Journal: Science Advances

Article Title: Genome-wide CRISPR screen identifies IRF1 and TFAP4 as transcriptional regulators of Galectin-9 in T cell acute lymphoblastic leukemia

doi: 10.1126/sciadv.ads8351

Figure Lengend Snippet: ( A ) Flow cytometry analysis of Galectin-9 expression in Jurkat, CCRF-CEM, Loucy, and HPB-ALL scrambled (Scr) and Galectin-9 KO T-ALL cell lines. Representative flow plots are shown (top), and the mean fluorescence intensity (MFI) was calculated of duplicate experiments (bottom) and normalized to the average MFI of Jurkat and CCRF-CEM scrambled control cell lines. A t test was used for statistical analysis. * P < 0.05; n.s., not significant. ( B ) Schematic overview of the genome-wide CRISPR screen for Galectin-9 in T-ALL cell line Jurkat. Created in BioRender. B.Y. (2025) https://BioRender.com/n03l034 . ( C ) Scatterplot showing log 2 fold changes of 20% low versus 20% high expressing Jurkat cells in the genome-wide CRISPR screen assessed by MAGeCK. Dot size indicates −log 10 of the mean FDR of both replicates of a one-sided significance test using a negative binomial model by the MAGeCK algorithm . ( D ) Heatmap depicting the log 2 fold change of 20% low versus 20% high Galectin-9 expressing Jurkat cells in the CRISPR genome-wide and validation screen. Only significant hits of the genome-wide screen and nontargeting controls were included and shown. ( E ) Log 2 fold changes of sgRNA abundance of significant hits IRF1 , TFAP4 , and ADD1 and positive control LGALS9 in the genome-wide CRISPR screen (top) and validation CRISPR screen (bottom). ( F ) Pearson correlation of IRF1 , TFAP4 , and ADD1 expression with LGALS9 expression in the TCGA and TARGET cohorts [UCSC Xena platform ( n = 14,726) ]. See table S1 for abbreviations of cancer types and the number of patients. ( G ) Boxplots depicting log2 IRF1 , TFAP4 , and ADD1 expression (normalized DESeq2 counts+1) in primary T-ALL (TARGET cohort, n = 265) split by low, mid, and high LGALS9 expression. An unpaired t test was used for statistical analysis. ** P < 0.01; *** P < 0.001; n.s., not significant.

Article Snippet: Human lentiviral CRISPR library was a gift from X. S. Liu (Addgene no. 1000000132).

Techniques: Flow Cytometry, Expressing, Fluorescence, Control, Genome Wide, CRISPR, Biomarker Discovery, Positive Control

Journal: Science Advances

Article Title: Genome-wide CRISPR screen identifies IRF1 and TFAP4 as transcriptional regulators of Galectin-9 in T cell acute lymphoblastic leukemia

doi: 10.1126/sciadv.ads8351

Figure Lengend Snippet: ( A ) Western blot quantification in single-cell–derived CRISPR KO clones in Jurkat T-ALL cells. Intensities were normalized to GAPDH and scrambled controls. Error bars indicate SEM of duplicates for which Western blots are shown. Data of scrambled 1+2 represent both scrambled controls together. ( B ) Bar plot showing Galectin-9 concentration secreted in cell culture supernatant after 2 days of culture in Jurkat scrambled control, IRF1 KO, and TFAP4 KO cell lines. Error bars indicate SEM of triplicates. ( C ) Bar plot depicting fold change of LGALS9 expression assessed by RT-qPCR compared to scrambled control in Jurkat scrambled, IRF1 KO, and TFAP4 KO cell lines. Error bars indicate SEM of triplicates. ( D and E ) Western blot (WB) quantification (D) and fold change of LGALS9 expression assessed by RT-qPCR (E) of IRF1-TFAP4 dual KO Jurkat cells. Single-cell–derived scrambled or TFAP4 KO cell lines were subjected to CRISPR editing of IRF1 and scrambled gRNAs. A bulk IRF1 KO sample was taken along as well and note order TFAP4 KO2/1 in WB. WB intensities were normalized to GAPDH and scrambled controls. Error bars indicate SEM of duplicates (D) or triplicates (E). Data of scrambled 1+2 represent both scrambled controls together. ( F ) Bar plot depicting fold change of LGALS9 expression assessed by RT-qPCR of Jurkat cells treated with a range of IFN-γ for 48 hours. Expression was normalized to vehicle-treated scrambled control. Error bars indicate SEM of triplicates. ( G ) Heatmap depicting TF activity analysis [RScenic ] in primary pediatric T-ALL patient samples from the TARGET cohort ( n = 265). Samples were equally split in LGALS9 high, mid, and low expressing groups. See table S2 for other regulons in the same hierarchical cluster as IRF1 and TFAP4. An unpaired t test was used for all statistical analysis. * P < 0.05; ** P < 0.01; *** P < 0.001; n.s., not significant.

