SKIL Antibody - #DF3088

Fig. 3. DNMT aberration-induced GPX4 suppression is co-regulated by KLF5. (A) Schematic diagram depicting mouse Gpx4 promoter luciferase reporter, Gpx4p-luc, the KLF5 binding motif, and its position relative to the transcription starting site. (B) Western blot. Calvarial tissues treated with control and TP (20 mg per mouse, 2 weeks) were assayed for KLF5, NCoR, SnoN, and SMRT, with β-actin serving as control. Two random samples per group were shown. Quantification beside blots was presented as mean ± SEM; n = 6; *P < 0.05, Student’s t test. (C) ChIP. Sham or TP-treated calvaria treated with or without SGI-1027 (2.5 mg/kg daily) were immunoprecipitated first with antibodies to KLF5, SMRT, NCoR, or SnoN, and then the immunoprecipitated DNA fragments were amplified through PCR with primer set specific for the KLF5 motif-containing region of the Gpx4 promoter, respectively. The non-immunoprecipitated DNA served as control (Input). PCR products were resolved on agarose gels. Quantifications on the right side were presented as mean ± SEM; n = 6; *P < 0.05, 2-way ANOVA. (D) Western blot. Primary osteoblasts (OBs) treated without or with TP (100 μg/ml) and the KLF5 inhibitor ML264 (10 μM) for 48 h were assayed for GPX4, with β-actin serving as control. Quantification below was presented as mean ± SD of 3 replicated experiments. *P < 0.05, 2-way ANOVA. (E) Luciferase assay. Primary OBs were transfected with a mouse Gpx4 promoter-luciferase reporter (Gpx4p-luc) and a Renilla luciferase reporter control, and then treated without or with TP (100 μg/ml) and SGI-1027 (10 μM) in the absence or presence of ML264 (10 μM) for 48 h. Luciferase activities of the Gpx4 promoter reporter were adjusted to Renilla luciferase activities and presented as box-and-whisker plots of 4 independent experiments. *P < 0.05, 3-way ANOVA.