Journal: bioRxiv

Article Title: Ubiquitin ligase CHFR impairs Tie2 signaling via K 48 -linked ubiquitylation and degradation of Akt1 in endothelial cells

doi: 10.64898/2026.03.31.715582

Figure Lengend Snippet: a HMEC were transfected with scrambled-siRNA (sc-siRNA) or CHFR-siRNA. At 72 h post-transfection, cells stimulated with rAng1 (400 ng/ml) for different time intervals were used for IB analysis to determine phosphorylation of Akt1 at S473 and T308 ( n = 3 independent experiments). b Control HMEC and CHFR knockdown HMEC stimulated with rAng1 as above were stained with phospho-473-Akt1 specific antibody. c Control HMEC and CHFR knockdown HMEC were transfected with the Akt1 biosensor plasmid and then stimulated with rAng1 as above to assess live cell Akt1 activity by measuring the FRET ratio ( p <0.0001) ( left panel shows basal Akt activity; right panel shows Akt activity in response to rAng1 challenge). d-f Control HMEC and CHFR-siRNA treated HMEC stimulated with rAng1 were used for IB analysis to determine tyrosine phosphorylation Tie2 ( d ), phosphorylation of GSK3β at S9 and phosphorylation of β-catenin ( e ), ( n = 3 independent experiments). a, d-e Shown are mean values ± SEM ( n = 3 independent experiments; two-way ANOVA followed by Tukey’s post-hoc test).

Article Snippet: Rabbit polyclonal antibody (pAb) against phospho-Tie2 (catalog #AF3909; IB, 1:1000), goat polyclonal antibody (pAb) against PDGFRβ (catalog #AF1042; IS, 1:100) were from R&D system.

Techniques: Transfection, Phospho-proteomics, Control, Knockdown, Staining, Plasmid Preparation, Activity Assay

Journal: bioRxiv

Article Title: Ubiquitin ligase CHFR impairs Tie2 signaling via K 48 -linked ubiquitylation and degradation of Akt1 in endothelial cells

doi: 10.64898/2026.03.31.715582

Figure Lengend Snippet: a HMEC were transfected with WT or K/R-mutant Akt1 constructs. At 36 h after transfection, cells were treated with LPS (5 μg/ml) for 0, 12, and 24h and then cells were used for IB to determine expression of Akt1 and VE-cadherin. Shown are mean values ± SEM (n = 3 independent experiments; two-way ANOVA followed by Tukey’s post hoc test). b TIME endothelial cells (telomerase-immortalized human dermal microvascular endothelial cell line) were transfected with WT or K/R mutant Akt1 constructs and stimulated with LPS (5 μg/ml) for 0 and 6 h. Confocal imaging showed that expression of K/R mutant Akt1 prevents degradation of VE-cadherin. c WT mice were injected (i.v.) with liposome-encapsulated pmCherry-tagged WT or K/R mutant Akt1 constructs. Lungs harvested 96 h after injection were subjected to cryosection and stained with EC marker antibody vWF (green). Confocal imaging confirms expression of pmCherry-Akt1 (red) plasmid in lung endothelial cells. d-f Liposome-mediated delivery of Akt1 (WT) or K/R-mutated Akt1 in WT mice prevents degradation of VE-cadherin, mitigates LPS-induced lung vascular leak (EBA uptake), and reduces PMN transmigration (MPO assay). Shown are mean values ± SEM ( n = 3 or n = 5 mice/group; two-way ANOVA followed by Tukey’s post hoc test). g Model for E3 ligase CHFR regulation of endothelial junctional barrier integrity. Under baseline condition, constitutive Ang1-Tie2 signaling in EC maintains endothelial junctional barrier through Akt1 activation-mediated inhibition of the transcription factor FoxO1 activation and Ang-2 expression. During vascular inflammatory conditions such as sepsis, TLR4 signaling induces the expression of E3 ligase CHFR in a FoxO1-dependent manner. Then the upregulated CHFR mediates K 48 -linked polyubiquitylation and degradation of Akt1 and VE-cadherin (Tiruppathi et al., 2023) to disassemble EC junctional barrier. CHFR-mediated loss of FoxO1 negative regulator Akt1 expression in EC leads to increased FoxO1 expression which in turn promotes sustained expression of Ang-2 in EC to induce life-threatening pulmonary edema.

