Journal: Nature Communications

Article Title: Tudor domain-containing protein 9-targeting siRNA nanoparticles alleviate Pseudomonas aeruginosa lung injury in preclinical models by promoting neutrophil cuproptosis

doi: 10.1038/s41467-026-70349-8

Figure Lengend Snippet: Male C57BL/6 mice were intranasally inoculated with 50 μL of bacterial suspension of PA strain PAO1 (OD 600 = 0.6; 7 × 10 6 CFU/mL in 0.9% sterile saline) for 1, 3, and 7 days. A Quantitative real-time PCR quantification of phosphodiesterase 4D ( PDE4D ), Tudor domain-containing protein 9 ( TDRD9 ), and integrin subunit alpha 7 ( ITGA7 ) in lung neutrophils. n = 6. mice per group. B Western blot analysis of PDE4D, TDRD9, and ITGA7 expression in lung neutrophils. n = 6 mice per group. C Western blot profiling of cuproptosis-related proteins (dihydrolipoamide S-acetyltransferase [DLAT], ferredoxin 1 [FDX1], lipoic acid synthetase [LIAS], copper-transporting ATPase A/B [ATP7A/B], solute carrier family 31 member 1 [SLC31A1], and tumor protein p53 [p53]) in neutrophils. n = 6 mice per group. D Immunofluorescence (IF) staining demonstrating co-localization of neutrophil marker LY6G (green) with Fe-S cluster proteins DLAT and FDX1 (red) in lung tissues. Nuclei were counterstained with DAPI (blue). Scale bar: 25 µm. n = 6 mice per group. Data represent median ± interquartile range (IQR), and analyzed by two-sided one-way ANOVA with Tukey’s multiple comparisons test ( A – D ). Source data are provided as a file.

Article Snippet: Soluble cluster of differentiation 80 (sCD80), p53, and programmed death ligand-1(PD-L1) in cell culture medium were measured using commercial human ELISA kits (sCD80: CSB-E15768h-IS; p53: Cat# CSB-E08334h-IS; PD-L1: CSB-E13644h; CUSABIO Co., Ltd.).

Techniques: Suspension, Sterility, Saline, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Immunofluorescence, Staining, Marker

Journal: Nature Communications

Article Title: Tudor domain-containing protein 9-targeting siRNA nanoparticles alleviate Pseudomonas aeruginosa lung injury in preclinical models by promoting neutrophil cuproptosis

doi: 10.1038/s41467-026-70349-8

Figure Lengend Snippet: A Neutrophils were pretreated with escalating doses of the copper ionophore elesclomol (ES) or dimethyl sulfoxide (DMSO; vehicle control) for 24 h. Dose-dependent cytotoxicity of ES in human neutrophils. n = 3 independent experiments. B Neutrophils were pretreated with 10 μM ES for 24 h and challenged with Pseudomonas aeruginosa (PA) strain PAO1 at a multiplicity of infection (MOI) of 10 for 1 h. Viability of neutrophils treated with 10 μM ES ± PAO1 infection. n = 3 independent experiments. C Western blot analysis of cuproptosis-related proteins under PAO1/ES co-treatment. n = 3 independent experiments. D PAO1-induced TDRD9 upregulation. n = 3 independent experiments. E Correlation heatmap between TDRD9 and cuproptosis regulators. F Neutrophils were pre-transducted with lentiviral-based short hairpin RNA (shRNA) knocking down Tudor domain-containing protein 9 ( TDRD9 ) (sh-TDRD9) and exposed to 20 μM tetrathiomolybdate (cuproptosis inhibitor; 2 h), 50 μM deferoxamine (DFO; iron chelator; 1 h), 10 μM ferrostatin-1 (ferroptosis inhibitor; 1 h), 5 μM Z-VAD-FMK (pan-caspase inhibitor; 1 h), 1 μM necrosulfonamide (necroptosis inhibitor; 1 h), and 500 μM 3-methyladenine (3-MA; autophagy inhibitor; 1 h). Neutrophil viability under pharmacological inhibition of alternative cell death pathways. n = 3 independent experiments. G Neutrophils were pre-transducted with sh-TDRD9, followed by treatment with 10 μM ES and PAO1. Quantitative analysis of colony-forming unit (CFU) in neutrophils. n = 3 independent experiments. H Neutrophils were pre-transducted with sh-TDRD9 or lentiviral TDRD9 overexpression construct (oe-TDRD9), followed by treatment with 10 μM ES and PAO1. Viability of PAO1/ES-treated neutrophils with TDRD9 knockdown/overexpression. n = 3 independent experiments. I Enzyme-linked immunosorbent assay (ELISA) analysis of tumor protein p53 (p53) levels in cell culture medium. n = 3 independent experiments. J Protein expression dynamics of cuproptosis mediators under genetic TDRD9 modulation. n = 3 independent experiments. Data represent median ± interquartile range (IQR), and analyzed by two-sided two-way ANOVA with Tukey’s multiple comparisons test ( A ), or two-sided one-way ANOVA with Tukey’s multiple comparisons test ( B – D , F – J ). Source data are provided as a file.

