agent vectashield mounting medium with dapi  (Vector Laboratories)

Journal: EMBO Molecular Medicine

Article Title: Typhoid toxin of Salmonella Typhi elicits host antimicrobial response during acute typhoid fever

doi: 10.1038/s44321-025-00347-8

Figure Lengend Snippet: ( A ) Representative images from three independent experiments of CACO2 intestinal cells either untreated, treated with wild-type typhoid toxin (TxWT) or H160Q DNase-deficient toxin (TxHQ) for 2 h prior to fluorescence microscopy at 96 h of γH2AX (cyan), APOC3 (yellow) and EdU (Magenta). EdU was incubated with cells 24 h before fixation. DAPI-stained nuclear outlines shown. Scale bars: 50 μm. ( B ) Bar chart showing proportion of APOC3 expressing cells ( n = 4). ( C ) Bar chart showing proportion of γH2AX-positive cells ( n = 5). ( D ) Bar chart showing proportion of cells incorporating EdU nucleotide analogue ( n = 3). ( E ) ELISA of APOC3 secreted into growth media harvested from cells in ( A ) ( n = 3). ( F ) Representative images from three independent experiments of CACO2 intestinal cells infected with wild-type or toxin-deficient ( ∆cdtB ) S . Javiana for 1 h prior to incubation in gentamicin-containing media and imaging at 96 h. Immunofluorescence performed as ( A ). Scale bars: 50 μm. Bar charts showing ( G ) proportion of APOC3 expressing cells ( n = 3), or ( H ) proportion of γH2AX-positive cells during infection from experiment in ( F ) ( n = 3). Data also analysed following infection with ∆cdtB expressing pTrc99A-cdtB (pCdtB). Statistical significance: one-way ANOVA with Tukey’s test in ( B – E , G , H ) analysing all pairs of >3 groups. Data are presented as mean ± SEM. Asterisks indicate significance: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. No significance (ns). Exact P values in Appendix Table . Circles and n represent biological repeats. .

Article Snippet: Coverslips were mounted and counterstained on 6 μl of VectaShield mounting agent with DAPI (Vector Lab, #H1200), and sealed with nail varnish before being imaged on Nikon’s Inverted Ti Eclipse equipped with an Andor Zyla sCMOS camera (2560 × 2160; 6.5 μm pixels).

Techniques: Fluorescence, Microscopy, Incubation, Staining, Expressing, Enzyme-linked Immunosorbent Assay, Infection, Imaging, Immunofluorescence

Journal: EMBO Molecular Medicine

Article Title: Typhoid toxin of Salmonella Typhi elicits host antimicrobial response during acute typhoid fever

doi: 10.1038/s44321-025-00347-8

Figure Lengend Snippet: ( A ) Fluorescence microscopy images of HepG2 intestinal cells, from three independent experiments, either untreated, treated with wild-type typhoid toxin (TxWT) or H160Q DNase-deficient toxin (TxHQ) for 2 h prior to imaging at 96 h of EdU (magenta) or APOC3 (yellow). DAPI-stained nuclear outlines shown. Scale bars: 50 μm. ( B ) Bar chart showing proportion of APOC3-positive cells ( n = 4), or ( C ) EdU-positive HepG2 cells ( n = 3), at 96 h. Circles indicate biological repeats. ( D ) Quantification of Salmonella CFUs following incubation with 50 mg/ml of purified APOC3 at 1 h, 2 h and 4 h ( n = 3). Statistical significance: Welch’s unpaired t-test for paired measures with unequal variances in ( B , C ), two-way ANOVA Sidak multiple comparisons ( D ) assessing 2 independent variables. Data are presented as mean ± SEM. Asterisks indicate significance: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. No significance (ns). Exact P values in Appendix Table . Circles and n represent biological replicates. Experiments in linked to Fig. . .

Article Snippet: Coverslips were mounted and counterstained on 6 μl of VectaShield mounting agent with DAPI (Vector Lab, #H1200), and sealed with nail varnish before being imaged on Nikon’s Inverted Ti Eclipse equipped with an Andor Zyla sCMOS camera (2560 × 2160; 6.5 μm pixels).

