Fig. 1: A kinome-wide screen uncovers protein kinases involved in RTEC cell death. | Nature Communications

Fig. 1: A kinome-wide screen uncovers protein kinases involved in RTEC cell death.

From: A kinome-wide screen identifies a CDKL5-SOX9 regulatory axis in epithelial cell death and kidney injury

Fig. 1

a Scheme depicting the assay conditions used in the primary siRNA screen. BUMPT cells were transfected with Kinome-wide siRNA library (Dharmacon), followed by cisplatin treatment and cell-titer-glo-based viability assay. b Results of primary RNAi screening, shown by plotting the relative survival post cisplatin treatment of individual siRNA-targeted genes obtained from triplicate samples. c Kinome map (KinMap) depicting kinases identified in the primary screen. d Validation of primary hits by distinct siRNAs (Sigma) in BUMPT cells. Survival data (MTT assay) are presented as individual data points (n = 4 biologically independent samples), from one out of three independent experiments, all producing similar results. e Further secondary screening was carried out in HK-2 cells, by RNAi-mediated knockdown of indicates genes, followed by MTT-based cellular viability assay. Data are presented as individual data points (n = 4 biologically independent samples), from one out of three independent experiments, all producing similar results. f Schematic representation of CDKL5, the top hit and other members of CMGC kinase family. g, h Tertiary screening was carried for the top hit (Cdkl5) by shRNA-mediated knockdown in BUMPT cells and “add back” of wild-type and mutant Cdkl5. Cellular viability assays (MTT) showed that shRNA-mediated Cdkl5 knockdown protects BUMPT cells from cisplatin-mediated cell death, an effect that was reversed by re-introduction of wild-type but not mutant Cdkl5. Data are presented as individual data points (n = 4 biologically independent samples), from one out of three independent experiments, all producing similar results. Representative western blot results demonstrating shRNA-mediated CDKL5 kinase knockdown and introduction of untagged wild-type, kinase-dead (KD), and TEY/AEF Cdkl5 constructs. Data are representative of three independent experiments. In all the bar graphs, experimental values are presented as mean ± s.e.m. The height of error bar = 1 s.e. and p < 0.05 was indicated as statistically significant. One-way ANOVA followed by Dunnett’s (d, e) or Tukey’s multiple-comparison test (h) was carried out, and statistical significance is indicated by *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a Source Data file.

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