Table 1.
Effect of Stat3 downregulation upon GJIC
|
A. Cells with extensive junctional communication | ||||||
|---|---|---|---|---|---|---|
|
Cell line |
Treatmentα |
Srcβ(%) |
Stat3β (%) |
GJICγ |
||
| 50% | 100+3d | 90% | 100+3d | |||
|
E10 |
- |
6±1 |
9±3 |
26±9 |
1.5±0.5 |
6.0±1 |
|
“ |
DMSO |
6±1 |
9±3 |
30±8 |
- |
6.0±1 |
|
“ |
CPA7 |
5±1 |
2±1.1 |
3±1 |
- |
0.2±0.1 |
|
“ |
sh-Stat3 |
N/A |
6±1.1 |
8±2 |
- |
1.0±0.2 |
|
QU-DB |
- |
7±1 |
10±2 |
20±4 |
1±0.2 |
6.3±1 |
|
“ |
DMSO |
7±1 |
10±2 |
22±3 |
- |
6.3±1 |
|
“ |
CPA7 |
5±1 |
2±0.5 |
2±0.5 |
- |
0.2±0.1 |
|
“ |
sh-Stat3 |
N/A |
5±3 |
8±2 |
- |
0.8±0.2 |
|
SK-LuCi6 |
- |
5±1 |
8±2 |
21±4 |
1.8±0.2 |
6.5±1 |
|
“ |
DMSO |
5±1 |
8±2 |
20±5 |
- |
6.5±1 |
|
“ |
CPA7 |
5±1.2 |
2.8±1.25 |
3±1 |
- |
0.3±0.1 |
|
“ |
sh-Stat3 |
N/A |
6±2 |
4±0.5 |
- |
1±0.2 |
|
“ |
Jak inhib.1 |
5.2±0.3 |
4.2±1.1 |
5±0.5 |
- |
0.5±0.2 |
|
“ |
Stat3C |
5.1±1 |
22±9 |
97±10 |
- |
8±1 |
|
B. Cells expressing activated Src | ||||||
|
A549 |
- |
95±11 |
93±12 |
320±32 |
0.1 ±0.1 |
0.3 ±0.1 |
|
“ |
DMSO |
95±11 |
93±12 |
320±32 |
- |
0.3 ±0.1 |
|
“ |
CPA7 |
93±10 |
8±1 |
12±2 |
- |
0.1 ±0.1 |
|
“ |
sh-Stat3 |
N/A |
12±3 |
11±4 |
- |
0.1±0.1 |
|
E10-Src |
DMSO |
98±12 |
98±15 |
350±28 |
- |
0.4 ±0.2 |
|
“ |
CPA7 |
95±11 |
5±1 |
15±4 |
- |
0.1 ±0.1 |
|
“ |
sh-Stat3 |
N/A |
9±3 |
20±3 |
- |
0.1±0.1 |
|
SK-LuCi6-Src |
DMSO |
100±12 |
100±12 |
420±33 |
- |
0.2±0.1 |
|
“ |
CPA7 |
98±10 |
3±1 |
9±1 |
- |
0.1 ±0.1 |
|
“ |
sh-Stat3 |
N/A |
11±3 |
17±3 |
- |
0.3±0.1 |
|
“ |
sh-Stat3+Das. |
4±1 |
6±1.1 |
14±3 |
- |
0.2±0.1 |
|
SK-Lu-1 |
DMSO |
85±5 |
90±11 |
311±23 |
- |
1±0.2 |
|
“ |
CPA7 |
82±4 |
6±1 |
8±3 |
- |
0.1 ±0.1 |
|
CALU-1 |
DMSO |
96±9 |
100±10 |
290±12 |
- |
0.1 ±0.1 |
|
“ |
CPA7 |
90±12 |
8±2 |
6±1 |
- |
0.1 ±0.1 |
|
SW-900 |
DMSO |
100±13 |
100±12 |
405±21 |
- |
0.1 ±0.1 |
|
“ |
CPA7 |
96±11 |
12±1 |
11±2 |
- |
0.1 ±0.1 |
|
CALU-6 |
DMSO |
95±11 |
93±10 |
300±18 |
- |
0.1 ±0.1 |
| “ | CPA7 | 93±10 | 10±1 | 15±5 | - | 0.1 ±0.1 |
αFor Stat3 inhibition, cells were treated with 50 μM CPA7, or the DMSO carrier for 24 hrs, or infected with a lentivirus vector expressing a Stat3-specific, shRNA [37]. For Stat3 upregulation, cells were infected with a retroviral vector containing Stat3C. Jak inhibitor-1 was used at 5μM [16].
β Stat3-tyr705 or Src-ptyr418 levels were measured by Western blotting. Numbers represent relative values obtained by quantitation analysis. Averages of at least three experiments ±SEM are shown. For Stat3, data from cells grown to 50% confluence or 3 days after confluence are presented [15], with the average of the values for DMSO-treated, Src-transduced, SK-LuCi6-Src cells grown to 50% confluence taken as 100%. The transcriptional activity values obtained paralleled the Stat3-705 phosphorylation levels indicated (Figure 4C and D, see Methods).
γGJIC was assessed by in situ electroporation at the indicated confluences (see Methods, Figure 8). Quantitation was achieved by dividing the number of cells into which the dye had transferred through gap junctions (denoted by dots, Figure 1B and 2A), by the number of cells at the edge of the electroporated area (denoted by stars). Numbers are averages ±SEM of at least three experiments, where transfer from more than 200 cells was examined.
N/A: Not applicable.