Journal: Cell reports

Article Title: Endolysosomal damage surveillance enables rapid inflammasome sensing of pathogens

doi: 10.1016/j.celrep.2025.116002

Figure Lengend Snippet: (A) Cell death (% LDH release) and IL-18 release in wild-type (WT) and CASP4 −/− HeLa cells primed with 10 ng/mL IFN-γ overnight and uninfected (medium) or infected with S. Typhimurium ( S . Tm; MOI of 50 unless otherwise indicated) at 6 h post-infection (p.i.). (B–D) Cell death in HeLa cells transfected with control or indicated siRNAs for 48–72 h, primed with IFN-γ, and then infected with S . Tm for 6 h. (E) Cell death in IFN-γ-primed WT and LGALS8 −/− HeLa cells infected with S . Tm at 6 h p.i. (F) Cell death in IFN-γ-primed WT and LGALS3 −/− HeLa cells infected with S . Tm at 6 h p.i. (G) Cell death in HeLa cells transfected with control or indicated siRNAs for 48–72 h, primed with IFN-γ, and then infected with S . Tm for 6 h. (H) Immunoblots for full-length (FL) and N-terminal domain (NT) GSDMD in the lysates and C-terminal domain (CT) GSDMD in the supernatants of IFN-γ-primed WT and LGALS8 −/− HeLa cells infected with S . Tm at 4 h p.i. (I) IL-18 in the supernatants of IFN-γ-primed WT and LGALS8 −/− HeLa cells infected with S . Tm at 6 h p.i. (J and K) Cell death and IL-18 secretion by HeLa cells of indicated genotypes unprimed or primed with 10 ng/mL IFN-γ overnight and infected with S. Tm (MOIs of 50 and 100) at 6 h p.i. (L) Cell death in IFN-γ-primed WT and LGALS8 −/− HeLa cells infected with WT or ΔsifA mutant S . Tm at 6 h p.i. (M and N) Cell death (M) and IL-18 secretion (N) by IFN-γ-primed WT and LGALS8 −/− HeLa cells infected with S . Tm at indicated time points p.i. (O) Immunoblotting of the lysates of WT HeLa cells, LGALS8 −/− HeLa cells, and LGALS8 −/− HeLa cells stably expressing FLAG-galectin-8 for indicated proteins. (P and Q) Cell death (P) and IL-18 secretion (Q) by IFN-γ-primed WT HeLa cells, LGALS8 −/− HeLa cells, and LGALS8 −/− HeLa cells stably expressing FLAG-galectin-8 infected with S . Tm at 6 h p.i. Combined data from 2 (D, J, K, and N) or 3 (A–C, E–G, I, L, M, P, and Q) experiments or one experiment representative of two (H and O) are shown. Data (biological replicates) are plotted in the graphs as mean ± SEM. ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001; two-way ANOVA with Sidak’s (A–D, G, L–N, and P) or Tukey’s (E, F, I–K, and Q) multiple comparison test. See also and .

Article Snippet: Mouse monoclonal anti- Salmonella Typhimurium LPS , Bio-Rad , Clone 1E6, Cat# 8210-0407; RRID:AB_619235.

Techniques: Infection, Transfection, Control, Western Blot, Mutagenesis, Stable Transfection, Expressing, Comparison

Journal: bioRxiv

Article Title: The Chlamydia trachomatis secreted effector protein CT181 binds to Mcl-1 to prolong neutrophil survival

doi: 10.1101/2025.03.16.643443

Figure Lengend Snippet: ( A ) A2EN or HeLa cells were infected at a MOI of 2.5 with wild-type, tmeA-lx , CT181:: bla , or CT144:: bla . At 48h, cells were lysed and replated on fresh HeLa cell monolayers and infectious forming units were quantified by immunofluorescence microscopy. ( B ) A2EN or HeLa cells were infected at a MOI of 2.5 for 60 min with wild-type, tmeA-lx , CT181:: bla , or CT144:: bla . The number of internal bacteria was determine using differential immunostaining. ( C) To measure inclusion size, A2EN cells were infected at an MOI of 2.5 for 24 h. Bacteria were stained with anti-LPS (green), the inclusion membrane was stained with an anti-IncE antibody (red), and DNA was stained with DAPI (blue). Inclusion diameter was measured in ImageJ. ( A-C ) Data are representative of three independent experiments. Statistical significance was determined using One-Way ANOVA with Dunnett’s multiple comparison post-test comparing the mutants to wild-type. ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05.

