Journal: iScience

Article Title: A unique poly(A) tail profile uncovers the stability and translational activation of TOP transcripts during neuronal differentiation

doi: 10.1016/j.isci.2023.107511

Figure Lengend Snippet: Reduced translational activity during neuronal differentiation is associated with the downregulation of TOP transcripts (A) Representative micrographs of P19 cells before differentiation (day 0, D0), and at 2 (D2), 4 (D4), and 6 (D6) days of differentiation. Scale bars: 100 μm. Schematic adaptation of “Neural cells” (composition and colors) from Servier Medical Art by Servier, licensed under a Creative Commons Attribution 3.0 Unported License ( https://creativecommons.org/licenses/by/3.0/ ). (B) Boxplot of the relative accumulation of Map2 transcripts before (D0) and during differentiation (D2, D4, and D6) measured by qPCR. n = 3 biological replicates. Data are expressed relative to the D0 condition. Each dot represents one replicate, the center values show the means, the boxes indicate the first and third quartiles and the bars indicate the 10 th and 90 th percentiles. Ordinary one-way analysis of variance (ANOVA), Dunnett’s multiple comparisons test. ∗∗∗p < 0.001. (C) Representative flow cytometry histogram (left panel) and quantification (right panel) of global protein synthesis measured by O-propargyl-puromycin (OPP) incorporation in cells before differentiation (D0) and after 6 days of differentiation (D6). Negative control (Neg) shows the background signal in the absence of OPP. n = 6 biological replicates. Data are expressed relative to the corresponding D0 condition. Each dot represents one replicate, the center values show the means, the boxes indicate the first and third quartiles and the bars the 10 th and 90 th percentiles. Two-tailed, two sample Student’s t test comparing D6 to D0. ∗∗∗p < 0.001. (D) Volcano plot of differential transcript accumulation in differentiated cells (D6) relative to undifferentiated cells (D0). Downregulated and upregulated transcripts are highlighted in green and brown, respectively. n = 3 biological replicates. Wald test corrected by Benjamini and Hochberg for multiple testing. Significance threshold q-val <0.05. (E) Table showing the 10 most enriched gene ontology terms in significantly downregulated transcripts during differentiation (from D0 to D6). Fisher’s exact test. (F) Venn diagram showing downregulated transcripts (from D0 to D6) in light green, and their intersection with TOP mRNAs in blue. Overrepresentation analysis was performed using a hypergeometric test. (G) Volcano plot shown in (D), highlighting TOP mRNAs in blue and Rpl4 (contig ENSMUST00000034966) in pink. See also Tables S1 , , and .

Article Snippet: Male mouse embryonal (E7) carcinoma P19 cells (ATCC, CRL-1825) were maintained in Dulbecco’s modified Eagle medium with 4,500 mg/L of glucose supplemented with 100 units/mL of penicillin/streptomycin (Life Technologies) and 10% fetal bovine serum (FBS, Gemini Bio-Products).

Techniques: Activity Assay, Flow Cytometry, Negative Control, Two Tailed Test

Journal: iScience

Article Title: A unique poly(A) tail profile uncovers the stability and translational activation of TOP transcripts during neuronal differentiation

