3 Results
3.1 Western Blot Analysis
(A)
(B)
Figure 1:
(A) Result of a Western Blot experiment performed with Sertoli cells, expression of connexin43 after incubation for three days with aciclovir at concentrations ranging from 0.3 to 10 mg/l.
(B) Results from three independently performed experiments. Western Blots were analyzed in three separate experiments. The blue line represents the blot shown in Figure 11A after densitometric analysis.
From the figures above, it can be seen that the amount of connexin43 protein present in the sample, after three days of culture with aciclovir, decreased in a dose-dependent manner, with the highest concentration of aciclovir tested, 10 mg/l, causing down-regulation of connexin43 by more than 50% (Figure 1A). The expression of connexin43 in the sample, compared to that of the control, decreased to 93.7 ± 11.3 % and 80.6 ± 9.8 % (n=3; mean ± SD) when the concentration of aciclovir was 0.3 mg/l and 1 mg/l, respectively. At the two higher concentrations of 3 mg/l and 10 mg/l, the expression of connexin43 was reduced to 59.0 ± 19.4 % and 49.8 ± 17.2 %, respectively (Figure 1B).
0 0.3 1 3 10 mg/l
Aciclovir-connexin43 (3-day culture)
43 kDa
(B)
Figure 2:
(A) Result of a Western Blot experiment performed with Sertoli cells, expression of connexin43 after incubation for three days with ganciclovir at concentrations ranging from 0.3 to 10 mg/l.
(B) Results from three independently performed experiments. Western Blots were analyzed in three separate experiments. The blue line represents the blot shown in Figure 11A after densitometric analysis.
From the figures above, it can be seen that, after three days of culture with ganciclovir, the level of connexin43 protein decreased in a dose-dependent manner. At the highest concentration tested (10 mg/l), ganciclovir caused down-regulation of connexin43 by more than 70% (Figure 2A). There were only slight changes in connexin43 levels when the concentration of ganciclovir was below 1 mg/l. The expression of connexin43 within the sample, compared with that of the control, decreased to 91.9 ± 5.8 % and 81.5 ± 10.4 % (n=3; mean ± SD) when the cells were cultured with ganciclovir at doses of 0.3 mg/l and 1 mg/l, respectively. More pronounced effects were apparent at the higher concentrations of 3 mg/l and 10 mg/l, with connexin43 expression reduced to 38.4 ± 2.1 % and 43.2 ± 10.8 %, respectively (Figure 2B).
0 0.3 1 3 10 mg/l Ganciclovir-connexin 43 (3-day culture)
43 kDa
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(B)
Figure 3:
(A) Result of a Western Blot experiment performed with Sertoli cells, expression of connexin43 after incubation for three days with penciclovir at concentrations ranging from 3 to 100 mg/l.
(B) Results from three independently performed experiments. Western Blots were analyzed in three separate experiments. The blue line represents the blot shown in Figure 11A after densitometric analysis.
Figures above show that, after three days of culture with penciclovir, the expression of connexin43 in the sample had been down-regulated in a dose-dependent manner. The strongest effect was detected at the highest concentration tested, 100 mg/l (Figure 3A). At the two lower concentrations of 3 mg/l and 10 mg/l, the expression of connexin43 decreased to 94.5 ± 8.3 % and 82.1 ± 20.6 %, respectively (n=3; mean ± SD). More pronounced changes were visible at the two higher concentrations of 30 mg/l and 100 mg/l, which demonstrated connexin43 expressions of 69.6 ± 16.8 % and 50.7 ± 16.5 %, respectively (Figure 3B).
0 3 10 30 100 mg/l Penciclovir-connexin 43 (3-day culture)
43 kDa
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(B)
Figure 4:
(A) Result of a Western Blot experiment performed with Sertoli cells, expression of connexin43 after incubation for three days with famciclovir at concentrations ranging from 3 to 100 mg/l.
(B) Results from three independently performed experiments. Western Blots were analyzed in three separate experiments. The blue line represents the blot shown in Figure 11A after densitometric analysis.
