Journal: PLoS ONE

Article Title: Lack of Effective Anti-Apoptotic Activities Restricts Growth of Parachlamydiaceae in Insect Cells

doi: 10.1371/journal.pone.0029565

Figure Lengend Snippet: S2 cells were either left untreated or were treated with SPG buffer, amoebal lysate, infectious Parachlamydiaceae ( Pa. acanthamoebae or P. amoebophila ; MOI 5) in the absence (Inf) or presence of the protein synthesis inhibitor doxycycline (Inf-Dox), heat-inactivated bacteria (Hi), UV- inactivated bacteria (UVi), a sterile-filtrate of the suspension of purified infectious bacteria (Filtrate) or a supernatant collected 48 h p.i. from an infected (MOI 5) apoptotic culture (Supernatant). For comparison, cells treated with infectious S. negevensis Z (MOI 5 or 50, as indicated) are shown. After 48 h incubation, DNA was stained with DAPI and the proportion of nuclei with altered morphology was determined. Mean values and standard deviations of six replicates (derived from three independent experiments) are shown. At least 500 nuclei per replicate were considered. Statistically significant differences compared to the untreated cells are indicated (ANOVA & Scheffé; ***, p≤0.001; **, p≤0.01; *, p≤0.05).

Article Snippet: The following fluorescently labelled (Cy3, Cy5, or Fluos) probes (Thermo Fisher Scientific, Germany) were applied in this study: EUK-516 ( 5′- ACCAGACTTGCCCTCC -3′ ) for the detection of eukaryotic host cells, Chls0523 ( 5′-CCTCCGTATTACCGCAGC-3′ ) in combination with the competitor probe ( 5′-CCTCCGTATTACCGCGGC-3′ ) as general probe for the detection of all chlamydiae used in this study, and UV7-763 ( 5′-TGCTCCCCCTTGCTTTCG-3′ ) and Simneg183 ( 5′-CAGGCTACCCCAGCTC-3′ ) for the specific detection of Pa. acanthamoebae UV7 and S. negevensis Z, respectively.

Techniques: Purification, Infection, Incubation, Staining, Derivative Assay

Journal: Journal of Bacteriology

Article Title: Nucleotide Parasitism by Simkania negevensis ( Chlamydiae )

doi: 10.1128/JB.00919-10

Figure Lengend Snippet: Transcription of SnNTT1 to SnNTT4 during multiplication of S. negevensis within Acanthamoeba castellani UWC1. Lanes 1, amplification of cDNA synthesized from whole RNA from amoebae harboring S. negevensis; lanes 2, PCR positive control using DNA isolated from S. negevensis growing in amoebae; lanes 3, PCR negative control (no cDNA added); lanes 4, PCR using whole RNA from amoebae containing S. negevensis (control for the absence of DNA in the purified and DNase-digested RNA); m, molecular size marker.

Article Snippet: The Sn NTT1 to Sn NTT4 genes were amplified with the Extensor Hi-Fidelity PCR Enzyme Mix (ABgene), and the primers used for the amplification of the genes were designed based on draft genome sequence data for S. negevensis strain Z (see Table S1 in the supplemental material).

Techniques: Amplification, Synthesized, Positive Control, Isolation, Negative Control, Purification, Marker