renal epithelial cell growth medium 2  (PromoCell)

Journal: iScience

Article Title: FTO inhibition attenuates renal fibrosis by downregulating ferroptosis activator ACSL4 and profibrotic factor TGFBI

doi: 10.1016/j.isci.2025.113515

Figure Lengend Snippet: FTO inhibition suppresses ferroptosis in kidney epithelial cells by ROS reduction Primary kidney epithelial cells were generated using mouse kidneys (A). Images of primary mouse kidney epithelial cells (B) and cell viability assay (C) showed that FB-23 and Lipro-1 protected primary mouse kidney epithelial cells from erastin-induced ferroptosis. FB-23 and Lipro-1 protected human kidney epithelial HK2 cells from erastin-induced decrease in cell viability (D) and increased ROS production (E) and membrane damage (F). Synergy map analysis revealed a high level of antagonism between erastin and FB23-2 (G). FTO protein expression was abolished by western blot in three independent FTO KO clones (H). Images of HK2 cells (I) and cell viability assay (J) showed FTO knockdown suppressed erastin-induced ferroptosis; cell membrane damage (K), lipid peroxidation (I), and ROS production (M) were decreased in FTO knockdown cells compared with WT cells. Both pharmacological (N) and genetic inhibition of FTO (O) significantly decreased the transcript level of kidney damage marker LCN2 in HK2, and LCN2 protein expression in FTO KO cells was diminished (P). ∗ p < 0.05; ∗∗ p < 0.01. Scale bars: 100 μm in (B and I). Data are represented as mean ± SD.

Article Snippet: Primary renal epithelial cells were generated from kidney tissues excised from 8- to 12-wk-old C57/BL6 female mice and cultured in Renal Epithelial Cell Growth Medium 2 (PromoCell C-26030) as previously described.

Techniques: Inhibition, Generated, Viability Assay, Membrane, Expressing, Western Blot, Clone Assay, Knockdown, Marker

Journal: iScience

Article Title: FTO inhibition attenuates renal fibrosis by downregulating ferroptosis activator ACSL4 and profibrotic factor TGFBI

doi: 10.1016/j.isci.2025.113515

Figure Lengend Snippet: FTO inhibition protects against ferroptosis in hESC-derived kidney organoids by downregulating ACSL4-mediated ferroptosis (A) Generation of kidney organoids from hESC cells. (B) Immunofluorescence staining for cell type-specific markers in kidney organoids. (C) Expression changes of stem cell markers and kidney epithelial markers during the differentiation period of 20 days determined by qRT-PCR. (D) FTO, ACSL4, and TGFBI expression levels were dramatically decreased in kidney organoids after lentiviral transfection of shFTO determined by western blot. FTO inhibition attenuated erastin-induced ferroptosis determined by LDH assay (E), lipid peroxidation determined by MDA assay (F), and ROS production (G) in kidney organoids. FTO inhibition significantly reduced TGFBI (H) and LCN2 (I) expression at transcript level determined by qPCR compared with vehicle control. Schematic diagram of the mechanisms of FTO upregulation (J) and downregulation (K) in RF. Scale bars: 50 μm in (A and B). ∗ p < 0.05; ∗∗ p < 0.01. Data are represented as mean ± SD.

Article Snippet: Primary renal epithelial cells were generated from kidney tissues excised from 8- to 12-wk-old C57/BL6 female mice and cultured in Renal Epithelial Cell Growth Medium 2 (PromoCell C-26030) as previously described.

Techniques: Inhibition, Derivative Assay, Immunofluorescence, Staining, Expressing, Quantitative RT-PCR, Transfection, Western Blot, Lactate Dehydrogenase Assay, Multiple Displacement Amplification, Control