Journal: American Journal of Physiology - Cell Physiology

Article Title: Cigarette smoke-induced autophagy impairment accelerates lung aging, COPD-emphysema exacerbations and pathogenesis

doi: 10.1152/ajpcell.00110.2016

Figure Lengend Snippet: Cigarette smoke (CS) exposure-induced autophagy impairment and aggresome formation activates cellular apoptosis and senescence. A: transmission electron microscopy (TEM) images of Beas2b cells exposed to various concentrations of cigarette smoke extract (CSE) for 6 h. I, air-exposed control cells displaying the nucleus (N) and nuclear membrane (NM) (×5,598 magnification). II, Beas2b cells exposed to 10% CSE show aggresome/autophagy bodies (AB) proximal to the nuclear membrane (NM) (×10,171 magnification). CSE treatment also shows some membrane disintegration along with the thinning of the nuclear membrane (NM) and perinuclear accumulation of aggresome/autophagy bodies (AB). III and IV (scale: 2 µm), higher magnification (×46,422, ×84,190) of I and II (scale: 100 nm). The thin white arrow indicates the nuclear membrane (NM) and thick white arrows indicate the localization of aggresome bodies (AB). B: Western blot analysis illustrating the levels of ubiquitinated proteins, sirtuin1 (Sirt1, senescence marker), and p53 (senescence/apoptosis mediator) in soluble protein fractions of control Beas2b cells and those treated with 250 µM cysteamine and/or 10% cigarette smoke extracts (CSE) for 6 h. Substantial accumulation of ubiquitinated proteins was observed in the insoluble protein fractions of Beas2b cells treated with 10% CSE, suggesting that ubiquitinated proteins are translocated from soluble to insoluble protein fractions upon 6 h of 10% CSE exposure. Cysteamine attenuates the accumulation of ubiquitinated proteins in insoluble fraction and partially restores normal Sirt1 and p53 levels in the soluble protein fraction. β-Actin levels of the soluble protein fraction indicate equal loading of the proteins. C: senescence-associated β-galactosidase (SA-β-gal) activity in Beas2b cells treated with cysteamine (6 h) and/or exposed to 10% CSE overnight was measured using a senescence cells histochemical staining kit. Senescent cells were visualized and identified via blue stain (black arrows), indicating positive SA-β-gal activity. Data are shown as means ± SE (n = 3) of percentage change in SA-β-gal-positive blue senescent cells in comparison to air-exposed controls (bottom) Scale: 56 µm. ***P < 0.001 and ****P < 0.0001.

Article Snippet: For in vitro studies, the human bronchial epithelial cell line Beas2b was cultured at 37°C with 5% CO 2 in DMEM/F-12 (Dulbecco's modified Eagle’s medium; Cellgro, Manassas, VA) supplemented with 10% fetal bovine serum (FBS; Cellgro) and 1% penicillin-streptomycin-amphotericin (Cellgro).

Techniques: Transmission Assay, Electron Microscopy, Control, Membrane, Western Blot, Marker, Activity Assay, Staining, Comparison

Journal: American Journal of Physiology - Cell Physiology

Article Title: Cigarette smoke-induced autophagy impairment accelerates lung aging, COPD-emphysema exacerbations and pathogenesis

doi: 10.1152/ajpcell.00110.2016

Figure Lengend Snippet: Cysteamine rescues CSE-induced autophagy impairment and aggresome formation. A: Beas2b cells were transduced with Premo autophagy tandem sensor LC3B-RFP-GFP construct. After 24 h posttransfection, Beas2b cells were preincubated with 250 µM cysteamine (Cys) and/or treated with 10% CSE for 6 h. Flow cytometry results show significant increase in GFP fluorescence (autophagosomes), RFP fluorescence (autolysosomes), and their colocalization (autophagosomes) in comparison to air-exposed cells. Preincubation with cysteamine reduced GFP fluorescence, RFP fluorescence, and their colocalization. The x-axis shows the log scale of LC3-GFP and y-axis shows the log scale of LC3-RFP fluorescence signals. The data represent the average (mean ± SE) of three replicates (right). B: Beas2b cells were pretreated with chloroquine (CQ, 90 µM, 12 h) or cysteamine (Cys, 250 µM, 6 h), and/or treated with 10% CSE for 6 h. Fluorescence microscopy images show autolysosomes (marked by the presence of puncta, second column), autophagosomes (marked by the presence of puncta, third column), and autophagosomes (marked by the colocalization of the puncta, fourth column). Scale bars, 56 μm. Fluorescence images were used to count the number of colocalized RFP-LC3B and GFP-LC3B puncta per image (fourth column). The data represent means ± SE of four replicates, and analysis is shown in D and E. Data suggest that CSE and CQ impair autophagy while cysteamine treatment restores CSE-impaired autophagy. C: Beas2b cells were treated with cysteamine (250 µM) and/or 10% CSE for 6 h. Cells were stained with ProteoStat aggresome dye and Hoechst nuclei dye. MG132 (5 μM; 12 h) treatment was used as a positive control for this experiment. Images were captured by ZOE Fluorescent Cell Imager (Bio-Rad) and quantitative analysis of aggresome bodies in each group is shown in D and E. Data suggest that CSE and MG132 induced aggresome/autophagy bodies while cysteamine treatment restored CSE-impaired autophagy, as seen by decrease in the number of aggresome/autophagy bodies. Scale bars, 56 μm. D and E: statistical analysis of microscopy data in B and C. Data are means ± SE (n = 3). **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Article Snippet: For in vitro studies, the human bronchial epithelial cell line Beas2b was cultured at 37°C with 5% CO 2 in DMEM/F-12 (Dulbecco's modified Eagle’s medium; Cellgro, Manassas, VA) supplemented with 10% fetal bovine serum (FBS; Cellgro) and 1% penicillin-streptomycin-amphotericin (Cellgro).

Techniques: Transduction, Construct, Flow Cytometry, Fluorescence, Comparison, Microscopy, Staining, Positive Control