Figure 5: Sahaquine-mediated HDAC6 inhibition results in selective α-tubulin hyperacetylation at nanomolar concentrations.

a Representative fluorescence micrographs of GBM α-tubulin acetylation (green) at lysine 40 in response to sahaquine (Sq, 100 nM, n = 121 cells), SAHA (100 nM, n = 54 cells) or primaquine (PQ, 10 µM, n = 30 cells) treatment for 24 h. b Representative fluorescence micrographs of GBM histone H3 acetylation (red) at lysine 9/lysine 14 in response to sahaquine (Sq, 100 nM, n = 120 cells), SAHA (100 nM, n = 90 cells) or primaquine (PQ, 10 µM, n = 83 cells) for 24 h. Nuclei (blue) were labeled with Hoechst 33342. Cells were imaged using a fluorescence microscope and fluorescence was analyzed in ImageJ. Shown are averages of fluorescence per cell (SD) normalized to the untreated controls (set to 1) from at least three independent experiments (***p < 0.001, Welch’s ANOVA with Games–Howell post hoc test). c Acetylated α-tubulin (n = 3) and d HDAC6 protein abundance (n = 4) in GBM cells treated with temozolomide (TMZ, 100 µM), sahaquine (Sq, 10 µM) or quercetin (Q, 100 µM) alone or in combination for 24 h, measured by Western blotting. Acetylated α-tubulin and HDAC6 were normalized to total α-tubulin and actin, respectively. Each point represents a value normalized to the untreated control (set to 1). Horizontal bars represent the mean from at least three independent experiments. e Representative fluorescence micrographs of GBM α-tubulin acetylation (green) at lysine 40 in response to temozolomide (TMZ, 100 µM, n = 76 cells), sahaquine (Sq, 10 µM, n = 56 cells), quercetin (Q, 100 µM, n = 32 cells) or SAHA (10 µM, n = 36 cells) alone or in combination for 24 h. Nuclei (blue) were labeled with Hoechst 33342. f Shown are averages (SD) of fluorescence per cell normalized to the untreated control (set to 1, n = 197 cells) from at least three independent experiments (***p < 0.001, Welch’s ANOVA with Games–Howell post hoc test)