Article Snippet: LPS antibody (Novus Cat#NBP1-28820) for enumeration of inclusions by immunofluorescence microscopy.

Techniques: Infection, Immunofluorescence, Microscopy, Bacteria, Immunostaining, Staining, Membrane, Comparison

Journal: Infection and Immunity

Article Title: Chlamydia pneumoniae Infects and Multiplies in Lymphocytes In Vitro

doi: 10.1128/iai.69.12.7753-7759.2001

Figure Lengend Snippet: FIG. 1. Fluorescence stain micrographs of C. pneumoniae-infected lymphocytes. (A and B) Human peripheral blood lymphocytes were infected with C. pneumoniae by gentle shaking and then incubated for 3 days at 37°C in 5% CO2. The infected cells were fixed on a glass slide by Cytospin and stained with FITC-conjugated anti-Chlamydia monoclonal antibody. Chlamydia inclusion bodies (arrow) were demonstrated in lymphocytes. Magnification, 1,000. (C and D) Immunofluoresence micrographs of C. pneumoniae-infected mouse spleen lymphocytes. The mouse spleen lymphocytes were infected with C. pneumoniae by gentle shaking, incubated for 3 days, and then stained with PE-conjugated anti-mouse IgG CD3 antibody and FITC-conjugated anti-Chlamydia antibody after treatment with the permeabilization kit. Red color indicates surface molecules of lymphocytes. Green to yellow color indicates C. pneumoniae. Magnification, 1,000. (E and F) Fluorescence micrographs of mouse T-cell-enriched lymphocyte cultures infected with C. pneumoniae. The purity of T cells in the cultures was more than 95% as determined by FACS analysis with anti-CD3 antibody. The T-cell cultures showed a few chlamydiae at 0 h (E). At 3 days after infection, there were many Chlamydia inclusion bodies (yellow spots) stained with FITC-conjugated anti-Chlamydia antibody in the culture (F). Magnification, 200.

Article Snippet: In some experiments, isolated mouse lymphocytes infected with C. pneumoniae were double stained with PE-conjugated anti-mouse CD3 antibody (BD Pharmingen) and FITC-conjugated anti-Chlamydia lipopolysaccharide (LPS) monoclonal antibody (Fitzgerald International Inc., Concord, Mass.).

Techniques: Fluorescence, Staining, Infection, Gentle, Incubation

Journal: Infection and Immunity

Article Title: Chlamydia pneumoniae Infects and Multiplies in Lymphocytes In Vitro

doi: 10.1128/iai.69.12.7753-7759.2001

Figure Lengend Snippet: FIG. 2. Transmission electron micrographs of C. pneumoniae-infected human lymphocytes. The analysis of C. pneumoniae-infected human lymphocytes (A and B, time zero after infection; C and D, 3 days after infection) by electron microscopy showed the attachment of chlamydiae to the surface of a lymphocyte (A), internalization of chlamydiae in a lymphocyte (B), and many Chlamydia particles of various sizes in a cell (C and D). Arrows indicate Chlamydia particles.

Article Snippet: In some experiments, isolated mouse lymphocytes infected with C. pneumoniae were double stained with PE-conjugated anti-mouse CD3 antibody (BD Pharmingen) and FITC-conjugated anti-Chlamydia lipopolysaccharide (LPS) monoclonal antibody (Fitzgerald International Inc., Concord, Mass.).

Techniques: Transmission Assay, Infection, Electron Microscopy

Journal: Infection and Immunity

Article Title: Chlamydia pneumoniae Infects and Multiplies in Lymphocytes In Vitro

doi: 10.1128/iai.69.12.7753-7759.2001

Figure Lengend Snippet: FIG. 3. Relative number of Chlamydia organisms determined by ELISA in lymphocyte cultures. The amount of Chlamydia LPS antigen in mouse lymphocyte cultures infected with C. pneumoniae was mea- sured by ELISA and converted to a relative number of Chlamydia organisms from the standard curve. The lymphocytes (106 cells) at time zero and 72 h after infection were collected, and Chlamydia LPS in lymphocytes was extracted (1.0 ml). The amount of LPS in extracts (200 l) was measured by ELISA. The data represent means stan- dard deviation (SD) for three experiments. , P 0.05 compared to time zero culture, analyzed by Student’s t test.

