Fig. 9: Close proximity of FXN and NBs within the cellular environment.

A–H Immunofluorescence to assess NB_4A7 and FXN in the cellular environment using HeLa Kyoto cells. (A–D) Transfection with an empty plasmid; neither FXN nor NB_4A7 was transiently overexpressed (FXN is endogenous). E–H Both FXN precursor and NB_4A7 were transiently overexpressed from pCDNA3.1 vectors. Detection of FXN and NB_4A7 was performed using anti-FXN (host species: mouse, ABCAM ab110328) and anti-HA MAB (host species: rabbit, Cell Signaling HA-Tag (C29F4)). Labeled secondary antibodies detected FXN at 488 nm (secondary Antibody, Alexa Fluor™ 488) and NB at 543 nm (secondary Antibody, Alexa Fluor™ 594). The merged fluorescence signals are shown (yellow, D and H). Nuclei were counterstained with Hoechst (blue). I, J Proximity Ligation Assay (PLA) in HeLa Kyoto cells. Dots indicate the proximity of FXN to NB_4A7. Cells were fixed, washed, permeabilized, and incubated with primary antibodies, anti-FXN (mouse) and anti-HA (rabbit). PLA reactions were carried out, and cells were mounted using in situ mounting medium with DAPI. Two independent experiments were performed, and the presence or absence of red dots was analyzed in at least five different fields. Fluorescence microscopy images were then acquired. In (I), an empty vector was used, while (J), NB_4A7, and FXN precursors were transiently overexpressed using pCDNA3.1 vectors. Immunofluorescence was performed 48 h after transfection. K Co-immunoprecipitation of NB:FXN. HEK-293T cells were transfected with either an empty vector or a vector encoding the NB_16C10 sequence for protein expression. IP: His indicates the lanes where co-immunoprecipitation was performed using an anti-His tag antibody. FXN was detected with an anti-FXN monoclonal antibody, while NB was detected using the anti-His antibody. GAPDH was used to assess the protein content in the input of the co-IP experiments. The symbols + and – indicate the presence or absence of the respective vectors for cell transfection (empty or encoding NB_16C10). The scale bar is the same for all panels and corresponds, in all cases, to 30.0 μm.