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Image Search Results
Journal: Science Advances
Article Title: EIF3D safeguards the homeostasis of key signaling pathways in human primed pluripotency
doi: 10.1126/sciadv.adq5484
Figure Lengend Snippet: ( A ) Rank plot from CRISPRi screening. Red and blue dots represent genes significantly increased or decreased (±1 SD) following 8 and 16 days of KD, respectively. n = 3. The full list is provided in data S1 and S2. ( B ) Protein expression in undifferentiated and differentiating PSCs (10 and 20 days post-FGF withdrawal) and HDFs. Vinculin (VCL) was used as a loading control. ( C ) Representative images of control and EIF3D KD iPSCs, 5 days post-KD induction. Scale bars, 100 μm. ( D ) Cell counts on days 3 ( P = 4.70 × 10 −4 ) and 5 ( P = 3.53 × 10 −7 ) post-KD induction (mean ± SD, n = 6). P values determined via unpaired t test. ( E ) Cell cycle phase distribution (mean ± SD, n = 3). G 1 : P = 2.14 × 10 −3 ; S: P = 4.43 × 10 −7 ; G 2 /M: P = 1.99 × 10 −4 , calculated by unpaired t test. ( F ) RNA expression of pluripotency and p53-related genes during EIF3D KD. n = 3. ( G ) Expression of pluripotency and p53-associated proteins during EIF3D KD. d, days; FC, fold change.
Article Snippet: We loaded 2 μg of cell lysate per detection, along with the following antibodies (see also table S3): mouse monoclonal anti-OCT3/4 (1:500, Santa Cruz Biotechnology), goat polyclonal anti-SOX2 (1:40, R&D Systems), goat polyclonal anti-NANOG (1:40, R&D Systems), mouse monoclonal anti-p53 (DO-7) (1:200, Novus Biologicals), goat polyclonal anti-p53 (1:100, R&D Systems), rabbit monoclonal anti-p21 (1:50, Cell Signaling Technology), rabbit monoclonal anti-MDM2 (1:50, Cell Signaling Technology),
Techniques: Expressing, Control, RNA Expression
![( A ) Principal components (PC) analysis of RNA-seq data. Each dot represents the average value of replicates. n = 3. ( B ) Volcano plots displaying DEGs between control and EIF3D KD iPSCs, 3 and 5 days after Dox addition. Understated-colored dots represent DEGs. Highlighted-colored dots denote key pluripotency markers. Red and blue dots indicate genes significantly up-regulated and down-regulated, respectively [|log 2 fold change (FC)| > 1, adjusted P < 0.05]. n = 3. See full gene list and FC in table S1. See also fig. S6. ( C ) The scheme of trilineage differentiation. Three days before differentiation induction, Dox was added to the control and sgEIF3D cell lines. On day 3, the cells were exposed to the specific culture conditions of each lineage and simultaneously maintained under the PSC condition for comparison. Dox continued to be added until day 8. ( D ) Phase contrast and immunocytochemistry images of control (top) and sgEIF3D (bottom) cells after differentiation, showing specified proteins (red). Nuclei visualized with Hoechst 33342 (blue). Scale bars, 100 μm. ( E ) RNA expression of pluripotency and lineage marker genes after differentiation (mean ± SD, n = 9). * P < 0.0001 for control versus sgEIF3D in the same condition determined by one-way analysis of variance (ANOVA).](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_0838/pmc11980838/pmc11980838__sciadv.adq5484-f2.jpg)
Journal: Science Advances
Article Title: EIF3D safeguards the homeostasis of key signaling pathways in human primed pluripotency
doi: 10.1126/sciadv.adq5484
Figure Lengend Snippet: ( A ) Principal components (PC) analysis of RNA-seq data. Each dot represents the average value of replicates. n = 3. ( B ) Volcano plots displaying DEGs between control and EIF3D KD iPSCs, 3 and 5 days after Dox addition. Understated-colored dots represent DEGs. Highlighted-colored dots denote key pluripotency markers. Red and blue dots indicate genes significantly up-regulated and down-regulated, respectively [|log 2 fold change (FC)| > 1, adjusted P < 0.05]. n = 3. See full gene list and FC in table S1. See also fig. S6. ( C ) The scheme of trilineage differentiation. Three days before differentiation induction, Dox was added to the control and sgEIF3D cell lines. On day 3, the cells were exposed to the specific culture conditions of each lineage and simultaneously maintained under the PSC condition for comparison. Dox continued to be added until day 8. ( D ) Phase contrast and immunocytochemistry images of control (top) and sgEIF3D (bottom) cells after differentiation, showing specified proteins (red). Nuclei visualized with Hoechst 33342 (blue). Scale bars, 100 μm. ( E ) RNA expression of pluripotency and lineage marker genes after differentiation (mean ± SD, n = 9). * P < 0.0001 for control versus sgEIF3D in the same condition determined by one-way analysis of variance (ANOVA).
