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, Images were digitally captured with GATAN Orius camera (Digital Micrograph Software). Magnification x12,000, scale bar: 1 µm

, speck-like aggregate formation and IL-1? secretion by WT MG-63 vs CASP1 -/-MG-63 cells exposed to S. aureus A. Analysis of mRNA levels of NLRP3 in WT MG-63

, MG-63 and CASP1 -/-MG-63 G2 cells were treated with 1 mg/mL ATP for 30 min

B. Wt, MG-63 or CASP1 -/-MG-63 G2 cells were exposed to S. aureus LAC (USA300) for 2 h. Following fixation and permeabilization of cells, NLRP3 expression at the protein level was determined by FACS using Rat Anti-Human NLRP3

, Alexa Fluor® 488-conjugated Monoclonal Antibody or isotype control antibody (blue colour) per 10 6 cells. NLRP3 protein expression of untreated (red colour) or infected (black colour) cells were analyzed with an Accuri C6 flow cytometer. Data were collected from 20,000 cells, and analyzed with CFlow software

, The major density of events is captured by the gate. The events that represent debris, cell fragments and pyknotic cells are eliminated. Values shown on the right side of the graph refer to the respective mean fluorescence intensities (MFIs), Cells are analyzed using FSC-A x SSC-A plot, vol.50

C. and D. Wt-mg-, 63 or CASP1 -/-MG-63 cells were exposed to 1 ?g/mL LPS for 30 min and to 5 mM ATP for 15 min (LPS+ATP) (D) or to S, vol.50, p.1

, Six hours post-infection cells were immunostained with rabbit anti PYCARD antibody (Coger France), followed by incubation with Alexa Fluor 488 labeled goat anti-rabbit antibody (Cell Signaling Ozyme) at a dilution of 1:20 for 2 h at room temperature (green staining, green arrows). Nuclear DNA was labeled with DAPI (blue staining). Samples were viewed with a Zeiss laser-scanning microscope equipped with a 63 × plan Apo-NA 1.4 immersion objective driven by Zen software, A. WT MG-63 or CASP1 -/-MG-63 cells were exposed to a fluorescent derivative of S. aureus SA113, which carries plasmid-encoded mCherry (red fluorescence, red arrows)

B. Wt, MG-63 or CASP1 -/-MG-63 G2 cells were exposed to S. aureus SA113 at MOI 50:1 for 6 h. To quantify the amount of ASC-speck forming cells the number of ASC speck out of 100 cells in the culture of WT MG-63 cells vs CASP1 -/-MG-63 G2 clone was enumerated. The results from three

C. Wt, MG-63 or CASP1 -/-MG-63 cells were exposed to S. aureus MW2 strain at MOI 50:1 for 2 h followed by antibiotic treatment as described. Six hours post-infection cells were lysed with 0.05% Triton X-100 in PBS, cell lysates were plated on BHI agar, and CFU were determined after overnight incubation

, After various times post-infection (6 h, 2 days, 5 days, 7, days and 11 days) cell supernatants were collected, centrifuged and the level of IL-1? was determined by a sandwich-ELISA (Thermofischer Life Technologies) as described in Experimental procedures. The values are presented as concentrations in pg/mL. Three independent assays were performed. The differences among the groups were assessed by the analysis of variance (ANOVA). P-values < 0.05 ( ? ) were considered to be significant. Tukey's Honestly Significant Difference test was applied for comparison of means between the groups, Figure 6. S. aureus strain-dependent release of IL-1? by infected MG-63 cells A

B. Wt, MG-63 cells were exposed to S. aureus strains LAC (USA300, p.2

, After various times post-infection (6 h, 2 days, 5 days, 7, days and 11 days) cell supernatants were collected, centrifuged and the level of IL-1? was determined by a commercial sandwich-ELISA (Invitrogen, France) as described in Experimental procedures. The values are presented as concentrations in pg/mL. Three independent assays were performed. The differences among the groups were assessed by analysis of variance (ANOVA)

