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. 2015 Feb 26;19(5):1133–1150. doi: 10.1111/jcmm.12518

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© 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

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Spatial alterations in autophagosomal LC3 in the presence of microtubule-interfering agents. Assessment of autophagic flux in BMSCs challenged with Staphylococcus epidermidis. Analysis of immunoreactivity of LC3 protein in BMSC cells is shown in A–D. BMSC mitochondria were visualized with immunofluorescence of Tom20, a mitochondrial marker. The images shown in the figure are representative ones of analyses of three specimens obtained from three experiments. (A1) LC3-projections (green channel) in control BMSCs. (B1) LC3-projections (green channel) in BMSCs fixed at 24 hrs following the bacterial challenge. (C1) Same as (B1), but BMSCs were incubated with 50 μM vinblastine for 6 hrs before fixation. Accumulation of LC3-positive puncta appeared in images displayed in B1 and C1. (A2–C2) Relative intensities of the LC3-immunofluorescence projections presented in the respective panels (A1–C1) Projection of the nuclear DNA appeared in blue (counterstaining of nuclei with Hoechst 33342) in A1, A3, B1, B3, C1, C3 and C4. Spatial interactions of bacterial DNA with LC3-positive puncta appears in turquoise as result of interference of green and blue colours (indicated with pink arrows in B1 and C3). Projections of Tom20 (red channel) and LC3 (green channel) are displayed in panels A3, B3 and C4. Co-localization of LC3-positive puncta with Tom20 appeared in yellow (as result of interference of green and red) and is indicated with white arrows in B3 and C4. The confocal images were taken with pinhole setup to obtain 0.3 μm Z-sections. For assessment of the amounts of LC3-positive puncta, only puncta with diameter ≥0.2 μm were counted. (D) Immunoblot assessment of effect of Rab7 siRNA on amount of LC3I/LC3II in BMSCs challenged with S. epidermidis. The cells were treated with lipofectamine in the absence (-) or presence (+) of 10 nM Rab7 siRNA overnight, (http://tools.lifetechnologies.com/content/sfs/manuals/cms_084833.pdf). The protein extracts were obtained from the BMSC cultures 24 hrs after a challenge with S. epidermidis.