Figure 2: In vitro functional analysis.
A) COS7 cells were transiently transfected with the vector pcDN3.1 TOX3/V5-His. i) Cells expressing the TOX3-V5/HIS tagged fusion protein stained green (anti-V5 primary antibody, secondary antibody coupled to Alexa488); ii) DAPI nuclear blue stain; iii) merged, co-localization of the TOX3 protein in the nucleus (magnification 630x, Zeiss Axiovert200M).
B) T24 bladder cancer cells were transiently transfected with an empty vector pcDN3.1 V5-His (mock) or pcDN3.1 TOX3/V5-His and whole cell protein extracts from three different time points post-transfection were analyzed by 4-12% gradient SDS-PAGE. Western blotting followed by incubation with the anti-V5 antibody showed a band with a molecular weight of about 75 kDa for the TOX3-V5/HIS tagged fusion protein. Lane 1 Marker, All Blue BioRad; lane 2,4,6 vector pcDN3.1 TOX3/V5-His, the TOX3-V5/HIS tagged fusion protein was already expressed 16h post-transfection; lane 3, 5, 7empty vector pcDN3.1 V5-His (mock).
C) Extracts from HEK cells over-expressing TOX3-V5/HIS were incubated with the anti-TOX3 antibody from A. Gosh.
D) T24 bladder cancer cells were untreated (T24 control) or transiently transfected with an empty vector pcDN3.1 V5-His (T24+mock) or the pcDN3.1 TOX3/V5-His vector (T24+TOX3). Cell viability was accessed by an MTT assaybetween 24h-96h post-transfection. Cells over expressing TOX3 (black) stopped proliferation, while control cells (gray) as well as mock transfected cells (white) proliferated further.
E) Cellular proliferation was also accessed in RealTime using the RTCA X-Celligence DP or SP instruments (Roche). 3000 T24 bladder cancer cells per well were seeded in triplicates on E-plates and transiently transfected with a vector encoding GFP (green fluorescent protein, T24+GFP) or an empty vector pcDN3.1 V5-His (T24+mock) or the pcDN3.1 TOX3/V5-His vector (T24+TOX3). Cells over expressing TOX3 stopped proliferation, while GFP transfected cells as well as mock transfected cellspreceded proliferation.