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. 2022 Nov 19;13:7108. doi: 10.1038/s41467-022-34831-3

Fig. 1. HDAC6 inhibition via TubA treatment increases grip strength, sarcolemmal localization of utrophin A and promotes reassembly of the DGC in mdx mice.

Fig. 1

a Protocol of TubA treatment. Three groups of 7-wk-old mice have been evaluated for 4 weeks either without treatment (group A, C57BL/10 mice: WT-CTL), or treated with daily injection for 30 consecutive days with DMSO (group B, C57BL/10ScSn-Dmdmdx/J mice; mdx-veh) or with TubA at 25 mg/kg/day (group C, C57BL/10ScSn-Dmdmdx/J mice; mdx-TubA). b To evaluate the level of tubulin acetylation (ac-tubK40) in TA muscles, Western blot analysis were performed. Quantifications of acetylated α-tubulin (c) and α-tubulin (α-tub, d) protein levels (n = 4–5 mice per group) were respectively normalized to α-tubulin and 2,2,2-Trichloroethanol (TCE). e Grip strength was measured on a grid measuring maximal hindlimb grip strength normalized on body weight (n = 5 mice per group). f Relative force gain was calculated by the difference between grip strength measured at the last day (day 30) and the day before starting treatment (day 0) and measured with a paired t-test (n = 5 mice per group). g Specific maximal force was evaluated by the best score of grip strength obtain in each animals at day 30 (n = 5 mice per group). To evaluate levels of utrophin A (Utr. A) and β-dystroglycan (β-DG) in TA muscles, Western blot analysis (h, k) and quantification (i, l) were performed (n = 4 mice per group). TCE was used as a loading control. Cross sections of EDL muscle were stained with an antibody against utrophin A (j, in gray) or against β-dystroglycan (m, in gray). Scale bars: 50 μm. n 4-d-old C2C12 myotubes pretreated for 24 h with different HDAC6 inhibitors TubA (5 μM) and tubacin (TBC, 5 μM) or with DMSO (CTL). o Representative Western blots showing utrophin A. GAPDH was used as a loading control. p Quantification of Utrophin A protein levels normalized with GAPDH (n = 3 independent experiments quantified). (c, d, e, f, g, i, l, p) Whiskers min to max; the line in the middle of the box is plotted at the median. *P < 0.05; **P < 0.01; n.s, not significant, P > 0.05; Mann–Whitney U test. kDa, relative molecular weight in kiloDalton.