human adenocarcinoma alveolar basal epithelial cells a549  (ATCC)

Journal: Nucleic Acids Research

Article Title: A 5′-UTR cis -acting RNA element targeted by RNase III is essential for DNA simple sequence repeat-dependent phase variation in Haemophilus influenzae

doi: 10.1093/nar/gkaf1332

Figure Lengend Snippet: hmw 5′-UTR undergoes RNase III processing. ( A ) Mapping of the 5′-end of hmw1A mRNA by RNA circularization using total RNAs extracted from Rd KW20 (pTBH03-P hmw- 86–028NP ) WT and Δ rnc strains. All RNA samples were divided into two aliquots; one was treated with CAP-CLIP to eliminate the 5′-triphosphate, and the other one was left untreated. Ligated RNAs were subjected to RT-PCR and amplification products were run in 2% agarose gels. Bands were excised from the gels and cloned into pGEM-T Easy. The 5′-ends were identified by Sanger sequencing. Percentage of clones showing the same nucleotide position is indicated at each corresponding nucleotide. ( B, C ) RNase III processing of the MB- hmw1 5′-UTR. ( B ) The MB- hmw1 5′-UTR was incubated in the presence (squares) or absence (circles) of RNase III prior to detection of the FAM fluorescence as a function of the temperature. ( C ) The MB- hmw1 5′-UTR was incubated in the presence or absence of a range of RNase III concentrations (0.5, 0.25, 0.125, 0.065 µl) prior to electrophoretic separation under denaturing conditions. FAM fluorescence was detected on a ChemiDoc™ MP Imaging System. ( D ) Prediction of the primary and RNase III processed hmw 5′-UTR secondary structures. ( E, F ) Inactivation of the rnc gene regulates hmw1 gene expression and HMW phenotypes. ( E ) The amount of hmw1A transcript (top), quantified by RT-qPCR, was lower in rRdSΔ rnc than in the rRdS WT strain. Results of at least three independent experiments ( n ≥ 3) in triplicate are shown as the mean (relative quantification, 2 −ΔCt × 100) values ± standard deviations (SD). Statistical comparison of means was performed by unpaired t test (*, P < 0.05). The bottom panel shows HMW1A protein detection by western blotting. Total protein extracts were obtained from bacterial cultures grown to exponential phase, which were also used for RNA extraction. A stain-free gel portion is shown as loading control (LC). Quantification data obtained with ImageLab 6.1 software are shown. ( F ) A549 epithelial cell invasion (left) and biofilm formation (right) by WT and Δ rnc rRdS strains. Results of at least three independent experiments ( n ≥ 3) in triplicate are shown as mean of log 10 CFU per well ± SD (cell invasion) and as the mean of OD 570 /OD 600 ratios ± SD (biofilm growth). Statistical comparisons of the means were performed by unpaired t test (****; P < 0.0001).

Article Snippet: A549 human alveolar basal epithelial cells (ATCC CCL-185) were maintained as described [ ], seeded to 1.5 × 10 5 cells/well on 24-well plates for 32 h, then serum starved for 16 h before infection.

Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Clone Assay, Sequencing, Incubation, Fluorescence, Imaging, Gene Expression, Quantitative RT-PCR, Quantitative Proteomics, Comparison, Western Blot, RNA Extraction, Staining, Control, Software

Journal: Nucleic Acids Research

Article Title: A 5′-UTR cis -acting RNA element targeted by RNase III is essential for DNA simple sequence repeat-dependent phase variation in Haemophilus influenzae

doi: 10.1093/nar/gkaf1332

Figure Lengend Snippet: Quantification of naturally occurring HMW SSR phase variation in intracellular bacterial populations upon epithelial invasion enrichment: SSR length contraction. ( A ) Schematic representation of sample generation for amplicon PacBio-SMRTbell 3.0. sequencing to quantify SSR count distribution at the bacterial population level upon A549 epithelial cell gentamicin protection serial cycling for invasion enrichment. ( B ) Epithelial invasion serial enrichment by the rRdS-24SSR and rRdS-SSR-invert strains. A 5-day serial cycling experiment (days 0 to 4; pools from cycles 1 to 4, C0 to C4) was performed, and increased rRdS-24SSR epithelial invasion was selected. Results of at least three independent experiments performed in triplicate ( n ≥ 3) are shown as mean of log 10 CFU per well ± SD. Statistical comparisons of the means were performed by one-way analysis of variance (ANOVA) and Tukey’s multiple-comparison test (***, P < 0.001; ****, P < 0.0001). ( C ) Violin plots illustrating the distribution of SSR counts in rRdS-24SSR and rRdS-SSR-invert strains during serial cycles of Gm protection assay (pools from cycles C0, C1, C2, C3, and C4, recovered on days 0, 1, 2, 3, and 4, respectively). Data corresponding to two independent experiments (replicates 1R and 2R) have been pooled for representation. The X -axis represents passage number; the Y -axis represents SSR number distribution.

Article Snippet: A549 human alveolar basal epithelial cells (ATCC CCL-185) were maintained as described [ ], seeded to 1.5 × 10 5 cells/well on 24-well plates for 32 h, then serum starved for 16 h before infection.

Techniques: Amplification, Sequencing, Comparison