A. Wt, After various time post-infection (2 days and 5 days) cell supernatants were collected, centrifuged and the level of IL-1? was determined by a sandwich-ELISA (Thermofischer Life Technologies) as described in Experimental procedures. The IL-1? values are presented as concentrations in pg/mL

, Tukey's Honestly Significant Difference test was applied for comparison of means between the groups. The values are expressed as means ± standard deviation (±SD)

, ( ? ) were considered to be significant. Three independent assays were performed

B. , MG-63 cells were exposed to USA300 LAC (pTX?16), which carries the control plasmid, the deletion mutant LAC?psm??hld (pTX?16) and the complemented strains expressing the four PSM? peptides (LAC?psm??hld-pTX??1-4), the two PSM? peptides (LAC?psm??hld-pTX??1-2), or the ?-toxin (LAC?psm??hld-pTX?hld) at MOI 50:1 for 2 h followed by antibiotic treatment as

, After various times post-infection (2 days, 5 days, 7 days and 9 days) cell supernatants were collected, centrifuged and the level of IL-1? was determined by a sandwich-ELISA (Thermofischer Life Technologies) as describe in Experimental procedures. The values are presented as concentrations in pg/mL. The differences among the groups

C. Wt, MG-63 or CASP1 -/-MG-63 cells were exposed to USA300 LAC (pTX?16), which carries the control plasmid, the deletion mutant LAC?psm??hld (pTX?16) and the complemented strains expressing the four PSM? peptides

, PSM? peptides (LAC?psm??hld-pTX??1-2), or the ?-toxin (LAC?psm??hld-pTX?hld) at MOI 50:1 for 2 h followed by antibiotic treatment as described. Six hours post-infection cells were lysed with 0.05% Triton X-100 in PBS

A. Wt, MG-63 or CASP1 -/-MG-63 G2 cells were grown in 12-well plates overnight, then were exposed to S. aureus SA113 strain at MOI 50:1 for 2 h followed by antibiotic treatment as described. Two hours, 6 h and 24 h post-infection cells were lysed with

, Experiments were performed in triplicate. Three independent assays were performed. The data are presented as means ± SD. Tukey's Honestly Significant Difference test was applied for comparison of means between the groups. P ? 0.01 (**), for the comparison of the number of internalized bacteria in CASP1 -/-MG-63 cells with those in WT MG-63 cells and P-values < 0.05 ( ? ) for the comparison of the number of internalized bacteria in CASP1 -/-MG-63 cells 6, 05% Triton X-100 in PBS, cell lysates were plated on BHI agar, and CFU were determined after overnight incubation. CFU values were normalized to 10 5 host cells

, 63 cells were grown on the slides of 12-well plates overnight, then cells were exposed to a fluorescent derivative of strain S. aureus SA113 fluorescent, which carries plasmid-encoded mCherry (red fluorescence), at MOI 50:1 for 2 h followed by antibiotic treatment as described. Six hours and 24 h postinfection cells were stained with rabbit anti-PYCARD antibody (Coger France), followed by incubation with Alexa Fluor 488 labeled goat anti-rabbit antibody, Cell Signaling Ozyme

, Phase contrast and fluorescent image of host cells bearing S. aureus were employed to demonstrate the intracellular localization of bacteria (Inserts). We have presented inset boxes

, WT MG-63 or CASP1 -/-MG-63 cells were exposed to a fluorescent derivative of S

, aureus SA113 at MOI 50:1 for 2 h followed by antibiotic treatment as described in Experimental procedures. Six hours post-infection nuclear DNA was labeled with DAPI

B. , Reconstituted 3D images of S. aureus-infected host cells were obtained by Z-stack, showing internalized S. aureus bacteria in the cytoplasm of infected cells

C. , Transmission electron micrographs of WT MG-63 or CASP1 -/-MG-63 G2 cells infected with SA113 strain at MOI 50:1 for 6 h. Cells were fixed as described in Experimental procedures. The pellets were mixed with 3% agar in sodium cacodylate, embedded in Epon-Araldite-DMP30 resin mixture and polymerized for 48 h. Sections were cut in Leica ultra microtome, stained with uranyl acetate and were analyzed with JEOL 1400 Electron Microscope (Jeol