Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet: PLY-EVs induce dendritic cell maturation and inflammatory cytokine release upon internalization (A) Confocal microscopy images showing the internalization of CFSE-labelled PLY (0.1) and naive EVs (green) by THP-1-monocyte-derived DCs at 24 h post-treatment. Scale bars, 25 μm. (B) Flow cytometry histograms ( N = 3) to quantify the DC uptake of CFSE-labeled PLY(0.5)EVs and naive EVs. (C) Dose-dependent uptake of PLY (0.1, 0.5) EVs by DCs. (D) Phase-contrast microscopy images of immature day 5 DCs coincubated with PLY (0.1, 0.5) EVs and naive EVs for 24 h. Arrows indicate matured DCs (magnified in inset). Scale bars, 50 μm. Images are representative of three independent experiments. (E–G) Flow cytometry histograms ( N = 3) to quantify the expression levels of (E) CD80, (F) CD86, and (G) CD83 on THP-1-monocyte-derived DCs treated with PLY(0.5) and naive EVs. (H and I) Flow cytometry histograms ( N = 2) showing the expression levels of DC maturation marker CD83 at 96 h post-incubation of primary human monocytes with (H) PLY(0.5) and naive EVs and (I) naive EVs pre-treated with recombinant PLY protein (naive EVs+rPLY). (J and K) Cytokine ELISA showing the levels of secreted TNF-α from (J) DCs treated with PLY (0.1) EVs or naive EVs alone ( N = 3) for 24 h and (K) DCs pre-treated with PLY (0.1,0.5) or naive EVs for 24 h followed by subsequent infection with S. pneumoniae , T4R strain ( N = 2). Recombinant PLY (0.5 μg/mL) was used as positive control. All data are represented as mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.005, and ∗∗∗ p < 0.001 by one-way ANOVA with Tukey’s multiple comparisons test. n.s., not significant. See also Figures S6–S9 .

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Confocal Microscopy, Derivative Assay, Flow Cytometry, Labeling, Microscopy, Expressing, Marker, Incubation, Recombinant, Enzyme-linked Immunosorbent Assay, Infection, Positive Control

Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet: Adoptive transfer of EVs from infected mice drives inflammation and pathology in a PLY-dependent manner (A) C57BL/6 mice were intranasally administered with 4 × 10 6 CFU of serotype 4 strain, T4 or the isogenic PLY mutant strain, T4Δply. At day 4 post-infection, EVs isolated from BALF were labeled and administered to healthy recipient mice at 35 μg/mice. The EV retention in murine respiratory tract was imaged by IVIS imaging and immune infiltration into lungs, and cytokine levels in BALF was measured. (B) Bacterial load in murine BALF ( N = 5 mice/group) upon infection with T4 and T4Δply strains was measured by CFU dilution assay. ∗∗ in (B) indicates p < 0.01 by Mann-Whitney test. (C) Quantification of relative total EV protein content from mice ( N = 3 mice/group) infected with T4 and T4Δply strains by BCA protein assay. PBS-treated mice served as control. ∗ and ∗∗ in (C) indicates p < 0.05 and p < 0.005, respectively, by unpaired t test. (D) IVIS imaging of mice intranasally administered with Nile-red-labeled EVs isolated from mice infected with T4 (EVs-T4) or T4Δply (EVs-T4Δply). EVs from PBS-treated mice (naive EVs) served as control. ROI intensity values indicate the total flux (photons/sec) recorded from the given region showing higher intensity of EVs from T4-infected mice in the respiratory tract. The color scale (photons/sec/cm 2 ) indicates the relative intensities of individual signals. (E and F) Flow cytometry analysis of inflammatory macrophages (F4/80 + ) and neutrophils (Ly6G + ) in BALF of mice ( N = 6 mice/group) administered with EVs from infected or untreated mice at 18 h. (G) TNF-α levels in the BALF of mice ( N = 5 mice/group) treated with EVs isolated from infected or untreated mice were measured post-sacrifice at 18 h by ELISA. ∗∗ and ∗∗∗ in (G) indicates p < 0.01 and p < 0.001, respectively, by unpaired t test. (H) Hematoxylin and eosin (H&E) staining of mouse lungs ( N = 6 mice/group) at 18 h post-administration of EVs from infected or PBS-treated mice. Mice treated with EVs from T4-infected mice showed tissue microlesions (MLEs) and immune cell infiltration in the alveolar interstitium indicative of PLY-induced tissue damage (magnified in the inset). BR, bronchiole; MLE, microlesions. Scale bars, 200 μm. Blind histopathological scoring was performed based on presence or absence of cellularity in alveolar interstitium and lesions. A score of “0” was given when no lesions were found, and a score of “1” was given to tissue showing increasing cellularity and lesions. Mouse BALF flow cytometry and histology data are representative of three independent experiments. All data are represented as mean ± SEM. See also Figure S12 .

