Autophagy induction in response to infection of <i>M. tuberculosis</i> in DC cell lines.

<p>(A, B, C) LC3 localization to mycobacteria in dendritic cell line. DC2.4 (A) or JAWSII (B) cells were infected with <i>M. tuberculosis</i> for 24 h and immunostained with anti-LC3 antibody. The proportion of LC3-positive mycobacteria in these cell lines is also shown (C). Data represent the mean and SD of three independent experiments. (D) Immunoblot analysis of LC3 processing in DC2.4 cells infected with <i>M. tuberculosis</i> or <i>M. bovis</i> BCG for 24 h. (E) Autophagic flux in <i>M. tuberculosis</i>-infected DC2.4 cells. DC2.4 cells treated with or without protease inhibitors, E64d (10 μg/ml) and pepstatin A (10 μg/ml), were infected with <i>M. tuberculosis</i> for 24 h at the indicated multiplicity of infection (MOI). *Non-specific band.</p

Atg5-dependent localization of LAMP1 and MHC class II to mycobacterial autophagosomes.

<p>(A-C) Thin-section electron micrograph of <i>M. tuberculosis</i> bacilli in p62- or Atg5-knockdown DC. DC2.4 cells transfected with siRNA for control (A), p62 (B) or Atg5 (C) were infected with <i>M. tuberculosis</i> for 24 h and observed by thin-section electron microscopy. Autophagosomes are indicated by arrowheads. (D) Proportion of mycobacteria in multi-membrane structures in DC2.4 transfected with siRNA for p62 or Atg5. (E, F) The proportion of LAMP1 (E) or MHC class II (F) localization to ubiquitin-positive mycobacteria is shown. JAWSII cells transfected with control or Atg5 siRNA for 24 h were infected with Alexa Fluor 405-labeled <i>M. tuberculosis</i> and immunostained with anti-LAMP1 and anti-ubiquitin antibodies or anti-MHC class II and anti-ubiquitin antibodies. Data represent the mean and SD of three independent experiments. *<i>p</i> < 0.05 (unpaired Student’s <i>t</i>-test).</p

Maturation of mycobacterial autophagosomes in DC2.4 cells.

<div><p>(A) Recruitment of p62 to LC3-positive mycobacteria. DC2.4 cells were infected with Alexa Fluor 405-labeled <i>M. tuberculosis</i> (blue) for 24 h and immunostained with anti-LC3 (green) and anti-p62 antibodies (red). (B) The proportion of p62 localization to LC3-positive mycobacteria in DC2.4 cells. </p> <p>(C) Recruitment of ubiquitin to p62-positive mycobacteria. DC2.4 cells were infected with Alexa Fluor 405-labeled <i>M. tuberculosis</i> (blue) for 24 h and immunostained with anti-p62 (red) and anti-ubiquitin antibodies (green). (D) The proportion of ubiquitin localization to p62-positive mycobacteria in DC2.4 cells. Data represent the mean and SD of three independent experiments.</p></div

Localization of autophagosome markers to <i>M. tuberculosis</i> in BMDC.

<p>Bone marrow-derived macrophages (BMM) or dendritic cells (BMDC) were infected with DsRed-expressing <i>M. tuberculosis</i> for 24 h and immunostained with anti-LC3 (A, B), anti-p62 (D, E) or anti-ubiquitin (G, H) antibody. The proportion of LC3-positive (C), p62-positive (F) or ubiquitin-positive (I) <i>M. tuberculosis</i> in BMM or BMDC is also shown. Data represent the mean and SD of three independent experiments.</p

Mycobacterial autolysosome biogenesis.

<p>(A, C) Localization of LAMP1 to p62-positive of ubiquitin-positive mycobacteria in DC2.4 cells. DC2.4 cells were infected with Alexa Fluor 405-labeled <i>M. tuberculosis</i> (blue) for 24 h and immunostained with anti-LAMP1 (red) and anti-p62 antibodies (green) (A) or anti-LAMP1 (red) and anti-ubiquitin antibodies (green) (C). (B, D) The proportion of LAMP1-localized p62-positive (B) or ubiquitin-positive (D) mycobacteria. (E) Localization of DQ-BSA to mycobacterial autophagosomes. DC2.4 cells were preloaded with DQ-BSA to label degradative vesicles. DC were infected with Alexa Fluor 405-labeled mycobacteria for 24 h and immunostained with anti-p62 antibody. (F) The proportion of DQ-BSA-labeled p62-positive mycobacteria in DC2.4 cells. Data represent the mean and SD of three independent experiments.</p

Localization of MHC class II to mycobacterial autophagosomes in DC.