doi: 10.1016/j.isci.2023.107511

Figure Lengend Snippet: TOP transcripts remain highly stable through differentiation despite global mRNA decay (A) Plot of RNA synthesis rates for the whole transcriptome of P19 cells before differentiation (D0, orange) and after 6 days of differentiation (D6, blue). The shaded area represents the range between the first and third quartiles. Median half-lives are indicated by dashed lines. (B) Plot as in (A) for RNA decay rate. Wilcoxon signed-rank test for unpaired samples comparing D6 to D0. ∗∗∗p < 0.001. (C) Boxplot of synthesis half-life for downregulated transcripts before differentiation (D0, orange) and after 6 days of differentiation (D6, blue) split between non-TOP and TOP transcripts. Two-tailed paired Student’s t test comparing D6 to D0. Two-tailed, two sample Student’s t test comparing non-TOP to TOP transcripts. ∗∗∗p < 0.001. (D) Boxplot showing the fold change of synthesis half-life during differentiation (D6 vs. D0) for downregulated transcripts split between non-TOP and TOP transcripts. Two-tailed, two sample Student’s t test comparing non-TOP to TOP transcripts. ∗p < 0.05. (E) Boxplot as in (C) for decay half-life. Wilcoxon signed-rank test for paired samples comparing D6 to D0. Wilcoxon signed-rank test for unpaired samples comparing non-TOP to TOP transcripts. ∗∗∗p < 0.001. (F) Boxplot as in (D) for decay half-life. Wilcoxon signed-rank test for unpaired samples comparing non-TOP to TOP transcripts. ns, non-significant. (G) Cumulative frequency plot comparing non-TOP (dashed line, green) and TOP (solid line, blue) downregulated transcripts according to their differential accumulation before (D0) and after P19 differentiation (D6). Kolmogorov-Smirnov test, two-sided, comparing non-TOP to TOP transcripts. ∗∗∗p < 0.001. For all plots, n = 3 biological replicates. For boxplots, each dot represents one transcript, the center values show the means, the boxes indicate the first and third quartiles and the bars indicate the 10 th and 90 th percentiles. See also Figure S1 .

Article Snippet: Male mouse embryonal (E7) carcinoma P19 cells (ATCC, CRL-1825) were maintained in Dulbecco’s modified Eagle medium with 4,500 mg/L of glucose supplemented with 100 units/mL of penicillin/streptomycin (Life Technologies) and 10% fetal bovine serum (FBS, Gemini Bio-Products).

Techniques: Two Tailed Test

Journal: iScience

Article Title: A unique poly(A) tail profile uncovers the stability and translational activation of TOP transcripts during neuronal differentiation

doi: 10.1016/j.isci.2023.107511

Figure Lengend Snippet: The timely processing of TOP mRNA poly(A) tails is associated with the expansion of neuronal progenitors (A) Representative micrographs of P19 cells treated with DMSO or MHY1485 (MHY), before differentiation (D0) and after two days of differentiation (D2). Scale bars: 100 μm. Schematic adaptation of “Neural cells” (composition and colors) from Servier Medical Art by Servier, licensed under a Creative Commons Attribution 3.0 Unported License ( https://creativecommons.org/licenses/by/3.0/ ). (B) Boxplot of the relative accumulation of Map2 transcripts before differentiation (D0) and after two days of differentiation (D2) in the presence of DMSO or MHY1485 (MHY). n = 3 biological replicates. Data are expressed relative to the D0 condition. Two-tailed, two sample Student’s t test, comparing D0 to D2 or DMSO to MHY1485. ∗p < 0.05; ∗∗∗p < 0.001. (C) Boxplot showing the proportion of proliferating cells before (D0) and after (D2) differentiation in the presence of DMSO or MHY148 (MHY). n = 6 biological replicates. Games-Howell analysis of unequal variance comparing D0 to D2 or DMSO to MHY1485. ∗p < 0.05; ∗∗∗p < 0.001. For all both boxplots, each dot represents individual replicate, the center values show the medians, the boxes indicate the first and third quartiles and the bars the 10 th and 90 th percentiles. See also Figure S6 .

Article Snippet: Male mouse embryonal (E7) carcinoma P19 cells (ATCC, CRL-1825) were maintained in Dulbecco’s modified Eagle medium with 4,500 mg/L of glucose supplemented with 100 units/mL of penicillin/streptomycin (Life Technologies) and 10% fetal bovine serum (FBS, Gemini Bio-Products).

Techniques: Two Tailed Test

Journal: iScience

Article Title: A unique poly(A) tail profile uncovers the stability and translational activation of TOP transcripts during neuronal differentiation

doi: 10.1016/j.isci.2023.107511

Figure Lengend Snippet:

Article Snippet: Male mouse embryonal (E7) carcinoma P19 cells (ATCC, CRL-1825) were maintained in Dulbecco’s modified Eagle medium with 4,500 mg/L of glucose supplemented with 100 units/mL of penicillin/streptomycin (Life Technologies) and 10% fetal bovine serum (FBS, Gemini Bio-Products).

Techniques: Recombinant, Cell Viability Assay, Flow Cytometry, Bicinchoninic Acid Protein Assay, Purification, RNA Sequencing, Microscopy, Control, Multiplexing, Plasmid Preparation, Software, Imaging, Sequencing