Journal: MedComm

Article Title: Ipriflavone From Aquilaria malaccensis Lam. Exosome‐Like Nanoparticles Targets Prolyl Hydroxylase Domain Protein 2 (PHD2) to Enhance Hypoxia‐Inducible Factor‐α (HIF‐α) Hydroxylation Thereby Alleviating Hypoxia‐Induced Gastrointestinal Mucosal Ferroptosis

doi: 10.1002/mco2.70722

Figure Lengend Snippet: Ipriflavone ameliorated hypoxia‐induced ferroptosis by inhibiting HIF‐α‐triggered PUFA‐PLs, NOX4, and ALOX5 in NGEC and HIEC. Under hypoxia (1% O 2 ), HIEC was incubated for 24 h and then treated with ipriflavone (10 µM) for another 24 h. In experiments with siRNA targeting HIF‐α , HIEC was transfected with siNC, siHIF‐1α, or siHIF‐2α under normoxia for 24 h and then subjected to hypoxia (1% O 2 ) for 48 h. (A) Volcano plots showing the changes in PUFA‐PLs and SFA/MUFA‐PLs, including PC, PE, and PI, between the indicated groups ( n = 3). The blue boxes indicate the PUFA‐PC and PUFA‐PE with consistent changes after ipriflavone treatment and HIF‐α ( HIF‐1α or HIF‐2α ) knockdown under hypoxia. NGEC and HIEC were transfected with a control vector or HIF‐1α or HIF‐2α plasmid under normoxia for 24 h, followed by hypoxia (1% O 2 ) for 24 h. The cells were then treated with DMSO or ipriflavone (10 µM) under hypoxia for another 24 h. (B) HIF‐1α, HIF‐2α, NOX4, and ALOX5 levels were assessed by Western blot. NGEC and HIEC were transfected with either a blank control vector or plasmids containing HIF‐1α or HIF‐2α under normoxic conditions for 24 h, followed by exposure to hypoxia (1% O 2 ) for 24 h. The cells were then treated with DMSO, zileuton (an ALOX5 inhibitor, 10 µM), or GLX351322 (a NOX4 inhibitor, 10 µM) for an additional 24 h under hypoxic conditions. (C) 4‐HNE levels ( n = 4) were then assessed using an enzyme‐linked immunosorbent assay (ELISA). NGEC and HIEC were transfected with a blank control vector or plasmids encoding NOX4 or ALOX5 for 24 h under normoxia, prior to a 24‐h exposure to hypoxia (1% O 2 ). Following this, the cells were incubated with DMSO or ipriflavone (10 µM) for an additional 24 h under hypoxia. (D) 4‐HNE levels ( n = 4) by ELISA. Representative replicates of three independent experiments are shown in B–D. One‐way ANOVA: hypoxia + NOX4, hypoxia + ALOX5, or hypoxia + NOX4 + ALOX5 versus hypoxia control, a p < 0.05 or less, and hypoxia + NOX4 + ipriflavone, hypoxia + ALOX5 + ipriflavone, or hypoxia + NOX4 + ALOX5 + ipriflavone versus hypoxia + ipriflavone, b p < 0.0001 (in D). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001; ns, not significant. MUFAs, monounsaturated fatty acids; PCs, phosphatidylcholines; PEs, phosphatidylethanolamines; PIs, phosphatidylinositols; PLs, phospholipids; PUFAs, polyunsaturated fatty acids; SFAs, saturated fatty acids.

Article Snippet: The cells were then exposed to hypoxic conditions (1% O 2 ) for 24 h and incubated for another 24 h under hypoxic conditions with DMSO, Fer‐1 (5 μM; S7243; Selleck), Lip‐1 (1 μM; S7699; Selleck), ipriflavone (10 μM), GLX351322 (a NOX4 inhibitor; 10 μM; HY‐100111; MedChemExpress, Shanghai, China), zileuton (an ALOX5 inhibitor; 10 μM; HY‐14164; MedChemExpress), or MG132 (10 μM; S2619; Selleck).

Techniques: Incubation, Transfection, Knockdown, Control, Plasmid Preparation, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Tuft cell cysteinyl leukotrienes are necessary for rhinovirus-induced mucus metaplasia, type 2 inflammation and airway hyperresponsiveness in immature mice

doi: 10.3389/fimmu.2025.1709008

Figure Lengend Snippet: RV treatment stimulates lung cysLT levels in C57BL/6 mice. Total lung cysLTs were measured by ELISA. (A) Left panel: CysLT levels in sham and RV-infected C57Bl/6, Alox5 -/- and Pou2f3 -/- mice. (Mean ± SD, n=4–15 per group, 1–6 experiments per condition). Right panel: CysLT levels in vehicle and montelukast-treated RV-infected C57Bl/6 mice. (Mean ± SD, n=4 per group, 1 experiment per condition). (B) Left panel: Alox5 mRNA expression in sham and RV-infected 57Bl/6, Alox5-/- and Pou2f3-/- mice. (Mean ± SD, n=6–12 per group, 2–3 experiments per condition.) Right panel: Alox5 mRNA expression in vehicle and montelukast-treated RV-infected C57Bl/6 mice. (Mean ± SD, n=7 per group, 2 experiments per condition.) For A and B, data were analyzed by Kruskal-Wallis test or Wilcoxon matched paired signed rank test. Dunn’s multiple comparisons test was used to pinpoint differences identified by the Kruskal-Wallis test. (C) ALOX5 and ALOX5AP transcripts in nasal samples from healthy children and infants with RV bronchiolitis. Shown are median, interquartile range (forming the box), and whiskers extending to the minimum and maximum values. Wald test p values were adjusted for multiple testing using the procedure of Benjamini and Hochberg.

