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. 2023 May 11;19(9):2695–2710. doi: 10.7150/ijbs.80735

Figure 2.

Figure 2

Effects of OGD/Rx to modulate HDAC9 nucleus/cytosol translocation and of siHDAC9 to revert OGD/Rx-induced HIF-1 and Sp1 deacetylation, in neuronal-like SH-SY5Y cells. A-B, Representative Western blots with quantification of TfR1 (A) and GPX4 (B) protein expression in SH-SY5Y cells treated with siRNA for HDAC9 or scrambled control at 72h after OGD/Rx. Bars represent mean ± SD (n = 5); *, p ≤ 0.05 versus control under normoxic conditions (CTL); #, p ≤ 0.05 versus siCTL under OGD/Rx conditions. C, Chromatin immunoprecipitation (ChIP) on Beclin promoter performed in NSC34 cells using anti-HDAC9 or anti-IgG used as negative control under control conditions or at 72 h after OGD/Rx. Bars represent mean ± SD (n = 4). D-E, Chromatin immunoprecipitation (ChIP) on TfR1 (C) and GPX4 (D) promoter performed in SH-SY5Y cells using anti-HDAC9 or anti-IgG used as negative control under control conditions or at 72 h after OGD/Rx. Bars represent mean ± SD (n = 5). F, Representative Western blot of HDAC9 protein levels in nucleus and cytosol of SH-SY5Y cells under control condition and at 72 h after OGD/Rx. Anti-GAPDH and anti-histone H3 (H3) were used to verify the purity of the cytoplasmic and nuclear fractions, respectively. (n = 5). G-L, Representative immunoprecipitation Western blot showing the interaction between HDAC9 and HIF-1 (G) or Sp1 (J) in SH-SY5Y cells under control conditions or at 72h after OGD/Rx; association between HIF-1 and lysine-acetylated (Lys-Ac) (H) or ubiquitine (Ub) (I) in SH-SY5Y cells treated with siHDAC9 or scrambled at 72h after OGD/Rx; association between Sp1 and Lys-Ac (K) or Ub (L) in SH-SY5Y cells treated with siHDAC9 or scrambled at 72h of OGD/Rx. The Input samples in panels G and J are SH-SY5Y cells subjected to OGD/Rx 72h, whereas in panels H, I, K and L are SH-SY5Y cells exposed to OGD/Rx 72h after siCTL transfection.