Article Snippet: Human lentiviral CRISPR library was a gift from X. S. Liu (Addgene no. 1000000132).

Techniques: Western Blot, Derivative Assay, CRISPR, Clone Assay, Concentration Assay, Cell Culture, Control, Expressing, Quantitative RT-PCR, Activity Assay

Journal: Science Advances

Article Title: Genome-wide CRISPR screen identifies IRF1 and TFAP4 as transcriptional regulators of Galectin-9 in T cell acute lymphoblastic leukemia

doi: 10.1126/sciadv.ads8351

Figure Lengend Snippet: ( A ) H3K27ac enrichment in the LGALS9 locus analyzed by ChIP-seq experiments in T-ALL cell lines. ( B ) Chromatin looping analyzed by H3K27ac HiChIP-seq experiments in Jurkat and CCRF-CEM cells. Only chromatin looping from the LGALS9 promoter is shown, and three enhancers detected in both cell lines are indicated by an arrow. ( C ) H3K27ac, IRF1, and TFAP4 enrichment in the LGALS9 locus analyzed by ChIP-seq experiments in Jurkat and CCRF-CEM cells. ( D ) Heatmap depicting H3K27ac (left), IRF1 (middle), and TFAP4 (right) enrichment on the LGALS9 promoter and three identified enhancers. Faint gray outline indicates that this region is not peak called in the respective sample. ( E ) Bar plot depicting fold change of LGALS9 expression assessed by RT-qPCR compared to scrambled control in single clone–derived CRISPR-edited Jurkat cells upon a 150-bp deletion in enhancer 1, a 1.6-kb deletion in enhancer 2, or scrambled controls. Error bars indicate SEM of triplicates, and an unpaired t test was used for statistical analysis; * P < 0.05; ** P < 0.01; n.s., not significant. ( F ) Schematic summary of the transcriptional regulation of LGALS9 expression. Created in BioRender. B.Y. (2025) https://BioRender.com/a56h547 .

Article Snippet: Human lentiviral CRISPR library was a gift from X. S. Liu (Addgene no. 1000000132).

Techniques: ChIP-sequencing, HiChIP, Expressing, Quantitative RT-PCR, Control, Derivative Assay, CRISPR

Journal: bioRxiv

Article Title: Genome wide CRISPR screen for Pasteurella multocida toxin (PMT) binding proteins reveals LDL Receptor Related Protein1 (LRP1) as crucial cellular receptor

doi: 10.1101/2022.08.04.502755

Figure Lengend Snippet: Mouse embryonic fibroblasts (MEF) stably expressing Flag-Cas9-EGFP were transduced with a lentiviral CRISPR-library. Cells were treated three times with PMT(C1165S)DTa and surviving cells grown. Genomic DNA was extracted, inserted sgRNA amplified and sequenced.

Article Snippet: We utilized a well-functioning and validate genome-wide mouse lentiviral CRISPR guide RNA library v2 (Addgene).

Techniques: Stable Transfection, Expressing, Transduction, CRISPR, Amplification

Journal: bioRxiv

Article Title: Genome wide CRISPR screen for Pasteurella multocida toxin (PMT) binding proteins reveals LDL Receptor Related Protein1 (LRP1) as crucial cellular receptor

doi: 10.1101/2022.08.04.502755

Figure Lengend Snippet: The volcano plot shows gene expression changes between cells treated with lethal PMT-DTa chimera and those only transduced with the CRISPR library. All reads were mapped locally using BWA-MEM [ ,6], then quantified with featureCounts [4], and finally fold changes between the condition were calculated by DESeq2 [7] (see galaxy history). The volcano plot was drawn with the bioinfokit toolkit [8]. Significantly enriched genes, having a positive fold change above 0.584 and a p-value lower or equal 5%, are shown in blue. The significance thresholds are marked by gray-dotted lines. The ten most significant genes are highlighted with their name. Non-significant genes are colored in gray.