Article Snippet: Rabbit polyclonal antibody (pAb) against phospho-Tie2 (catalog #AF3909; IB, 1:1000), goat polyclonal antibody (pAb) against PDGFRβ (catalog #AF1042; IS, 1:100) were from R&D system.

Techniques: Transfection, Mutagenesis, Construct, Expressing, Imaging, Injection, Staining, Marker, Plasmid Preparation, Transmigration Assay, MPO Assay, Activation Assay, Inhibition

Journal: bioRxiv

Article Title: SEPSIS-INDUCED LIPID DROPLET ACCUMULATION ENHANCES ANTIBACTERIAL INNATE IMMUNITY

doi: 10.1101/2025.01.29.635463

Figure Lengend Snippet: BMDMs were infected with E. coli (MOI 100) for 1 hour, followed by gentamicin treatment to remove extracellular bacteria. (A) Confocal images showing Oil Red O-labeled LDs (red) in infected BMDMs. Nuclei were stained with DAPI (blue). Scale bar: 10 μm.(B) Quantification of LDs at 1 and 24 h post-infection (hpi). (C) Relative mRNA expression of lipid metabolism genes ( plin2, plin3, fasn, dgat1, acat1, cd36, pnpla2/atgl, abca1 ) at 6 hpi via RT-qPCR. Data normalized to bactin . (D) Intracellular E. coli CFUs at 1 and 24 hpi. (E) mRNA expression of ptgs-2/cox-2, 5-lo, and 15-lo in noninfected (NI) and infected BMDMs at 6 hpi. (F) Levels of PGE 2 , LTB 4 , and RvD1 in supernatants at 24 hpi (EIA assay). (G) Lactate levels in supernatants at 24 hpi (enzymatic assay). (H) Nitrite (NO) levels in supernatants at 24 hpi (Griess method). (I) Expression of pro-inflammatory ( il-1b, inos ) and anti-inflammatory ( il-10, ch3l3, arg1 ) genes at 6 hpi via RT-qPCR. (J) Levels of cytokines (CCL2, CXCL1, IL-10, IL-1β, IL-6, IL-12p40, TNF) at 24 hpi (ELISA). (K) Levels of pan-IFN-α, IFN-β, and IFN-γ at 24 hpi (ELISA). Data represent mean ± SEM from three independent experiments. Significant difference (p < 0.05) compared to NI group.

Article Snippet: CXCL1 / KC, CCL2 / MCP-1, IL-1β, IL-6, IL-10, IL-12p40, TNF-α, IFN-β and IFN-γ in cell-free culture supernatants and peritoneal lavage were measured using mouse Duoset ELISA kit (R&D Systems, USA) according to manufacturer’s instructions.

Techniques: Infection, Bacteria, Labeling, Staining, Expressing, Quantitative RT-PCR, Enzyme Immunoassay, Enzymatic Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: SEPSIS-INDUCED LIPID DROPLET ACCUMULATION ENHANCES ANTIBACTERIAL INNATE IMMUNITY

doi: 10.1101/2025.01.29.635463

Figure Lengend Snippet: BMDMs were infected with E. coli (MOI 100) with or without 10 μM DGAT1 inhibitor (A922500) pretreatment. (A) PGE 2 and LTB 4 levels in cell-free supernatants at 24 hpi were measured by EIA. (B) Lactate levels in supernatants at 24 hpi were assessed using an enzymatic assay. (C) CCL2, IL-1β, and IL-6 levels in supernatants at 24 hpi were determined by ELISA. (D) LDH release in supernatants indicated cellular viability. (E) IFN-β levels in supernatants at 24 hpi were measured by ELISA. (F) CAMP, IGTP/IRGM3, iNOS, and Plin2 expression in cell lysates were analyzed by Western blotting, using β-actin and β-tubulin as loading controls. (G) Nitrite (NO) levels in supernatants at 24 hpi were quantified by the Griess method. Data represent the mean ± SEM of three independent experiments. (*) indicates a value significantly different (p<0.05) from that of the respective noninfected group.

