Page 253 - Haematologica Vol. 107 - September 2022

LETTER TO THE EDITOR
co-expressed with M-ETV6-Myc, both proteins were mainly detected in the cytoplasm (Figure 3A). A significant shift of WT-ETV6-Flag from nucleus to cytoplasm was confirmed also by quantitative analysis which detected 37.0±16,4% of the WT protein in the cytoplasm when co- expressed with WT-ETV6-Myc, while when transfected in combination with all M-ETV6 tested the protein amount reached a value of up to 81.1±10.8% (Figure 3B), supporting our hypothesis that the mutated forms act through a dominant negative effect on WT protein, retaining this
form in the cytoplasm and consequently affecting its functions.
In order to further support the dominant negative effect, we tested the transcriptional activity using the luciferase assay. As above (Online Supplementary Figure S2), ex- pression of WT-ETV6 alone represses the luciferase ac- tivity whereas expression of M-ETV6 abolishes the inhibition. Consistent with a dominant negative effect, co- expressing the WT- with the M-ETV6, we did not detect any significant difference in comparison with the effect of
A
B
C
Figure 3. Alteration of wild-type function and intracellular distribution after co-transfection with mutant forms. (A) Western blot analysis performed in HEK293T 48 hours after transfection. Mutant forms and control wild-type (WT) are Myc-tagged, instead the co-transfected WT form is Flag-tagged. Hsp90 and SP1 were used as cytoplasmatic and nuclear markers, respectively. (B) Semi-quantitative analysis of WT-ETV6 Flag-tagged protein ratio was obtained using ImageJ. Histogram shows nuclear (N) and cytoplasmatic (C) ratios of respective variant. Error bars represent standard deviation of 3 independent experiments. *P<0.05, **P<0.01, ***P<0.001, vs. WT-ETV6, Student’s t-test. (C) Luciferase assay performed on HEK293T 48 hours after single transfection (black) or co-transfection (grey) of respective variant with WT form to analyse functional loss of WT form due to the presence of mutated forms. Firefly luciferase cloned downstream MMP3 promoter was used as reporter and Renilla luciferase under the control of CMV promoter as normalizer. The experiment shows co-transfection cause only a partial reduction of firefly/renilla ratio, accordingly to the dominant negative hypothesis. P214L used as control mutation (striped column). Error bars represent standard deviation of 3 independent experiments.
Haematologica | 107 September 2022
2252