Article Snippet: We utilized a well-functioning and validate genome-wide mouse lentiviral CRISPR guide RNA library v2 (Addgene).

Techniques: Gene Expression, Transduction, CRISPR

Journal: Molecular Cancer Research

Article Title: Metabolic Regulator IAPP (Amylin) Is Required for BRAF and RAS Oncogene-Induced Senescence

doi: 10.1158/1541-7786.mcr-20-0879

Figure Lengend Snippet: Figure 1. A lentiviral-based shRNA screen identifies IAPP as a new modulator of OIS. A, Schematic overview of genetic screen workflow. BRER cells were transduced with library, drug selected for stable integration and treated for 21 days with 4-OHT. Genomic DNA extracted from emergent clones was used to identify each shRNA contained therein. See also Supplementary Figs. S1 and S2 for BRER characterization and screening results. B, Representative photos of shIAPP cells and control cells after 7 dayswithout (EtOH) or with (þ4-OHT) BRAF activation. C, Flow cytometric analysis of cell-cycle distribution for cells treated as in B. D, Proliferation curves for BRER cells expressing vector (pRS19, left), control shRNA (pLKO-1 shLuc, right), or IAPP-specific shRNAs in the continuous presence of 4-OHT or solvent (EtOH) as indicated. Data are representative of three experiments, with biological triplicates for each time point. P < 0.05, P < 0.01, P < 0.001 (Tukey’s multiple comparisons test). E, RT-qPCR analysis of IAPP mRNA in HFTE cells transduced with shRNA to IAPP or indicated controls. Error represents SEM. Significance was determined using t test.

Article Snippet: BRER cells were infected with the DECIPHER Lentiviral shRNA Library, Human Mod 1: Signaling Pathway Targets (Addgene, #28285).

Techniques: shRNA, Transduction, Clone Assay, Control, Activation Assay, Expressing, Plasmid Preparation, Solvent, Quantitative RT-PCR

Journal: Molecular Cancer Research

Article Title: Metabolic Regulator IAPP (Amylin) Is Required for BRAF and RAS Oncogene-Induced Senescence

doi: 10.1158/1541-7786.mcr-20-0879

Figure Lengend Snippet: Figure 2. Loss of IAPP expression permits bypass BRAF-induced senescence. A, Western blot analysis of cell cycle and proliferation markers following 4 days of BRAF activation in vector control or shIAPP expressing cells. See Supplementary Fig. S3 for independent shRNA knockdown data. B, SAHF formation in BRER cells with and without shIAPP after 4 days 4-OHT treatment. Representative of more than three experiments. C, SA-b-Gal expression in vector control and shIAPP expressing BRER cells following 10 days 4-OHT treatment. Represen- tative of three experiments. D, Quantification of C from three independent experiments with a minimum of 450 cells assessed for each condition. Error bars represent SEM. Significance was determined with Student t test.

Article Snippet: BRER cells were infected with the DECIPHER Lentiviral shRNA Library, Human Mod 1: Signaling Pathway Targets (Addgene, #28285).

Techniques: Expressing, Western Blot, Activation Assay, Plasmid Preparation, Control, shRNA, Knockdown

Journal: G3: Genes|Genomes|Genetics

Article Title: RNA sequencing-based screen for reactivation of silenced alleles of autosomal genes

