Figure 2 from SMYD3 Impedes Small Cell Lung Cancer Sensitivity to Alkylation Damage through RNF113A Methylation–Phosphorylation Cross-talk

Abstract

<p>Identification of RNF113A as a novel methylated substrate of SMYD3. <b>A,</b> Recombinant SMYD3 was used for <i>in vitro</i> methylation reactions using radiolabeled S-adenosylmethionine and potential substrates. Top, autoradiogram of the methylation assay. Bottom, Coomassie stain of proteins in the reaction. <b>B,</b> H1092 SCLC cells were modified to express Cas9/sgRNA targeting MAP3K2 (sg<i>MAP3K2</i>) or control nontargeting sgRNA (sgControl). The cells were grafted subcutaneously to immunocompromised NSG mice. Once tumor volume reached 100 mm<sup>3</sup>, indicated animal groups were treated with CP and control groups received placebo (vehicle) treatment. Quantification of xenograft tumor volume growth is shown (<i>n</i> = 5 mice for each treatment group). <i>P</i> values were calculated by two-way ANOVA with the Tukey test for multiple comparisons. Data are represented as mean ± SEM. Representative immunoblot analysis of indicated cell lysates is shown. Tubulin is used as a loading control. <b>C,</b> Representative image showing recombinant SMYD3 <i>in vitro</i> methylation reaction on protein arrays (ProtoArray—containing more than 9,500 potential substrates) using radiolabeled S-adenosylmethionine as a methyl donor. Magnification shows the signals identified in square 43 of the Protoarray, corresponding to the indicated spotted proteins and controls. <b>D,</b><i>In vitro</i> methylation assay as in <b>A</b> using potential substrates identified by ProtoArray. Top, autoradiogram of the methylation assay. Bottom, Coomassie stain of proteins in the reaction. <b>E,</b> Identification of RNF113A K20 trimethylation by bottom-up MS-based proteomic analysis of RNF113A methylated <i>in vitro</i> by SMYD3. Note that deuterated SAM was used as a methyl donor. <b>F,</b><i>In vitro</i> methylation assay as in <b>A</b> with recombinant SMYD3 and WT RNF113A as well as K20A or K21A mutant proteins. Top, autoradiogram of the methylation assay. Bottom, Coomassie stain of proteins in the reaction. <b>G,</b> Detection of RNF113A methylation in 293T cells using the RNF113A K20me3 antibody after ectopic expression of SMYD3 and WT or K20A mutant RNF113A. In all panels, a representative of at least three independent experiments is shown unless stated otherwise.</p>

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