Journal: bioRxiv

Article Title: A cilia-dependent inflammatory programme links bacterial detection to kidney disease

doi: 10.64898/2026.04.24.720658

Figure Lengend Snippet: (A) qPCR quantification of Ccl2 mRNA in Kif3a i-shRNA MDCK cells treated for 6 hours with TLR-1/2 (Pam3), TRL-2/6 (Pam2), TLR-4 (Lipopolysaccharide, LPS) or TLR-5 agonist (Flagellin, FLA). (B) qPCR quantification of Ccl2 mRNA in luciferase (blue), Kif3a (red) or Ift88 (green) i-shRNA MDCK cells treated for 6 hours by NOD1 (Tri-DAP) or NOD2 agonist (MDP). (C) qPCR quantification of Ccl2 mRNA in doxycycline (DOX) inducible luciferase (blue), Kif3a (red) or Ift88 (green) shRNA MDCK cells treated for 6 hours with 13µM ADPh. (D) qPCR quantification of Ccl2 mRNA in Kif3a i-shRNA MDCK cells treated for 4 hours with 500nM DF-006. (E-F) qPCR quantification of Alpk1 (E) and Tifa (F) mRNA in doxycycline (DOX) inducible luciferase (blue), Kif3a (red) or Ift88 (green) shRNA MDCK cells. (G) qPCR quantification of Ccl2 mRNA in Kif3a i-shRNA MDCK cells treated for 30min with 13µM ADPh in presence of digitonin, and stopped 5 hours and a half later. (H) Percentage of cells with TIFAsome formation in HeLa cells upon ADPh, UPEC heat-killed supernatant stimulation from WT (snUTI89 WT) or lacking Hlde (sn UTI89 ΔHlde), an essential enzyme to generate ADPh. (I-J) qPCR quantification of Ccl2 mRNA expression (expressed in percentage of the maximum value) depending on treatment duration (0min, 30min, 1h, 2h, 4h or 6h) after ADPh (I) and UPEC (J) stimulation of Kif3a i-shRNA MDCK cells. (K) qPCR quantification of Ccl2 mRNA in Kif3a i-shRNA MDCK cells treated for 6 hours with UPEC heat-killed supernatant lacking Hlde (sn UTI89 ΔHlde). (A-B, E-F) Ratio paired t test: *P<0.05, **P<0.01, ***P<0.001, ****P<0.00001. Each dot represents an experimental n. AU: arbitrary units. (D, G-H, K) Paired one-way RM ANOVA (log-normal) with Geisser Greenhouse correction followed by Tukey test: *P<0.05, **P<0.01, ***P<0.001, ****P<0.00001. Each dot represents an experimental n. AU: arbitrary units.

Article Snippet: Tri-DAP (tlrl-tdap, InvivoGen) was dissolved in sterile water at 10 mg/mL and added to the cell culture medium at 10μg/mL final concentration during 6 hours.

Techniques: shRNA, Luciferase, Expressing

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: TLR8 agonists remodel the tumor immune microenvironment through PF4-dependent T cell recruitment and ancillary mechanisms

doi: 10.1007/s00262-026-04329-8

Figure Lengend Snippet: Dynamics of immune cell subsets in response to agonist stimulation. A Schematic of the experimental workflow. Immune profiling by flow cytometry was performed 48 h after a single intratumoral injection of PRR agonists. B Percentage of CD45 + leukocytes among live cells following intratumoral administration of different agonists: TLR3 agonist poly IC (25 µg/tumor), TLR7 agonist imiquimod hydrochloride (25 µg/tumor), TLR8 agonist TL8-506 (10 µg/tumor), TLR9 agonist CpG ODN 2395 (50 µg/tumor), STING agonist ADU-S100 ammonium salt (25 µg/tumor), NOD1 agonist Tri-DAP (10 µg/tumor), and NOD2 agonist murabutide (5 µg/tumor). C Proportion of cDCs within the CD45 + population. D Percentage of ZsGreen + cells among DCs. E Proportion of macrophages within the CD45 + population. F Percentage of ZsGreen + cells among macrophages. G Proportion of CD8 + T cells within the CD45 + compartment. H Proportion of cCD4 + T cells within the CD45 + compartment. I Proportion of Tregs within the CD45 + compartment, pooled from two independent experiments. J Ratio of cCD4 + T cells to Tregs. (K) Ratio of CD8 + T cells to Tregs. Data represent the mean ± SEM ( n = 8 mice per group). Statistical comparisons were performed using one-way ANOVA. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001

Article Snippet: When tumors reached approximately 200 mm 3 , PRR agonists were administered via intratumoral injection at the following doses: TLR3 agonist poly(I:C) (Sigma-Aldrich, #42,424–50-0; 25 μg/tumor); TLR7 agonist imiquimod hydrochloride (MedChemExpress, #HY-B0180A; 25 μg/tumor); TLR8 agonist TL8-506 (InvivoGen, #tlrl-tl8506; 10 μg/tumor); TLR8 agonist motolimod (MedChemExpress, #HY-13773; 50 μg/tumor); TLR9 agonist CpG ODN 2395 (Class C) (InvivoGen, #tlrl-2395–1; 50 μg/tumor); STING agonist ADU-S100 ammonium salt (MedChemExpress, #HY-12885B; 25 μg/tumor); NOD1 agonist Tri-DAP (InvivoGen, #tlrl-tdap; 10 μg/tumor); and NOD2 agonist murabutide (InvivoGen, #tlrl-mbt; 5 μg/tumor).

