Journal: The Journal of Biological Chemistry

Article Title: The Ser/Thr kinase p90RSK promotes kidney fibrosis by modulating fibroblast–epithelial crosstalk

doi: 10.1074/jbc.RA119.007904

Figure Lengend Snippet: p90RSK induces fibroblast-mediated tubular epithelial apoptosis in vivo and in vitro. A, obstructed kidney sections from FSP-1–specific p90RSK transgenic mice (RSK-Tg) and their littermates (RSK-wt) were subjected to immunofluorescence staining of cleaved caspase-3 (red) and lectin (green, proximal tubular epithelial marker), 200×. Scale bar, 50 μm. B, immune staining of TUNEL (green) and lectin (red), 400×. Scale bar, 20 μm. C, quantitation of apoptotic epithelial cells. ***, p < 0.001; n = 5 microscopic fields per mouse × 5 mice/group. D, illustration of primary fibroblast (RSK-Tg and RSK-wt) and epithelial cell (HKC-8) coculture. After serum starvation overnight in the coculture, 50 nm staurosporine or 150 μm H2O2 was added to the lower chamber for additional 4 or 16 h, respectively. Then, HKC-8 cells were harvested and subjected to Western blotting for cleaved caspase-3 and GAPDH. E, Western blotting of cleaved caspase-3 after staurosporine treatment. F, quantitation of cleaved caspase-3 abundance. *, p < 0.05; **, p < 0.01; n = 3 experiments. G, Western blotting of cleaved caspase-3 after H2O2 treatment. H, quantitation of cleaved caspase-3 abundance. **, p < 0.01; n = 3 experiments. CLK, control kidney; staur, staurosporine. Error bars, S.E.

Article Snippet: Hydrogen peroxide production assay HKC-8 and fibroblasts were cocultured in serum-free medium for 4 h, followed by measurement of hydrogen peroxide content in the coculture medium by a hydrogen peroxide assay kit (Bioassay Systems, Hayward, CA).

Techniques: In Vivo, In Vitro, Transgenic Assay, Immunofluorescence, Staining, Marker, TUNEL Assay, Quantitation Assay, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: The Ser/Thr kinase p90RSK promotes kidney fibrosis by modulating fibroblast–epithelial crosstalk

doi: 10.1074/jbc.RA119.007904

Figure Lengend Snippet: ROS mediates fibroblast p90RSK-induced tubular epithelial apoptosis. A, obstructed kidneys were subjected to immune staining for 4-HNE. Scale bar, 50 μm. B, quantitation of ROS level in the obstructed kidneys. **, p < 0.01; ***, p < 0.001; n = 5 mice/group. Primary fibroblasts from FSP-1–specific p90RSK transgenic mice (RSK-Tg) or littermates (RSK-wt) were in coculture with HKC-8 cells for 4 h. C, then, H2O2 level in the coculture medium were measured. **, p < 0.01; ***, p < 0.001; RSK-Tg versus RSK-wt, n = 3 experiments. D, 50 nm staurosporine and 50 nm ROS-specific inhibitor YCG063 were added into the coculture for 4 h, followed by Western blotting for cleaved caspase-3 and GAPDH in HKC-8 lysates. E, quantitation of epithelial cleaved caspase-3 abundance. *, p < 0.05; **, p < 0.01; n = 3 experiments. F, 150 μm H2O2 and 50 nm YCG063 were added into the coculture for 16 h, followed by Western blotting for cleaved caspase-3 and GAPDH. G, quantitation of cleaved caspase-3 abundance in epithelial cells. **, p < 0.01; n = 3 experiments. Staur, staurosporine; T, tubular epithelial cells. Error bars, S.E.

Article Snippet: Hydrogen peroxide production assay HKC-8 and fibroblasts were cocultured in serum-free medium for 4 h, followed by measurement of hydrogen peroxide content in the coculture medium by a hydrogen peroxide assay kit (Bioassay Systems, Hayward, CA).

