Fig. 4: HDAC8 ablation activates a HIF1α/JNK/c-Jun axis.

a, h, i Western blot of HDAC8 (a) or HIF1α (h, i) on lysates of rat SCs cultured in conditions mimicking the conversion into the repair phenotype under normoxia or hypoxia (a, CoCl₂ for 16 h; i, hypoxic chamber for 5 h) or on lysates of contralateral (Co) and crushed (Cr) sciatic nerves of HDAC8 KO (H8KO) and control (Ctrl) mice at 1 dpl (h), after subcellular fractionation (Cyt = cytoplasmic, Nuc = nuclear) and quantification of the nuclear fraction normalized to Lamin A/C (h) or of the cytoplasmic fraction normalized to GAPDH (h, i) in cells where HDAC8 was downregulated by shRNA (H8sh) compared to control shRNA (Csh) (i), or in H8KO compared to Ctrl nerves (h). b–g, j HIF1α (b–d, g), c-Jun (e), phospho-c-Jun (p-c-Jun, f) and phospho-JNK1/2 (p-JNK1/2, j) Western blot on lysates of rat SCs cultured as above (a) in normoxia (N, b, c, g) or hypoxia (H, 16 h (b, d–f, j) with CoCl₂ or (c) in hypoxia chamber), and (g) incubated with the proteasome inhibitor MG-132 or its vehicle for 4 h, and quantification normalized to GAPDH, β-actin or eEF1A1 in cells incubated with lentiviruses carrying H8sh (b, c, g) or a HIF1α shRNA (HFsh, d–f, j) or Csh. k, l Phospho-c-Jun (p-c-Jun, k) or c-Jun (l) Western blots on lysates of rat SCs cultured as above (a) in hypoxia and incubated with H8sh or Csh lentivirus, and with a JNK inhibitor (JNKi) or its vehicle (V), and quantification normalized to GAPDH. Dashed lines=samples run on the same gel but not on consecutive lanes. Three (a) or 6 (d) independent experiments, representative images are shown. h N = 4 (Ctrl) or 3 (H8KO) animals. N = 3 (i, j, k, l), 4 (f) or 6 (b, c, e, g) independent experiments. Paired (b, c, e, g, i, j, k, l) or unpaired (f, h) two-tailed (black asterisks) or one-tailed (gray asterisks, n.s.) Student’s t-tests, p values: *<0.05, **<0.01, n.s. = non-significant, values = mean, error bars = s.e.m. Source data are provided as a Source data file.