Characterization of the HDAC6 c.2566C>T/p.P856S variant. A: Electropherograms of HDAC6 exon 25 region containing the c.2566C>T variant, in patients 1 and 2 (Pt1, Pt2) and their mother (I-2). B: Levels of HDAC6 transcript, normalized to that of the endogenous GAPDH cDNA, in controls (Ct; mean of five subjects) and patients 1 and 2 (Pt1, Pt2), obtained from 3 independent experiments. Two-tailed Student’s t-tests showed no significant differences: Pt2 vs. Ct p = 0.811; Pt2 vs. Pt1 p = 0.813; Pt1 vs. Ct p = 0.896. C: Exemplifying Western-blot analysis of fibroblast lysates from control subjects (Ct1, Ct2) and patients 1 and 2 (Pt1, Pt2), using antibodies against acetylated α-tubulin (upper panel), α-tubulin (middle panel) and α-GAPDH, as loading control (lower panel). The graph represents the ratio acetylated α-tubulin/α-tubulin obtained by densitometric analysis from 3 independent experiments: 100% corresponds to the mean value of four control subjects. Two-tailed Student’s t-test Pt2 vs Ct p = 0.002, Pt1 vs Ct p = 0.33. D: Immunocytochemistry on fibroblasts from a control subject (Ct) and patients 1 and 2 (Pt1, Pt2), using antibodies against α-tubulin and acetylated α-tubulin. Scale bars are reported on the right for each row. E: Percentages of multilobated nuclei in control, Patient1 and Patient2. A total of 15 digital images (at least 600 cells for each patient) representative of the whole sections were collected and analyzed for each sample; the arrow indicates a typical multilobated nucleus. Two-tailed Student’s t-test between Control vs. Pt1 showed no significant differences (p = 0.4970); Pt2 vs. Control p = 0.000099; Pt2 vs. Pt1 p = 0.000018 (both highly significant).