Fig 2: PRO-Br specifically binds to HDAC6 (A) PRO-Br/PRO can dose-dependently inhibit the activity of HDAC6. (B) Table shows the 50% inhibitory concentration (IC50) values of PRO-Br and PRO. Estimated free energies values of PRO-Br/PRO are as shown in table. (C–F) Visualization of the binding and key interacting amino acids between PRO-Br/PRO and HDAC6.

Fig 3: Tubacin and HDAC6 deficiency inhibit soft agar colony formation by cells transformed by FGFR3K644E and MYC(A) Representative images of colonies formed in soft agar by wild type MEFs or HDAC6 KO MEF cells expressing empty vector, FGFR3K644E, MYC or MYC plus FGFR3K644E. (B) Mean number of soft agar colonies per plate (n = 3) formed by the indicated cell lines. p-values were calculated using a one-way ANOVA analysis. ***p < 0.0001 (C) Immunoblot analysis of the indicated proteins in wildtype immortal MEFs or HDAC6 knockout (KO) MEFs that were infected with empty vector, or with expression vectors for FGFR3K644E or MYC. GAPDH is used as a loading control. * indicates ectopic MYC and - indicates endogenous MYC.

Fig 4: Effect of HDAC6 inhibition on markers of autophagy protein levels in corpus cavernosum tissue. (A) Representative immunoblot images for ATG5, ATG7, LC3B, Ulk1, and their respective loading controls GAPDH and LC3II. Quantification of normalized band intensity density of (B) ATG5, (C) LC3-II/LC3-I ratio, (D) LC3-I, (E) ATG7, (F) P-ULK, (G) ULK1, and (H) P-ULK1 normalized to ULK1. Data are presented as mean ± SEM for n = 5 mice per group.

Fig 5: H19 inhibition regulates HDAC6 levels in C2C12 cells. A Differentiated C2C12 cells were transfected with either the scramble or H19 siRNA (1–5 nM) and after 48 h, total RNA was isolated and the transcript levels of H19 were assessed by qRT-PCR. 18S rRNA was used as the loading control. B Differentiated C2C12 cells were transfected with either the scramble or H19 siRNA as in “A” and after 48 h, cells were lysed and 90 µg cell lysates were used to assay HDAC activity as described in the “Material and methods” section. HDAC activity is expressed as µM of substrate deacetylated per µg of protein. C Total RNA was isolated from the C2C12 cells transfected as in “A” and the transcript levels of HDAC 1–6 were evaluated by qRT-PCR. 18S rRNA was used as the loading control. C2C12 cells transfected as in “A” were lysed and lysates (20–30 µg) were subjected to Western Blot analyses to evaluate the levels of HDAC 1–6. HSC70 was used as the loading control (D–I). J 20 µg of lysate from scramble and H19 siRNA transfected C2C12 cells was subjected to western blot analysis to probe the expression of pHDAC6-Ser22. HSC70 was used as the loading control. Figures presented are representative images and values are means ± SD of at least three independent experiments. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ns: non-significant