Article Snippet: We loaded 2 μg of cell lysate per detection, along with the following antibodies (see also table S3): mouse monoclonal anti-OCT3/4 (1:500, Santa Cruz Biotechnology), goat polyclonal anti-SOX2 (1:40, R&D Systems), goat polyclonal anti-NANOG (1:40, R&D Systems), mouse monoclonal anti-p53 (DO-7) (1:200, Novus Biologicals), goat polyclonal anti-p53 (1:100, R&D Systems), rabbit monoclonal anti-p21 (1:50, Cell Signaling Technology), rabbit monoclonal anti-MDM2 (1:50, Cell Signaling Technology),
Techniques: RNA Sequencing, Control, Comparison, Immunocytochemistry, RNA Expression, Marker
Journal: Science Advances
Article Title: EIF3D safeguards the homeostasis of key signaling pathways in human primed pluripotency
doi: 10.1126/sciadv.adq5484
Figure Lengend Snippet: ( A ) Scheme of differentiation of naïve PSCs into primed state. ( B ) Differentiation of naïve PSCs to primed state. Induction of differentiation from naïve to primed PSCs, 3 days post-Dox addition, by altering culture conditions. Representative images of specified cell lines and days are shown. Scale bars, 100 μm. ( C ) Scheme of the comparison between naïve and primed PSCs with EIF3D KD. ( D ) Representative images of control and sgEIF3D naïve PSCs, 5 days post-Dox addition. Scale bars, 100 μm. ( E ) Protein expression in control and EIF3D KD naïve (N) and primed (P) PSCs, 5 days post-Dox addition. ( F ) Hierarchical clustering of the averaged values of RNA-seq data. ( G ) Scatterplots of log 2 FC (control and EIF3D KD) from the Wald tests of primed 5 and 3 days (left) and primed 5 days and naïve 5 days (right) post-Dox addition. Colors show up (red) and down (blue) from the result of primed control and EIF3D KD 5 days after Dox addition. Pearson’s correlation values and P values of t test were shown on top. ( H ) The DEG numbers between control versus EIF3D KD. ( I ) RNA expression of EIF3D and pluripotency marker genes after 7 days of Dox addition (mean ± SD, n = 3). * P < 0.05 determined by unpaired t test.
Article Snippet: We loaded 2 μg of cell lysate per detection, along with the following antibodies (see also table S3): mouse monoclonal anti-OCT3/4 (1:500, Santa Cruz Biotechnology), goat polyclonal anti-SOX2 (1:40, R&D Systems), goat polyclonal anti-NANOG (1:40, R&D Systems), mouse monoclonal anti-p53 (DO-7) (1:200, Novus Biologicals), goat polyclonal anti-p53 (1:100, R&D Systems), rabbit monoclonal anti-p21 (1:50, Cell Signaling Technology), rabbit monoclonal anti-MDM2 (1:50, Cell Signaling Technology),
Techniques: Comparison, Control, Expressing, RNA Sequencing, RNA Expression, Marker
Journal: Science Advances
Article Title: EIF3D safeguards the homeostasis of key signaling pathways in human primed pluripotency
doi: 10.1126/sciadv.adq5484
Figure Lengend Snippet: ( A ) Quantification of de novo protein synthesis by detecting incorporated puromycin. n = 3. P = 6.75 × 10 −3 determined by unpaired t test. a.u., arbitrary units. ( B ) Representative polysome profiles of sgEIF3D primed PSCs compared to control lines on day 3 post-Dox addition. ( C ) Area under the curve (AUC) quantification for specified ribosomal fractions (mean ± SD, n = 3). Ratios 60 S /40 S : P = 0.011; 80 S /40 S : P = 0.012; polysomes (PS)/40 S : P = 0.094, calculated using unpaired t test. ( D ) Categorization of ORFs with varying translation efficiency (TE) during EIF3D KD. Displayed are all ORFs translated in human iPSCs (all) and those down-regulated (down) or up-regulated (up) by EIF3D KD over 3 days. ( E ) Volcano plot showing up-regulated DTEGs (uDTEG; red) and down-regulated DTEGs (dDTEGs; blue) (|log 2 FC| > 1, adjusted P < 0.05). See full gene list, FC, and adjusted P value in data S3 and S4. ( F ) Pathway enrichment analysis of dDTEGs using WikiPathways. ( G ) Volcano plots displaying log 2 FC of transcripts in specified pathways according to WikiPathways (WP437, WP399, WP481, WP382, and WP366 for EGF, WNT, insulin, MAPK, and TGFβ pathways, respectively). Understated-colored dots represent DTEGs. Highlighted-colored dots denote transcripts in specified pathways. Red and blue dots indicate genes whose TEs are significantly up-regulated and down-regulated, respectively (|log 2 FC| > 1, adjusted P < 0.05). n = 3. See full gene list, FC, and adjusted P value in data S5 to S9. ( H ) Phosphorylation status of key proteins in the signaling pathways across the timeline of EIF3D KD.
