rnai screens  (Broad Institute Inc)

Journal: Nature Communications

Article Title: A functional screen uncovers circular RNAs regulating excitatory synaptogenesis in hippocampal neurons

doi: 10.1038/s41467-025-58070-4

Figure Lengend Snippet: A Workflow of the circRNA functional screen. Heatmap of candidate circRNAs (30 most process-enriched circRNAs plus Cdr1-as and Hipk3 ) for which siRNA pools (3 independent siRNAs per circRNA) were designed. Immunocytochemistry for pre (synapsin-1) and postsynaptic (PSD-95) markers was performed at DIV16, followed by an automated count of synaptic co-clusters based on a GFP mask which labels transfected cells. B Representative Images of siControl condition as well as two selected circRNA candidates (si- circPhf21a , si- circRERE2 ) which display increased synaptic density upon knockdown. Red: Hoechst; green: PSD-95; blue: Synapsin-1; turquoise: GFP. Upper panel: overview images. Middle and lower panel: Magnification of boxed insets depicting either synaptic co-clusters (middle, marked by arrows) or dendritic segments with spines (lower). 8 images were taken (i.e., cells per condition per replicate). C Volcano Plot showing log 2 fold-changes in synapse co-cluster density (x-axis) vs. −log 10 p -value (y-axis) for individual siRNA pools directed against circRNA candidates (two-sided). Significant up-or downregulated siRNA pools ( p < 0.05), as well as siRERE_1 ( p < 0.14), are labeled. D Synapse co-cluster density (normalized to the GFP-only condition present on the same plate) for siRNA pools directed against indicated circRNA candidates (two-sided). Violin plots with embedded boxplots, including the median, interquartile range, and whiskers from minimum to maximum. Pairwise comparisons (emmeans/GLMM): siRERE_2 vs. siControl1: logFC = 0.37764 ± 0.0874, t -ratio = 4.32097, p = 0.000582117. siPhf21a vs. siControl2: logFC = 0.33387 ± 0.0874, t -ratio = 3.82025, p = 0.0045. P < 0.001 = ***, P < 0.01 = **. C/D: GLMM statistical modeling, n = 4 independent biological replicates, 8 cells per condition. All comparisons are provided in Supplemental Table . Created in BioRender. https://BioRender.com/d06j864 . Source data are provided as a Source Data file.

Article Snippet: Silencer Select siRNA Custom RNAi Screen (Thermo Fisher) pools of 3× siRNAs against each investigated circRNA BSJ were generated, each staggered by 2–3 nt.

Techniques: Functional Assay, Immunocytochemistry, Transfection, Knockdown, Labeling

Journal: Nature Communications

Article Title: A functional screen uncovers circular RNAs regulating excitatory synaptogenesis in hippocampal neurons

doi: 10.1038/s41467-025-58070-4

Figure Lengend Snippet: Electrophysiological characterization of circRERE kd neurons. A , B mEPSC frequency. p = 0.0004, ScramSh = 1.185 ± 0.1411, CircRERE sh = 0.5992 ± 0.1411, t = 4.154, df = 22. 95% confidence interval −0.8788 to −0.2935. (two-sided unpaired t -test). C , D mEPSC amplitudes p = 0.7452. ScramSh = 1.625 ± 0.1516, CircRERE sh = 15.44 ± 0.1516, t = 0.5322, df = 22. 95% CI −3.950 to 2.337 (two-sided unpaired t -test). E Representative traces illustrating mEPSC frequency decreases with circRERE sh compared to ScramSh. F , G mEPSC decay time in circRERE shRNA (orange bars) or shRNA control (blue bars), p = 0.0485. ScramSh = 5.297 ± 0.2493, CircRERE sh = 4.776 ± 0.2493, t = 2.089, df = 22. 95% CI, −1.038 to −0.003845 (two-sided unpaired t -test). A , C , F N = 12, whole-cell patch-clamp recordings of primary hippocampal neurons, boxplots including the median, interquartile range, and whiskers from minimum to maximum. P < 0.05 *, P < 0.001 ***. H Representative images from rat hippocampal neurons (DIV16) transfected with ScramSh or circRERE sh and immunostained for surface GluA1 (green). Turquoise: GFP; Red: Hoechst. Upper panel: Overview image. Lower panel: Boxed inset at higher magnification showing GluA1 puncta (green, arrows) located at the dendritic surface. I Quantification of GluA1 surface puncta density in neurons transfected with indicated circRERE shRNA constructs. N = 4 independent biological replicates, 10 cells per condition per experiment. Violin plots with embedded boxplots, including the median, interquartile range, and whiskers from minimum to maximum. GLMM statistical modeling (two-sided). Emmeans: GFP = -0.881 ± 0.132, ScramSh = −0.491 ± 0.132, circRERE2 Sh = −0.983 ± 0.132, circRERE sh = −1.059 ± 0.132. Contrasts: ScramSh/ circRERE2 Sh = 0.493 ± 0.174, t -ratio = 2.838, p = 0.0263. ScramSh/ circRERE sh = 0.569 ± 0.174, t -ratio = 3.275, p = 0.0071. circRERE2 Sh/ circRERE sh = 0.076 ± 0.174, t -ratio = 0.438, p = 0.9719. GLMM statistical modeling. P < 0.05 = *, P < 0.01 = **. J Representative images from rat hippocampal neurons (DIV16) transfected with ScramSh or circRERE Sh and immunostained for total GluA1 (green). Magenta: GFP. K Quantification of total GluA1 mean gray value intensity in cells transfected with indicated circRERE shRNA constructs. GLMM statistical modeling (two-sided) performed post log 2 transformation of raw-values. Emmeans: GFP = 10.5 ± 0. 0378, ScramSh = 10.3 ± 0.0378, circRERE2 Sh = 10.4 ± 0.0378, circRERE sh = 10.6 ± 0.0378. Contrasts: ScramSh/ circRERE2 Sh = −0.0839 ± 0.0534, t -ratio = −1.570, p = 0.3978. ScramSh/ circRERE Sh = −0.3040 ± 0.0534, t -ratio = −5.692, p < .0001. circRERE2 Sh/ circRERE sh = −0.2202 ± 0.0534, t -ratio = −4.122, p < 0.0003. p < .0001 = ***. P < 0.0001 = ****. Source data are provided as a Source Data file.