Article Snippet: C57Bl/6 and Alox5 -/- mice (B6.129S2- Alox5 tm1Fun /J) ( ) were purchased from Jackson Laboratories (Bar Harbor, ME).

Techniques: Enzyme-linked Immunosorbent Assay, Infection, Expressing

Journal: Frontiers in Immunology

Article Title: Tuft cell cysteinyl leukotrienes are necessary for rhinovirus-induced mucus metaplasia, type 2 inflammation and airway hyperresponsiveness in immature mice

doi: 10.3389/fimmu.2025.1709008

Figure Lengend Snippet: Localization of Alox5, Alox5ap and Dclk1 in airway epithelium. (A) Immunofluorescent staining for Alox5, Alox5ap and Dclk1 in uninfected wild-type C57Bl/6 mice. Lungs from uninfected wild-type mice show no Dclk1 staining in the airway epithelium and round Alox5+ cells were present in the subepithelium. The white bars are 100 microns. (B-D) . Immunofluorescence staining for Alox5 (B) , Alox5ap (C) and Dclk1 [ (D) , each shown in red]. For (B-D) , RV-infected mice show rare triangular or bottle-like shaped cells, a morphology typical of tuft cells. There was no staining in Pou2f 3-/- mice (middle panels) or Alox5-/- mice (right panels). DAPI-stained nuclei are shown in blue. The white bars are 100 microns.

Article Snippet: C57Bl/6 and Alox5 -/- mice (B6.129S2- Alox5 tm1Fun /J) ( ) were purchased from Jackson Laboratories (Bar Harbor, ME).

Techniques: Staining, Immunofluorescence, Infection

Journal: Frontiers in Immunology

Article Title: Tuft cell cysteinyl leukotrienes are necessary for rhinovirus-induced mucus metaplasia, type 2 inflammation and airway hyperresponsiveness in immature mice

doi: 10.3389/fimmu.2025.1709008

Figure Lengend Snippet: Heterologous RV infection induces mucous metaplasia in the lungs of C57Bl/6 mice but not Alox5-/- or Pou2f3-/- mice. (A) Lungs were harvested on day of life 20, processed for histology and stained with periodic acid-Schiff (PAS) and counterstained with hematoxylin. Heterologous RV infection increased PAS staining (magenta). Montelukast-treated C57Bl/6, Alox5-/- and Pou2f3-/- mice showed reduced PAS staining. The black bar is 200 microns. (B) PAS staining was quantified by point counting using NIH ImageJ. Heterologous RV infection increased the fraction of epithelia stained with PAS. Left panel: Fraction of epithelium stained with PAS in sham and RV-infected C57Bl/6, Alox5-/- and Pou2f3-/- mice. (Mean ± SD, n=4–8 per group, 2–3 experiments per condition.) Right panel: Fraction of epithelium stained with PAS in vehicle and montelukast-treated RV-infected C57Bl/6 mice. (Mean ± SD, n=5 per group, 2 experiments per condition.) (C) Muc5ac mRNA levels measured by qPCR. Left panel: Muc5ac mRNA expression in sham and RV-infected C57Bl/6, Alox5-/- and Pou2f3-/- mice. (Mean ± SD, n=11-17, 3–4 experiments per condition.) Right panel: Muc5ac mRNA expression in vehicle and montelukast-treated RV-infected C57Bl/6 mice. (Mean ± SD, n=4, 1 experiment per condition). (D) Muc5ac protein levels measured by ELISA. Left panel: Muc5ac protein expression in sham and RV-infected C57Bl/6, Alox5-/- and Pou2f3-/- mice. (Mean ± SD, n=3–6 per group, 1–2 experiments per condition.) Right panel: Muc5ac protein expression in vehicle and montelukast-treated RV-infected C57Bl/6 mice. (Mean ± SD, n=6 per group, 2 experiments per condition.) For B-D, data were analyzed by Kruskal-Wallis test or Wilcoxon matched paired signed rank test. Dunn’s multiple comparisons test was used to pinpoint differences identified by the Kruskal-Wallis test.

Article Snippet: C57Bl/6 and Alox5 -/- mice (B6.129S2- Alox5 tm1Fun /J) ( ) were purchased from Jackson Laboratories (Bar Harbor, ME).

