Journal: PLOS Pathogens

Article Title: Gelatinase regulates the egress of intracellular replicating populations during Enterococcus faecalis infection

doi: 10.1371/journal.ppat.1013738

Figure Lengend Snippet: (A) Quantification of intracellular replicating (eFluor - ) and non-replicating (eFluor + ) E. faecalis strains from macrophage lysates at 2, 6, and 20 hpi using flow cytometry. WT OG1RF fixed with 4% PFA prior to infection (Fixed WT) was included as non-proliferating controls. Representative histograms from n = 2-4 are shown. (B) Proportion (%) of intracellular replicating E. faecalis (eFluor - ) population from WT and mutant E. faecalis -infected macrophage lysates. Bars represent mean ± SD of n = 4, except fixed WT (n = 2). Statistical significance of each strain against WT was assessed using one-way ANOVA with Dunnett’s multiple comparisons test. Only comparisons with p < 0.05 are annotated. ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001. (C) Representative confocal microscopy images of RAW264.7 macrophages infected by E. faecalis strains pre-stained with eFluor 670 (magenta) at 20 hpi from n = 3. Samples were fixed and post-stained for Enterococcus -specific Group D antigen (green), double-stranded DNA (dsDNA; blue) and F-actin (white).

Article Snippet: Intracellular E. faecalis was labelled with 1:500 rabbit anti-Streptococcus Group D polyclonal antibody (anti-AgD; American Research Products, Cat# 12-6231D, USA) for 15 min at room temperature, followed by 1:1000 Alexa Fluor 488-conjugated goat anti-Rabbit IgG (Invitrogen, Cat# A11034, USA) for 15 min at room temperature.

Techniques: Flow Cytometry, Infection, Mutagenesis, Confocal Microscopy, Staining

Journal: PLOS Pathogens

Article Title: Gelatinase regulates the egress of intracellular replicating populations during Enterococcus faecalis infection

doi: 10.1371/journal.ppat.1013738

Figure Lengend Snippet: (A) LDH cytotoxicity quantification of culture supernatants from E. faecalis -infected macrophages at 2, 6 and 20 hpi using the antibiotic protection assay. Bars represent mean ± SD from n = 5. Statistical significance against WT was assessed using one-way ANOVA with Dunnett’s multiple comparisons test. A subset of this data comparing macrophage cytotoxicity from WT infection at 2, 6 and 20 hpi is shown in . (B-C) Quantification of extracellular egressed bacteria from culture supernatants of RAW264.7 macrophages infected with (B) fsrABDC/gelE deletion mutants and (C) gelE -complemented OG1RF strains at 6 to 12 hpi. Each data point represents mean ± SD of n = 3-4. At each timepoint, statistical significance against WT was assessed using two-way ANOVA with Dunnett’s multiple comparisons test. LOD = limit of detection. (D) Representative Z-projections of WT- and Δ gelE -infected RAW264.7 macrophages at 6, 9 and 12 hpi from (B) , captured with confocal microscopy and stained for Enterococcus specific Group D antigen (yellow), dsDNA (cyan), and F-actin (magenta) (n = 2). Orthogonal Z-axis projections along the blue and red lines are shown in the adjacent colored boxes. White and yellow arrows indicate matched top view and Z-axis projections of selected macrophages with dense intracellular E. faecalis . For all graphs, only comparisons with p < 0.05 are annotated. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.

Article Snippet: Intracellular E. faecalis was labelled with 1:500 rabbit anti-Streptococcus Group D polyclonal antibody (anti-AgD; American Research Products, Cat# 12-6231D, USA) for 15 min at room temperature, followed by 1:1000 Alexa Fluor 488-conjugated goat anti-Rabbit IgG (Invitrogen, Cat# A11034, USA) for 15 min at room temperature.

