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# First Part

pCDNA3.1-IL7-AS-S2 was transfected in 786-O cells for scratch test and the width of the scratch was measured after 6 hours, 24 hours respectively to observe the difference of cell migration rate. Meanwhile, we used the plasmid of pCDNA3.1-IL7-AS-S2 to quantify IL7-AS-S2 expression and calculate the copy numbers of IL7-AS-S2 by using the formula listed below.

copies/ul= (6.02×1023)×(plasmids concentrations ng/ul×10-9)/(DNA length×660)

The standard curve of IL7-AS-S2 was made by EXCEL. We find that IL7-AS-S2 increases the rate of cell migration in a copies-dependent manner.

(Fig1. The effect of IL7-AS-S2 on the correlative between growth area ratio of 786-O cells at 6 or 12 hours and copy numbers of IL7-AS-S2.)

# Second Part

## In different concentration, Analysis of control and I7-AS-S2

Is there any significant difference between control-1μg and I7-AS-S2-1μg After 6hours?

Among them, 1 indicates control, 2 indicates I7-AS-S2, there is a significant difference between them . For this data, analysis variance is carried out. After the normal test and the variance homogeneous test of the data, the results of the variance test are as follows:

When the significant level was 0.001 (1.36e-5 < 0.1), the original hypothesis was rejected. It was considered that there was a significant difference between the I7-AS-S2 and control

Is there any significant difference between control-1μg and I7-AS-S2-1μg After 12hours?

When the significant level was 0.001 (9.44e-06 < 0.1), the original hypothesis was rejected. It was believed that there was a significant difference between the the I7-AS-S2 and control

Is there any significant difference between control-2μg and I7-AS-S2-2μg after 6hours?

When the significance level was 0.001 (0.000312 < 0.1), the original hypothesis was rejected. It was considered that there was a significant difference between control-2μg and I7-AS-S2-2μg

Is there any significant difference between control-2μg and I7-AS-S2-2μg after 12hours?

When the significant level was 0.001 (2.2e-05 < 0.1), the original hypothesis was rejected. It was considered that there was a significant difference between control-2μg and I7-AS-S2-2μg.

Is there any significant difference between control-4μg and I7-AS-S2-4μg after 12 hours

When the significant level was 0.001 (8.64e-09 < 0.1), the original hypothesis was rejected. It was believed that there was a significant difference between control-4μg and I7-AS-S2-4μg.

Is there any significant difference between control-4μg and I7-AS-S2-4μg after 12 hours?

When the significant level was 0.001 (0.000675 < 0.1), the original hypothesis was rejected. It was considered that there was a significant difference between control-4μg and I7-AS-S2-4μg

## 2. Significant differences were made for different concentration changes.

There was no significant difference between control and I7-AS-S2 in the concentration of 1μg and 2 μg.

1μg.

2μg.

From the above analysis, it can be seen that there is no significant difference between control and I7-AS-S2 from 6 hours to 12 hours under the condition of 1 and 2μg concentration.

There was a significant difference between control and I7-AS-S2 in the concentration of 4μg

From the above analysis, it can be seen that there is a significant difference between between control and I7-AS-S2 from 6 hours to 12 hours under the condition of 4 μg concentration.

## Two factors interaction differences

There is no interaction between control (I7-AS-S2 ) and concentration (1μg 2μg 4μg) after 6hours.

A indicates control or I7-AS-S2, B indicates concentration level, A, B factors have an impact on subsequent concentration, and A: B even if the A B interaction has no significant effect, because 0.1072 > 0.1 significant level.

There is no interaction between control (I7-AS-S2) and concentration (1μg 2μg 4μg) after 12hours.

A indicates control or I7-AS-S2, B indicates concentration level, A, B factors have an impact on subsequent concentration, and A: B even if the A B interaction has no significant effect, because 0.537 > 0.1 significant level.

## Summary

Through the above analysis, we can draw a few points:

- control and I7-AS-S2 really affect final migration rate.
- When the original concentration is 4μg, effect of control and I7-AS-S2 on the migration rate is different.
- There are significant differences in the effects of different concentrations as same as whether adding I7-AS-S2 on the experimental results, but there is no obvious interaction between the two, which can be said to be independent of each other.