Journal:

Article Title: Deviant Expression of Rab5 on Phagosomes Containing the Intracellular Pathogens Mycobacterium tuberculosis and Legionella pneumophila Is Associated with Altered Phagosomal Fate

doi:

Figure Lengend Snippet: Growth of L. pneumophila and M. tuberculosis in THP-1, HeLa Tet-off, and HeLa-Rab5c cells. Monolayers of THP-1 macrophage-like cells, HeLa Tet-off cells, and HeLa-Rab5c cells expressing Rab5c were coincubated with L. pneumophila at a high MOI (2 × 108/ml) or a low MOI (2 × 107/ml) for 1 h (A), with L. pneumophila (2 × 108/ml) for 2 h (C), with M. tuberculosis (106/ml) for 2 h (B), or with M. tuberculosis (107/ml) for 2 h (D) at 37°C, washed, and incubated in fresh medium at 37°C. At sequential times thereafter, the monolayers were lysed and combined with the culture supernatant, and the number of CFU was determined by plating serial dilutions on CYE (A and C) or 7H11 (B and D) agar plates. The capacity of the bacteria to grow extracellularly in the culture medium was assessed by inoculating L. pneumophila (C) or M. tuberculosis (D) into wells containing only the culture medium or into parabiotic chambers in which the bacteria were separated from the HeLa cells by a 0.2-μm-pore-size filter. Although the bacteria are taken up much less efficiently by HeLa cells, they multiply, once inside, with a similar doubling time in HeLa cells and THP-1 cells (A and B). Overexpression of Rab5c does not alter the intracellular growth rate of L. pneumophila or M. tuberculosis in HeLa cells (C and D). The bacteria do not grow in the absence of cell monolayers or when separated from the monolayer in a parabiotic chamber (C and D). Data shown are the means ± the standard deviations of triplicate determinations.

Article Snippet: We cloned the human rab5c gene into pTRE, transfected the recombinant plasmids into HeLa Tet-off cells (Clontech) by calcium phosphate precipitation, and selected stably transfected clones with hygromycin in the presence of tetracycline.

Techniques: Expressing, Incubation, Over Expression

Journal:

Article Title: Deviant Expression of Rab5 on Phagosomes Containing the Intracellular Pathogens Mycobacterium tuberculosis and Legionella pneumophila Is Associated with Altered Phagosomal Fate

doi:

Figure Lengend Snippet: Phagosomes containing wild-type L. pneumophila but not avirulent L. pneumophila exclude Rab5c. Suspensions of wild-type (A) or avirulent (B and C) L. pneumophila were spun down onto monolayers of pTRE/rab5c-HeLa Tet-off cells at 4°C, incubated at 37°C for 15 min, and either fixed immediately (A and B) or washed, incubated in fresh culture medium for 6 h, and then fixed (C). Cells were processed for cryoimmunoelectron microscopy. Rab5c has been stained using 15-nm gold particles (arrowheads), and L. pneumophila LPS has been stained using 5-nm gold particles (arrows). Rab5c is absent from the wild-type L. pneumophila phagosome (A), despite the presence of Rab5c immunogold staining on vesicles adjacent to the phagosome. Rab5c is present on the avirulent L. pneumophila phagosome at 15 min (B) but is absent by 6 h (C). Magnifications, ×72,827 (A), ×38,235 (B), and ×50,664 (C).

Article Snippet: We cloned the human rab5c gene into pTRE, transfected the recombinant plasmids into HeLa Tet-off cells (Clontech) by calcium phosphate precipitation, and selected stably transfected clones with hygromycin in the presence of tetracycline.

Techniques: Incubation, Microscopy, Staining

Journal: Neuroscience

Article Title: Macroautophagy and the proteasome are differently involved in the degradation of alpha-synuclein wild type and mutated A30P in an in vitro inducible model (PC12/TetOn)

doi: 10.1016/j.neuroscience.2011.08.030

Figure Lengend Snippet: Development of an inducible α-syn expression system in PC12 cells. (A) Representative Western blot showing human α-syn-inducible expression. PC12 cells were transfected with a tetracycline responsive plasmid (pBI-G) carrying human α-syn(WT or A30P) full-length cDNAs as described in methods. The dynamic range of α-syn expression was tested by addition of the antibiotic doxycycline (doxy.) for 96 h to culture medium in a dose ranging between 0.1 and 1 μg/ml. The bar graph ( right ) is the densitometric analysis of three independent Western blot experiments, using α-tubulin immunoreactivity as normalization standard. (B) Immunocytochemistry of human α-syn expression (WT or A30P) in PC12/TetOn cells. The upper picture for each cell line is the basal expression in absence of doxycycline (−doxy), while the lower picture shows α-syn immunoreactivity after 48-h induction by 1 μg/ml doxycycline (+doxy) (magnification 10×; bar=10 μm). Alpha-syn expression at higher magnification (40×) before and after doxycycline induction (1 μg/ml for 48 h) is also presented (right-Bar=5 μm). (C) Western blot showing α-syn(WT) and (A30P) cellular bioavailability over time. The bar graphs ( right ) represent the densitometric analysis of three independent Western blot assays.

Article Snippet: A commercial PC12/TetOn cell line was purchased from Clontech (Palo Alto, CA, USA) ( ).

Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation, Immunocytochemistry

Journal: Neuroscience

Article Title: Macroautophagy and the proteasome are differently involved in the degradation of alpha-synuclein wild type and mutated A30P in an in vitro inducible model (PC12/TetOn)

doi: 10.1016/j.neuroscience.2011.08.030

Figure Lengend Snippet: Neuroprotective effect of human α-syn against oxidative stress without cellular toxicity. PC12/TetOn cells were seeded in 96-well plates at 100×10 3 cells/well and grown overnight. Next day, cells were exposed to doxycycline 0.1 μg/ml or 1 μg/ml for 48 h. Afterward, H 2 O 2 150 μM was added for further 72 h and then cell viability was assessed by MTT or cells were fixed with 4% PFA and stained with 0.05% thioflavin-T as described in methods. (A) Viability of PC12/TetOn cells expressing different levels of human α-syn(WT) challenged by H 2 O 2 ; (B) Viability assay of PC12/TetOn expressing α-syn(WT) after addition of doxycycline 1 μg/ml for increasing time intervals; (C) Evaluation of viability of PC12/TetOn expressing different levels of human α-syn(A30P) after oxidative challenge by H 2 O 2 . (D) Viability of PC12/TetOn stimulated with doxycycline 1 μg/ml to express human α-syn(A30P) for increasing time intervals. * P <0.05; ** P <0.01, one-way ANOVA followed by Tukey's post-hoc test. (E) Absence of α-syn aggregate formation assessed by thioflavin-T staining in PC12/TetOn cells after 48 h stimulation with doxycycline 1 μg/ml. The upper pictures show the basal condition (−doxy), while the +doxy conditions are the stimulated cells. (F) Representative dot blot assessing A11 reactivity in uninduced (−doxy) and induced (+doxy) condition for both cell lines at two different time-points (48 and 96 h) from doxycycline addition. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.

Article Snippet: A commercial PC12/TetOn cell line was purchased from Clontech (Palo Alto, CA, USA) ( ).

Techniques: Staining, Expressing, Viability Assay, Dot Blot

Journal: Neuroscience

Article Title: Macroautophagy and the proteasome are differently involved in the degradation of alpha-synuclein wild type and mutated A30P in an in vitro inducible model (PC12/TetOn)

doi: 10.1016/j.neuroscience.2011.08.030

Figure Lengend Snippet: Macroautophagy activation after doxycycline addition and α-syn expression in PC12/TetOn cells. Cells were seeded at 50×10 3 cell/well in a multiwell chamber slide and grown overnight. The next day, doxycycline was added (1 μg/ml) for 48 h and then macroautophagic organelle formation was monitored by LC3-II immunoreactivity (highlighted by arrows), while nuclei were stained by Hoechst 33342 . As positive control (CT+), PC12/TetOn mock-transfected with empty pBI-G vector were seeded as previously described but then were kept for 18 h in a starving condition (serum deprivation[SD]). (A–C) Control condition showing LC3-II immunoreactivity in uninduced PC12/TetOn cells (−doxy) or in PC12/TetOn mock transfected cells cultured in presence of complete medium (CM). Bar=10 μm (magnification 20). (D–F) LC3-II immunoreactivity as above but after 48 h induction by doxycycline (+doxy) or 18 h SD. Bar=10 μm (magnification 20×). (G–I) Control condition as in (A–C) but at higher magnification (60×; bar=2.5 μm). (J–L) Same situation as in (D–F) (60× magnification; bar=2.5 μm).

Article Snippet: A commercial PC12/TetOn cell line was purchased from Clontech (Palo Alto, CA, USA) ( ).

Techniques: Activation Assay, Expressing, Staining, Positive Control, Transfection, Plasmid Preparation, Cell Culture

Journal: Neuroscience

Article Title: Macroautophagy and the proteasome are differently involved in the degradation of alpha-synuclein wild type and mutated A30P in an in vitro inducible model (PC12/TetOn)

doi: 10.1016/j.neuroscience.2011.08.030

Figure Lengend Snippet: Inhibition of macroautophagy by 3-MA leads to α-syn(WT) accumulation, toxicity, and oligomer formation in PC12/TetOn. (A) PC12/TetOn cells expressing α-syn(WT) were exposed to 0.1 or 1 μg/ml of doxycycline for 48 h, and then the UPS inhibitor MG132 or the macroautophagy inhibitor 3-MA was added for further 18 h. Alpha-syn(WT) expression level was then quantified by Western blotting, using α-tubulin as internal standard. The blot quantification (three replicates for each condition) is shown in (B). **: P <0.01, one-way ANOVA followed by Tukey's post-hoc test. (C) The same experiment was carried out using PC12/TetOn overexpressing α-syn(A30P). The graph shown in (D) reports the quantification by densitometric analysis (three replicates for each condition). **: P <0.01, one-way ANOVA followed by Tukey's post-hoc test. (E) Cell viability assessment for the PC12/TetOn cells expressing α-syn(WT). The experimental scheme was the same described in (A). *: P <0.05; ** P <0.01, one-way ANOVA followed by Tukey's post-hoc test. (F) Cell viability assay for PC12/TetOn cells expressiong α-syn(A30P) after doxycycline stimulation. The experiment was carried out as detailed in (A); *: P <0.05, one-way ANOVA followed by Tukey's post-hoc test. (G) Representative dot blot showing the effect of 3-MA treatment on α-syn oligomer formation detected by A11 antibody. PC12/TetOn cells were incubated with doxycycline (1 μg/ml) for 48 h, and then 3-MA 10 mM was added for further 18 h. Oligomeric species formation was assessed by A11 reactivity. The bar graph in (H) shows the densitometric quantification of three independent replicates, performed by normalizing the A11 signal to α-tubulin immunoreactivity in the same blot (not shown). For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.

Article Snippet: A commercial PC12/TetOn cell line was purchased from Clontech (Palo Alto, CA, USA) ( ).

Techniques: Inhibition, Expressing, Western Blot, Viability Assay, Dot Blot, Incubation