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

Figure 3.

Figure 3

Sik3 deletion in brown adipocytes increases thermogenic gene expression. (A) Sequence of wild-type (WT) and CRISPR-edited Sik3 mutations, the guide RNA (gRNA), and the protospacer adjacent motif (PAM) are shown (upper). The wild-type non-targeting control (NTC), and two CRISPR-edited HIB-1B clones with frameshifted Sik3 mutations (SC19 and SC13) were generated (lower). (B) Western blot analysis of SIK3, type IIa HDACs, PGC1α and mitochondrial OXPHOS proteins ATP5A, UQCRC2, SDHB, NDUFSB8, COXIV in control (NTC) and Sik3−/− (SC19 and SC13) brown adipocytes. p-HDAC4 Ser246, p-HDAC5 Ser259, p-HDAC7 Ser155. OXPHOS: oxidative phosphorylation complex. (C) Expression of Sik3, Ucp1 and Pgc1α mRNA in control (NTC) and Sik3−/− (SC19 and SC13) brown adipocytes. Student's t-test, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 vs NTC vehicle (Veh); $p < 0.01, $$$p < 0.001, $$$$p < 0.0001 vs NTC Iso. (D–E) Cell mitochondrial stress test of control (NTC) and Sik3−/− (SC19 and SC13) brown adipocytes. Oligo: oligomycin; FCCP: carbonyl cyanide p-trifluoro-methoxyphenyl hydrazone; Rot/AA: rotenone and antimycin A. The basal, proton leak, maximal, and non-mitochondrial respiration were presented. Student's t-test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 vs NTC, (F) SIK3 deletion/mutation constructs. ΔPKA: Δ432-805, PKA phosphorylation domain. ΔKD/PKA: Δ147-850, kinase domain (KD), ubiquitin-associated domain (UBA), and PKA phosphorylation domain. S884A: Ser884 to Ala phosphorylation-resistant mutation. S884D: Ser884 to Asp phosphorylation-mimetic mutation. (G) Expression of Ucp1 mRNA in Sik3−/− brown adipocytes (SC19) transfected with GFP, SIK3, or SIK3 deletion/mutation plasmids. At day 3 of differentiation, cells were recovered in fresh medium for 3 h and treated with vehicle (Veh) or 100 nM Iso for 3 h. Student's t-test, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; ns: no statistical difference vs SIK3 Iso.