Fig. 3: HDAC6 inhibition with BAS-2 increases mtROS that leads to cell death and disruption to mitochondrial cristae structure in TNBC cells.

A Representative confocal images of live BT-549 cells after treatment with BAS-2 (10 µM and 30 µM) for 24 h stained with MitoSOX Red to indicate mitochondrial ROS. Lower panels show zoomed regions as indicated by yellow boxes. Scale bars show 10 µm (upper panels) and 1 µm (lower). MitoSOX Red intensity is max-adjusted and coloured using the ‘Royal’ colour map in ImageJ/FIJI of the scale (left to right) 0-100%. B Quantification of live-cell confocal images of BT-549 cells after BAS-2 treatment (24 h) where points represent mean pixel intensity of MitoSOX red channel from segmented cell regions. Data show 5-11 technical replicates (segmented cell regions) from one biological replicate. C Representative histograms of MitoSOX Red signal from MDA-MB-231 or BT-549 cells after 24 h BAS-2 treatment. D MitoSOX Red mean fluorescence intensity (MFI) of BT-549 and MDA-MB-231 cells after 24 h treatment with BAS-2 normalised to DMSO controls (n = 3 independent experiments). Bars represent mean ± SEM. E Cell death in MDA-MB-231 cells after pre-treatment with the mitochondrial ROS scavenger MitoTEMPO for 1 h and subsequent treatment with BAS-2 for 24 h, as measured by Annexin V-FITC and propidium iodide (PI) staining, where cells negative for both were designated ‘healthy’. Values normalised to control and mean ± SEM graphed (n = 3 independent experiments). F Representative live-cell STED images of BT-549 cells after pre-treatment with MitoTEMPO (mtT, 50 µM), treatment with BAS-2 (10 µM) and staining with PKMO (300 nM, 30 min) for mitochondrial membrane (orange) or picogreen for DNA (cyan). Line profiles showing PKMO signal are shown below indicating the path of the white dotted arrows. Scale bars show 1 µm. PKMO intensity is max-adjusted and coloured using the ‘mpl-inferno’ colour map in FIJI/ImageJ of the scale (left to right) 0-100%. G Box-and-whisker plots showing mitochondrial diameters from 3 confocal images per condition (images not shown) of the treatments indicated (mtT, 50 µM; BAS-2, 10 µM) from one biological replicate. Plots represent the data from 4400-5500 automatically segmented mitochondria, 1000-2000 on average per confocal image. Mitochondria were analysed as described in methods using the Mitochondria Analyzer FIJI plugin⁶⁰. Diameters were measured on a per-mito basis. H Mean PKMO signal on a per-mito basis from 4400-5500 segmented mitochondria from 3 confocal images and one biological replicate. Box-and-whisker boxes show 25th to 75th percentiles with the median at the centre and whiskers indicate the range from minimum to maximum. Significance by one-way ANOVA. Source data are provided as a Source Data file.