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. 2021 Aug 11;321(3):C585–C595. doi: 10.1152/ajpcell.00195.2021

Figure 2.

Figure 2.

ATF4 activation contributes to palmitate induced NNMT upregulation. A: AML12 were treated with palmitate (0.4 mM) for 16 h. Total RNA was extracted, and ATF4 gene expression quantified using real time-qPCR. Data are expressed as means ± SD, n = 4 separate experiments. Differences between the two groups were determined using Student’s t test (***P < 0.001 vs. control). B: AML12 cells were treated with the indicated concentrations of palmitate for 16 h, ATF4 protein abundance was determined by Western blotting. The signal of ATF4 protein band was measured by densitometry and then divided by the signal of its corresponding actin abundance in the same sample. Data are expressed as means ± SD, n = 3 separate experiments. Student’s t test was used for statistical evaluation (*P < 0.05; ***P < 0.001 vs. control). C: AML12 cells were pretreated with GSK2006414 (20 nM), a selective inhibitor of PERK, for 2 h before 0.4 mM palmitate exposure for 16 h. NNMT expression was quantified by real time-qPCR for mRNA and Western blotting for protein, respectively. Data are expressed as means ± SD, n = 3 separate experiments. Differences between the two groups were determined using Student’s t test (**P < 0.01; ***P < 0.001 vs. control). D and E: AML12 cells were transfected with either scramble siRNA (siCtrl) or ATF4 siRNA (siATF4) for 24 h and treated with palmitate (PA at 0.4 mM) or tunicamycin (TUM at 5 µM) for 16 h. ATF4 (D) and NNMT (E) expression was quantified and data were expressed as means ± SD, n = 6 separate experiments. Differences between the two groups were determined using Student’s t test (*P < 0.05; **P < 0.01 vs. corresponding control cells). F: AML12 cells were pretreated with myriocin (20 µM), a specific inhibitor of serine palmitoyltransferase (SPT), for 2 h before 0.4 mM palmitate exposure for 16 h. ATF4 and NNMT gene expression was quantified by real time-qPCR. Data are expressed as means ± SD, n = 3 separate experiments. Differences between the two groups were determined using Student’s t test (*P < 0.05; **P < 0.01; ***P < 0.001 vs. control). ATF, activating transcription factor; NNMT, nicotinamide N-methyltransferase.