Figure 1.
Flow Chart of SiLAD technology.
Figure 2.
Evaluation of the developed 35S labeling protocol.
(A) Phosphor-image of one 2-D gel of A549 cell at 1 hr time point. Labeling time was 15 min. pH-range 4–7 iso-electric focusing strips were used to separate protein in the first dimension and 12.5% polyacrylamide gels in the second dimension. (B) The CBB staining image for the same gel with A. (C) Statistical analysis of the spots numbers shown in the two kinds of images. PH-I stands for the images were got from Phosphor-imaging, while CBB means the CBB staining. The 18 gray circles in each block denote the spots numbers of 18 corresponding images (each time running at least 6 gels, 5gels for 5 time points and another gel for the proteins extracted from asynchronous cells as a control, repeated 3 times). Black error bars represent average spots numbers±s.e.m. for each kind of image. This chart was made by SPSS13. (D) Zoom in view of two spots, the spot labeled by upward arrow is protein PGDH, while the spot pointed with downward arrow is PRDX4. (E) Zoom in view of two spots only shown in the phosphor-image.
Figure 3.
Dynamic metabolized changes of the proteins.
(A, D, G and I) Zoom in view of the spots dynamic changes at different time points. Panel A, D, and G show three differential changed proteins in A549 model. The differential protein shown in panel I was from rat liver hepatectomy model. PH-I stands for the images were got from Phosphor-imaging, while CBB means the CBB staining. (B and E) Shown the dynamic expression changes of the spots labeled in panel A and D, respectively. The x axis is time (t), and the y axis is the protein synthesizing speed (vexpressed), and the area of each rectangle means the total amount of protein synthesized during each 15 minutes interval (sexpressed). The midpoint of each rectangle's top edge was used to interpolate these five points with piecewise cubic Hermite polynomial, as shown in the diagram as the green line. (C and F) Shown the dynamic total amount changes of the spots labeled in A and D. The x axis also is time (t), and the y axis is the total protein metabolized speed (vexisted), consequently the area of each rectangle means the total amount of protein (sexisted) variation during each time interval. This green line indicates the function of the variation speed of the total amount of existed protein (sexisted) with respect to time (t), during these six hours. (H) Presence %Volumes changes of the protein labeled in chart G. Ph-R denotes phosphorylation rate, i.e. the percentage of phosphorylated proteins accounted for the total sum in each time point. The value of PH-R represents mean±s.e.m. for 3 independent experiments. Error bars represent mean±s.e.m., n = 3. Chart B, C, E and F were made by the software MatLab, and chart H was made by SPSS13.
Figure 4.
SiLAD profiles are served to characterize the specific state of the cell cycle progress.
Curve 1 was from the CBB spot in Figure 3D. Curve 2 was from the spot in Figure 3G PH-I image labeled with downward arrow. Curve 3 was from the same image with Curve 2, but was the spot labeled with upward arrow. The right panels A and B were the defined bar codes for the two time points labeled with spotted line A and B in the left panel. The curve was made by the software Origin6.
Figure 5.
Comparison among BrdU labeling, Flow Cytometry and SiLAD to check cell cycle stage.
Black line a means only to determine the cell cycle phase roughly. Curve line b means detecting the specific time point exactly. BrdU L is stands for the BrdU labeling and FCM is Flow Cytometry.
Figure 6.
Evaluation the synchronized efficiency of serum starved A549 cells.
(A) FACS analysis of A549 cells synchronization by serum starvation for 48 hr. (B) Western blot analysis of expression of CyclinD1 at the indicate time lengths after serum re-stimulation. (C) FACS to detect the cell cycle progress after serum re-stimulation.