Techniques: Fluorescence, Microscopy, Imaging, Staining, Incubation, Purification

Journal: EMBO Molecular Medicine

Article Title: Typhoid toxin of Salmonella Typhi elicits host antimicrobial response during acute typhoid fever

doi: 10.1038/s44321-025-00347-8

Figure Lengend Snippet: ( A ) Representative fluorescence microscopy images of CACO2 cells either untreated or treated with 20 ng/ml TxWT or TxHQ for 2 h before imaging at 96 h. Images show γH2AX (cyan), LYZ (yellow) and EdU (magenta) with outlines of DAPI-stained nuclei. EdU nucleotide was incubated with cells for 24 h prior to fixation at 96 h. Magnified inset shows cell cycle-arrested cell producing LYZ. Scale bars: 50 μm. ( B ) Bar chart showing proportion of LYZ-expressing from cells in ( A ) ( n = 3). ( C ) ELISA of LYZ secreted into growth media harvested from cells in ( A ) ( n = 3). ( D ) Representative transmission electron microscopy (TEM) images from three independent experiments of S . Javiana either untreated or incubated with 1 mg/ml LYZ, 100 μg/ml LFN, LYZ and LFN, LYZ and 1 mM EDTA in M9 minimal media for 2 h. ( E ) Bar chart showing proportion of S . Javiana with spherical morphology from ( D ) ( n = 3). ( F ) Representative TEM from ( D ) highlighting changes in cell morphology indicative of S . Javiana spheroplast formation with LYZ and LFN. ( G ) LYZ and LFN treatment of S . Javiana in the presence of cephalexin. Left: schematic of cell elongation due to cephalexin and spheroplast formation. Right: Representative fluorescence microscopy images, from three independent experiments, of S . Javiana pFPV-mCherry in LB at OD 600 1.0 either untreated (left), or treated with cephalexin (right) prior to 20 min incubation with cephalexin only (control), LYZ, or LYZ and LFN. Top row: fluorescent images of DAPI-stained (blue) mCherry S . Javiana (red). Red arrows indicate elongated cephalexin-treated S . Javiana and yellow arrows spheroplasts incapable of mCherry retention due to LYZ and LFN. Bottom row: corresponding phase contrast images. Scale bars: 5 μm. Untreated, LYZ, and LYZ/LFN images reused in Fig. to show alongside additional controls. ( H ) Bar chart showing number of elongated S . Javiana (>5 μm) per field of view in ( G ) ( n = 3). Statistical significance: Welch’s unpaired t test in ( B , C ) for paired measures with unequal variances; one-way ANOVA with Dunnett’s post hoc test in ( E ) for analysing >3 groups versus control, or with Brown–Forsythe test in ( H ) for unequal variances (>3 groups). Data are presented as mean ± SEM. Asterisks indicate significance * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. No significance (ns). Exact P values in Appendix Table . Circles and n represent biological repeats. .

Article Snippet: Coverslips were mounted and counterstained on 6 μl of VectaShield mounting agent with DAPI (Vector Lab, #H1200), and sealed with nail varnish before being imaged on Nikon’s Inverted Ti Eclipse equipped with an Andor Zyla sCMOS camera (2560 × 2160; 6.5 μm pixels).

Techniques: Fluorescence, Microscopy, Imaging, Staining, Incubation, Expressing, Enzyme-linked Immunosorbent Assay, Transmission Assay, Electron Microscopy, Control

Journal: EMBO Molecular Medicine

Article Title: Typhoid toxin of Salmonella Typhi elicits host antimicrobial response during acute typhoid fever

doi: 10.1038/s44321-025-00347-8

Figure Lengend Snippet: ( A ) ELISA of LYZ using growth media from untreated or etoposide-treated (ETP) CACO2 cells at 96 h, or 10% FBS used to supplement growth media as control ( n = 3). ( B ) ELISA of LYZ using plasma from human participants in TYGER study at baseline (non-infect) or at typhoid diagnosis following S . Typhi infection (typhoid). Circles represent participants and biological repeats ( n = 3). ( C ) Fluorescence microscopy images of cephalexin-treated S . Javiana pFPV-mCherry treated with LYZ, LFN, LYZ-LFN or LYZ-EDTA, from three independent experiments, before imaging mCherry Salmonella and DAPI-staining by fluorescence microscopy (top panel) or phase contrast (bottom panel). Elongated bacteria >5 μm (yellow arrows), spheroplasts with loss of mCherry (green arrows), and spheroplasts with mCherry retention (white arrows). Untreated, LYZ, and LYZ/LFN images reused in Fig. . Scale bars: 5 μm. Bar charts showing the proportion of long bacteria (>5 μm) following cephalexin treatment of ( D ) S . Typhi or ( E ) S . Typhimurium in the absence (unt) or presence of LYZ, LFN, LYZ and LFN, LYZ and EDTA ( n = 3). Statistical significance: one-way ANOVA Tukey’s multiple comparison ( A ) analysing all pairs of >3 groups: Welch’s unpaired t test for paired measures with unequal variances in ( B ); one-way ANOVA with Brown–Forsythe ( D , E ) for unequal variances (>3 groups). Data are presented as mean ± SEM. Asterisks indicate significance: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. No significance (ns). Exact P values in Appendix Table . Circles represent biological replicates. Experiments in EV2 linked to Fig. .