Article Snippet: We loaded 2 μg of cell lysate per detection, along with the following antibodies (see also table S3): mouse monoclonal anti-OCT3/4 (1:500, Santa Cruz Biotechnology), goat polyclonal anti-SOX2 (1:40, R&D Systems), goat polyclonal anti-NANOG (1:40, R&D Systems), mouse monoclonal anti-p53 (DO-7) (1:200, Novus Biologicals), goat polyclonal anti-p53 (1:100, R&D Systems), rabbit monoclonal anti-p21 (1:50, Cell Signaling Technology), rabbit monoclonal anti-MDM2 (1:50, Cell Signaling Technology),
Techniques: Control
Journal: Science Advances
Article Title: EIF3D safeguards the homeostasis of key signaling pathways in human primed pluripotency
doi: 10.1126/sciadv.adq5484
Figure Lengend Snippet: ( A ) Venn diagrams display the overlap between DTEGs with significant TE (|log 2 FC| > 0.58) and p53 regulators. Genes with higher TE (|log 2 FC| > 1) are highlighted in red. See full gene list, FC, and adjusted P value in data S10. ( B ) Protein expression of p53 regulators translationally controlled by EIF3D. High TE (|log 2 FC| > 1) includes RBBP6, SSU72, and TCP1; moderate TE (|log 2 FC| > 0.58) includes KDM5C, WDR5, WDR82, and REST. ( C ) Luciferase reporter assays showing the effects of RBBP6 ( P = 2.89 × 10 −9 ) and GAPDH ( P = 0.85) 5′UTRs on translation in control and EIF3D KD primed PSCs (mean ± SD, n = 6). P values determined via unpaired t test. ( D ) Representative images of specified cells, 5 days post-KD induction. Scale bars, 100 μm. ( E ) Cell counts of the cells depicted in (mean ± SD, n = 6). ( F ) Relative gene expression in cells from . Values normalized to GAPDH and compared to control without Dox. n = 3. ( G ) Expression of specified proteins in cells from .
Article Snippet: We loaded 2 μg of cell lysate per detection, along with the following antibodies (see also table S3): mouse monoclonal anti-OCT3/4 (1:500, Santa Cruz Biotechnology), goat polyclonal anti-SOX2 (1:40, R&D Systems), goat polyclonal anti-NANOG (1:40, R&D Systems), mouse monoclonal anti-p53 (DO-7) (1:200, Novus Biologicals), goat polyclonal anti-p53 (1:100, R&D Systems), rabbit monoclonal anti-p21 (1:50, Cell Signaling Technology), rabbit monoclonal anti-MDM2 (1:50, Cell Signaling Technology),
Techniques: Expressing, Luciferase, Control, Gene Expression
Journal: Science Advances
Article Title: EIF3D safeguards the homeostasis of key signaling pathways in human primed pluripotency
doi: 10.1126/sciadv.adq5484
Figure Lengend Snippet: ( A ) Overview of chemical compound and growth factor treatments. Control cells were treated with DMSO. ( B ) Representative images of iPSCs treated with specified factors. Scale bars, 100 μm. ( C ) Cell counts on day 5 (mean ± SD, n = 9). * P < 0.0001 compared to the control; † P < 0.05 versus 5F determined by one-way ANOVA. ( D ) Sample clustering from (A), alongside control and sgEIF3D PSC core transcription factor (TF) KD iPSCs, as well as trilineage differentiated cells, highlighting selected PSC marker genes. n = 3. ( E ) Schematic representation of the model describing how EIF3D-mediated translational regulation balances homeostasis of critical signaling pathways in primed pluripotency.