Techniques: Infection, Bacteria, Confocal Microscopy, Staining

Journal: PLOS Pathogens

Article Title: Gelatinase regulates the egress of intracellular replicating populations during Enterococcus faecalis infection

doi: 10.1371/journal.ppat.1013738

Figure Lengend Snippet: (A-B) Total CFU from wound homogenates of (A) mono-infection or (B) competitive infection at 1, 3, and 5 dpi. Median of 3-5 animals per group from one independent experiment is shown. Dotted lines show inoculum CFU for mono-infection. (C) Flow cytometry analysis of intracellular E. faecalis (stained for Group D antigen) in various immune cells at 5 dpi. Median from 6-9 animals per group from 2 independent experiments is shown. (D-E) Quantification of (D) intracellular CFU at 1, 3 and 5 dpi or (E) total CFU at 5 dpi from enzymatically dissociated wounds. Median from 4-5 animals per group from one independent experiment is shown. For A-D, only comparisons with p < 0.05 are annotated. At each timepoint, statistical significance between infection groups was assessed using Mann-Whitney test, except for C, which was assessed using Kruskal-Wallis test with Dunn's multiple comparisons test. * = p < 0.05, ** = p < 0.01, *** = p < 0.001. n.s. = not significant.

Article Snippet: Intracellular E. faecalis was labelled with 1:500 rabbit anti-Streptococcus Group D polyclonal antibody (anti-AgD; American Research Products, Cat# 12-6231D, USA) for 15 min at room temperature, followed by 1:1000 Alexa Fluor 488-conjugated goat anti-Rabbit IgG (Invitrogen, Cat# A11034, USA) for 15 min at room temperature.

Techniques: Infection, Flow Cytometry, Staining, MANN-WHITNEY

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Chronic psychological stress promotes breast cancer pre-metastatic niche formation by mobilizing splenic MDSCs via TAM/CXCL1 signaling

doi: 10.1186/s13046-023-02696-z

Figure Lengend Snippet: Chronic unpredictable mild stress (CUMS) promotes the formation of an immunosuppressive microenvironment in breast cancer. A Flowchart of the mouse experiments. B The representative mouse movement trajectories of each group in the open field tests were shown. Total immobility duration, total distance in the open field tests, as well as the percentage of sucrose water consumed in the sucrose preference tests were calculated ( n = 6). C Representative pictures and growth curves of tumors in each group ( n = 6). D In vivo imaging of lung metastatic lesions and the quantification of bioluminescence intensity in each group ( n = 5, upper panel). Representative images of lung metastasis lesions in gross and HE staining (black circle indicates the metastatic lesions of the lung, and the scale bars indicate 1000 μm) and the quantification of metastatic lesion area in each group ( n = 3, lower panel). E Representative multiplexed immunofluorescence images showing the tumor immune microenvironment of control (scale bar 2000 μm) and CUMS groups (scale bar 800 μm), using antibodies CD11b, Ly6G, FOXP3, CD8, and CD206. The proportion of CD11b + CD206 + , CD11b + Ly6G + , CD8 + , and FOXP3 + were quantified ( n = 6). F Flow cytometry analysis of F4-80 + CD206 + -TAMs population in the tumor tissue. Data are represented as the Mean ± SD. For statistical analysis, unpaired t-tests ( B , D , F ) and repeated measures analysis of variance ( C ) were applied. * P < 0.05, # P < 0.01

Article Snippet: Primary antibodies included Pan-Keratin antibody (Cell Signaling Technology Cat# 4545, RRID:AB_490860), CD11b antibody (Abcam Cat# ab133357, RRID:AB_2650514), CD8 antibody (Abcam Cat# ab217344, RRID:AB_2890649), FoxP3 antibody (Cell Signaling Technology Cat# 12,653, RRID:AB_2797979), CD206 antibody (Cell Signaling Technology Cat# 24,595, RRID:AB_2892682), Ly6g antibody (Abcam Cat# ab238132, RRID:AB_2923218).

Techniques: In Vivo Imaging, Staining, Immunofluorescence, Control, Flow Cytometry