Article Snippet: Coverslips were mounted and counterstained on 6 μl of VectaShield mounting agent with DAPI (Vector Lab, #H1200), and sealed with nail varnish before being imaged on Nikon’s Inverted Ti Eclipse equipped with an Andor Zyla sCMOS camera (2560 × 2160; 6.5 μm pixels).

Techniques: Enzyme-linked Immunosorbent Assay, Control, Clinical Proteomics, Biomarker Discovery, Infection, Fluorescence, Microscopy, Imaging, Staining, Bacteria, Comparison

Journal: EMBO Molecular Medicine

Article Title: Typhoid toxin of Salmonella Typhi elicits host antimicrobial response during acute typhoid fever

doi: 10.1038/s44321-025-00347-8

Figure Lengend Snippet: ( A ) Representative immunoblot from three independent experiments of S . Javiana in LB broth either untreated, or cultured with 1 mg/ml endogenous LYZ, 100 μg/ml LFN, LYZ and LFN, or 1 mg/ml recombinant LYZ (rec. LYZ) for 2 h. Whole cell lysates (WCLs) or supernatants immunoblotted with antibodies to virulence effectors SipB or SopE, T3SS component InvG, T1SS component TolC or the intracellular loading control DnaK. Molecular weight (MW) markers left. *indicates unidentified cross-reactive protein in SopE blot of whole cell lysate. Quantification of immunoblot band intensity quantified from n = 3 of experiment ( A ) for: ( B ) SopE, or ( C ) SipB. ( D ) Same experiment as ( A ) using attenuated S . Typhi BRD948, n = 2. ( E ) Immunoblot of whole cell lysates or supernatants of three independent experiments from S . Javiana cultured in LB only (untreated) or treated for 2 h with indicated concentrations of LYZ, or denatured LYZ (denat. LYZ). Immunoblotted with antibodies to SipB or DnaK. MW in kDa, left. ( F ) Quantification of immunoblot band intensity from n = 3 of experiment in ( E ). ( G ) S . Javiana (SJ) CFUs calculated on LB agar plates at 24 h post-infection of CACO2 cells already treated for 72 h with TxWT or TxHQ ( n = 4). ( H ) Salmonella infection of LYZ-depleted intoxicated cells. HCT116 cells were transfected with non-targeting or LYZ siRNA (siNT; siLYZ) for 48 h before treatment with TxWT and further 48 h incubation (96 h total). At 96 h, cells were infected with S . Javiana (SJ) and CFUs quantified at 2 h or 24 h on LB agar plates ( n = 4). ( I ) Localisation of endocytosed LYZ during S . Javiana infection ( n = 1). HCT116 cells were infected with S . Javiana pFPV-mCherry (mCherry Salmonella ) for 30 min (MOI 100) when 100 µg/ml LYZ-488 was added to infected cells to allow endocytosis and incubated for 2 h in gentamicin-containing media ( n = 3). Outlines of DAPI-stained nuclei. Arrows indicate colocalisation. Scale bar: 5 μm. Statistical significance: one-way ANOVA with Dunnett’s post hoc test ( B , C , F ) for analysing >3 groups versus control; Welch’s unpaired t test ( G ) for paired measures with unequal variances; two-way ANOVA Sidak’s multiple comparison in ( H ) assessing two independent variables. Data are presented as mean ± SEM. Asterisks indicate significance: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. No significance (ns). Exact P values in Appendix Table . Circles and n represent biological repeats. .

Article Snippet: Coverslips were mounted and counterstained on 6 μl of VectaShield mounting agent with DAPI (Vector Lab, #H1200), and sealed with nail varnish before being imaged on Nikon’s Inverted Ti Eclipse equipped with an Andor Zyla sCMOS camera (2560 × 2160; 6.5 μm pixels).