Article Snippet: Soluble cluster of differentiation 80 (sCD80), p53, and programmed death ligand-1(PD-L1) in cell culture medium were measured using commercial human ELISA kits (sCD80: CSB-E15768h-IS; p53: Cat# CSB-E08334h-IS; PD-L1: CSB-E13644h; CUSABIO Co., Ltd.).

Techniques: Control, Infection, Western Blot, shRNA, Inhibition, Over Expression, Construct, Knockdown, Enzyme-linked Immunosorbent Assay, Cell Culture, Expressing

Journal: Nature Communications

Article Title: Tudor domain-containing protein 9-targeting siRNA nanoparticles alleviate Pseudomonas aeruginosa lung injury in preclinical models by promoting neutrophil cuproptosis

doi: 10.1038/s41467-026-70349-8

Figure Lengend Snippet: A Neutrophils were pre-transducted with lentiviral-based short hairpin RNA (shRNA) knocking down TDRD9 (sh-TDRD9) and lentiviral PD-L1 overexpression construct (oe-PD-L1), followed by treatment with 10 μM elesclomol (ES) for 24 h and Pseudomonas aeruginosa (PA) strain PAO1 at a multiplicity of infection (MOI) of 10 for 1 h. Quantitative analysis of colony-forming unit (CFU) in neutrophils. n = 3 independent experiments. B PD-L1 overexpression rescued TDRD9 knockdown-induced pathway suppression. n = 3 independent experiments. C Enzyme-linked immunosorbent assay (ELISA) analysis of PD-L1 and sCD80 levels in cell culture medium. n = 3 independent experiments. D Viability restoration by PD-L1 overexpression in TDRD9-depleted neutrophils. n = 3 independent experiments. E Cuproptosis-related protein dynamics under TDRD9 knockdown ± PD-L1 overexpression. n = 3 independent experiments. F ELISA analysis of tumor protein p53 (p53) levels in cell culture medium. n = 3 independent experiments. G Neutrophils were pre-transducted with oe-PD-L1 and lentiviral-based shRNA knocking down CD80 (sh-CD80), followed by treatment with 10 μM elesclomol (ES) for 24 h and infection with PAO1 at an MOI of 10 for 1 h. Signaling pathway-related protein expressions under PD-L1 overexpression ± CD80 knockdown. n = 3 independent experiments. H ELISA analysis of PD-L1 and sCD80 levels in cell culture medium. n = 3 independent experiments. I Viability reduction by CD80 knockdown in PD-L1-overexpressed neutrophils. n = 3 independent experiments. J , K Cuproptosis-related protein dynamics under PD-L1 overexpression ± CD80 knockdown. n = 3 independent experiments. L PD-L1-CD80 interaction using co-immunoprecipitation assay. n = 3 independent experiments. Data represent median ± interquartile range (IQR), and were analyzed by two-sided one-way ANOVA with Tukey’s multiple comparisons test ( A – K ). Source data are provided as a file.

