Rapid degradation of premature translational termination products.

<p><i>A.</i> Denaturing immunoprecipitation of peptidyl-puromycins. 293-K^b cells were radiolabeled for 30 minutes with [³⁵S]-Met, 20 µM puromycin, and 20 µM MG132. Polypeptides were precipitated with TCA, solubilized, then subjected to a denaturing immunoprecipitation using either non-specific rabbit serum (negative IP control) or anti-puromycin serum. Results are representative of three independent experiments. <i>B.</i> 293-K^b cells were pulse labeled with [³⁵S]-Met and 20 µM puro and chased as described in Fig. 3B. Solubilized TCA precipitates were subjected to denaturing immunoprecipitation using anti-puromycin serum. [³⁵S] in the anti-puromycin immunoprecipitates was measured by liquid scintillation counting (<i>n</i> = 4; mean ± s.e.m.).</p

Puromycin treatment leads to increased levels of polyubiquitinated proteins.

<p>293-K^b cells were treated for 4 hours with media alone, 20 µM MG132, or the indicated concentrations of cycloheximide and puromycin (µM). Lysates were subjected to Western blotting with FK2 (<i>upper panel</i>), a monoclonal antibody specific for mono- and polyubiquitinated protein conjugates. Beta-tubulin was probed as a loading control (<i>lower panel</i>). Results are representative of three independent experiments.</p

Treatment with puromycin increases the fraction of rapidly degraded polypeptides.

<p><i>A.</i> 293-K^b cells were labeled with [³⁵S]-Met for 10 minutes in the presence of 0 to 40 µM MG132. [³⁵S] incorporation was measured as in Fig. 1A and normalized to controls without proteasome inhibitor (<i>n</i> = 3; mean ± s.e.m.) <i>B.</i> 293-K^b cells were pulse labeled with [³⁵S]-Met +/−20 µM puro and +/−20 µM MG132 for 5 minutes, then chased from 0 to 50 minutes in the presence of excess cold methionine, CHX and +/−20 µM MG132. DMSO is a solvent control for MG132. The chase was terminated at the indicated time points by the addition of TCA to cell suspensions to precipitate polypeptides. TCA precipitates were solubilized and [³⁵S] was measured by liquid scintillation counting (<i>n</i> ≥4; mean ± s.e.m.) <i>C</i> and <i>D</i>. Solubilized TCA precipitates from cells radiolabeled in the absence (<i>C</i>) or presence (<i>D</i>) of 20 µM puro were separated by tricine SDS-PAGE on 10% gels. Gels were dried and exposed to a PhosphorImager plate overnight. Note that for <i>D</i>, the darkness of the image has been enhanced in order to see the contrast in degradation rates between <i>C</i> and <i>D</i> more clearly.</p

Time-dependent inhibition of MHC class I pathway function following puromycin treatment.

<p><i>A</i> and <i>B.</i> 293-K^b cells were stripped of cell surface MHC I peptides as in Fig. 5. Recovery of cell surface K^b was conducted in the presence of varying concentrations of puro for one (<i>A</i>) and four (<i>B</i>) hours. During the final 30 minutes of the recovery, cells were treated with either distilled water ((−) peptide) or 5 µM SIINFEKL peptide ((+) peptide) to promote the export of K^b to the cell surface <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051968#pone.0051968-Day1" target="_blank">[31]</a>. Flow cytometry was used to measure total cell surface K^b and the MFI was normalized to untreated cells in the absence of exogenous SIINFEKL peptide (<i>n</i> = 3; mean ± s.e.m.; * <i>p</i><0.05 for (−) peptide vs. (+) peptide).</p

Effects of puromycin on the recovery of cell surface MHC class I-peptide complexes.

<p><i>A</i>–<i>E</i>. 293-K^b cells expressing the TRx9 reporter were stripped of cell surface MHC I peptides as in Fig. 5. Recovery of cell surface MHC class I-peptide complexes was conducted in the presence of varying concentrations of puro from 0 to 180 minutes. Flow cytometry was used to measure reporter eGFP fluorescence (<i>A</i>), as well as cell surface K^b-SIINFEKL complexes (<i>B</i>) and total cell surface K^b (<i>D</i>). To quantitate differences in the kinetics of MHC class I-peptide complex recovery, we normalized the MFI values of puro-treated cells to untreated cells for K^b-SIINFEKL (<i>C</i>) and total K^b (<i>E</i>) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051968#pone.0051968-Qian1" target="_blank">[4]</a> (<i>n</i> = 5; mean ± s.e.m.) For <i>C</i> and <i>E</i>, * <i>p</i><0.05 compared to untreated samples. <i>F.</i> MHC class I-peptide complex recovery after 1 hour in the presence of varying concentrations of puromycin. MFI values are normalized to untreated cells (<i>n</i> = 3; mean ± s.e.m.) <i>G</i>. MHC class I-peptide complex recovery after 4 hours in the presence of varying concentrations of puromycin. MFI values are normalized to untreated cells (<i>n</i> = 3; mean ± s.e.m.).</p

MHC class I-peptide complex recovery assay using a fluorescent reporter encoding antigenic peptides.