Article Snippet: We loaded 2 μg of cell lysate per detection, along with the following antibodies (see also table S3): mouse monoclonal anti-OCT3/4 (1:500, Santa Cruz Biotechnology), goat polyclonal anti-SOX2 (1:40, R&D Systems), goat polyclonal anti-NANOG (1:40, R&D Systems), mouse monoclonal anti-p53 (DO-7) (1:200, Novus Biologicals), goat polyclonal anti-p53 (1:100, R&D Systems), rabbit monoclonal anti-p21 (1:50, Cell Signaling Technology), rabbit monoclonal anti-MDM2 (1:50, Cell Signaling Technology),
Techniques: Control, Marker
Journal: iScience
Article Title: Roles for the long non-coding RNA Pax6os1 / PAX6-AS1 in pancreatic beta cell function
doi: 10.1016/j.isci.2024.111518
Figure Lengend Snippet: Pax6os1 silencing upregulates beta cell signature genes in MIN6 cells (A) Differential expressed genes by Pax6os1 knockdown as determined by RNA-seq performed in MIN6 cells 72 h post-transfection with siRNA targeting Pax6os1 . n = 4. (B) KEGG pathway enrichment analysis relative to (A). Significantly enriched KEGG pathways ( p < 0.05) are presented, and the bar shows the fold enrichment of the pathway. (C) mRNA levels of beta cell signature genes and markers characteristic of other endocrine cell lineages in control and Pax6os1 knockdown cells. n = 7. (D) Fold change of insulin secreted relative to 3 mM glucose. n = 5. (E) Total insulin content. n = 14. (F) Pax6os1 subcellular distribution. (G) Schematic representation of Pax6os1 pull-down and MS. (H) Relationship in abundance ratios above the 1.1 cut between the two experimental replicates performed. Top 5 hits are labeled. Short/branched chain-specific acyl-CoA dehydrogenase, mitochondrial 1 (ACADSB), eukaryotic translation initiation factor 3 subunit D (EIF3D), inosine-5′-monophosphate dehydrogenase 2 (IMPDH2), histone 1.0 (H1.0), and uncharacterized protein Rab8a (Rab8a). Data are represented as the mean ± SEM. ∗ p < 0.05, Student’s t test.
Article Snippet:
Techniques: Knockdown, RNA Sequencing Assay, Transfection, Control, Labeling
Journal: iScience
Article Title: Roles for the long non-coding RNA Pax6os1 / PAX6-AS1 in pancreatic beta cell function
doi: 10.1016/j.isci.2024.111518
Figure Lengend Snippet: PAX6-AS1 directly interacts to EIF3D, H3, and H4 (A) Subcellular localization of PAX6-AS1 in EndoC-βH1 cells. n = 3. (B) Schematic representation of the RNA antisense pull-down. (C) PAX-AS1 mRNA enrichment vs. input in cells hybridized with probes targeting the lncRNA. (D) Western blots showing EIF3D, H3, and H4 in cells hybridized with probes against our lncRNA or luciferase. (E) Mature and nascent insulin mRNA expression as determined by qPCR in control and PAX6-AS1 depleted cells. n = 5. (F) Determination of insulin mRNA stability as determined by actinomycin D treatment in control and PAX6-AS1 KO cells. n = 6–3. Data are represented as the mean ± SEM. ∗ p < 0.05, Student’s t test.
Article Snippet:
Techniques: Western Blot, Luciferase, Expressing, Control
Journal: iScience
Article Title: Roles for the long non-coding RNA Pax6os1 / PAX6-AS1 in pancreatic beta cell function
doi: 10.1016/j.isci.2024.111518
Figure Lengend Snippet:
Article Snippet:
Techniques: Virus, Expressing, shRNA, Recombinant, Fluorescence, Enzyme-linked Immunosorbent Assay, Mass Spectrometry, Binding Assay, Plasmid Preparation, Labeling, Software