From the figures above, little difference can be seen between the treated group and the control group in terms of the different concentrations of famciclovir tested. In the two lower concentrations in particular, connexin43 expression remained at nearly 100% that of the control (Figure 4A, B). At the highest concentration tested (100 mg/l), the expression of connexin43 still remained at around 80%. When the concentration of famciclovir increased from 30 mg/l to 100 mg/l, the values of the expression were reduced to 84.6% ± 21.3% and 83.5% ± 1.7%, respectively (n=3; mean ± SD).
0 3 10 30 100 mg/l Famciclovir-connexin43 (3-day culture)
43 kDa
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(B)
Figure 5:
(A) Result of a Western Blot experiment performed with Sertoli cells, expression of N-cadherin after incubation for three days with aciclovir at concentrations ranging from 0.3 to 10 mg/l.
(B) Results from three independently performed experiments. Western Blots were analyzed in three separate experiments. The blue line represents the blot shown in Figure 11A after densitometric analysis.
Only slight effects were observed after the cells were cultured with aciclovir for three days (Figure 5A) and the level of N-cadherin protein slightly decreased in a concentration-dependent manner (Figure 5B). The expression of N-cadherin were 94.3% ± 2.0% of the control value in the 0.3 mg/l group, 87.8% ± 1.9% in the 1 mg/l group, 82.5% ± 1.7% in the 3 mg/l group and 75.3% ± 1.7% in the 10 mg/l group (n=3; mean ± SD).
0 0.3 1 3 10 mg/l
Aciclovir-N-cadherin (3-day culture)
130 kDa
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(B)
Figure 6:
(A) Result of a Western Blot experiment performed with Sertoli cells, expression of N-cadherin after incubation for three days with ganciclovir at concentrations ranging from 0.3 to 10 mg/l.
(B) Results from three independently performed experiments. Western Blots were analyzed in three separate experiments. The blue line represents the blot shown in Figure 11A after densitometric analysis.
There were no obvious changes in N-cadherin after three days of culture with ganciclovir compared to the control group (Figure 6A). When measured with Bio1D, the mean values at different concentrations were all above 80% but under 100% of the control values (Figure 6B).
At concentrations between 0.3 mg/l and 10 mg/l, the expressions of N-cadherin protein were 99.9 ± 8.6 %, 90.2 ± 8.4 %, 80.2 ± 8.7 % and 84.4 ± 10.8 %, respectively (n=3; mean ± SD).
0 0.3 1 3 10 mg/l
Ganciclovir-N-cadherin (3-day culture)
130 kDa
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(B)
Figure 7:
(A) Result of a Western Blot experiment performed with Sertoli cells, expression of N-cadherin after incubation for three days with penciclovir at concentrations ranging from 3 to 100 mg/l.
(B) Results from three independently performed experiments. Western Blots were analyzed in three separate experiments. The blue line represents the blot shown in Figure 11A after densitometric analysis.
No large differences were observed after three days of culture with penciclovir (Figure 7A). In Figure 7B, all the values are above 80%, but below 100% of the control values, apart from the value of around 50% at a dose of 10 mg/l, which was probably caused by a mistake during the conduction of the Western Blot. At the two higher doses tested (30 mg/l and 100 mg/l), the expression of N-cadherin decreased to 89.0 ± 1.6 % and 88.1 ± 4.9 %, respectively (n=3; mean
± SD).
0 3 10 30 100 mg/l Penciclovir-N-cadherin (3-day culture)
130 kDa
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(B)
Figure 8:
(A) Result of a Western Blot experiment performed with Sertoli cells, expression of N-cadherin after incubation for three days with famciclovir at concentrations ranging from 3 to 100 mg/l.
(B) Results from three independently performed experiments. Western Blots were analyzed in three separate experiments. The blue line represents the blot shown in Figure 11A after densitometric analysis.
From the figures above, after 3 days of culture no large differences could be seen among the different concentrations of famciclovir tested (Figure 8A). In Figure 8B, the expression of N-cadherin remained between 80% and 100%, even at the highest concentration tested, 100 mg/l. The mean values of N-cadherin expression were 94.4% ± 3.8 %, 88.2% ± 5.0 %, 87.4% ± 9.3 % and 85.3% ± 5.1 % at concentrations from 3 mg/l to 100 mg/l, respectively (n=3; mean ± SD).
0 3 10 30 100 mg/l Famciclovir-N-cadherin (3-day culture)
130 kDa
(A)
(B)
Figure 9:
(A) Result of a Western Blot experiment performed with Sertoli cells, expression of vimentin after incubation for three days with aciclovir at concentrations ranging from 0.3 to 10 mg/l.