<p>(A, C) Localization of MHC class II to p62-positive or ubiquitin-positive mycobacteria in JAWSII cells. JAWSII cells were infected with Alexa Fluor 405-labeled <i>M. tuberculosis</i> (blue) for 24 h and immunostained with anti-MHC class II (green) and anti-p62 antibodies (red) (A) or anti-MHC class II (green) and anti-ubiquitin antibodies (red) (C). (B, D) The proportion of MHCII-localized p62-positive (B) orubiquitin-positive (D) mycobacteria in JAWSII cells. Data represent the mean and SD of three independent experiments.</p

p62-dependent ubiquitination of mycobacteria in DC.

<p>(A) The proportion of LC3, p62 or ubiquitin recruitment to mycobacteria in DC treated with 3-MA. DC2.4 were infected with DsRed-expressing <i>M. tuberculosis</i> for 24 h with or without 3-MA and immunostained with anti-LC3, anti-p62 or anti-ubiquitin antibodies. Data represent the mean and SD of three or four independent experiments. *<i>p</i> < 0.05 (unpaired Student’s <i>t</i>-test). (B) Immunoblot analysis on the silencing effects of p62 and Atg5. DC2.4 cells were transfected with siRNA for p62 or Atg5 for 48 h and subjected to immunoblot analysis using indicated antibodies. (C, D) The proportion of p62 or ubiquitin recruitment to mycobacteria in DC. DC2.4 cells transfected with siRNA for p62 or Atg5 were infected with DsRed-expressing <i>M. tuberculosis</i> for 12 h and immunostained with anti-p62 (C) or anti-ubiquitin (D) antibody. Data represent the mean and SD of three independent experiments. *<i>p</i> < 0.05 (unpaired Student’s <i>t</i>-test).</p

Augmentation of autophagy induced by LPS in Rab39a-KD macrophages.

<p>(A) Immunostaining analysis of LPS-induced autophagy in Rab39a-KD macrophages. Raw264.7 macrophages transfected with control or Rab39a siRNA were treated with LPS for 24 h and immunostained with anti-LC3 antibody. An arrowhead indicates an LC3-dot fluorescence. (B) Proportion of Raw264.7 macrophages with LC3-dots induced by LPS stimulation for 24 h. Data represent the mean and SD of three or four independent experiments. *<i>p</i> < 0.05 (unpaired Student’s <i>t</i>-test). (C) Immunoblot analysis of LC3 processing in macrophages treated with LPS. Raw264.7 macrophages transfected with control or Rab39a siRNA were treated with LPS for 24 h and subjected to immunoblot analysis using indicated antibodies. (D) Autophagic flux in Rab39a-KD macrophages treated with LPS. Raw264.7 macrophages treated with control or Rab39a siRNA were treated with LPS in the absence or presence of protease inhibitors, E64d and pepstatin A, for 24 h. Cell lysates were subjected to immunoblot analysis using indicated antibodies.</p

Rab39a Interacts with Phosphatidylinositol 3-Kinase and Negatively Regulates Autophagy Induced by Lipopolysaccharide Stimulation in Macrophages

<div><p>Rab39a has pleiotropic functions in phagosome maturation, inflammatory activation and neuritogenesis. Here, we characterized Rab39a function in membrane trafficking of phagocytosis and autophagy induction in macrophages. Rab39a localized to the periphery of LAMP2-positive vesicles and showed the similar kinetics on the phagosome to that of LAMP1. The depletion of Rab39a did not influence the localization of LAMP2 to the phagosome, but it augments the autophagosome formation and LC3 processing by lipopolysaccharide (LPS) stimulation. The augmentation of autophagosome formation in Rab39a-knockdown macrophages was suppressed by Atg5 depletion or an inhibitor for phosphatidylinostol 3-kinase (PI3K). Immunoprecipitation analysis revealed that Rab39a interacts with PI3K and that the amino acid residues from 34^th to 41^st in Rab39a were indispensable for this interaction. These results suggest that Rab39a negatively regulates the LPS-induced autophagy in macrophages.</p> </div

Involvement of Caspase-1 and classical autophagy pathway in autophagy induced by LPS

<p>(A) Autophagy induced by LSP in Caspase-1-KD macrophages. The proportion of macrophages with LC3-dot (upper panel) and immunoblot analysis of LC3 processing (lower panel) are shown. (B) Autophagy induced by LSP in Atg5-KD macrophages. The proportion of macrophages with LC3-dot (upper panel) and immunoblot analysis of LC3 processing (lower panel) are shown. (C) Autophagy induced by LPS in the presence of a PI3K inhibitor, 3-MA. Transfected macrophages were stimulated by LPS in the presence or absence of 3-MA at 10 mM for 24 h. The proportion of macrophages with LC3-dot (upper panel) and immunoblot analysis of LC3 processing (lower panel) are shown. Data represent the mean and SD of three or four independent experiments. *<i>p</i> < 0.05 (unpaired Student’s <i>t</i>-test).</p