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. 2020 Aug 2;36:101669. doi: 10.1016/j.redox.2020.101669

Fig. 5.

Fig. 5

Effect of the C667/669A HDAC4 mutation. (A) Ratio of HDAC4 protein distribution between nucleus and cytosol. HEK293 cells were transfected with WT- or RI-HDAC4. Nuclear and cytosolic extracts were analyzed by Western blot (n = 7) (Unpaired t-test, *p < 0.05). (B) BIAM switch redox assay with Nox4-tet-on cells. Cells were transfected with RI-HDAC4 (C667/669A). After 24 h cells were treated with or without tetracycline (1 μg/ml, 24 h) and DPI (3 μM, 3 h). (C) Nox4-tet-on cells, were transfected with WT- or RI-HDAC4-myc and treated with or without tetracycline (Tet) (1 μg/ml, 24 h). Ratio of phosphorylated HDAC4 (pHDAC4) and HDAC4 (myc tag) is depicted (n > 3) (One-way ANOVA with Sidak post-hoc test, *p < 0.05). One representative Western blot is depicted. (D) HMECs were transfected with GFP, WT- or RI-HDAC4 for 48 h and seeded on matrigel. Representative images after 4 h of tube formation are shown. Numbers of nodes were counted and total tube length was measured with an image angiogenesis analyzer (Carpentier, 2012) in ImageJ and summarized in the statistics (n = 7). (One-way ANOVA with Tukey post-hoc test, *p ≤ 0.05). All bar graphs show means ± SEM.