Fig. 4

HTL-001 disrupts HOX-PBX interactions in glioblastoma cells. U87 and LN18 cells were treated with 25 µM and 54 µM HTL-001, respectively for 2 h. Cells were washed, fixed and permeabilised, after which they were stained with an anti-PBX1 antibody and an anti-HOX antibody (either against HOXA1, HOXB5, HOXC4 or HOXC9). Co-localisation of HOX and PBX were visualised for immunofluorescence by staining cells with fluorescence-labelled secondary antibodies against the primary antibodies, using confocal microscopy. A In HTL-001 treated cells, HOX-PBX dimer formation decreased significantly with the most significant decrease seen in the nucleus. B Intensity graphs were generated using NIS-Elements Confocal Software to visualise expression intensity of HOX (indicated by green lines), PBX (indicated by red lines) and DAPI for the nuclear region of the cell (indicated by blue lines). HTL-001 induced cytoplasmic localisation of HOX and PBX proteins and disrupted co-localisation patterns between HOX and PBX. C Colocalization of HOX and PBX proteins and colocalization of HOX/PBX dimers in the nucleus was quantified using ImageJ. Both colocalization of HOX and PBX proteins and localisation of HOX/PBX dimers in the nucleus were lost following HTL-001 treatment