KLF5 Antibody - #AF7542

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.

Fig. 6 ACSS2 induces KLF5-mediated p65 activation and downstream cell pyroptosis in LPS-induced kidney tubular injury. A RNA-seq analysis of kidney tissues from ACSS2 gene knockout (KO) mice and wild-type mice was performed. The colored dots indicated differentially expressed genes (DEGs). B Relative protein expression levels of KLF5 in the mouse kidney (n = 3). C Representative immunofluorescent images of KLF5 and LTL in the renal cortex of mice (green, LTL; red, KLF5; blue, DAPI; Scale bars, 100 μm). D Western blotting was used to compare expression of KLF5 in HK-2 cells treated with LPS (1 μg/mL) or vehicle and with ACSS2 siRNA or scrambled siRNA for 24 hours (n = 3). E Immunofluorescence staining was used to detect KLF5 (green, KLF5; blue, DAPI; scale bars, 50 μm). F and G HK-2 cells were pre-treated with ML264 (10 μmol/L) or vehicle for 1 hour, then with LPS (1 μg/ml) or vehicle for 24 hours. Western blotting was used to analyze (F) the expression of KLF5 or (G) the phosphorylation of p65, the expression of NLRP3, and the cleavage of GSDMD (n = 3). H The levels of cleaved GSDMD were determined by immunofluorescence staining (green, cleaved GSDMD; blue, DAPI; scale bars, 50 μm). Data were presented as mean ± SD. *P 

Fig. 6 ACSS2 induces KLF5-mediated p65 activation and downstream cell pyroptosis in LPS-induced kidney tubular injury. A RNA-seq analysis of kidney tissues from ACSS2 gene knockout (KO) mice and wild-type mice was performed. The colored dots indicated differentially expressed genes (DEGs). B Relative protein expression levels of KLF5 in the mouse kidney (n = 3). C Representative immunofluorescent images of KLF5 and LTL in the renal cortex of mice (green, LTL; red, KLF5; blue, DAPI; Scale bars, 100 μm). D Western blotting was used to compare expression of KLF5 in HK-2 cells treated with LPS (1 μg/mL) or vehicle and with ACSS2 siRNA or scrambled siRNA for 24 hours (n = 3). E Immunofluorescence staining was used to detect KLF5 (green, KLF5; blue, DAPI; scale bars, 50 μm). F and G HK-2 cells were pre-treated with ML264 (10 μmol/L) or vehicle for 1 hour, then with LPS (1 μg/ml) or vehicle for 24 hours. Western blotting was used to analyze (F) the expression of KLF5 or (G) the phosphorylation of p65, the expression of NLRP3, and the cleavage of GSDMD (n = 3). H The levels of cleaved GSDMD were determined by immunofluorescence staining (green, cleaved GSDMD; blue, DAPI; scale bars, 50 μm). Data were presented as mean ± SD. *P