Article Snippet: Silencer Select siRNA Custom RNAi Screen (Thermo Fisher) pools of 3× siRNAs against each investigated circRNA BSJ were generated, each staggered by 2–3 nt.

Techniques: shRNA, Control, Patch Clamp, Transfection, Construct, Transformation Assay

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: BHLHE41, a transcriptional repressor involved in physiological processes and tumor development

doi: 10.1007/s13402-024-00973-3

Figure Lengend Snippet: A . BHLHE41 is an essential gene in several cancer cell types. Boxplots showing the dependency scores calculated using data from the Cancer Dependency Map project ( https://depmap.org ). RNAi-based viability assays in Ewing sarcoma ( n =10), multiple myeloma ( n = 16), non-Hodgkin lymphoma ( n = 26) and acute myeloid leukemia ( n = 22) cell lines. DEMETER2 scores were calculated using data from RNAi-based screens. The mention “all” refers to the 974 cell lines included in the projects Achilles, DRIVE and Marcotte. A DEMETER score = 0 indicates that the gene is not essential. A lower score indicates that the gene is more likely to be essential in a given cell line. The figure shows the scores for the BHLHE41 gene in the indicated samples. B. A high expression of BHLHE41 is associated with a poor survival in patients with AML and MM. The link between BHLHE41 and outcome in hematological malignancies was investigated using GenomicScape webtool ( http://www.genomicscape.com/microarray/survival.php ). High BHLHE41 expression (red) is associated with a significant poor overall survival in a cohort of newly-diagnosed cytogenetically normal AML (GSE12417—Gene expression omnibus, n =163) and a cohort of newly diagnosed MM patients (HM cohort, n =206, Array Express public database (E-MTAB372)) compared to patients with low BHLHE41 expression (blue)

Article Snippet: We used public datasets of RNAi viability assay screening (Dependency Map data, Broad Institute, www.depmap.org ) [ ] to determine whether BHLHE41 is an essential gene in several cancers.

Techniques: Expressing, Microarray, Gene Expression

Journal: Nature Communications

Article Title: The tyrosine kinase KDR is essential for the survival of HTLV-1-infected T cells by stabilizing the Tax oncoprotein

doi: 10.1038/s41467-024-49737-5

Figure Lengend Snippet: A Waterfall plot representing Z scores for each shRNA in the kinome-wide shRNA screen for identification of survival factors in MT-2 cells. B Genes from the shRNA screen are listed with a mean Z -score of either <−1.5 (positive regulator) or >1.5 (negative regulator). C MT-2 cell viability in the 96-well plate from the kinome-wide shRNA screen containing the five KDR shRNAs (boxed area). D HTLV-1 transformed cells were treated with SU 1498 in a concentration-dependent manner for 24 h and stained with Annexin V-FITC and propidium iodide for acquisition by flow cytometry. Representative pseudocolor plots indicate the gating strategy for quantifying the percentage of each single-stained and dual-stained cells and highlighting the percentage of pre-apoptotic and post-apoptotic cell death in response to SU 1498 treatment. E Immunofluorescence was performed using HTLV-1 transformed cells treated with SU 1498 for 24 h and stained with MitoTracker Deep Red. The experiment is representative of two independent experiments with similar results. F FLICA caspase assays were performed using MT-2 and C8166 cells treated with SU 1498 at the indicated concentrations for 24 h. The histograms represent the percentage of active caspase 3/7 in treated cells. G Immunoblotting was performed with the indicated antibodies using lysates from MT-2 and C8166 cells treated with the indicated concentrations of SU 1498 for 24 h. The experiment is representative of three independent experiments with similar results. H Graphical representation of cell cycle analysis of HTLV-1 transformed cells treated with the indicated concentrations of SU 1498 for 24 h and the proportions of cells at different phases of the cell cycle (sub G1, G1/M, S, G2/M). The results are expressed as the mean ± SD of three independent experiments. **** P < 0.0001; *** P < 0.001; ** P < 0.01; * P < 0.05. Two-way ANOVA with Dunnett’s multiple comparisons test. P values from left to right: P = 0.7880, P = 0.4538, P = 0.1483, P < 0.0001, P = 0.0010, P < 0.0001, P = 0.2411, P < 0.0001, P = 0.9872, P = 0.8933, P = 0.0005, P < 0.0001, P = 0.9264, P < 0.0001, P = 0.0002, P < 0.0001, ns not significant. I Immunoblotting was performed with the indicated antibodies using lysates from Jurkat cells treated with the indicated concentrations of SU 1498 for 24 h. The experiment was independently repeated three times with similar results. Representative data from one experiment is shown. Source data are provided as a Source data file.

Article Snippet: The kinome-wide shRNA screen was performed with the MISSION® LentiExpress Kinome RNAi screening platform (Millipore-Sigma) consisting of pre-arrayed lentiviral particles in 41 × 96-well plates.

Techniques: shRNA, Transformation Assay, Concentration Assay, Staining, Flow Cytometry, Immunofluorescence, Western Blot, Cell Cycle Assay