doi: 10.1093/g3journal/jkab428

Figure Lengend Snippet: Hit validation for 5-aza-dC. (A and B) Confirmation of Screen-seq results for 5-aza-dC-treated cells using an orthogonal method to measure AI. cDNA samples from day 7 of screening were assessed using ddPCR with allele-specific fluorescent probes. (A) Scatterplots for 20,000 droplets targeting the readout gene, Col6a5 . 5-aza-dC concentration is shown in the plots. Black : empty droplets; blue : droplets with the Cast paternal allele amplified (labeled by FAM fluorophore); red : droplets with the 129 maternal allele amplified (labeled by HEX fluorophore). Ratio of red: blue droplets are shown. AI value written in red is the maternal AI. Note that the double-positive droplets (orange) contained both maternal and paternal templates; a small number of such double-positives is expected with higher concentrations of biallelic template. These droplets are ignored in the quantitative analysis. (B) left— summary of AI measurements shown in (A) for Col6a5 on day 7; right— summary of AI measurements for Dnajc12 on day 7. (C and D) Biological replicate of Abl.1 cells were treated with 5-aza-dC and AI was measured using ddPCR. (C) Scatterplots representation as shown in (A) after 2 days of exposure. (D) Summary of AI measurement for Col6a5 (left) and Dnajc12 (right) after 2, 5, and 7 days of exposure (denoted by color). Gray vertical dashed lines for Col6a5 dose-response were used to determine “low,” “medium,” and “high” 5-aza-dC concentrations for the future experiments. Results for readout gene Adnp2 are in . (E–G) Analysis of Dnmt1 knock-down (KD) in Abl.1 cells. (E) Real-time quantitative PCR (RT-qPCR) analysis of Dnmt1 relative expression (expression in the empty vector control, normalized to Nono , taken as 1.0). Abl.1 cells were transduced with an empty plKO vector (control) or with two separate Dnmt1 shRNA knockdown constructs ( Dnmt1 KD construct 1 or 2) and grown for 2 days. Transduced cells were then selected by growing in the presence of a selection antibiotic for an additional 17 days. RT-qPCR quantification was performed on cells collected 19 days after transduction. Mean and SEM for three technical replicates are shown. (F) Representative scatterplots show AI measurement for Col6a5 in the transduced Abl.1 cells. AI was measured using ddPCR. (G) Summary of the AI measurement for Col6a5 (left) and Dnajc12 (right) after Dnmt1 KD.

Article Snippet: Two shRNA vectors targeting Dnmt1 (SHR000038801.1_TRC001.1 and SHR000373188.1_TRC005.1) and a control empty vector (NUL003.3_TRC021.1) packaged in lentiviral vectors obtained from the Genetic Perturbation Platform at the Broad Institute were tested.

Techniques: Biomarker Discovery, Concentration Assay, Amplification, Labeling, Knockdown, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Plasmid Preparation, Control, Transduction, shRNA, Construct, Selection

Journal: Molecular Therapy. Nucleic Acids

Article Title: Non-targeting control for MISSION shRNA library silences SNRPD3 leading to cell death or permanent growth arrest

doi: 10.1016/j.omtn.2021.09.004

Figure Lengend Snippet: Expression of SHC016 in MC38CEA results in apoptosis and cell-cycle arrest at the G2/M phase The expression of non-targeting shRNA SHC002 or SHC016 was induced by doxycycline (dox; 100 ng/mL) for the last 4, 3, or 2 days of the 5-day culture. (A) Cell viability assessed by MTT assay. Absorbance values of the cells without induced transgene expression were taken as 100%. (B) Analysis of death via apoptosis assessed by annexin V/PI staining and flow cytometry analysis; alive cells, annexin V − /PI − ; apoptotic, annexin V + /PI − ; and late apoptotic and necrotic, annexin V + /PI + and annexin V − /PI + (the latter usually did not exceed 3% of the cells expressing either shRNA). (C) Activity of caspase-3 and -7 measured via chemiluminescence assay. Chemiluminescence values of the cells without induced transgene expression were taken as 100%. (D) Flow cytometry analysis of the cell cycle. (B−D) The cells treated for the last 2 days of the experiment with etoposide (eto; 2 μM) were used as a positive control. (E) Western blot (WB) analysis of p53 levels in MC38CEA and MEF cells incubated for 48 h with or without dox. Luminescent signals were collected for approximately 3 min (MC38CEA) or 10 s (MEF). Representative images and quantifications of WB signals of three independent experiments are shown. Whole WB and Ponceau S-stained membranes are given in Figure S4 . (F) qRT-PCR analysis of mRNA levels of p53 target genes in MC38CEA and MEF cells incubated for 1 or 2 days with or without dox. The relative levels of the transcripts in uninduced cells were taken as 1. (A−F) Data are shown as mean values (MVs) from 3 independent experiments. Error bars represent standard deviation (SD).

