Fig. 4: Activity of the HDAC4 target OAT1 in the spinal cord mediates mechanical hypersensitivity.

a Immunoblots of OAT1 and β-Actin from dorsal spinal cord segments L3–5. b Relative protein levels of OAT1 and β-Actin normalized to values from saline-injected control mice (n = 5). c Representative images of dorsal horn sections of spinal segments L3–5 of mice 24 h after injection, OAT1 (green), NeuN (red, neuronal marker), Hoechst (blue, nuclear marker). Lower panels are a higher magnification. Arrows indicate OAT1-positive and NeuN positive cells. Scale bars are 40 µm. d Relative OAT1 immunofluorescence intensity in dorsal horn following injections as in (c) (n = 4). e–g Co-localization analysis of OAT1-positive cells in the dorsal horn following injections 24 h earlier. Graph represents the percentage of OAT1-positive cells within the total cell population (e, Hoechst-positive), within the neuronal population (f, NeuN-positive), and within non-neuronal cells (g, NeuN-negative) (n = 3). h Relative OAT1 immunofluorescence intensity in dorsal horn 21 days after SNI, normalized to the contralateral side (n = 3). i Relative OAT1 immunofluorescence intensity in dorsal horn laminae I–V of mice intra-spinally injected as indicated, followed by intraplantar injections of CFA or saline for 24 h (LacZ, HDAC4 3SA n = 4; HDAC4 wt n = 3). j, k Relative fluorescence intensities of the nuclear HDAC4 (j) and OAT1 (k) signal in neurons of the dorsal horn from female mice, following intraplantar injections of CFA or saline for 24 h, normalised to contralateral (saline) sides (n = 4). l Mechanical sensitivity of female mice intra-spinally injected as indicated, followed by unilateral intraplantar injections of CFA or saline for 24 h (l, m; LacZ = 7; HDAC4 wt n = 6; HDAC4 3SA n = 8). m Relative OAT1 immunofluorescence intensity in dorsal horn laminae I–V of female mice intra-spinally injected as in (l), followed by intraplantar injections of CFA or saline for 24 h prior to tissue harvest (n = 5–7). Statistically significant differences were determined by two-tailed Student’s t-test (d–h, j, k), one-way ANOVA followed by Dunnett’s post hoc test (b, F_(4,20) = 4.281, m, F_{(2, 15)} = 10.567), or two-way ANOVA followed by Dunnett’s post hoc test (i, F_(2,8) = 9.016; l, F_{(2, 19)} = 1.087). **p < 0.01; ^#, *p < 0.05. Asterisks (*) refer to statistical comparisons between conditions and hashtags (^#) to comparisons relative to basal values within each condition. Each point represents the mean value derived from one mouse. Graphs represent mean + SEM. See also Supplementary Fig. 4.