Techniques: Staining, Quantitation Assay, Transgenic Assay, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: The Ser/Thr kinase p90RSK promotes kidney fibrosis by modulating fibroblast–epithelial crosstalk

doi: 10.1074/jbc.RA119.007904

Figure Lengend Snippet: ROS-activated β-catenin mediates fibroblast p90RSK-induced tubular epithelial apoptosis. A, HKC-8 cells were incubated with 1.5 μm H2O2 for 1 h, followed by immunofluorescence staining of β-catenin (green) and phalloidin (red). Scale bar, 25 μm. B, ROS-specific inhibitor YCG063 (50 nm) was added into the fibroblast–epithelial coculture, followed by Western blotting for β-catenin and GAPDH in epithelial lysates. C, quantitation of β-catenin abundance. *, p < 0.05; **, p < 0.01; n = 3 experiments. D, Western blotting for active β-catenin and GAPDH in epithelial lysates. E, HKC-8 cells were transfected with control or β-catenin siRNAs, followed by coculture with RSK-Tg or RSK-wt fibroblasts with or without 50 nm staurosporine for 4 h. HKC-8 lysates were subjected to Western blotting for cleaved caspase-3 and GAPDH. Nuclear extracts of HKC-8 were probed with β-catenin and PCNA. F, quantitation of cleaved caspase-3 abundance in epithelial lysates. ***, p < 0.001; n = 3 experiments. staur, staurosporine; consiRNA, control siRNA; ctnsiRNA, β-catenin siRNA. Error bars, S.E.

Article Snippet: Hydrogen peroxide production assay HKC-8 and fibroblasts were cocultured in serum-free medium for 4 h, followed by measurement of hydrogen peroxide content in the coculture medium by a hydrogen peroxide assay kit (Bioassay Systems, Hayward, CA).

Techniques: Incubation, Immunofluorescence, Staining, Western Blot, Quantitation Assay, Transfection

Journal: The Journal of Biological Chemistry

Article Title: The Ser/Thr kinase p90RSK promotes kidney fibrosis by modulating fibroblast–epithelial crosstalk

doi: 10.1074/jbc.RA119.007904

Figure Lengend Snippet: ROS-activated FOXO1 mediates fibroblast p90RSK-induced tubular epithelial apoptosis. A, HKC-8 cells were incubated with 1.5 μm H2O2 for 4 h, followed by immunofluorescence staining of FOXO1 (green) and phalloidin (red). Scale bar, 25 μm. B, HKC-8 cells were in coculture with primary RSK-Tg or RSK-wt fibroblasts for the period as indicated, then the nuclear extracts from HKC-8 cells were subjected to Western blotting for FOXO1 and PCNA. C, quantitation of nuclear FOXO1 abundance. ***, p < 0.001; RSK-Tg versus RSK-wt; n = 3 experiments. D, FOXO1 activity was also evaluated by FKHR ELISA assay. *, p < 0.05; **, p < 0.01; n = 3 experiments. E, immune staining of FOXO1 in the obstructed kidneys from FSP-1–specific p90RSK transgenic mice (RSK-Tg) and their littermates (RSK-wt). Scale bar, 50 μm. F, 50 nm YCG063 (ROS-specific inhibitor) was added into the coculture for 24 h, followed by FKHR ELISA assay in HKC-8 nuclear extracts. *, p < 0.05; **, p < 0.01; n = 3 experiments. G, 1 μm FOXO1-specific inhibitor AS1842856 and staurosporine (50 nm) were added into the coculture for 4 h, followed by Western blotting for cleaved caspase-3 and GAPDH in HKC-8 lysates. H, quantitation of cleaved caspase-3 abundance. ***, p < 0.001; n = 3 experiments. I, HKC-8 cells were transfected with FOXO1 siRNA, followed by Western blotting for FOXO1 and GAPDH. J, HKC-8 cells were transfected with 80 nm control or FOXO1 siRNAs, followed by coculture with RSK-Tg or RSK-wt fibroblasts with or without 50 nm staurosporine for 4 h, and Western blotting for cleaved caspase-3 and GAPDH. K, quantitation of cleaved caspase-3 abundance. ***, p < 0.001; n = 3 experiments. Staur, staurosporine; consiRNA, control siRNA; FOXsiRNA, FOXO1 siRNA; T, tubular epithelial cells. Error bars, S.E.

Article Snippet: Hydrogen peroxide production assay HKC-8 and fibroblasts were cocultured in serum-free medium for 4 h, followed by measurement of hydrogen peroxide content in the coculture medium by a hydrogen peroxide assay kit (Bioassay Systems, Hayward, CA).

Techniques: Incubation, Immunofluorescence, Staining, Western Blot, Quantitation Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Transgenic Assay, Transfection