Integrin dependent protein tyrosine phosphorylation is a key regulatory event in collagen IV mediated adhesion and proliferation of human lung tumor cell line, Calu-1  Nishit K Mukhopadhyay, PhD, David Gilchrist, BS, Gavin J Gordon, PhD, Chang-Jie Chen, PhD, Raphael Bueno, MD, Michael L Lu, PhD, Ravi Salgia, MD, PhD, David J Sugarbaker, MD, Michael T Jaklitsch, MD  The Annals of Thoracic Surgery  Volume 78, Issue 2, Pages 450-457 (August 2004) DOI: 10.1016/j.athoracsur.2004.01.042

Fig 1 (A) Adhesion, (B) proliferation, and (C) morphology of Calu-1 cells when deposited on collagen type IV (•), fibronectin (○), and bovine serum albumin (■) coated matrix. The data were expressed as either percentage of control (A, % of total number of attached cells compared to total) or the actual cell number (B). (C) Represents the time-lapsed video microscopy of Calu-1 cells on fibronectin (left) or on Collagen IV (right). Pictures were taken every 3 hours as described in Materials and Methods. The arrow indicates a long actin extension. The Annals of Thoracic Surgery 2004 78, 450-457DOI: (10.1016/j.athoracsur.2004.01.042)

Fig 2 (A) Western blot analysis of β1-integrin subtypes in nonsmall-cell lung cancer cell lines. Calu-1 and H520 are squamous cell lines; H23 and H441 are adenocarcinoma lines. Antibody specific to β1-integrin (141720) revealed a 135-kDa band expressed approximately eight- to tenfold higher in Calu-1 cells compared with the other three lines, and a 120-kDa band less concentrated in Calu-1 but equivalently expressed in the other three lines. The control lane was loaded with 15 μL (equivalent to 15 μg) of whole cell extract (from human epidermoid carcinoma cell line supplied by Transduction Laboratories). The blot was stripped and reprobed for p85 to confirm equal loading. (B) Competition assay with increasing concentrations (1 to 10 μg) of blocking β1-integrin antibody (P5D2), for 60 minutes incubation at 4°C. Normal mouse IgG was used as a negative control. (C) Competition assay with 1 to 10 μg of blocking α2 (solid bar) and αV (open bar) antibodies. The data represent the means of triplicate experiments. (Coll. IV = collagen IV; Cont. Ab. = control antibody.) The Annals of Thoracic Surgery 2004 78, 450-457DOI: (10.1016/j.athoracsur.2004.01.042)

Fig 3 Effect of peroxyvanadate treatment on adhesion of Calu-1 cells to collagen type IV. Serum-starved Calu-1 cells (9.6×104) were treated with peroxyvanadate (100 μmol/L) for time intervals (2.5 to 10 minutes) as described in the Materials and Methods section. (A) The cells were washed to remove the vanadate and an adhesion assay was performed for 60 minutes. (B) Whole cell extract proteins (50 μg) from each time point of peroxyvanadate treated cells were loaded on a 10% SDS-PAGE, transferred to nitrocellulose membranes and blotted with 4G10 antibodies to compare the tyrosine phosphorylation pattern. With increasing time of exposure to peroxyvanadate, there is increased tyrosine phosphorylation of multiple intracellular proteins. The phosphotyrosine blot was reprobed for β-actin to verify equal loading. The Annals of Thoracic Surgery 2004 78, 450-457DOI: (10.1016/j.athoracsur.2004.01.042)

Fig 4 Effect of genistein on type IV collagen-mediated adhesion and proliferation of Calu-1 cells. (A) There is no difference in the attachment rate of Calu-1 cells after 60-minute exposure to collagen IV matrix with either pretreatment by 60 μmol/L genistein (•) for 3 days or dimethyl sulphoxide exposure (○). Each point in the curve indicates the mean of triplicate counts. (B) Inhibition of Calu-1 cell proliferation by 60 μmol/L genistein for 3 days when added to bovine serum albumin (BSA)-coated plates (•) or collagen IV-coated plates (■) compared with BSA-coated (○) or collagen IV-coated plates without genistein (□). Triplicate wells of each time point were counted for cell growth by hemocytometer. (C) Phosphotyrosine blot of whole cell extracts of control and genistein treated cells shows suppressed phosphorylation of several proteins ranging from 30 to 200 kDa (as indicated by arrows). β-actin was used as a loading control. The Annals of Thoracic Surgery 2004 78, 450-457DOI: (10.1016/j.athoracsur.2004.01.042)

Fig 5 Effect of peroxyvanadate, genistein, and β1-integrin antibodies on collagen IV induced morphology of Calu-1 cells. (A) Cells attached to collagen IV for 60 minutes. (B) Cells attached to collagen IV after control antibody (Ab; IgG) treatment (1 μg per 106 cells). (C) Cells adhered to collagen IV after nonblocking β1-integrin antibody (I41720) treatment (1 μg per 106 cells). (D) Cells on collagen type IV matrix following the blocking β1-integrin Ab (P5D2) treatment (1 μg per 106 cells). (E) Effect of 60 μmol/L genistein treatment for 3 days on Calu-1 cells attached to collagen IV matrix. (F) Effect of 100 μmol/L peroxyvanadate exposure for 2.5 minutes on Calu-1 cells attached to collagen IV matrix. The Annals of Thoracic Surgery 2004 78, 450-457DOI: (10.1016/j.athoracsur.2004.01.042)

Fig 6 Immunoprecipitation and immunoblotting analysis of Calu-1 whole cell lysates following adhesion to collagen IV matrix. FAK tyrosine phosphorylation increased within 30 minutes of exposure to collagen IV matrix (top). The blot was striped and reblotted for FAK to verify equal loading (bottom). (IP = immunoprecipitation; FAK = polyclonal focal adhesion kinase; Wb = Western blotting.) The Annals of Thoracic Surgery 2004 78, 450-457DOI: (10.1016/j.athoracsur.2004.01.042)