Article Snippet: Engineered shRNA libraries have facilitated the development of high-throughput methods using arrayed or pooled RNAi screens to identify proteins involved in different cellular processes or novel specific therapeutic targets., , , A popular lentiviral TRC shRNA library has been developed by the RNAi Consortium at the Broad Institute and marketed by Merck (Darmstadt, Germany; previously Sigma-Aldrich, St. Louis, MO, USA) under the trade name MISSION.

Techniques: Expressing, shRNA, MTT Assay, Staining, Flow Cytometry, Activity Assay, Chemiluminescence Immunoassay, Positive Control, Western Blot, Incubation, Quantitative RT-PCR, Standard Deviation

Journal: Molecular Therapy. Nucleic Acids

Article Title: Non-targeting control for MISSION shRNA library silences SNRPD3 leading to cell death or permanent growth arrest

doi: 10.1016/j.omtn.2021.09.004

Figure Lengend Snippet: Expression of SHC016 in human cell lines results in inhibition of cell proliferation and/or viability (A) Cell viability assessed by MTT assay. The expression of non-targeting shRNA SHC002 or SHC016 was induced by dox (100 ng/mL) for the last 5, 4, 3, or 2 days of the 6-day culture. The measurements taken each day (A549, U251) or on the 5th day of dox treatment (PC3, MCF7, HeLa) are shown. Full data for PC3, MCF7, and HeLa are available in Figure S6 . Absorbance values of the cells without induction of shRNA expression were taken as 100%. (B) DNA synthesis and cell viability assessed via double BrdU/eFluor 520 viability staining were performed 3 (A549) or 5 (U251, PC3, MCF7, HeLa) days after inducing SHC002 or SHC016 expression. BrdU was added to the medium for the last 6 h of culture. The difference of the time schedule was dictated by shorter time needed to manifest cytostatic/cytotoxic effects of SHC016 in A549 and substantially shorter doubling time of A549 than of other cell lines ; dead, all eFluor 520 + cells; BrdU + , eFluor 520 − /BrdU + cells; and BrdU − , eFluor 520 − /BrdU − cells. (A and B) Data are shown as MV from 3 independent experiments. (A) Error bars represent SD. (C) Analysis of clonal growth capacity of A549, U251, and HeLa cells by colony formation assay. Pictures of culture plates with stained clones were taken 7–10 days after plating the cells. Expression of SHC002 or SHC016 was induced with dox, 24 h after seeding the cells. The number of colonies in the corresponding, uninduced cultures was taken as 100%. Data are shown as MV ± SD from 3 (U251 and HeLa) or 4 (A549) independent experiments.

Article Snippet: Engineered shRNA libraries have facilitated the development of high-throughput methods using arrayed or pooled RNAi screens to identify proteins involved in different cellular processes or novel specific therapeutic targets., , , A popular lentiviral TRC shRNA library has been developed by the RNAi Consortium at the Broad Institute and marketed by Merck (Darmstadt, Germany; previously Sigma-Aldrich, St. Louis, MO, USA) under the trade name MISSION.

Techniques: Expressing, Inhibition, MTT Assay, shRNA, DNA Synthesis, Staining, Colony Assay, Clone Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: Non-targeting control for MISSION shRNA library silences SNRPD3 leading to cell death or permanent growth arrest

doi: 10.1016/j.omtn.2021.09.004

Figure Lengend Snippet: SHC016 expression cassette is eliminated from the cell cultures (A) Comparison of the viability of MCF7 and HeLa cells 5 days after induction of either SHC002 or SHC016 shRNA expression in the absence or presence of selection antibiotic, puromycin (1 μg/mL), which was added for the last 72 h of culture. Absorbance values of the cells without induction of shRNA expression were taken as 100%. Data are shown as MV ± SD from 3 independent experiments. (B) Comparison of DNA synthesis and cell viability assessed via BrdU/eFluor 520 viability staining of HeLa cells expressing for 5 days either SHC002 or SHC016 in the absence or presence of selection antibiotic, puromycin. Dead, all eFluor 520 + cells; BrdU + , eFluor 520 − /BrdU + cells; and BrdU − , eFluor 520 − /BrdU − cells. (C) PCR analysis of the abundance of the elements of pLKO vectors in the long-term cultures of MC38CEA and U251 cells. The cells were transduced with the empty vector (SHC001) or SHC002 or SHC016. DNA was isolated after indicated times, and the sequence from puromycin-resistance gene (puro) and the sequence comprising the shRNA insertion site (pLKO) were amplified. Inverted images of DNA electrophoresis gels are shown. S, size standard; GeneRuler 100 bp DNA Ladder, Thermo Scientific. The sizes of amplified fragments are as follows: puro, 123 bp; pLKO empty, 136 bp; and pLKO coding for either non-targeting RNA, 170 bp.