Article Snippet: Soluble cluster of differentiation 80 (sCD80), p53, and programmed death ligand-1(PD-L1) in cell culture medium were measured using commercial human ELISA kits (sCD80: CSB-E15768h-IS; p53: Cat# CSB-E08334h-IS; PD-L1: CSB-E13644h; CUSABIO Co., Ltd.).

Techniques: shRNA, Over Expression, Construct, Infection, Knockdown, Enzyme-linked Immunosorbent Assay, Cell Culture, Co-Immunoprecipitation Assay

Journal: Nature Communications

Article Title: Tudor domain-containing protein 9-targeting siRNA nanoparticles alleviate Pseudomonas aeruginosa lung injury in preclinical models by promoting neutrophil cuproptosis

doi: 10.1038/s41467-026-70349-8

Figure Lengend Snippet: A A spherical 3D human lung organoid (HLO) structure was successfully established in Matrigel, and its growth was observed under an optical microscope for 7 days. Representative bright-field images of HLOs cultured for 7 days. Scale bars: 200 μm (top), 100 μm (bottom). n = 3 independent experiments. B Hematoxylin and eosin (HE) staining comparing histological features of native human lung tissue and HLOs. Scale bar: 60 μm. n = 3 independent experiments. C Immunohistochemistry analysis of Ki67, NK2 homeobox 1 (NKX2.1), and SRY-box transcription factor 9 (SOX9) expression in native lung tissue and HLOs. Scale bar: 60 μm. n = 3 independent experiments. D HLOs were incubated with 0.1 μM hyaluronic acid (HA)-coated peptide nanoparticle (NP) system for targeted small interfering RNA (siRNA) delivery against Tudor domain-containing protein 9 ( TDRD9 ) (HA-si-TDRD9 NPs) or HA-si-NC NPs, followed by infection with Pseudomonas aeruginosa (PA) strain PAO1. HE staining showing HA-si-TDRD9-mediated preservation of HLO morphology following PA challenge. n = 3 independent experiments. E Bacterial growth curves in HLO cultures. n = 3. F Quantitative real-time PCR analysis of TDRD9 mRNA levels in HLOs. n = 3 independent experiments. G Western blot analysis of TDRD9, programmed death ligand-1(PD-L1), cluster of differentiation 80 (CD80), and mitogen-activated protein kinase (MAPK) expression. n = 3 independent experiments. H TUNEL assay quantifying apoptotic cells in HLOs. Scale bar: 25 μm. n = 3 independent experiments. I Western blot of cleaved caspase-3, -8, and -9 levels. n = 3 independent experiments. J Enzyme-linked immunosorbent assay (ELISA) quantification of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and IL-6 levels in HLO supernatants. n = 3 independent experiments. Data represent median ± interquartile range (IQR), and analyzed by two-sided two-way ANOVA with Tukey’s multiple comparisons test ( E ), or two-sided one-way ANOVA with Tukey’s multiple comparisons test ( F –J ). Source data are provided as a file.

Article Snippet: Soluble cluster of differentiation 80 (sCD80), p53, and programmed death ligand-1(PD-L1) in cell culture medium were measured using commercial human ELISA kits (sCD80: CSB-E15768h-IS; p53: Cat# CSB-E08334h-IS; PD-L1: CSB-E13644h; CUSABIO Co., Ltd.).

Techniques: Microscopy, Cell Culture, Staining, Immunohistochemistry, Expressing, Incubation, Small Interfering RNA, Infection, Preserving, Real-time Polymerase Chain Reaction, Western Blot, TUNEL Assay, Enzyme-linked Immunosorbent Assay