70344

Figure Lengend Snippet: Increased protein lactylation in the PECs of ARDS mice, with Eno1 as a substrate. (A) Histogram of the ratio distribution of quantifiable Klac sites in the PECs of control and LPS‐induced ARDS mice. (B) GO items associated with Klac sites (ARDS/Control > +∞) located proteins. (C) Venn diagram of predicted CXCL12 3′ UTR binding proteins and proteins in picked GO items and the average protein copies of 13 screened proteins in PECs. (D) Ribbon diagram of the crystal structure of mouse Eno1 and lactylation at the K193 residue. (E) Mass spectrometry of Eno1 lactylated at K193. (F) Purified mouse Eno1 (100 µg/mL) was incubated with different concentrations of lactyl‐CoA for 1 h at 37°C. Then, the mixtures were added to protein loading buffer for denaturation, and K‐lactylation was assessed by western blotting. (G) The K193 site in Eno1 is conserved in different species. Conserved K193 sites are marked in red in the sequences among different species. (H) Western blotting analysis of the K193 lactylated level in PECs isolated from the lungs of control and LPS‐induced ARDS mice. (I) In vitro, the K193 lactylation levels in PBS‐ and LPS‐activated MPMECs were measured by western blotting.

Article Snippet: The following primary antibodies were used: anti‐L‐lactyl lysine (PTM‐1401, PTM Biolabs, China), anti‐mouse Eno1 (A1033, ABclonal, China), anti‐K193 lactylated Eno1 (custom designed, ABclonal, China), and anti‐mouse β‐actin (ZF0033, ZFdows Bio, China).

Techniques: Control, Binding Assay, Residue, Mass Spectrometry, Purification, Incubation, Western Blot, Isolation, In Vitro

Journal: MedComm

Article Title: Global Lactylome Reveals Lactylation‐Dependent Mechanisms Underlying CXC Motif Chemokine Ligand 12 Expression in Pulmonary Endothelium During Acute Respiratory Distress Syndrome

doi: 10.1002/mco2.70344

Figure Lengend Snippet: K193 lactylated Eno1 released the bound CXCL12 mRNA to accelerate translation. (A) Eno1 expression in MPMECs transfected with siRNAs. (B) CXCL12 mRNA and (C) protein expression in Eno1 KD cells after LPS stimulation ( n = 3). (D) Molecular docking and ΔG binding energy calculation (E) RIP‐qPCR detection of Eno1‐bound CXCL12 transcripts in MPMECs treated with LPS ( n = 3). (F) Click‐iT AHA assays to detect the CXCL12 translation efficacy in MPMECs treated with LPS ( n = 3). (G) Schematics of the reporter and effector constructs. Dual‐luciferase analysis in HEK293T cells transfected with the reporter expressing the CXCL12‐3′ UTR and constructs expressing the WT, K193Q, or K193R Eno1 ( n = 3). (H) CXCL12 concentrations in culture media from MPMECs transfected with shEno1, shEno1+oe‐WT, or shEno1+oe‐K193R ( n = 3). * p < 0.05, ** p < 0.01, **** p < 0.001.

Article Snippet: The following primary antibodies were used: anti‐L‐lactyl lysine (PTM‐1401, PTM Biolabs, China), anti‐mouse Eno1 (A1033, ABclonal, China), anti‐K193 lactylated Eno1 (custom designed, ABclonal, China), and anti‐mouse β‐actin (ZF0033, ZFdows Bio, China).

Techniques: Expressing, Transfection, Binding Assay, Construct, Luciferase

Journal: MedComm

Article Title: Global Lactylome Reveals Lactylation‐Dependent Mechanisms Underlying CXC Motif Chemokine Ligand 12 Expression in Pulmonary Endothelium During Acute Respiratory Distress Syndrome

doi: 10.1002/mco2.70344

Figure Lengend Snippet: K193 lactylation augmented Eno1 enzymatic activity. (A) The levels of lactate in the culture medium of PBS‐ and LPS‐treated MPMECs over time ( n = 3). (B) PBS‐ and LPS‐treated MPMECs lysates were prepared to measure Eno1 enzymatic activity by monitoring phosphoenolpyruvate concentrations over time ( n = 3). (C) Eno1 enzymatic activity was measured in lysates from shEno1+oe‐WT and shEno1+oe‐K193R MPMEC ( n = 6). * p < 0.05. In C, *compared with the untreated shEno1+oe‐WT group, * p < 0.05; # compared with the lactate‐treated shEno1+oe‐K193R group, # p < 0.05.

Article Snippet: The following primary antibodies were used: anti‐L‐lactyl lysine (PTM‐1401, PTM Biolabs, China), anti‐mouse Eno1 (A1033, ABclonal, China), anti‐K193 lactylated Eno1 (custom designed, ABclonal, China), and anti‐mouse β‐actin (ZF0033, ZFdows Bio, China).

Techniques: Activity Assay