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We thus evaluated the role of LA-induced [Ca2+]m efflux in peroxynitrite generation in isolated mitochondria from HM cells.
We thus evaluated whether mitochondrial complexes I and III were responsible for the LA-induced peroxynitrite generation in HM cells.
Human mesangial (HM) cells grown in high glucose show largely increased mitochondrial membrane potential and ROS production [13].
To show that PIMCE is a contributing factor to diabetic nephropathy, we determined whether PIMCE occurs in primary cultured HM cells.
In the current work, we observed increased nitrotyrosylation in multiple proteins of LA-treated HM cells (Figure 2), demonstrating that the LA responsive peroxynitrite generation enhanced nitrosative stress.
As shown in Figure 5A, treatment of HM cells with 17-DMAG (90 nM) or the hsp90β1 RNAi for 48 h effectively downregulated hsp90β1 expression.
In our current work, we have demonstrated that LA (an 18∶2 n-6 PUFA) induced Ca2+ efflux from mitochondria and caused peroxynitrite production in HM cells.
The effects of 17-DMAG and hsp90β1 RNAi on LA-induced [Ca2+]i mobilization and peroxynitrite generation in treated HM cells were assessed as described above.
These data indicated that at least part of the [Ca2+]i response to LA resulted from [Ca2+]m efflux, which might be essential to the peroxynitrite generation in HM cells.
HM cells in 100 mm dishes were grown for 24 h in RPMI 1640 supplemented with 14% FBS prior to treatment with hsp90β1 RNAi or 17-DMAG.
Because nitrosative injury as indexed by protein nitrotyrosine was dramatically increased in diabetic nephropathy [11], we wanted to know whether the ROS generated along with LA-induced [Ca2+]i mobilization in HM cells was peroxynitrite.
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