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. 2020 Sep 15;10(25):11479–11496. doi: 10.7150/thno.49870

Figure 4.

Figure 4

DAPK1-mediated Pellino1 Ser39 phosphorylation contributes to Pellino1 turnover, which is instrumental for the LPS-induced caspase-8 recruitment of TRIF-RIP1 signalosome and tubular damage under hypoxia. (A) Cellular ubiquitination assays comparing the poly-Ub levels of Pellino1 in Flag-tagged wild-type Pellino1 (WT)-, mutant Pellino1 Ser39A (S39A)- or Pellino1 Ser39D (S39D)-expressed HK-2 cells exposed to 50 ng/mL LPS with or without hypoxia. (B) CHX pulse-chase experiments determining the turnover of Pellino1 protein in Flag-tagged wild-type Pellino1 (WT)-, mutant Pellino1 Ser39A (S39A)- or Pellino1 Ser39D (S39D)-expressed HK-2 cells exposed to 50 ng/mL LPS with or without hypoxia in the presence or absence of 20 µg/mL CHX treatment for the indicated times. (C) Coimmunoprecipitation assay measuring the interaction of caspase-8 and TRIF and RIP1 in HK-2 cells exposed to 50 ng/mL LPS with or without hypoxia in the presence of 10 µM DAPK1 inhibitor (DAPK1-i) treatment. V, vehicle. (D) Coimmunoprecipitation assay examining the interaction of caspase-8 and TRIF and RIP1 in HK-2 cells exposed to 50 ng/mL LPS with or without hypoxia in the presence of DAPK1 siRNA transfection. (E) Cellular ubiquitination assays comparing the poly-Ub levels of Pellino1 in Flag-tagged wild-type Pellino1 (WT)-expressed HK-2 cells exposed to 50 ng/mL LPS with or without hypoxia in the presence of 10 µM DAPK1 inhibitor (DAPK1-i) treatment. (F) Cellular ubiquitination assays comparing the poly-Ub levels of Pellino1 in Flag-tagged wild-type Pellino1 (WT)-expressed HK-2 cells exposed to 50 ng/mL LPS with or without hypoxia in the presence of DAPK1 siRNA transfection. (G) Top panel: Western blotting analyses examining abundance of Pellino1 protein in Pellino1 knockout HK-2 cells (Pellino1 KO) where Pellino1 was deleted from the genome by CRISPR-Cas9 editing. Bottom panel: Western blotting analyses evaluating levels of DAPK1 protein expression in HK-2 cells with or without DAPK1 siRNA transfection. (H) MTT assay comparing cell viability of Pellino1 knockout HK-2 cells exposed to 50 ng/mL LPS with or without hypoxia in the presence or absence of DAPK1 siRNA transfection. Experiments were performed three times and data are expressed as mean ± s.d. *P<0.05, one-way ANOVA, post hoc comparisons, Tukey's test. n.s. no significant. (I) Representative pictures and quantification from Hoechst33342 and PI double-staining assay of Pellino1 knockout HK-2 cells exposed to 50 ng/mL LPS with or without hypoxia in the presence or absence of DAPK1 siRNA transfection. Experiments were performed three times and data are expressed as mean ± s.d. *P<0.05, one-way ANOVA, post hoc comparisons, Tukey's test.