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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].
We thus evaluated the role of LA-induced [Ca2+]m efflux in peroxynitrite generation in isolated mitochondria from HM cells.
To show that PIMCE is a contributing factor to diabetic nephropathy, we determined whether PIMCE occurs in primary cultured HM cells.
As shown in Figure 2H and I, LA treatment caused significantly increased nitrotyrosine levels in a spectrum of proteins in 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.
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To test this hypothesis, we first treated parental hTERT-HM cells with either vehicle or R1881.
As described in our previous study, hTERT-HM cells stably expressing control shRNA or shRNA against AR were designated as hTERT shAR) and hTERT shCTRL), whereas cells overexpressing mock or AR cDNA were designated as hTERT AR), hTERT(Mock).
To study the hormonal regulation of AR in myometrial cell apoptosis, we treated parental hTERT-HM cells with CPT, UV light or anti-Fas antibody in the presence of vehicle, synthetic AR agonist R1881 or AR antagonist MDV3100 for 0 48 h.
Though our results on the antiapoptotic actions of AR were collected from in vitro myometrial hTERT-HM cell model, further studies shall apply in vivo animal models (e.g. the female AR knockout mice).
Prior to gene targeting, HM-1 cells were cultured in ES cell medium supplemented with 6-thiguanine (Sigma) for 7 days to select for Hprt-deficient cells.
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