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Adoptive Transfer Assay, Infection, Mutagenesis, Isolation, Labeling, Imaging, Dilution Assay, MANN-WHITNEY, Bicinchoninic Acid Protein Assay, Control, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Staining

Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet:

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Virus, Mutagenesis, Isolation, Recombinant, Modification, Saline, Labeling, Staining, Electron Microscopy, Lysis, Western Blot, Buffer Exchange, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, Clone Assay, Software, Membrane

Journal: bioRxiv

Article Title: ZCCHC8 is required for the degradation of pervasive transcripts originating from multiple genomic regulatory features

doi: 10.1101/2021.01.29.428898

Figure Lengend Snippet: (A) Schematic depicting the CRISPR/Cas9 strategy for disrupting Zcchc8 in SIMS mouse salivary gland cells. Single cell cloning produced three separate knockout clones with two-base-pair deletions ∼12-15 bp downstream of the TSS. The resulting frameshift produced a translational stop codon at amino acid position 16. (B) Western blot confirmation of ZCCHC8 ablation in three separate clones. Western blots were probed with a rabbit, polyclonal anti-ZCCHC8 antibody from Proteintech and a mouse, monoclonal anti-α-Tubulin antibody from Sigma. The composite image of the blot was produced using a LI-COR Odyssey CLx imaging system controlled by the LI-COR Image Studio software. (C) Immunofluorescence confirmation of ZCCHC8 ablation in three separate clones compared to wild-type and knockout of the unrelated gene Btbd7 (negative control). The ZCCHC8 immunofluorescence (greyscale) dynamic range was equal for all images. The same anti-ZCCHC8 antibody as in (B) was used. Note the presence and loss of nuclear ZCCHC8 staining compared to the continued presence of antibody cross-reactivity to primary cilia before and after Zcchc8 knockout. Actin fibers (red) were stained with a rhodamine-labeled phalloidin from Thermo Fisher. Actin images were processed using a rolling-ball background subtraction method. Scale bar = 20 μm.

Article Snippet: The membranes were then washed 3 times for 15 min each in TBST at room temperature and imaged on a LI-COR Odyssey CLx imaging system controlled by the LI-COR Image Studio software.

Techniques: CRISPR, Clone Assay, Produced, Knock-Out, Western Blot, Imaging, Software, Immunofluorescence, Negative Control, Staining, Labeling

Journal: bioRxiv

Article Title: Sterols lower energetic barriers of membrane bending and fission necessary for efficient clathrin mediated endocytosis

doi: 10.1101/2021.01.31.428633

Figure Lengend Snippet: A) Overview of recovery conditions. Following AY9944 sterol depletion, HEK293T hCLTA-Tq2 EN cells were exposed to cholesterol-loaded MβCD (MβCD-Chol, and chased in LPDS media) [Top] , supplemented continuously with lipoprotein-rich 15% FBS in the presence of AY9944 [Middle] , or incubated continuously in 7.5% LPDS media in the absence of AY9944 to allow endogenous cholesterol synthesis [Bottom] . B) Mid-plane live-cell confocal microscopy of HEK293T hCLTA-Tq2 EN following addition of transferrin-conjugated to AF-555 (Tfn-555) during recovery as described in (A). Representative images from different fields of view shown. Tfn and clathrin distributions in normal and arrested CME [Inset] . Scale bar = 10 μm. C) Corresponding cellular sterol profiles quantified by GC/MS (Mean ± SD). N = 3 biological replicates from independent experiments. Dotted and dashed lines represent total sterol abundance of untreated FBS and LPDS cultured cells, respectively.

Article Snippet: HEK293T cells were then incubated with transferrin (Tfn) conjugated to Alexa FluorTM 555 (25 ug/mL, Invitrogen) for 30 mins at 37°C, followed by rinsing in ice-cold PBS, and fixation in 4% paraformaldehyde for 20 min. For high-content imaging analysis, 24 well glass bottom plates (Cellvis) were imaged using CX7 Rescue High-Content Screening (HCS) Navigator software (PerkinElmer, Waltham, MA) and standard HCS imaging protocol.

Techniques: Incubation, Confocal Microscopy, Gas Chromatography-Mass Spectrometry, Cell Culture

Journal: bioRxiv

Article Title: Sterols lower energetic barriers of membrane bending and fission necessary for efficient clathrin mediated endocytosis

doi: 10.1101/2021.01.31.428633

Figure Lengend Snippet: The ability of structurally diverse sterols to rescue CME inhibition due to sterol depletion by AY9944 treatment was evaluated by direct delivery of sterols to the PM via MβCD carrier. A) Summary of physical properties and phase separation behavior of sterols. Topological Polar Surface Area (TPSA) computed surface sum over all polar atoms. Ordered lipid ( L o ) domain (raft) stabilizing (+) or disrupting (−) sterols indicated relative to cholesterol (++). Refer to Table S2 for additional details. B) Sterol profiles of AY9944 treated HEK293T cells following 1h incubation with sterol loaded MβCD (Mean ± SD). N = 4 independent biological replicates from two MβCD-sterol preparations. C) Tfn uptake relative to controls cultured in 7.5% LPDS for 48 h (Mean ± SD). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; one-way ANOVA (F(12, 51) = 11.23, p < 0.0001) and Dunnett’s test versus LPDS control (N = 5 biological replicates from 3 independent experiments, ~1,500 cells per replicate). Cholesterol, desmosterol, 7DHC, and lathosterol support formation of L o domains in model membranes as designated. D) Representative confocal images taken mid-plane following 30 min incubation with AF-555 conjugated transferrin (Tfn). Scale bar = 20 μm. (c), Commercially available, pre-loaded MβCD-Chol.

Article Snippet: HEK293T cells were then incubated with transferrin (Tfn) conjugated to Alexa FluorTM 555 (25 ug/mL, Invitrogen) for 30 mins at 37°C, followed by rinsing in ice-cold PBS, and fixation in 4% paraformaldehyde for 20 min. For high-content imaging analysis, 24 well glass bottom plates (Cellvis) were imaged using CX7 Rescue High-Content Screening (HCS) Navigator software (PerkinElmer, Waltham, MA) and standard HCS imaging protocol.

Techniques: Inhibition, Incubation, Cell Culture, Control