Figure 3.

Regulatory impact of HDAC5 over-expression in RAW264.7 cells for cytokine response and NF-κB activation. (A) Cells were transfected with the HDAC5 expression plasmid pHDAC5 or remained non-transfected (w/o plasmid). Expression levels of HDAC5 mRNA were determined by qPCR relative to the untreated samples. (B) Lysates from transfected and non-transfected cells (10⁵) were analysed by Western blot for the HA-tag and β-actin as loading control. (C) Cells transfected with pHDAC5 were stimulated with LPS for 16 hrs before TNFα, MCP-1, IL-10 or IL-6 was determined from the supernatant. Median values and ranges of n = 2–8 from 2–4 independent experiments. (D) Cells transfected with NF-κB reporter plasmid in combination with pHDAC5 as indicated were incubated for 1 hr in the absence or presence of LPS. NF-κB activation was measured in relative light units. (E) Cells co-transfected with pHDAC5 and the plasmids coding for a frame shift mutant (pHDAC5-FS) or a mutant bearing an amino acid exchange (pHDAC5-F885) were stimulated with LPS for 16 hrs and production of TNFα was measured in the supernatant. (C–E) Median values and ranges of n = 4–11 from 2–4 independent experiments. *P < 0.05, ***P < 0.001 by Kruskal–Wallis test and Mann–Whitney test as post-test (A, E) or by Mann–Whitney test (C, D).