Article Snippet: Engineered shRNA libraries have facilitated the development of high-throughput methods using arrayed or pooled RNAi screens to identify proteins involved in different cellular processes or novel specific therapeutic targets., , , A popular lentiviral TRC shRNA library has been developed by the RNAi Consortium at the Broad Institute and marketed by Merck (Darmstadt, Germany; previously Sigma-Aldrich, St. Louis, MO, USA) under the trade name MISSION.

Techniques: Expressing, Comparison, shRNA, Selection, DNA Synthesis, Staining, Transduction, Plasmid Preparation, Isolation, Sequencing, Amplification, Nucleic Acid Electrophoresis

Journal: BMC Genomics

Article Title: Functional microRNA screening using a comprehensive lentiviral human microRNA expression library

doi: 10.1186/1471-2164-12-546

Figure Lengend Snippet: Overview of the miRNA library . (A) 660 annotated and 422 candidate miRNAs were cloned from their genomic loci into a lentiviral expression construct. From each construct, an individual batch of virus supernatant was created and portioned into individual tubes organized in a 96-well format. (B) Overlap between miRNAs included in the lentiviral library and miRNAs annotated in version 14 and version 17 of miRBase. The library contains constructs with variants or duplicates of some miRNA loci, which brings the total to 1120 constructs.

Article Snippet: InteRNA Technologies BV and TI Pharma funded construction of the lentiviral miRNA expression library.

Techniques: Clone Assay, Expressing, Construct, Virus

Journal: BMC Genomics

Article Title: Functional microRNA screening using a comprehensive lentiviral human microRNA expression library

doi: 10.1186/1471-2164-12-546

Figure Lengend Snippet: Overexpression of miRNAs in various cell lines after lentiviral transduction

Article Snippet: InteRNA Technologies BV and TI Pharma funded construction of the lentiviral miRNA expression library.

Techniques: Over Expression

Journal: BMC Genomics

Article Title: Functional microRNA screening using a comprehensive lentiviral human microRNA expression library

doi: 10.1186/1471-2164-12-546

Figure Lengend Snippet: miRNA overexpression kinetics . A375 cells were infected with either let-7a or miR-372-encoding virus and expression was measured by miRNA qPCR. (A, B) Expression of let-7a and miR-372 is measured 0, 4, 12, 24 and 72 hours after infection. Data is plotted as fold overexpression (A) or relative to U6 snRNA expression (B). (C) Three days after infection, samples were passaged and cultured either with or without puromycin selection. Expression of let-7a and miR-372 was measured 5, 9 and 20 days after infection. For both miRNAs, expression dipped after three days but stabilized thereafter, resulting in a sustained overexpression.

Article Snippet: InteRNA Technologies BV and TI Pharma funded construction of the lentiviral miRNA expression library.

Techniques: Over Expression, Infection, Virus, Expressing, Cell Culture, Selection

Journal: BMC Genomics

Article Title: Functional microRNA screening using a comprehensive lentiviral human microRNA expression library

doi: 10.1186/1471-2164-12-546

Figure Lengend Snippet: Lentiviral transduction of miRNAs in a high-content setup . Five different cell lines were infected with either empty vector miR-372, or let-7a. Cells were fixed 96 hours after infection and stained with Hoechst. Samples were analyzed with automated image analysis software. Four parameters were assessed: 1, number of nuclei per field; 2) nuclear size; 3) nuclear shape; 4) chromatin condensation. Graphs on the right show the quantification of the number of nuclei per field. Error bars represent standard deviations of triplicate wells. Four fields were counted per well.

Article Snippet: InteRNA Technologies BV and TI Pharma funded construction of the lentiviral miRNA expression library.