Techniques: Western Blot, Cell Culture, Recombinant, Control, Molecular Weight, Infection, Transfection, Incubation, Staining, Comparison

Journal: EMBO Molecular Medicine

Article Title: Typhoid toxin of Salmonella Typhi elicits host antimicrobial response during acute typhoid fever

doi: 10.1038/s44321-025-00347-8

Figure Lengend Snippet: ( A ) Fluorescence microscopy images of CACO2 cells treated with fresh media (untreated), 20 ng/ml TxWT or 8 µM etoposide (ETP) for 2 h, −/+ caffeine, at 48 h. Representative images, from three independent experiments, show γH2AX (magenta) and outlines of DAPI-stained nuclei. Scale bars: 50 μm. ( B ) Same experiment as ( A ) with imaging of APOC3 (yellow) and LYZ (cyan) ( n = 3). ( C ) Bar chart showing proportion of APOC3-positive cells or ( D ) LYZ-positive puncta per field of view, from experiment in ( B ) ( n = 3). ( E ) LYZ expression in CACO2, HCT116 and RKO intestinal epithelial cells at 72 h following no treatment (unt), or treatment with etoposide, TxHQ or TxWT ( n = 2). Immunoblots performed with indicated antibodies. MW in kDa, left. Statistical significance: Welch’s unpaired t test for paired measures with unequal variances in ( C , D ). Data presented as mean ± SEM. Asterisks indicate significance: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Exact P values in Appendix Table . Circles and n represent biological replicates. .

Article Snippet: Coverslips were mounted and counterstained on 6 μl of VectaShield mounting agent with DAPI (Vector Lab, #H1200), and sealed with nail varnish before being imaged on Nikon’s Inverted Ti Eclipse equipped with an Andor Zyla sCMOS camera (2560 × 2160; 6.5 μm pixels).

Techniques: Fluorescence, Microscopy, Staining, Imaging, Expressing, Western Blot

Journal: EMBO Molecular Medicine

Article Title: Typhoid toxin of Salmonella Typhi elicits host antimicrobial response during acute typhoid fever

doi: 10.1038/s44321-025-00347-8

Figure Lengend Snippet: ( A ) Fluorescence microscopy, from there independent experiments, of parental and TP53 -/- HCT116 cells treated with TxWT prior to imaging at 72 h of p53 (cyan), LYZ (yellow) and EdU (magenta). EdU was incubated with cells 24 h before fixation. DAPI-stained nuclear outlines shown. Scale bars: 50 μm. Bar chart quantifying ( B ) LYZ-positive cells ( n = 3), or ( C ) LYZ secretion from cells ( n = 3), from experiment ( A ). ( D ) Immunoblot showing LYZ expression and p53 responses in parental and TP53 −/− HCT116 cells at 72 h following 2 h treatment with TxHQ or TxWT ( n = 2). Antibodies indicated. MW markers left. ( E ) Immunoblot showing LYZ expression and p53 responses in HCT116 cells at 24 h following treatment with TxHQ or TxWT and incubation in complete media (−) or with addition of oxidative stress inhibitors CCCP, MitoQ, NADC or DPI ( n = 3). Antibodies indicated. MW markers left. Bar chart quantifying ( F ) p21 or ( G ) LYZ, band intensities from n = 3 of the experiment in ( E ). ( H ) Fluorescence microscopy images of oxidative stress in TxWT-treated HCT116 cells from three independent experiments, alone (control), or in the presence of added 1 µg/ml LYZ ( + exogenous LYZ). Oxidative damage to DNA determined using antibodies to 8-OHdG (yellow). Untreated control HCT116 cells and outlines of DAPI-stained nuclei shown. Arrows indicate nuclear 8-OHdG. Scale bars: 50 μm. ( I ) Bar chart quantifying 8-OHdG-positive nuclei from cells in experiment ( H ) ( n = 3). Statistical significance: Welch’s unpaired t test for paired measures with unequal variances in ( B , C ); one-way ANOVA Dunnett’s post hoc test ( F , G , I ) for analysing >3 groups versus control. Data are presented as mean ± SEM. Asterisks indicate significance: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. No significance (ns). Exact P values in Appendix Table . Circles and n represent biological replicates. .

Article Snippet: Coverslips were mounted and counterstained on 6 μl of VectaShield mounting agent with DAPI (Vector Lab, #H1200), and sealed with nail varnish before being imaged on Nikon’s Inverted Ti Eclipse equipped with an Andor Zyla sCMOS camera (2560 × 2160; 6.5 μm pixels).

Techniques: Fluorescence, Microscopy, Imaging, Incubation, Staining, Western Blot, Expressing, Control