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. 2023 Jun 13;74:101753. doi: 10.1016/j.molmet.2023.101753

Figure 4.

Figure 4

HDAC4 is involved in SIK inhibition to promote thermogenic gene expression. (A) Type IIa HDAC inhibitors blunt Iso-stimulated Ucp1 expression. At day 3 of differentiation, HIB-1B cells were pretreated with Tmp195 and Tmp269 for 16 h and then treated with 100 nM Iso for 3 h. Student's t-test, ∗∗∗∗p < 0.0001. (B–D) Knockdown of Hdac4 or Pgc1α, but not Hdac5 or Hdac7, blunts the increase of Ucp1 expression after SIK inhibition. After transfection of shRNA plasmids, HIB-1B cells were differentiated for three days and replenished with fresh medium overnight. On the following day, cells were pretreated with 1 μM HG for 3 h, and then treated with 100 nM Iso for 3 h. Student's t-test, ∗∗p < 0.01; ∗p < 0.05. (E) HDAC4 interacts with PGC1α. HEK293FT cells were transiently transfected with indicated plasmids and treated with 1 μM Iso for 6 h. Protein lysates were immunoprecipitated with anti-Flag antibody-conjugated beads and blotted with indicated antibodies. (F) PGC1α-Flag, GCN5, NAA10-Myc plasmids were co-transfected either with HDAC4 or vector into HEK293FT cells. Cells were treated with 5 mM nicotinamide (NAM, SIRT1 inhibitor) overnight followed by 1 μM Iso for 6 h. PGC1α were immunoprecipitated with anti-Flag beads and blotted with anti-acetylated lysine (Ac-Lys) or PGC1α antibodies.