Fig 1: Essential role of UCHL1 in OC formation. (a) Immunoblot analysis of UCHL1 protein abundance during OCgenesis. Left, representative images; right, quantification of band intensity normalized on actin (mean ± SD, n = 3 independent experiments, Kruskal Wallis test with Dunn multiple comparison vs BMMs, *p < 0.05); (b) qRT-PCR analysis of Uchl1, Acp5, and Ctsk mRNAs during OCgenesis (mean ± SD normalized on actin mRNA; n = 3 independent experiments, Kruskal Wallis test with Dunn multiple comparison vs BMMs, *p < 0.05, **p < 0.01); (c) Effect of continuous pharmacological inhibition of UCHL1 by LDN57444 at the indicated doses (administered together with RANKL at the start of differentiation and at every change of medium) or vehicle (DMSO) on OC formation as assessed by TRAP staining. Left, representative images; right, quantification of the number of OCs per well (mean ± SD, n = 3 independent experiments, RM one-ANOVA test with Dunnett’s multiple comparison vs BMMs, *p < 0.05, **p < 0.01); (d) Effect of transient pharmacological inhibition of UCHL1 by LDN57444 at day 3 of differentiation (24 h, 20 µM) on OCgenesis as assessed by TRAP staining. Left, representative images (scale bar 500 µm); right, quantification of the number of OCs per well (mean ± SD, n = 4 independent experiments, Welch’s t-test, *p < 0.05); (e) Analyses of Uchl1 expression in silenced preOCs (sh1, sh2) vs control (mock) by qRT-PCR (left, mean ± SD, n = 3 independent experiments, Welch’s t-test) and immunoblot (right, mean ± SD, n = 4 independent experiments, paired t-test, *p < 0.05); (f) Effect of Uchl1 silencing by lentiviral expression of specific (sh1, sh2) or mock shRNAs on OC formation assessed as above. Left, representative images; right, quantification of the number of OCs per well (mean ± SD, n = 3 independent experiments, paired t-test, ****p < 0.0001).