nanodlr stop & glo buffer containing substrate for nanoluciferase  (Promega)

Journal: eLife

Article Title: Insufficiency of 40S ribosomal proteins, RPS26 and RPS25, negatively affects biosynthesis of polyglycine-containing proteins in fragile-X associated conditions

doi: 10.7554/eLife.98631

Figure Lengend Snippet: ( A ) Scheme of two RNA molecules used to perform MS-based screening. The FMR1 RNA contains the entire length of the 5’ untranslated region (5’UTR) of FMR1 with expanded CGG ( x99 ) repeats forming a hairpin structure (red). The open-reading frame for the polyglycine-containing protein starts at a repeats-associated non-AUG initiated (RAN) translation-specific ACG codon. RAN translation can also be initiated from a GUG near-cognate start codon, which is not indicated in the scheme. The GC-rich RNA contains TMEM107 mRNA enriched with G and C nucleotide residues (GC content >70%; similar to FMR1 RNA) with the open-reading frame starting at the canonical AUG codon (blue). Both RNAs are tagged with three MS2 stem-loop aptamers (grey) interacting with an MS2 protein tagged with an in vivo biotinylating peptide used to pull down proteins interacting with the RNAs. ( B ) The volcano plot representing proteins captured during MS-based screening showing the magnitude of enrichment (log2 fold change) and the statistical significance (F02Dlog p-value); red dots indicate proteins significantly enriched (p < 0.05) on FMR1 RNA compared to GC-rich RNA. Three proteins, RPS26, LUC7L3, and DHX15, tested in a subsequent validation experiment are marked. DDX3X is also indicated as this protein has been previously described in the context of interaction with FMR1 mRNA. ( C ) The scheme of RNA used for the transient overexpression of FMR99xG (mutant, long, 99 polyglycine tract-containing protein). The construct contains the entire length of the 5’UTR of FMR1 with expanded CGG repeats forming a hairpin structure (red) tagged with enhanced green fluorescent protein (eGFP) (green). Western blot analysis of FMR99xG and Vinculin for HEK293T cells with insufficient DHX15, RPS26, and LUC7L3 induced by specific short interfering RNA (siRNA) treatment. To detect FMR99xG, the 9FM antibody was used. The upper bands were used for quantification. The graph presents the mean signal for FMR99xG normalized to Vinculin from N = 3 biologically independent samples with the standard deviation (SD). An unpaired Student’s t-test was used to calculate statistical significance: *, p < 0.05; ***, p < 0.001; ns, non-significant. ( D ) Results of luciferase assay for cells with overexpression of FMR16xG fused with nanoluciferase pretreated with different concentrations of siCtrl or siRPS26 (see legend). The graph presents luminescence signals (means from N=8 with SDs) normalized to siCtrl treated cells. Western blot represents the siRNA-concentration dependent effect on RPS26 downregulation. An unpaired Student’s t-test was used to calculate statistical significance: ****, p < 0.0001; ns, non-significant. ( E ) Results of microscopic quantification of FMR99xG-positive aggregates (white arrows) in HeLa cells transfected with the FMR99xG construct on the background of normal (siCtrl) or reduced amount of RPS26 (siRPS26). Representative images were pseudo-colored and merged; green, GFP-positive inclusions; blue, nuclei stained with Hoechst 33342; scale bars, 50 μm. The histogram presents a number of FMRpolyG-GFP inclusions per nucleus in cells treated with siCtrl (blue) or siRPS26 (red) in N=10 biologically independent samples with SDs. In average, 1100 cells were identified per single image and final value were normalized to 1 for control siRNA treated cells. An unpaired Student’s t-test was used to calculate statistical significance: *, p < 0.05. ( F ) The influence of RPS26 silencing on apoptosis evoked in HeLa cells after 28 hr or 43 hr of FMR99xG overexpression or in mock treated cells. Apoptosis was measured as luminescence signals (relative luminescence units; RLU) derived from Annexin V fusion protein bound to phosphatidylserine (PS) exposed on the outer leaflet of cell membranes, an indicator for early apoptosis. The graph presents relative mean values from N = 6 biologically independent samples treated with either siCtrl (red) or siRPS26 (blue) with the SD normalized to mock controls (cells transfected only with the delivering reagent and cultured for 28 hr or 43 hr). An unpaired Student’s t-test was used to calculate statistical significance: *, p<0.05; **, p<0.01; ***, p < 0.001. Note, that significantly higher apoptosis was observed in cells expressing FMR99xG compare to control (mock), and that pretreatment with siRPS26 significantly reduced this phenotype. Figure 1—source data 1. PDF containing tiff files representing western blot results with labeled lanes matching the content of . Figure 1—source data 2. Folder containing raw tiff files representing western blot results matching the content of .

Article Snippet: Next, the NanoDLR Stop & Glo (Promega) buffer containing substrate for nanoluciferase was added and after 10 min incubation, the nanoluciferase-based luminescence was measured by the same equipment.

