Fig 2: JMJD1C regulates AMPK in a CAMKK2-dependent manner. (A,B) JMJD1C does not affect activation of LKB1 or levels of its activation partners STRAD and MO25 in NRCMs. The cells were infected with adenovirus expressing Jmjd1c, shJmjd1c, or the control constructs for 24 h, followed by Ang II treatment for an additional 24 h. n = 3 in each group. (C) Effects of JMJD1C on CAMKK2 expression. NRCMs were treated as in (A). **p < 0.01 vs. Ad-Ctrl or shCtrl. n = 3 in each group. (D,E) Effects of JMJD1C on CAMKK2 mRNA expression. NRCMs were treated as in (A). **p < 0.01 vs. Ad-Ctrl or shCtrl. (F) Chromatin immunoprecipitation (ChIP) assay showing the binding of JMJD1C, JMJD2A, and JMJD3 on the FHL1, ß-MHC, and CAMKK2 promoters. IgG was used for negative control, and the enrichment was normalized to the input. ***p < 0.001 vs. IgG. (G) ChIP assay showing that JMJD1C overexpression reduced H3K9me1 enrichment at the CAMKK2 promoter in cardiomyocytes. ***p < 0.001 (H). The inhibition of CAMKK2 blocks the effects of JMJD1C on AMPK activation in NRCMs. The cells were treated with adenovirus expressing shJmjd1c or shCtrl; then the cells were treated with Ang II and the CAMKK2 inhibitor STO-609 (20 µM) for an additional 24 h. All results are from three independent experiments. **p < 0.01 vs. Ad-shCtrl+PBS; ##p < 0.01 vs. Ad-shCtrl+STO-609. n = 3 in each group.

Fig 3: (A, B) Western blots of proteins related to senescence and inflammation at PID 15 in shNegative, shSirt1, and shSirt1+LKB1 K48R cells. (A) The aforementioned proteins were assessed by western blot in PID 15 shNegative, Sirt1 knockdown, and Sirt1 knockdown + LKB1 K48R expressing 3T3-L1 cells. Upregulation of cyclin kinase inhibitor p21CIP, inflammatory chemokine receptor IL8b, and TGF-beta was observed in Sirt1 knockdown cells, and LKB1 K48R prevented these changes. (n=4, *p<0.05 vs. shNegative).

Fig 4: (A, B) Western blots of proteins related to and AMPK, LKB1, and Sirt1 signaling and mitochondrial dysfunction at PID 15 in shNegative, shSirt1, and shSirt1+LKB1 K48R cells. p-T172 AMPK and AMPK alpha2 were increased despite decreased p-MARK (LKB1 substrate) in Sirt1 knockdown cells. LKB1 K48R increased p-MARK and decreased p-T172 AMPK. It also significantly upregulated mitochondrial complex I proteins. The ratio of phospho-to-total MARK: A decrease in this ratio indicates a decrease in LKB1 activity caused by Sirt1 knockdown and upregulated by LKB1 K48R expression. Complex 1 expression normalized by HSP90: As seen in (A), expression of LKB1 K48R not only ameliorates LKB1 activity, it also increases mitochondrial complex I expression. (n=4, *p<0.05 vs shNegative). AMPK activity was increased by shSirt1 cells: AMPK activity was assessed by using 32P-ATP in vitro in the SAMS peptide assay. (*p<0.05 vs shNegative, n=6).

Fig 5: (A) Differentiation of 3T3-L1 cells in shNegative and Sirt1 knockdown cells. Oil O red staining image (x100). Red staining was extracted with isopropanol and absorbance was measured at 400 nm. There were no apparent effects of Sirt1 knockdown on fat accumulation. (B, C) Proteins indicating adipocyte differentiation and AMPK, LKB1, and Sirt1 signaling assessed by western blot. Sirt1 knockdown was evident at 10 PID; however, pACC/tACC and pAMPK/tAMPK showed no apparent suppression of AMPK activity. pSerLKB1/LKB1 showed suppression of LKB1 at 6 and 10 PID. A similar suppression trend was observed in PGC1α/HSP90. n=4, *p<0.05 vs shNegative. (D) Effect of Sirt1 knockdown on LKB1 activity and ATP/ADP ratio. LKB1 activity was assessed by LKBtide phosphorylation in immuno-precipitated samples. Sirt1 knock-down causes LKB1 activity downregulation. (n-6, *p<0.05). ATP/ADP ratios were significantly lower with Sirt1 knock-down (n=6. P<0.05). Similar experiments were repeated three times.