FIG. 4.

Cigarette smoke exposure increases markers of autophagy in pulmonary endothelial cells. Pulmonary microvasculature endothelial cells were transfected with ptfLC3, which encodes LC3 fused to both RFP and GFP proteins; visualization via fluorescence microscopy of autophagic vacuoles (RFP⁺/GFP⁺; RFP⁺/GFP⁻), autophagosomes (RFP⁺/GFP⁺), and autolysosomes (RFP⁺/GFP⁻). The endothelial cells were exposed to vehicle (a, d), CSE (b, d) for 2 h, or serum deprivation as a positive control for 30 min (c). (a–c) Immunofluorescence of RFP and GFP was used to assess the level of RFP-positive autophagic vesicles. Yellow arrows indicate co-expression of RFP and GFP that is indicative of autophagosomes, whereas white arrows show RFP staining that is indicative of autophagolysosomes. (d) Expression of LC3I and II was assessed by Western blotting, with increased LC3II indicative of autophagy. (H.C. and E.O.H; Unpublished data). CSE, cigarette smoke extract; GFP, green fluorescent protein; LC3, light chain-3; RFP, red fluorescent protein.