25 research outputs found

    Representative morphologies of <i>csnk-1(lf)</i>, <i>bli-3(lf)</i> and <i>doxa-1(lf)</i> single mutants.

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    Representative morphologies of csnk-1(lf), bli-3(lf) and doxa-1(lf) single mutants.</p

    Depletion of wildtype <i>csnk-1</i> transcripts in L4 <i>csnk-1(lf)</i> mutants.

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    (A) Positions and sequences of PCR primers for detecting all or wildtype-only csnk-1 transcripts. Partial wildtype and mutant csnk-1 sequences are aligned to show the specificity of the primers for wildtype-only transcripts. (B) Relative total csnk-1 transcript levels. (C, D) Relative wildtype-only csnk-1 transcript levels in wildtype, csnk-1(mac494lf) or csnk-1(mac495lf) animals. tba-1 was the loading control. Statistics: two-tailed unpaired Student’s t-test. *: p p p (TIFF)</p

    Effects of <i>csnk-1</i> mutations or variable feeding RNAis on the Sisi phenotype.

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    Mammalian orthologs or homologs are indicated in the parentheses. (TIFF)</p

    Representative morphologies of double homozygous mutants between <i>csnk-1(lf)</i> and <i>bli-3(lf)</i> or <i>doxa-1(lf)</i>.

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    bli-3(lf) csnk-1(lf) double homozygous mutants were derived from bli-3(lf) csnk-1(lf)/hT2 heterozygous mutants. csnk-1(lf); doxa-1(lf) double homozygous mutants were derived from csnk-1(lf)/hT2; doxa-1(lf)/hT2 heterozygous mutants. Arrows point to typical blisters. All images are of the same scale. (TIFF)</p

    Genomic target sequences for generating <i>tsp-15</i> knockin and <i>csnk-1</i> knockout strains using the CRISPR/Cas9 method.

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    ND: not determined. For tsp-15 repair template, the letter in red was for introducing the missense mutation and letters in blue were for introducing silent mutations. (TIFF)</p

    Raw data for Figures and Tables.

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    Oxidative stress response is a fundamental biological process mediated by conserved mechanisms. The identities and functions of some key regulators remain unknown. Here, we report a novel role of C. elegans casein kinase 1 gamma CSNK-1 (also known as CK1γ or CSNK1G) in regulating oxidative stress response and ROS levels. csnk-1 interacted with the bli-3/tsp-15/doxa-1 NADPH dual oxidase genes via genetic nonallelic noncomplementation to affect C. elegans survival in oxidative stress. The genetic interaction was supported by specific biochemical interactions between DOXA-1 and CSNK-1 and potentially between their human orthologs DUOXA2 and CSNK1G2. Consistently, CSNK-1 was required for normal ROS levels in C. elegans. CSNK1G2 and DUOXA2 each can promote ROS levels in human cells, effects that were suppressed by a small molecule casein kinase 1 inhibitor. We also detected genetic interactions between csnk-1 and skn-1 Nrf2 in oxidative stress response. Together, we propose that CSNK-1 CSNK1G defines a novel conserved regulatory mechanism for ROS homeostasis.</div

    <i>csnk-1(lf)</i> and <i>skn-1(gf)</i> mutations exhibit synergy on the Sisi phenotype in 50 mM NaI.

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    csnk-1(lf) and skn-1(gf) mutations exhibit synergy on the Sisi phenotype in 50 mM NaI.</p

    Characterization of <i>csnk-1</i>.

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    (A) csnk-1 gene structure (based on wormbase.org). The positions of mac397, mac494 and mac495 mutations are indicated. The position of the sgRNA used for CRISPR/Cas9-based mutagenesis is shown as a red bar. (B) Percentage of L1 larva that grew into adults on plates with 5 mM NaI. Results were based on two biological replicates. 100 L1 larva were analyzed in each replicate. Statistics: two-tailed unpaired Student’s t-test. *: p csnk-1p::GFP transgene in adults. (C) Fluorescent picture of a transgenic adult. The head, tail and vulva are indicated. (D-H) Higher-resolution pictures of transgenic adults. Cells with obvious GFP expression are indicated.</p

    Effects of <i>csnk-1</i> transgenes on the Sisi phenotype of <i>csnk-1(lf)</i> mutants in 5 mM NaI.

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    Effects of csnk-1 transgenes on the Sisi phenotype of csnk-1(lf) mutants in 5 mM NaI.</p

    CSNK-1 interacts with DOXA-1.

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    (A, B, C) Confocal pictures of a transgenic L3 larva co-expressing dpy-7p::doxa-1::GFP and dpy-7p::csnk-1::mCherry transgenes. Enclosed areas are enlarged in lower panels. Arrows indicate typical subcellular structures co-labeled by GFP and mCherry. (D, E) Western blotting showing the specific interaction between DOXA-1::HA and FLAG::CSNK-1 expressed in HEK293T Cells. Immunoprecipitation was performed using an anti-HA antibody (D) or an anti-FLAG antibody (E). (F, G) Western blotting showing that His::TF::DOXA-1 or His::TF::CSNK-1 purified from BL21 bacteria can specifically pull-down FLAG::CSNK-1 (F) or DOXA-1::HA (G) expressed in HEK293T Cells, respectively. Coomassie blue (CB) staining of purified proteins is shown in lower panels. (H, I) Relative fluorescent intensities of L4 larva stained with DCFDA or Amplex Red. Results were based on three biological replicates, with 2–5 samples per replicate. Each datapoint represents one sample. Colors represent different replicates. Statistics: two-tailed unpaired Student’s t-test. ***: p jrIs1 reporter in wildtype or csnk-1(lf) L4 larva. Picture exposure time was 432.6 ms. (K) Relative oxidized/reduced HyPer reporter signals. Results were based on three biological replicates, with 2–3 samples per replicate. Each data point represents one sample. Colors represent different replicates. Statistics: two-tailed unpaired Student’s t-test. **: p < 0.01. Error bars: standard deviation.</p
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