Techniques: In Vivo, Biomarker Discovery, Over Expression, Mutagenesis, Construct, Western Blot, Small Interfering RNA, Standard Deviation, Luciferase, Concentration Assay, Transfection, Staining, Control, Derivative Assay, Cell Culture, Expressing, Labeling

Journal: eLife

Article Title: Insufficiency of 40S ribosomal proteins, RPS26 and RPS25, negatively affects biosynthesis of polyglycine-containing proteins in fragile-X associated conditions

doi: 10.7554/eLife.98631

Figure Lengend Snippet: ( A ) The volcano plot represents a stable isotope labeling using amino acids in cell culture (SILAC)-based quantitative proteomic analysis identifying proteins sensitive to RPS26 insufficiency. It shows the magnitude of protein-level changes (log2 fold change) vs. the statistical significance (F02Dlog p-value) 48 hr post siRPS26 treatment. Data were collected from three independent biological replicates for each group. Grey dots indicate proteins non-responding to RPS26 depletion (N=1506). Red dots indicate proteins responding to RPS26 insufficiency (p<0.05); EIF5 and PDCD4 are examples of Negative (N=223; green) and Positive (N=158; orange) responders, respectively. Protein groups were further analyzed in B, C, and D. ( B ) Gene ontology (GO) analysis performed for positive responders of RPS26 insufficiency from the proteomic experiment shown in A. The graph presents significantly enriched GO terms (p<0.05); statistical significance was calculated using Fisher’s Exact test with the Bonferroni correction. Note that for negative responders, no GO terms were significantly enriched. ( C ) Data validation from the proteomic experiment described in A. Western blot analyses of PDCD4, ILF3, FMRP, and EIF5 proteins normalized to Vinculin for HEK293T cells treated with siRPS26. The graphs present means from N = 4 biologically independent samples with SDs . An unpaired Student’s t-test was used to calculate statistical significance: *, p<0.05; **, p<0.01; ***, p < 0.001; ns, non-significant. ( D ) The percentage of GC content in 5’UTRs and coding sequences across extending sequence windows initiated from the start codon within three groups of transcripts: Negative responders (green), Positive responders (orange), and Background (BG) understood as the total transcriptome (red). To avoid biases in the GC-content mean, transcripts with 5’UTR sequences shorter than 20 nucleotides were excluded from the analysis, yielding the following sample sizes: Negative responders (N=213), Positive responders (N=147), and BG (N=20,862). For example, position F02D6 in 5' UTR sequences corresponds to a 6-nucleotide fragment (window from F02D6 to F02D1 positions upstream of ATG), while position F02D7 corresponds to a 7-nucleotide fragment (window from F02D7 to F02D1 positions). The solid line shows the mean GC content at a given position (i.e., within the window), and the shade indicates the standard error of the mean. < i>P-values <0.01 are denoted by green or yellow dots. These reflect pairwise comparisons of GC content between transcript groups (compared to BG) and were determined using a two-tailed paired t-test with Bonferroni correction. ( E ) Box plot showing the results of a one-tailed Mann-Whitney U test comparing 5'UTR sequence lengths between positive and negative responders relative to the background transcriptome. To ensure a balanced comparison, the background transcriptome is represented by 1000 randomly selected transcripts from the complete human transcriptome. The plot highlights the median (indicated by the central line), the upper and lower quartiles (represented by the boxes), and the highest and lowest values, which are determined within a range of 1.5 times the interquartile range (IQR) (whiskers). ( F ) Results of luciferase assay post RPS26 silencing showing differences in expression of transgene containing 5’UTR of FMR1 with 16xCGG fused with nanoluciferase from which RAN translation was initiated from either ACG (wild type) or GTG or CTG near cognate codons. The signals for activity of nanoluciferase fused with FMR16xG were normalized to firefly luciferase signals (means from N=4 with SDs are presented on the graph). Note that firefly luciferase was translated from canonical ATG start codon. An unpaired Student’s t-test was used to calculate statistical significance: **, p<0.01; ns, non-significant. ( G ) Western blot analysis of FMR99xG post RPS26 silencing and treatment with control ASO (ASO_CTRL) or ASO targeting ACG codon, RAN translation initiation codon in 5’UTR of FMR1 (ASO_ACG). The graph represents the means of N=3 with SDs. An unpaired Student’s t-test was used to calculate statistical significance: *, p<0.05; **, p<0.01; ***, p < 0.001; ns, non-significant. Figure 4—source data 1. PDF containing tiff files representing western blot results with labeled lanes matching the content of . Figure 4—source data 2. Folder containing raw tiff files representing western blot results matching the content of .

Article Snippet: Next, the NanoDLR Stop & Glo (Promega) buffer containing substrate for nanoluciferase was added and after 10 min incubation, the nanoluciferase-based luminescence was measured by the same equipment.

Techniques: Quantitative Proteomics, Cell Culture, Multiplex sample analysis, Biomarker Discovery, Western Blot, Sequencing, Two Tailed Test, One-tailed Test, MANN-WHITNEY, Comparison, Luciferase, Expressing, Activity Assay, Control, Labeling