fluorescent puromycin incorporation  (Jena Bioscience)

Journal: Science Advances

Article Title: Hyperactive 20 S proteasome enhances proteostasis and ERAD in C. elegans via degradation of intrinsically disordered proteins

doi: 10.1126/sciadv.adx3014

Figure Lengend Snippet: ( A ) Dimensionality reduction via three-dimensional PCA was performed on TMT-MS data to depict profile differences between n = 5 WT and α3ΔN mutants. ( B ) Volcano plot of DEPs detected log 2 FC α3ΔN/WT versus −log 10 FDR. All points above the dotted line are statistically significant (FDR < 0.05). Blue points above the x axis dotted line represent DEPs down-regulated (FC < −1.5), and orange dots represent DEPs up-regulated (FC > 1.5). ( C ) GSEA of DEPs found in α3ΔN mutants, and x axis represents the normalized enriched score for each of the categories according to ClusterProfiler in R; FDR < 0.05. NADH, reduced form of nicotinamide adenine dinucleotide. ( D ) Rates of protein translation in live C. elegans determined by 6-FAM-puromycin, lysing and separating free 6-FAM-puro from labeled nascent proteins with desalting columns, followed by fluorescent quantification in a plate reader. Bars represent mean RFU ± SEM of n = 7, *** P < 0.01, ordinary one-way analysis of variance (ANOVA). RFU, relative fluorescence units.

Article Snippet: To assess protein synthesis, we used a fluorescent puromycin incorporation method, using 2 mM 6-FAM-dC-puromycin (Jena Bioscience), according to Wang et al. ( ) with some modifications for C. elegans .

Techniques: Labeling, Fluorescence

Journal: Science Advances

Article Title: Hyperactive 20 S proteasome enhances proteostasis and ERAD in C. elegans via degradation of intrinsically disordered proteins

doi: 10.1126/sciadv.adx3014

Figure Lengend Snippet: ( A ) Survival of WT animals and α3ΔN mutants upon tunicamycin treatment (50 μg/ml) (data from n = 3 independent experiments); P < 0.01 determined by the log-rank test. Representative images ( B ) and fluorescent quantification ( C ) of vit-2::GFP + rol-6(su1006) and α3ΔN; vit-2::GFP + rol-6(su1006) worms expressing the vitellogenin 2 protein fused to GFP VIT-S::GFP from the outcrossed RT99 strain with defective accumulation of VIT-2. Scale bar, 500 μm. Images of day 3 worms at ×4 magnification. (C) Accumulation over time. We tracked the fluorescent intensity of vit-2::GFP versus α3ΔN; vit-2::GFP worms, revealing a decrease in VIT-2 ::GFP accumulation in α3ΔN; vit-2::GFP mutants. Statistical analysis ( **P < 0.01) via multiple two-tailed t tests. Data shown as mean ± SEM of three independent batches of ≥10 worms each for a total n of ≥30 worms. ( D and E ) VIT-2 ::GFP validation as proteasome substrate by quantifying GFP fluorescence after addition of proteasome inhibitor MG132 (50 μM) to the vit-2::GFP (D) and α3ΔN; vit-2::GFP (E) adult worms. Data represent mean values ± SEM ( n = 6 protein lysates), * P < 0.05 by two-tailed t test with Welch correction. ( F ) Representative fluorescent images of postreproductive day 7 vit-2::GFP and α3ΔN; vit-2::GFP adults treated with 10 μM ML240 (cdc48 inhibitor) or control (DMSO). Scale bars, 500 μM. ( G ) Quantification of GFP intensity from (F). Data shown as mean ± SEM from three independent batches (≥10 worms per batch; n ≥ 30 total). Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test (*** P < 0.01). a.u., arbitrary units; ns, not significant.

Article Snippet: To assess protein synthesis, we used a fluorescent puromycin incorporation method, using 2 mM 6-FAM-dC-puromycin (Jena Bioscience), according to Wang et al. ( ) with some modifications for C. elegans .

Techniques: Expressing, Two Tailed Test, Biomarker Discovery, Fluorescence, Control

Journal: Science Advances

Article Title: Hyperactive 20 S proteasome enhances proteostasis and ERAD in C. elegans via degradation of intrinsically disordered proteins

doi: 10.1126/sciadv.adx3014

Figure Lengend Snippet: ( A ) Representative imaging of day 5 transgenic animals expressing ERAD substrate nhx-2p::sGFP::ATZ in intestinal epithelium. Scale bar, 500 μm. ( B ) ATZ aggregate quantification: Comparison of ATZ aggregate counts in nhx-2p::sGFP::ATZ (n = 35) and α3ΔN; nhx-2p::sGFP::ATZ ( n = 42) worms on day 5. Data represent mean values ± SEM, *** P < 0.001 by two-tailed unpaired t test. The bright pharyngeal signal, indicated with an asterisk *, is a genetic marker and should not be mistaken for ATZ aggregates. ( C ) Representative images of nhx-2p::sGFP::ATZ at day 7 after treatment with the proteasome inhibitor MG132 (100 μM). Images show increased accumulation of ATZ aggregates in both nhx-2p::sGFP::ATZ and α3ΔN; nhx-2p::sGFP::ATZ worms upon proteasome inhibition. Scale bars, 500 μm. ( D ) Quantification of ATZ aggregates from (C). Data represent mean values ± SEM ( n = ≥15 worms per group). P < 0.01 determined by one-way ANOVA followed by Tukey’s post hoc test. ( E to G ) Representative side-by-side fluorescent micrographs of nhx-2p ::sGFP::ATZ animals grown on control RNAi (vector) or RNAi against sel-11 (E), npl-4.1 + npl-4.2 (F), or cdc-48.1 + cdc-48.2 (G). Worms were maintained on RNAi plates until day 5 posthatching, then collected, and imaged for aggregate counting. Scale bars, 500 μm. ( H to J ) Quantification of sGFP::ATZ aggregates per worm in WT and α3ΔN backgrounds after RNAi of sel-11 (H; WT n ≥ 43, α3ΔN n ≥ 35), npl-4.1 + npl-4.2 (I; WT n ≥ 40, α3ΔN n ≥ 35), or cdc-48.1 + cdc-48.2 (J; n = 45 each). Data are mean ± SEM from ≥3 independent cohorts. Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test (*** P < 0.01).

Article Snippet: To assess protein synthesis, we used a fluorescent puromycin incorporation method, using 2 mM 6-FAM-dC-puromycin (Jena Bioscience), according to Wang et al. ( ) with some modifications for C. elegans .

Techniques: Imaging, Transgenic Assay, Expressing, Comparison, Two Tailed Test, Marker, Inhibition, Control, Plasmid Preparation