Article Snippet: In some experiments, isolated mouse lymphocytes infected with C. pneumoniae were double stained with PE-conjugated anti-mouse CD3 antibody (BD Pharmingen) and FITC-conjugated anti-Chlamydia lipopolysaccharide (LPS) monoclonal antibody (Fitzgerald International Inc., Concord, Mass.).

Techniques: Enzyme-linked Immunosorbent Assay, Infection

Journal: Infection and Immunity

Article Title: Chlamydia pneumoniae Infects and Multiplies in Lymphocytes In Vitro

doi: 10.1128/iai.69.12.7753-7759.2001

Figure Lengend Snippet: FIG. 5. Fluorescence micrographs and relative number of Chla- mydia organisms in T-lymphocyte cell line Molt 3 cultures infected with C. pneumoniae. See the legend to Fig. 3 for details. The cultures (106 cells) at time zero and 72 h after infection were collected and stained with FITC-conjugated anti-Chlamydia antibody, or Chlamydia LPS in cultures was extracted (1.0 ml). Fluorescence micrographs show the culture at time zero and 72 h after infection with viable C. pneu- moniae. Relative number of Chlamydia organisms was calculated from the LPS concentrations determined by ELISA. The data shown as the relative bacterial number represent means SD for three experi- ments. Open bars, infected with viable bacteria; solid bars, infected with UV-killed bacteria. , P 0.05 compared to time zero culture, analyzed by Student’s t test. Magnification, 1,000.

Article Snippet: In some experiments, isolated mouse lymphocytes infected with C. pneumoniae were double stained with PE-conjugated anti-mouse CD3 antibody (BD Pharmingen) and FITC-conjugated anti-Chlamydia lipopolysaccharide (LPS) monoclonal antibody (Fitzgerald International Inc., Concord, Mass.).

Techniques: Fluorescence, Infection, Staining, Enzyme-linked Immunosorbent Assay, Bacteria

Journal: BMC Gastroenterology

Article Title: Chlamydia trachomatis antigens in enteroendocrine cells and macrophages of the small bowel in patients with severe irritable bowel syndrome

doi: 10.1186/1471-230X-10-19

Figure Lengend Snippet: Fluorescent microscope images of small bowel preparations from patients with IBS A . Chlamydia LPS in EEC-like cells with apical nuclei and strong basal immunofluorescence (arrows). (Monoclonal FITC-conjugated antibody with Evans blue; original magnification × 63). B . Chlamydia LPS in a few cells within the epithelium (thick arrows) and l. propria (thin arrows). (Monoclonal FITC-conjugated antibody with Evans blue; original magnification × 63). C . Chlamydia trachomatis MOMP-positive immunofluorescence within 2 EEC-like cells (arrows) and 4 cells within l. propria (arrowheads). (Mouse MOMP-antibody and FITC-conjugated rabbit anti-mouse antibody; original magnification × 63). Hoechst (DAPI conjugated) for nuclear staining. D-F . Immunostainings for (D) Chlamydia LPS (FITC, green), ( E ) chromogranin A (Alexia 568, red), and ( F ) merged showing co-localisation of chromogranin A and Chlamydia LPS in enteroendocrine cells and Chlamydia LPS in l. propria . G-I . Immunostainings for ( G ) Chlamydia LPS (FITC, green,) ( H ) CD68 (Alexia 350. blue), and ( I ) merged showing co-localisation of CD68 and Chlamydia LPS in macrophages (arrows). Three enteroendocrine cells are also positive for Chlamydia LPS (arrowheads).

Article Snippet: Immunofluorescence was performed using a genus-specific mouse monoclonal antibody to Chlamydia lipopolysaccharide (LPS)-FITC conjugated with Evans blue (RDI-PROAC1FT, Fitzgerald Industries International, Concord, MA, USA) and C. trachomatis major outer membrane protein (MOMP) as primary antibody (GeneTex, San Antonio, TX, USA) with a polyclonal rabbit anti-mouse antibody-FITC conjugated (Dako, Glostrup, Danmark) as the secondary antibody.

Techniques: Microscopy, Immunofluorescence, Staining