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

Journal: Frontiers in Immunology

Article Title: Tuft cell cysteinyl leukotrienes are necessary for rhinovirus-induced mucus metaplasia, type 2 inflammation and airway hyperresponsiveness in immature mice

doi: 10.3389/fimmu.2025.1709008

Figure Lengend Snippet: Cytokine mRNA expression in RV-infected mice. Lungs were harvested on day 20 and processed for mRNA expression. Left panels: mRNA expression in sham and RV-infected C57Bl/6, Alox5-/- and Pou2f3-/- mice. (Mean ± SD, n=7-21, 2–4 experiments per condition.) Right panels: mRNA expression in vehicle and montelukast-treated RV-infected C57Bl/6 mice. (Mean ± SD, n=7-12, 2–4 experiments per condition.) Data were analyzed by Kruskal-Wallis test or Wilcoxon matched paired signed rank test. Dunn’s multiple comparisons test was used to pinpoint differences identified by the Kruskal-Wallis test.

Article Snippet: C57Bl/6 and Alox5 -/- mice (B6.129S2- Alox5 tm1Fun /J) ( ) were purchased from Jackson Laboratories (Bar Harbor, ME).

Techniques: Expressing, Infection

Journal: Frontiers in Immunology

Article Title: Tuft cell cysteinyl leukotrienes are necessary for rhinovirus-induced mucus metaplasia, type 2 inflammation and airway hyperresponsiveness in immature mice

doi: 10.3389/fimmu.2025.1709008

Figure Lengend Snippet: Cytokine protein expression in RV-infected mice. Lungs were harvested on day 20 and processed for protein expression by ELISA. (A) Type 2 and innate cytokines. (B) Type 1 cytokines. Left panels: Protein expression in sham and RV-infected C57Bl/6, Alox5 -/- and Pou2f3 -/- mice. (Mean ± SD, n=3–16 per group, 1–4 experiments per condition.) Right panels: Protein expression in vehicle and montelukast-treated RV-infected C57Bl/6 mice. (Mean ± SD, n=3–11 per group, 1–3 experiments per condition.) Data were analyzed by Kruskal-Wallis test or Wilcoxon matched paired signed rank test. Dunn’s multiple comparisons test was used to pinpoint differences identified by the Kruskal-Wallis test.

Article Snippet: C57Bl/6 and Alox5 -/- mice (B6.129S2- Alox5 tm1Fun /J) ( ) were purchased from Jackson Laboratories (Bar Harbor, ME).

Techniques: Expressing, Infection, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Tuft cell cysteinyl leukotrienes are necessary for rhinovirus-induced mucus metaplasia, type 2 inflammation and airway hyperresponsiveness in immature mice

doi: 10.3389/fimmu.2025.1709008

Figure Lengend Snippet: RV-induced airways responsiveness is blocked in Alox5-/- and Pou2f3-/- mice and montelukast-treated C57Bl/6 mice. Airway responsiveness was assessed by measuring changes in total respiratory system resistance in response to increasing doses of nebulized methacholine. (Mean ± SD, n=5–10 for each condition from two separate experiments.) Total respiratory system resistance (Rrs) data at 40 mg/mL methacholine were analyzed by Kruskal-Wallis test. Dunn’s multiple comparisons test was used to pinpoint differences identified by the Kruskal-Wallis test.

Article Snippet: C57Bl/6 and Alox5 -/- mice (B6.129S2- Alox5 tm1Fun /J) ( ) were purchased from Jackson Laboratories (Bar Harbor, ME).

Techniques:

Journal: Discover Oncology

Article Title: Lipid metabolism-related genes correlate with immune microenvironment and regulate the efficacy of immunotherapy via ferroptosis in melanoma

doi: 10.1007/s12672-025-04163-x

Figure Lengend Snippet: Screening and functional validation of differentially-expressed ferroptosis-related genes. (A) Venn diagrams of the differentially-expressed ferroptosis-related genes that have prognostic potential. (B) The expression of ALOX5, ACSL4, ACACA, ABCC1 in the two subgroups in training cohort, as well as Kaplan-Meier analysis of the survival of patients in the two subgroups. (C) The knockdown efficiency of ASCL4 and ALOX5 in melanoma cells, as well as cell viability of melanoma cells in response to RSL3 treatment. (D) Lipid ROS levels of indicated cells measured by flow cytometry using C11-BODIPY. Data represent the mean ± SEM of triplicates. P value was calculated by two-tailed Student’s t-test and log-rank tests. ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns, non-significant. n (cluster 1) = 208, n (cluster 2) = 246. n (low) = 227, n (high) = 227

Article Snippet: The primary antibodies and dilutions for western blotting (WB) and immunofluorescence (IF) staining analysis are listed below: ACSL4 (22401-1-AP, Proteintech, Wuhan, China; 1:100 for IHC, 1:100 for IF and 1:5 000 for WB), ALOX5 (10021-1-Ig, Proteintech; 1:400 for IHC, and 1:500 for WB), PTGS2 (66351-1-Ig, Proteintech; 1:400 for IF), CD8α(ab22378,, Abcam; 1:300 for IF), ACTIN (66009-1-Ig, Proteintech; 1:10 000 for WB).

Techniques: Functional Assay, Biomarker Discovery, Expressing, Knockdown, Flow Cytometry, Two Tailed Test