Figure 6 from Translational Activation of ATF4 through Mitochondrial Anaplerotic Metabolic Pathways Is Required for DLBCL Growth and Survival
<p>ATF4 translation and protein level respond to nutrient level and are regulated by autophagy. <b>A,</b> FCs of mean fluorescence intensity (MFI) of the GFP reporter expressed from the ATF4-5′UTR-GFP translation reporter in Karpas 422 cells containing control or SIRT3 shRNAs under...
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2025
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| Yhteenveto: | <p>ATF4 translation and protein level respond to nutrient level and are regulated by autophagy. <b>A,</b> FCs of mean fluorescence intensity (MFI) of the GFP reporter expressed from the ATF4-5′UTR-GFP translation reporter in Karpas 422 cells containing control or SIRT3 shRNAs under different culture conditions. Top, experiments were done as in the schema. Briefly, cells were cultured with fresh medium for 48 hours and then replenished with same volume of fresh medium or maintained without replenishment (as a control) for another 16 hours. The <i>y</i>-axis denotes GFP signal intensity relative to control cells without expression of reporter, determined with MFI of GFP signals from flow cytometer. <b>B,</b> FCs of MFI of the GFP reporter expressed from the ATF4-5′UTR-GFP translation reporter in Karpas 422 cells with control or SIRT3 shRNAs under control or glutamine starvation or tunicamycin (10 μg/mL) treatment. Both starvation and tunicamycin treatment were maintained for 15 hours at day 4 after viral transduction. <b>C,</b> Western blot results show the GFP expression from the ATF4-5′UTR-GFP translation reporter in Karpas 422 cells from <b>B</b>. <b>D,</b> The heatmap shows that relative abundances of amino acids were detected by metabolic profiling from Karpas 422 cells transduced with SIRT3 or control shRNAs. The metabolite levels were mean value from five to six replicate samples obtained on day 6 after infection. <b>E,</b> Relative activities of the ATF4-5′UTR-GFP reporter in Karpas 422 were cultured for 48 hours, replenished with fresh media or with the indicated nutrients, and then assessed for ATF4 translation reporter activity 16 hours later. The <i>y</i>-axis denotes GFP signal intensity relative to control cells without expression of reporter, determined with MFI of GFP signals from flow cytometer. NT, not treated; Q, glutamine. <b>F,</b> Western blots show ATF4 protein levels from the indicated cell lines cultured as in <b>A</b> and <b>E</b>, and then replenished with NEAA or not replenished, after which immunoblots were performed for ATF4, with tubulin and actin as loading controls, in control or NEAA-added conditions. <b>G,</b> The heatmap shows the amino acid abundance measured with LC/MS from Karpas 422 cells transduced with SIRT3 or control shRNA and cultured with or without DMKG supplementation. The values show the average logFC from three replicates in amino acid abundance as compared with DSMO-treated control shRNA–transduced cells. <b>H,</b> Western blot results show ATF4 level changes in control or ATG5 knockdown Karpas 422 cells under glutamine starvation condition. Samples were collected at different time points, and protein level changes were quantified with densitometry results. <b>I,</b> Western blots show the ATF4 and autophagy changes in Karpas 422 cells treated with DMSO or YC8-02 (YC; 3 μmol/L) for 40 hours. Cell lysates were subjected to Western blot using the indicated antibodies. Protein levels were quantified with densitometry results. <b>J,</b> The barplot shows the relative cell viability after OCI-LY1 and Karpas 422 cells were treated with DMSO, YC8-02 (OCI-LY1: 6 μmol/L; Karpas 422: 2 μmol/L), GCN2IN6 (OCI-LY1: 7.5 μmol/L; Karpas 422: 10 μmol/L), and combinations for 72 hours. Cells were subject to flow cytometry for viability tests (DAPI staining) and counting. *, <i>P</i> < 0.05; **, <i>P</i> < 0.01. NS, not significant. Error bars represent the mean ± SD of three or more replicates.</p> |
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