<p><i>A.</i> Schematic of the modified NP-SIINFEKL-eGFP reporter (adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051968#pone.0051968-Princiotta1" target="_blank">[2]</a>) containing eight additional tandem repeats of SIINFEKL (nine total) and its five flanking amino acids from the native ovalbumin sequence, NP-[SIINFEKL]₉-eGFP (Tandem Repeat x9 or TRx9). <i>B.</i> Validation of MHC I peptide stripping and recovery in TRx9-expressing cells. Biexponential scatter plots show single cell profiles of the mean fluorescence intensity (MFI) for eGFP on the <i>x</i>-axis and 25-D1.16 on the <i>y</i>-axis. Plots show fluorescence profiles immediately pre- (<i>left</i>) and post- (<i>middle</i>) peptide stripping, and after a 4 hour recovery (<i>right</i>).</p

Puromycin stimulates the production of truncated polypeptides in a dose-dependent manner.

<p><i>A.</i> 293-K^b cells were radiolabeled with [³⁵S]-Met for 10 minutes in the presence of a linear range of puromycin concentrations from 0 to 20 µM. Radiolabeled polypeptides were visualized as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051968#pone-0051968-g001" target="_blank">Fig. 1B</a>. In the later panels, we analyze the [³⁵S] signals from the four regions indicated to the right of the gel. <i>B</i>. PhosphorImager signal intensities (arbitrary units) from selected lanes in <i>A</i>. The left side of the graph corresponds to the top of the gel while the right side of the graph corresponds to the bottom of the gel at the dye front. The highlighted regions correspond to the parts of the gel indicated in <i>A</i>. <i>C</i>. The effects of puromycin concentration on [³⁵S] signal for each of the highlighted gel regions in <i>A</i> and <i>B</i>. Results are representative of three independent experiments.</p

A pulse of DUX4 negatively regulates the status of multiple translational regulators and broadly suppresses nascent protein synthesis.

(A) Immunoblot analysis 0–7 days following a 4-hour pulse of DOX in MB135iDUX4 myoblasts. Alpha, beta, and gamma correspond to the phosphorylated forms of 4EBP1. GAPDH serves as loading control. (B) Schematic of cap-dependent translation initiation complex (top) and immunoblot analysis of m7GTP pull-downs (bottom). (C) Immunoblot analysis of MB135 myoblasts treated with mTORC1 inhibitors Everolimus or Torin2. (D) Schematic of experimental time course for metabolic labeling with 35S or HPG. (E) Autoradiograph of samples pulsed with active DUX4 (top, left) or DNA-binding mutant F67A (top, right); Coomassie stain of total protein (middle); quantification of relative 35S signal normalized to paired 0-hour condition (bottom). Source data available in S1 Data. (F) Immunofluorescence (left) and flow cytometry (right) of HPG/Click-iT labeled proteins (scale bars, 50 μm). DOX, doxycycline; DUX4, double homeobox protein 4; HPG, L-homopropargylglycine; mTORC1, mechanistic target of rapamycin complex 1; m7GTP, 7-methylguaniosine 5′-triphosphate; 4EBP1, 4E-binding protein 1.</p

Differential RNA-seq analysis of polysome profiling.

Source data for Figs 3F, 3H, 3I and 4A, and S8B. (XLSB)</p

Oxidative stress, DNA damage, and hypoxia-induced cell stress pathways moderately suppress MHC-I and PSMB9.

(A) Immunoblot analysis of MB135 myoblasts treated with hydrogen peroxide (H2O2) to induce oxidative stress, (B) etoposide to induce DNA damage, or (C) cobalt chloride (CoCl2) to mimic hypoxia. Cells were treated with stress-inducing reagents for 24 hours, followed by a 16-hour incubation in media resupplemented with cell stress–inducing reagent plus IFNγ. Beta-actin serves as loading control. IFNγ, interferon gamma; MHC-I, major histocompatibility complex class I. (TIF)</p