Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Persistent Escherichia coli infection in renal tubular cells enhances calcium oxalate crystal–cell adhesion by inducing ezrin translocation to apical membranes via Rho/ROCK pathway

doi: 10.1007/s00018-022-04414-y

Figure Lengend Snippet: Establishment of persistent bacterial infection in renal tubular cells. A Schematic summary for induction of persistent E. coli infection in MDCK cells. B Cell morphology was observed under a phase-contrast light microscope at the end of each passage. C Intracellular bacteria were quantified by colony plate count on LB agar plate and reported as CFU/ml. D At the end of Passage 3, cell death was quantitated by flow cytometry using annexin V/propidium iodide staining. All quantitative data are reported as mean ± SEM derived from three independent experiments

Article Snippet: Quantification of intracellular bacteria At the end of each passage, cell morphology was examined and imaged under an inverted phase-contrast light microscope (Eclipse Ti-S) (Nikon; Tokyo, Japan).

Techniques: Infection, Light Microscopy, Bacteria, Flow Cytometry, Staining, Derivative Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Persistent Escherichia coli infection in renal tubular cells enhances calcium oxalate crystal–cell adhesion by inducing ezrin translocation to apical membranes via Rho/ROCK pathway

doi: 10.1007/s00018-022-04414-y

Figure Lengend Snippet: Expression and localization of ezrin in non-infected and persistently infected cells. Non-infected and infected MDCK cells (at the end of Passage 3) were subjected to evaluation for expression and localization of ezrin. A, B Western blot analysis of ezrin in whole cell lysate. A-tubulin served as a loading control. C, D Western blot analysis of ezrin in apical membrane and cytosolic fractions. E, F Immunofluorescence staining of ezrin with permeabilization. After immunostaining, the cells were examined under a laser-scanning confocal microscope. Fluorescence intensity data were measured from at least 10 high-power fields (HPFs) per sample in each experiment. All quantitative data are reported as mean ± SEM derived from three independent experiments. *p < 0.05 vs. non-infected cells; A.U. = arbitrary unit

Article Snippet: Quantification of intracellular bacteria At the end of each passage, cell morphology was examined and imaged under an inverted phase-contrast light microscope (Eclipse Ti-S) (Nikon; Tokyo, Japan).

Techniques: Expressing, Infection, Western Blot, Membrane, Immunofluorescence, Staining, Immunostaining, Microscopy, Fluorescence, Derivative Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Persistent Escherichia coli infection in renal tubular cells enhances calcium oxalate crystal–cell adhesion by inducing ezrin translocation to apical membranes via Rho/ROCK pathway

doi: 10.1007/s00018-022-04414-y

Figure Lengend Snippet: Effects of Y-27632, a ROCK inhibitor, on ezrin phosphorylation and apical surface expression, and COM crystal–cell adhesion in persistently infected cells. Non-infected and infected MDCK cells (at the end of Passage 3) were treated with 20 µM Y-27632 and further incubated for 24 h. The cells were then examined for ezrin phosphorylation and apical surface expression, and COM crystal-binding capability. A–C Western blot analysis of p-ezrin and total ezrin in whole cell lysate. A-tubulin served as a loading control. D, E Immunofluorescence staining of ezrin with permeabilization. After immunostaining, the cells were examined under a laser-scanning confocal microscope. Fluorescence intensity data were measured from at least ten high-power fields (HPFs) per sample in each experiment. F, G COM crystal-cell adhesion assay. Numbers of the adhered COM crystals were counted from at least 20 HPFs per sample in each experiment. All quantitative data are reported as mean ± SEM derived from three independent experiments. *p < 0.05 vs. non-infected cells; #p < 0.05 vs. infected cells; A.U. = arbitrary unit

Article Snippet: Quantification of intracellular bacteria At the end of each passage, cell morphology was examined and imaged under an inverted phase-contrast light microscope (Eclipse Ti-S) (Nikon; Tokyo, Japan).

Techniques: Expressing, Infection, Incubation, Binding Assay, Western Blot, Immunofluorescence, Staining, Immunostaining, Microscopy, Fluorescence, Cell Adhesion Assay, Derivative Assay