Figure 1.
STC1 Enhanced Calcium Wave Propagation Following Mechanical Injury.
A) Confluent A549 monolayers were labeled with Fluo-4 and pre-incubated with or without 500 ng/mL STC1 for 10 min prior to mechanical disruption. Shown are images obtained −1, 0, 10, 20 and 30 s post-disruption. Images in each group were manipulated equally using the threshold function in Adobe Photoshop in order to false color the wave for clarity (red). Arrow points to leading edge of wave. B) Mean distance traveled by calcium wave over time in control and STC1 treatment groups from A. Error Bars = SD. * = p<0.05. n = 5 movies. C) Mean maximum distance reached by calcium wave with or without pretreatment with 500 ng/mL STC2. NS = Not significant. n = 3.
Figure 2.
Mechanically Injured Cells Released and Responded to Extracellular ATP.
A) Mean ATP content of conditioned media from control or mechanically stimulated A549 monolayers. RLU, relative luciferase units. Error Bars = SD. * = p<0.05. n = 3. B) ATP (50 µM) alone was added to confluent Fluo-4 labeled A549 cells (vertical line). Calcium response was measured by fluorescent microscopy. n = 4 movies.
Figure 3.
Trinucleotides from Injured Cells Were Required to Initiate Calcium Response.
A) Conditioned medium from viable (No Injury), mechanically disrupted (Injury) or mechanically disrupted lysate treated with 250 mU/mL apyrase for 10 min. (Injury + Apyrase) was placed on a confluent layer of Fluo-4 labeled A549 cells. Left column: Before addition of conditioned medium. Right column: 10 s after treatment with conditioned medium. Magnification = 4×. Inset for each: Pixel intensity profile for entire field of view. Inset images for each treatment group were manipulated equally using the threshold function in Adobe Photoshop in order to false color the peaks for clarity (red). n = 3 movies. B) Calcium response of 10 individual cells following addition of Injury or Injury + Apyrase (250 mU/mL for 10 min) treated conditioned medium. Black Arrow: Addition of conditioned medium. Red Arrow: Background Intensity Measurement. C) Average maximum intensity for individual cells after addition of No Injury, Injury, or Injury + Apyrase conditioned medium. * = p<0.05; n = 30.
Figure 4.
STC1 Enhanced ATP-Induced Calcium Response.
A) Mean calcium response of Fluo-4 labeled confluent monolayers of A549 cells were analyzed by live cell microscopy after the addition of 50 µM ATP to control (ATP) or monolayers pretreated with 500 ng/mL STC1 for 10 min (ATP + STC1). Error bars = SD. * = p<0.05. n = 3 movies. B) Mean calcium response of Fluo-4 labeled A549 monolayers analyzed by fluorescent spectroscopy after addition of various concentrations of ATP and STC1. Data are displayed as average signal intensity at 25 s after addition of ATP. Error bars = SD. * = p<0.05. n = 4 for each condition. C) Continuous assays from B). n = 3 for B, C. D) Measurement of individual cells from A revealed prolonged calcium oscillations in STC1 pretreated samples.
Figure 5.
Inhibition of PLC Reduced Mechanical and ATP Induced Calcium Response.
A) Fluo-4 labeled A549 cells were mechanically stimulated in the absence (control) or presence of either a P2X inhibitor (50 µM NF023), PI-PLC pathway inhibitor (50 µM D609), or PLC inhibitor (12.5 µM U73122) t = 20 s post-injury. n = 5. B) Quantification of distance traveled by calcium wave from A at t = 20 s. Error bars = SD. * = p<0.05. n = 5. C) Fluo-4 labeled A549 cells pre-treated for 30 min with NF023, D609, or U73122 were incubated with 500 ng/mL STC1 for 10 min prior to scrape. Maximum distance of the calcium wave is displayed. Error bars = SD. * = p<0.05. n = 3.
Figure 6.
STC1 and Extracellular ATP Were Required for Calcium Wave Propagation following Mechanical Stimulation.
A) Fluo-4 labeled A549 monolayers were mechanically stimulated in the presence of isotype antibody (control), an STC1 blocking antibody (anti-STC1; 1 µg/mL) or apyrase (250 mU/mL) and assayed by live cell microscopy. B) Distance of calcium wave propagation from A. Error bars = SD. * = p<0.05. C) Quantification of signal intensity adjacent to scrape site following mechanical stimulation. Error bars = SD. * = p<0.05.
Figure 7.
STC1 Enhanced the Release of ATP in vitro and in vivo.
A) A549 cells were stimulated with 500 ng/mL STC1 for 10 mins. Conditioned medium was collected and assayed for ATP. Adherent cells were lysed and measured for ATP and DNA content. ATP values were normalized to DNA fluorescence. * = p<0.05; n = 3. B) A549 cells were stimulated with 10 µM ATP for 2, 5 and 10 mins. Assay of conditioned medium and cell lysates of wild type and STC1 over-expressing MEFs. * = p<0.05; n = 3. C) Serum was isolated from wild type and STC1 transgenic mice and assayed for ATP content. * = p<0.05; n>17.