Journal: Aging Cell

Article Title: The HIV proteins Tat and Nef promote human bone marrow mesenchymal stem cell senescence and alter osteoblastic differentiation

doi: 10.1111/acel.12308

Figure Lengend Snippet: Nef impedes the autophagic efflux in MSCs: prevention by rapamycin. MSCs were treated with the HIV proteins Tat and Nef for up to 20 days. The level of autophagic activity was determined by LC3-II staining of autophagic vesicles, as stated in Experimental procedures. Total fluorescence of LC3-II was evaluated, and results are expressed as per cent of control cells (40× magnification) (A). Representative micrographs of LC3-II-labelled and DAPI-stained cells are shown (100× magnification) (B). Whole-cell lysates were extracted from MSCs at day 10 and day 20 of treatment with the HIV proteins and analysed by immunoblotting. Representative immunoblots of LC3 and tubulin (loading control) and the relative quantification of LC3II/I ratio are shown (C). Representative immunoblots of p62 and tubulin (loading control) are shown (D). Whole- cell lysates were extracted from MSCs at day 20 of treatment with histidine-tagged Nef, and Beclin-1 was immunoprecipitated. Representative immunoblots of Nef (using an anti-his-tag antibody) and beclin-1 are shown (E). Rapamycin (Rapa) was added from day 15 to day 20. The level of autophagic activity was determined by with LC3-II staining of autophagic vesicles, as stated in Experimental procedures. Total fluorescence of LC3-II was evaluated and results are expressed as % of control cells (F). Representative immunoblots of LC3 and tubulin (loading control) and the relative quantification of LC3II/I ratio are shown (G). Senescence was evaluated by SA-β- galactosidase activity and expressed as the total percentage of SA-β-galactosidase cells at pH6 (H). Lysosomal accumulation was assessed by LysoTracker fluorescence probe and expressed as percentage of control cells (I). ROS production was assessed on day 20 by the oxidation of CM-H 2 DCFDA (J). Results are mean ± SEM. All experiments were performed in duplicate or triplicate. * P < 0.05, ** P < 0.01,*** P < 0.001, vs control or vs Tat- and/or Nef-treated cells without rapamycin. NS, nonsignificant.

Article Snippet: Autophagosomes density was visualized by immunofluorescence with an antibody directed against LC3II (LC3, M152-3, MBL international, Woburn MA, USA) in MSCs cultured with the HIV proteins for 20 days (Leica TCS SP microscope, 40× and 100× magnifications).

Techniques: Activity Assay, Staining, Fluorescence, Control, Western Blot, Quantitative Proteomics, Immunoprecipitation

Journal: Cells

Article Title: Enhanced Autophagic Flux, Suppressed Apoptosis and Reduced Macrophage Infiltration by Dasatinib in Kidneys of Obese Mice

doi: 10.3390/cells11040746

Figure Lengend Snippet: Sample size calculation.

Article Snippet: After blocking, an overnight membrane incubation with the primary antibodies—rabbit polyclonal antibody for LC3 and rabbit polyclonal antibody for SQSTM1/P62 as markers for autophagic activity, mouse monoclonal antibody for caspase-3 as an apoptosis marker and β-actin as a control protein (Biospes; YPA1652, ABclonal; A11250, Santa Cruz sc-7272 and Abcam ab227387, respectively)—was performed overnight at 4 °C (1:500, 1:1000, 1:100 and 1:5000 dilutions, respectively).

Techniques:

Journal: Cells

Article Title: Enhanced Autophagic Flux, Suppressed Apoptosis and Reduced Macrophage Infiltration by Dasatinib in Kidneys of Obese Mice

doi: 10.3390/cells11040746

Figure Lengend Snippet: ( A – D ) Protein analysis by Western blotting. ( A ) LC3 I and II (Molecular weight: 18 kDa and 16 kDa, respectively). ( B ) p62 (Molecular weight: 62 kDa). ( C ) Caspase-3: 32 kDa). ( D ) β-actin: 42 kDa). ( E – H ): LC3II, LC3II/LC3I ratio, P62 and caspase3 proteins relative quantitation /β-actin ratio) by Western blotting. Data are mentioned as mean ± SE; different letters = significant difference). LC3: Microtubule-associated proteins 1A/1B light chain 3B. M: Marker. MW: molecular weight.

Article Snippet: After blocking, an overnight membrane incubation with the primary antibodies—rabbit polyclonal antibody for LC3 and rabbit polyclonal antibody for SQSTM1/P62 as markers for autophagic activity, mouse monoclonal antibody for caspase-3 as an apoptosis marker and β-actin as a control protein (Biospes; YPA1652, ABclonal; A11250, Santa Cruz sc-7272 and Abcam ab227387, respectively)—was performed overnight at 4 °C (1:500, 1:1000, 1:100 and 1:5000 dilutions, respectively).

Techniques: Western Blot, Molecular Weight, Quantitation Assay, Marker

Journal: Cells

Article Title: Enhanced Autophagic Flux, Suppressed Apoptosis and Reduced Macrophage Infiltration by Dasatinib in Kidneys of Obese Mice

doi: 10.3390/cells11040746

Figure Lengend Snippet: ( A – D ): Immunohistochemical staining for LC3 (×400) in the kidney tissues of vehicle, Dasatinib control, Obesogenic diet and Obesogenic diet + Dasatinib groups ( A – D , respectively). Scale bar = 50 µm. ( E ) The mean percentage of LC3 immunopositive area. Results are presented as mean ± standard error. Different letters mean significant difference. Arrows: LC3 + ve cells. LC3 = Microtubule-associated protein light chain 3.

Article Snippet: After blocking, an overnight membrane incubation with the primary antibodies—rabbit polyclonal antibody for LC3 and rabbit polyclonal antibody for SQSTM1/P62 as markers for autophagic activity, mouse monoclonal antibody for caspase-3 as an apoptosis marker and β-actin as a control protein (Biospes; YPA1652, ABclonal; A11250, Santa Cruz sc-7272 and Abcam ab227387, respectively)—was performed overnight at 4 °C (1:500, 1:1000, 1:100 and 1:5000 dilutions, respectively).

Techniques: Immunohistochemical staining, Staining, Control