(B) Results from three independently performed experiments. Western Blots were analyzed in three separate experiments. The blue line represents the blot shown in Figure 11A after densitometric analysis.
From the figures above, it can be seen that the quantity of vimentin protein decreased in a concentration-dependent manner after three days of culture with aciclovir (Figure 9A). In Figure 9B, it can be seen that the mean value remained above 80% at the two lower concentrations tested; 99.5 ± 5.3 % and 88.7 ± 0.9 % at 0.3 mg/l and 1 mg/l, respectively. More pronounced reductions of the vimentin content were visible at the two higher concentrations tested: to 76.5 ± 9.3 % and 68.3 ± 7.6 % of the control values at 3 mg/l and 10 mg/l, respectively (n=3; mean ± SD).
0 0.3 1 3 10 mg/l Aciclovir-vimentin (3-day culture)
58 kDa
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Figure 10:
(A) Result of a Western Blot experiment performed with Sertoli cells, expression of vimentin after incubation for three days with ganciclovir at concentrations ranging from 0.3 to 10 mg/l.
(B) Results from three independently performed experiments. Western Blots were analyzed in three separate experiments. The blue line represents the blot shown in Figure 11A after densitometric analysis.
In Figure 10A, although the level of vimentin present at the concentration of 0.3 mg/l was slightly higher than that of the control group, vimentin otherwise appeared to decrease in a concentration-dependent manner. The expression of vimentin decreased at a lower rate at the two lower concentrations tested (0.3 mg/l and 1 mg/l) than at the two higher concentrations tested (3 mg/l and 10 mg/l). In Figure 10B, the level of vimentin present at the concentration of 0.3 mg/l remained almost the same as that of the control, but it was reduced to 80.0 ± 4.5 %, 79.8 ± 10.6 %, 76.9 ± 4.5 % (n=3; mean ± SD) at the three higher concentrations tested (1 mg/l, 3 mg/l, 10 mg/l), respectively.
0 0.3 1 3 10 mg/l Ganciclovir-vimentin (3-day culture)
58 kDa
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Figure 11:
(A) Result of a Western Blot experiment performed with Sertoli cells, expression of vimentin after incubation for three days with penciclovir at concentrations ranging from 3 to 100 mg/l.
(B) Results from three independently performed experiments. Western Blots were analyzed in three separate experiments. The blue line represents the blot shown in Figure 11A after densitometric analysis.
In Figure 11A, vimentin appeared to slightly decrease in a concentration-dependent manner after three days of culture with penciclovir, except that the level of vimentin present at the concentration of 10 mg/l was slightly higher than that of the control group. Figure 11B shows at the highest concentration tested (100 mg/l), the expression of vimentin was 79.5 ±6.0 % that of the control. At 10 mg/l and 30 mg/l, the expression of vimentin was 83.0 ± 17.9 % and 88.5 ± 1.0 %, respectively (n=3; mean ± SD). The lowest concentration of penciclovir, 3 mg/l, did not have any obvious effects on the level of vimentin present.
0 3 10 30 100 mg/l
Penciclovir-vimentin (3-day culture)
58 kDa
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(B)
Figure 12:
(A) Result of a Western Blot experiment performed with Sertoli cells, expression of vimentin after incubation for three days with famciclovir at concentrations ranging from 3 to 100 mg/l.
(B) Results from three independently performed experiments. Western Blots were analyzed in three separate experiments. The blue line represents the blot shown in Figure 11A after densitometric analysis.
Figures above show that very similar levels of vimentin were present in all samples of cells incubated with various concentrations (Figure 12A, B). There were no obvious differences between the treated group and the control group. After three days of culture with famciclovir at the concentrations tested, the expression of vimentin remained between 80% and 100% that of the control, with the mean value across this period remaining above 90%. When the concentration of famciclovir increased from 10 mg/l to 100 mg/l, the values of the expression were reduced to 96.7% ± 2.7%, 92.3% ± 5.0% and 91.2% ± 3.3%, respectively (n=3; mean ± SD).
0 3 1 30 100 mg/l
Famciclovir-vimentin (3-day culture)
58 kDa