Figure 1.
STAT3 activation by TRK oncogenes.
(A) Western blot analysis of PC12 cells transfected with empty pRC/CMV vector, TRKA, TRK-T3, and TRK cDNAs. NGF treatment (50ng/ml, 10′) is indicated. Cell lysates and immunocomplexes were separated by SDS PAGE as described in Material and Methods and immunoblotted with indicated antibodies. In the bottom panel two distinct exposures of the same blot were used for documenting TRKA or TRK oncoproteins phosphorylation. (B) HeLa cells were co-transfected with pM67 and pRL-TK in combination with the indicated TRK oncogene cDNAs in the absence (left graph) or presence (right graph) of STAT3 cDNA and assayed for STAT3-dependent luciferase activity 48 hours later. Activity is expressed as the ratio of luciferase/renilla activity, and reported as fold-inductions over empty vector (left panel) or STAT3 (right panel) (RLA: Relative Luciferase Activity). The data represent the mean values ± SD of triplicate samples. Similar results were obtained in three independent experiments. (C) MAPK involvement in TRK-induced Stat3 phosphorylation. Western blot analysis of PC12 cells transfected with empty pRC/CMV vector, TRK-T3 or TRK cDNAs. NGF (10′, 50ng/ml) and UO126 (16 hr, 10 µM) treatments are indicated. Immunoblotting was performed with the indicated antibodies.
Figure 2.
Analysis of Stat3 in NIH3T3 cells expressing TRK oncoproteins.
(A) Western blot analysis of NIH3T3 cells transiently transfected with TRK oncogenes. Cells were transfected as described in Materials and Methods, cell lysates and immunocomplexes separated by SDS PAGE and immunoblotted with the indicated antibodies. (B) NIH3T3 cells were co-transfected with pM67 and pRL-TK in combination with the indicated TRK oncogene and STAT3 constructs and assayed for STAT3-dependent luciferase activity 48 hours later. Activity is expressed as the ratio of luciferase/renilla activity, and reported as fold-inductions over STAT3 basal activity (RLA: Relative Luciferase Activity). The data represent the mean values ± SD of triplicate samples. Similar results were obtained in two independent experiments. (C) Cell extracts from NIH3T3, NF861 and NF797 cells were immunoblotted with the indicated antibodies. Images were acquired by Biorad ChemiDoc and densitometric analysis of the bands was performed by Image Quant software. Data are expressed as ratio STAT3/vinculin and normalized for NIH3T3 sample. (D) RT PCR analysis of Stat3 expression. Stat3 and HPRT fragments were obtained as described in Material and methods and separated on 2% agarose gel. Images were acquired by Biorad GelDoc and densitometric analysis of the bands was performed by Image Quant software. Densitometric values are expressed as ratio Stat3/HPRT and normalized for NIH3T3 sample. Data represent the mean values ± SD of four independent experiments. (E) Cytoplasmic and nuclear extracts from NIH3T3, NF861 and NF797 cells were obtained as described in Material and methods and subjected to Western blot analysis with the indicated antibodies. (F) NIH3T3, NF861 and NF797 cells were co-transfected with pM67 and pRL-TK in combination with the indicated TRK oncogene, alone (upper panel) or in combination with STAT3 construct (bottom panel) and assayed for STAT3-dependent luciferase activity 48 hours later. Activity is expressed as the ratio of luciferase/renilla activity, and reported as fold-inductions over STAT3 activity in NIH3T3 cells. Experiments were performed in triplicates; data represent the mean values of three independent experiments. RLA: Relative Luciferase Activity.
Figure 3.
Reduction of Stat3 level is associated to morphological transformation induced by TRK oncogenes activity.
(A) NIH3T3, NF861 and NF797 cells were treated with K252a (200nM) for the indicated time; cell lysates were processed and immunoblotted with the indicated antibodies. Densitometric analysis of the bands is reported in the bottom panel. Images were acquired by Biorad ChemiDoc and analysed with Image Quant software. Data are reported as ratio of STAT3/vinculin and normalized over untreated samples for any cell line. (B) Cell extracts from NIH3T3, NF861 and NF797 cells treated with K252a (200nM, 16 hr) were analyzed by Western blot with the indicated antibodies. (C) Cell extracts from NIH3T3, NF861 and NF797 cells treated with UO126 (10 µM, 16 hr) were analyzed by Western blot with the indicated antibodies. (D) Phase contrast images (top) and α-tubulin immunostaining (bottom) of NIH3T3 cells, NF797 cells treated or not with K252a (200nM, overnight) or UO126 (10 µM).
Figure 4.
Role of Stat3 in TRK-induced cell growth of NIH3T3 derived foci.
(A) Phase contrast images of NF861, NF797 and NIH3T3 cells treated or not with S3I-201 (100 µM, 48 hr). (B) Cell viability of NF861 and NF797 cells treated with DMSO (0.3%) or S3I-201 (100 µM) determined by the Alamar Blue cell viability assay. (C) Western blot analysis of NIH3T3, NF861 and NF797 cells treated with S3I-201 (100 µM, 48 hr). Samples were immunoblotted with the indicated antibodies.