Techniques: Transduction, Infection, Plasmid Preparation, Staining, Software

Journal: BMC Genomics

Article Title: Functional microRNA screening using a comprehensive lentiviral human microRNA expression library

doi: 10.1186/1471-2164-12-546

Figure Lengend Snippet: Arrayed screening using the lentiviral library . (A) A375 cells were seeded in a 96-well plate and infected with virus containing an empty vector, let-7a, or miR-372. Five days after infection, cells expressing let-7a were significantly less numerous than cells expressing empty vector, while cells expressing miR-372 were significantly more numerous (left panel, * indicates p < 10 -4 in a two-tailed t-test). Values are average number of cells per well ± standard deviation, n = 6. Each measurement of both let-7a and miR-372 samples is separated from the empty vector samples by more than three standard deviations (Z-score, right panel). (B) Results of an arrayed screen on cell viability of A375 cells. Cells in individual wells were infected with single miRNA constructs and subjected to an MTS assay 5 days after infection. The screen was performed in duplicate. For each measurement a B-score was calculated. Correlation between duplicate B-scores is shown. The green dots represent let-7a-1, -2, and -3, while the red dot represents miR-372.

Article Snippet: InteRNA Technologies BV and TI Pharma funded construction of the lentiviral miRNA expression library.

Techniques: Infection, Virus, Plasmid Preparation, Expressing, Two Tailed Test, Standard Deviation, Construct, MTS Assay

Journal: BMC Genomics

Article Title: Functional microRNA screening using a comprehensive lentiviral human microRNA expression library

doi: 10.1186/1471-2164-12-546

Figure Lengend Snippet: A pooled screen for invasive capacity of miRNAs using the lentiviral library . (A) Schematic of the pooled invasion screen setup. In brief, after clonal infection (not shown here), cells with 40 individual miRNA constructs were pooled and either subjected to two rounds of an invasion assay or used as the control fraction. Of both fractions, integrated constructs were quantified by massively parallel sequencing to determine enrichment. (B) miRNAs were chosen for confirmation based on a combination (E × R > 1) of enrichment (E) after invasion and their abundance in the invasive fraction; expressed as the scaled number of reads (R). 23 out of 45 miRNAs were confirmed to enhance invasive capacity of HCT15 cells in final individual invasion assays.

Article Snippet: InteRNA Technologies BV and TI Pharma funded construction of the lentiviral miRNA expression library.

Techniques: Infection, Construct, Invasion Assay, Control, Sequencing

Journal: Genes & Development

Article Title: TRPS1 acts as a context-dependent regulator of mammary epithelial cell growth/differentiation and breast cancer development

doi: 10.1101/gad.331371.119

Figure Lengend Snippet: TRPS1 loss impairs cell proliferation and lactogenic differentiation, mediated by increased interferon signaling. ( A ) Cell proliferation of control and TRPS1 knockout HC11 clones, as quantified using IncuCyte imaging for 140 h. Data represent mean ± standard error of the mean (SEM). ( B ) RT-qPCR analysis of β-casein ( Csn2 ) expression levels upon stimulation with DIP mix (dexamethasone, insulin, and prolactin) in control and TRPS1 knockout HC11 clones. Data represent mean + SD, n = 2. One-way ANOVA: (***) P < 0.001. ( C ) Pathway enrichment analysis performed on RNA-seq of HC11 control and TRPS1 knockout cells, using the MSigDB hallmark gene set collection . FDR < 0.05 was considered significant (blue bars). ( D ) Heat map reflecting gene expression changes at the genes listed in the interferon α and interferon γ gene sets indicated in C between HC11 control and TRPS1 knockout cells. Key factors in these signaling pathways are indicated. ( E ) Western blot analysis of STAT1 and STAT2 expression levels in HC11 control and TRPS1 knockout cells transduced with a nontargeting shRNA or a pool of shRNAs targeting Stat1 . β-actin was used as a loading control. ( F ) RT-qPCR analysis of β-casein ( Csn2 ) expression levels upon stimulation with DIP mix in control and TRPS1 knockout HC11 clones transduced with a nontargeting shRNA or a pool of shRNAs targeting Stat1 . Data represent mean + SD, n = 3. One-way ANOVA: (***) P < 0.001.

Article Snippet: Broad Institute's Mission TRC-1 mouse library lentiviral shRNAs were used targeting Trps1 or Stat1 ( Supplemental Table S2B ).

Techniques: Control, Knock-Out, Clone Assay, Imaging, Quantitative RT-PCR, Expressing, RNA Sequencing, Gene Expression, Protein-Protein interactions, Western Blot, Transduction, shRNA