Techniques: Flow Cytometry, Injection

Journal: Infection and Immunity

Article Title: Chlamydia trachomatis restricts signaling through NOD2 until late in the pathogen’s developmental cycle

doi: 10.1128/iai.00472-25

Figure Lengend Snippet: Pre-treatment with NOD1- and NOD2-stimulatory ligands prior to infection with C. trachomatis significantly impacts inclusion size in NLR-expressing cell lines. HepG2, C-33A, and SiHa cells were pre-treated with Tri-DAP, MDP, or both, prior to infection with C. trachomatis L2 strain Bu/434. At 24 hpi, cells were fixed, inclusions were labeled, counted, and measured. zStacks of MOMP-labeled objects were obtained from a Zeiss 700 confocal microscope, and approximate volume measurements were calculated for all inclusions present within 10 imaging fields. Data presented represent all MOMP-labeled objects counted resulting in inclusions > 20 µm 3 ; 3 µm across with red lines indicating the mean of each group within a data set. Significance was assessed via one-way ANOVA with multiple comparisons. ****; P < 0.0001, ***; P < 0.001, **; P < 0.01, ns; not significant.

Article Snippet: MDP and L-Ala-γ-D-Glu-mDAP (Tri-DAP) were purchased from InvivoGen.

Techniques: Infection, Expressing, Labeling, Microscopy, Imaging

Journal: Infection and Immunity

Article Title: Chlamydia trachomatis restricts signaling through NOD2 until late in the pathogen’s developmental cycle

doi: 10.1128/iai.00472-25

Figure Lengend Snippet: Pre-treatment with NOD2-stimulatory ligands delays the development of C. trachomatis in NLR-expressing cell lines. HepG2 ( A ), C-33A ( B ), and SiHa ( C ) cells were either left untreated or pre-treated with the NOD1-stimulatory ligand Tri-DAP, the NOD2-stimulatory ligand MDP, or both Tri-DAP and MDP. After 24 hours, cells were infected with C. trachomatis , and the development of infectious EBs was assessed at the indicated time points (30, 48, and 72 hpi). Lines delineate the mean of three separate biological replicates with each replicate displayed as a colored dot. Significance was assessed via one-way ANOVA with multiple comparisons. **; P < 0.01, *; P < 0.05, ns; not significant.

Article Snippet: MDP and L-Ala-γ-D-Glu-mDAP (Tri-DAP) were purchased from InvivoGen.

Techniques: Expressing, Infection

Journal: Experimental & Molecular Medicine

Article Title: The dual roles of peptidoglycans: NOD1 and NOD2 inversely regulate bone metabolism

doi: 10.1038/s12276-025-01522-0

Figure Lengend Snippet: Mice were intraperitoneally administered 1.25 mg/kg of TriDAP or MDP, or PBS, on days 0 and 4. a On day 7, micro-CT images were obtained of the distal femurs of those mice. b BV/TV, Tb.Th, Tb.N and Tb.Sp. were analyzed using a CT analyzer. BV/TV, trabecular bone volume; Tb.Th, trabecular thickness; Tb.N, trabecular number; Tb.Sp., trabecular separation. c Distal femur sections from mice treated with PBS or TriDAP were stained with H&E and photographed with an inverted phase-contrast microscope. d Mice were intraperitoneally administered calcein one day before the first TriDAP administration and after the second TriDAP administration. Resin blocks of those femurs were sectioned. The images were obtained using a fluorescent microscope. e The distance between two labels represents the mineral apposition rate (MAR). Scale bars, 500 μm. f The MAR was quantified using OsteoMeasure. * P < 0.05.

Article Snippet: TriDAP, C12-iE-DAP, C14-Tri-LAN-Gly, MDP and Pam3CSK4 were obtained from InvivoGen.

Techniques: Micro-CT, Staining, Microscopy

Journal: Experimental & Molecular Medicine

Article Title: The dual roles of peptidoglycans: NOD1 and NOD2 inversely regulate bone metabolism

doi: 10.1038/s12276-025-01522-0

Figure Lengend Snippet: a MC3T3-E1 cells were treated with 1 µg/ml TriDAP for 15, 30, 60 or 90 min. The cells were lysed and subjected to western blotting using specific antibodies to IκBα or p65. b MC3T3-E1 cells were treated with 1 µg/ml TriDAP for 15 min and then fixed and stained sequentially with specific antibodies to p65 and Hoechst 33342. Cell images were obtained using a confocal microscope. Scale bar, 20 μm. c , e MC3T3-E1 cells were transfected with an NF-κB-Luc plasmid and treated with 0, 0.1, 1 or 10 µg/ml TriDAP ( c ) or MDP ( e ) for 24 h. The cells were lysed and subjected to a luciferase assay. d MC3T3-E1 cells were treated with 1 µg/ml of MDP for 15, 30, 60 or 90 min. The cells were lysed and subjected to western blotting using specific antibodies to IκBα. f MC3T3-E1 cells were transfected with 6× OSE-Luc plasmids. The cells were pretreated with 10 μM TPCK for 1 h, followed by stimulation with 10 μg/ml TriDAP for an additional 23 h. The cells were lysed and subjected to a luciferase assay. * P < 0.05. n.s. not significant.

Article Snippet: TriDAP, C12-iE-DAP, C14-Tri-LAN-Gly, MDP and Pam3CSK4 were obtained from InvivoGen.

Techniques: Western Blot, Staining, Microscopy, Transfection, Plasmid Preparation, Luciferase