Article Snippet: CXCL1 / KC, CCL2 / MCP-1, IL-1β, IL-6, IL-10, IL-12p40, TNF-α, IFN-β and IFN-γ in cell-free culture supernatants and peritoneal lavage were measured using mouse Duoset ELISA kit (R&D Systems, USA) according to manufacturer’s instructions.

Techniques: Infection, Enzymatic Assay, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot

Journal: bioRxiv

Article Title: SEPSIS-INDUCED LIPID DROPLET ACCUMULATION ENHANCES ANTIBACTERIAL INNATE IMMUNITY

doi: 10.1101/2025.01.29.635463

Figure Lengend Snippet: BMDMs were transfected with 5 µM self-deliverable AUMsilence oligos for DGAT1 or scramble RNA (SCR). After 48 h, BMDMs were infected with E. coli (MOI 100). (A) Oil Red O-stained LDs (red) in infected BMDMs at 24 hpi, with nuclei stained by DAPI (blue). Scale bar: 10 μm. (B) LD quantification in infected BMDMs (≥100 cells per group across 10 fields/experiment). (C) Relative intracellular bacterial CFU at 24 hpi. (D) PGE 2 levels in supernatants at 24 hpi by EIA. Levels of (E) IL-6 and (G) IFN-β in supernatants at 24 hpi by ELISA. (H) Nitrite (NO) levels in supernatants at 24 hpi by the Griess method. (I, J) Relative mRNA expression of inos/nox2 and ptgs-2/cox-2 genes in noninfected (NI) and infected BMDMs at 6 hpi, normalized to β-actin (mean 2−ΔΔCt ± SEM; n = 3). (K) Lactate levels in supernatants at 24 hpi by enzymatic assay. (L) Cellular viability assessed by LDH release in supernatants. Data represent the mean ± SEM of three independent experiments. (*) indicates a significant difference (p < 0.05) compared to the respective noninfected group or SCR E. coli-infected group.

Article Snippet: CXCL1 / KC, CCL2 / MCP-1, IL-1β, IL-6, IL-10, IL-12p40, TNF-α, IFN-β and IFN-γ in cell-free culture supernatants and peritoneal lavage were measured using mouse Duoset ELISA kit (R&D Systems, USA) according to manufacturer’s instructions.

Techniques: Transfection, Infection, Staining, Enzyme-linked Immunosorbent Assay, Expressing, Enzymatic Assay

Journal: bioRxiv

Article Title: SEPSIS-INDUCED LIPID DROPLET ACCUMULATION ENHANCES ANTIBACTERIAL INNATE IMMUNITY

doi: 10.1101/2025.01.29.635463

Figure Lengend Snippet: Sham or septic (CLP) mice were orally treated with A922500 (DGAT1 inhibitor, 3 mg/kg) or vehicle. At 6 h or 24 h post-surgery, peritoneal lavage was collected. (A) In vivo experimental design for DGAT1 inhibition. (B) Oil Red O-stained lipid droplets (red) in peritoneal leukocytes, counterstained with Mayer’s hematoxylin. Scale bar: 20 μm. (C) Lipid droplet enumeration in peritoneal leukocytes (≥100 cells/group across 10 fields/experiment). (D) PGE 2 , LTB 4 , and RvD1 levels in peritoneal lavage at 24 h post-surgery by EIA. Levels of (E) pan-IFN-α, (F) IFN-β, (G) IFN-γ, (H) IL-6, (I) CCL2, (J) nitrite, and (K) cathelicidin (CAMP) in peritoneal lavage at 6 h and 24 h post-surgery by ELISA. Data (4–6 mice/group) are presented as means ± SEM, analyzed by one-way ANOVA with Tukey’s post hoc test. *p < 0.05 compared to sham; #p < 0.05 compared to untreated CLP. CLP: cecal ligation and puncture.

Article Snippet: CXCL1 / KC, CCL2 / MCP-1, IL-1β, IL-6, IL-10, IL-12p40, TNF-α, IFN-β and IFN-γ in cell-free culture supernatants and peritoneal lavage were measured using mouse Duoset ELISA kit (R&D Systems, USA) according to manufacturer’s instructions.

Techniques: In Vivo, Inhibition, Staining, Enzyme-linked Immunosorbent Assay, Ligation