Journal: Cell host & microbe
Article Title: Thymidine starvation promotes c-di-AMP-dependent inflammation during pathogenic bacterial infection.
doi: 10.1016/j.chom.2022.03.028
Figure Lengend Snippet: Figure 5. C-di-AMP promotes airway neutrophil infiltration (A–C) ELISA quantification of cytokines IL-6 (A), CXCL10 (B), and CCL5 (C) in BALF collected from the WT or Sting/ mice intranasally instilled with 15 mg Pam3CSK4 and 15 mg c-di-AMP or Pam3CSK4 alone. (D) Neutrophil numbers in BALF collected from the WT mice with intranasal instillation of 15 mg Pam3CSK4 and 15 mg c-di-AMP or Pam3CSK4 alone. (E) Ifnb1 expression in pBMDMs infected with dacA overexpression (NC/dacA) and its control (NC/vector) Newman strain relative to uninfected control. Mean values of triplicates are plotted and error bars indicate ±SD. p values were calculated using two-way ANOVA analysis. (F) CFU recovery from the right lung of WT mice intranasally infected with NC/dacA or NC/vector Newman strain at 8 hpi. (G and H) Neutrophil numbers (G) and active neutrophil elastase concentration (H) in BALF collected from mice infected in (F). Neutrophils are identified and enumerated by FACS. For all the mouse infections, biological replicates are plotted, and horizontal black bars are the median of the data. p values were calculated using Mann-Whitney analysis. Asterisks indicate statistical significance (*, p < 0.05; **, p < 0.01; ***, p < 0.001), and ns indicates no significant difference.
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Mouse CXCL10/IP-10/CRG-2 DuoSet ELISA R&D systems Cat# DY466-05 Mouse CCL5 Quantikine ELISA Kit R&D systems Cat# MMR00 Experimental Models: Cell Lines Murine: J2 immortalized BMDMs (Sauer et al., 2010) N/A Murine: WT, Tmem173-/-, cGAS-/- primary BMDMs This paper N/A Murine: ISRE-L929 cells (Woodward et al., 2010) N/A Experimental Models: Organisms/Strains Mouse: C57BL/6J The Jackson Laboratory JAX: 000664 Mouse: C57BL/6J Tmem173-/- (Brunette et al., 2012) N/A Mouse: C57BL/6J cGAS-/- (Brunette et al., 2012) N/A Oligonucleotides Mouse Hprt TaqMan probe Thermo Fisher Assay ID: Mm03024075_m1 Mouse Ifnb1 TaqMan probe Thermo Fisher Assay ID: Mm00439552_s1 Mouse Cxcl10 TaqMan probe Thermo Fisher Assay ID: Mm00445235_m1 Mouse Il6 TaqMan probe Thermo Fisher Assay ID: Mm00446190_m1 Oligonucleotides used in S. aureus mutant construction See Table S3 N/A Oligonucleotides used in construction of dacA or lmpde overexpression vectors See Table S3 N/A Oligonucleotides used in mouse genotyping See Table S3 N/A Recombinant DNA pIMAY (Monk et al., 2012) N/A pIMAY-derivative plasmids This paper N/A pBAV1K-E* (Topp et al., 2010) N/A pBAV1K-thyA expression plasmid This paper N/A pBAV1K-E*-dacA expression plasmid This paper N/A pEPSA5 (Whitney et al., 2017) N/A pEPSA5-Lmpde expression plasmid This paper N/A Software and Algorithms Prism 6.0 GraphPad https://www.graphpad.com/scientific-software/prism/; RRID:SCR_002798 Morpheus Broad Institute https://software.broadinstitute.org/morpheus/; RRID:SCR_017386 FlowJo v10.7 FlowJo https://www.flowjo.com/solutions/flowjo; RRID:SCR_008520 Adobe Photoshop Adobe https://www.adobe.com/products/photoshop.html; RRID:SCR_014199 Adobe Illustrator 2019 Adobe http://www.adobe.com/products/illustrator.html; RRID:SCR_010279 Image studio v5.2 LI-COR Biosciences https://www.licor.com/bio/image-studio-lite/; RRID:SCR_015795 Clustal Omega server Kyoto University Bioinformatics Center https://www.genome.jp/tools-bin/clustalw ApE (A plasmid Editor) M. Wayne Davis https://jorgensen.biology.utah.edu/wayned/ape/; RRID:SCR_014266 Other Synergy HTX Multi-Mode Reader BioTek N/A Cytek Aurora Cytek N/A Odyssey Fc Imaging System LI-COR Biosciences N/A CFX Connect Real-Time PCR Detection System Bio-Rad Cat# 1855200 (Continued on next page) e2 Cell Host & Microbe 30, 961–974.e1–e6, July 13, 2022
Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Infection, Over Expression, Control, Plasmid Preparation, Concentration Assay, MANN-WHITNEY
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