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Novus Biologicals dgcr8 knockout mefs
Dgcr8 Knockout Mefs, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 90/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/dgcr8+knockout/DGCR8+knockout+MEF+cells/pm41634336-225-0-5
Average 90 stars, based on 4 article reviews

dgcr8 knockout mefs - by Bioz Stars, 2026-11

90/100 stars

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Article Title: Biogenesis of mammalian microRNAs by a non-canonical processing pathway
Article Snippet: .. Hannon) ( ) and DGCR8 knockout (DGCR8 −/− ) cells (Novus Biologicals) were grown on a gelatin layer in Knockout Dulbecco's modified Eagle's medium (Gibco) supplemented with 15% ES cell FBS (Gibco), 1% non-essential amino acids, 1% l -glutamine, 1% penicillin/streptomycin/Amphotericin B, 0.1% ESGRO-LIF and 0.008% beta-mercaptoethanol. .. Cells were transfected with 3 μg of minigene plasmid using Lipofectamine 2000 (Invitrogen) as per the manufacturer's instructions and RNA was collected after 48 h. RNA was collected for all experiments using Trizol reagent (Invitrogen).

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Figure 4. Post-transcriptional Regulation of HMGA2 Isoforms (A) Left: Normalized luciferase (Renilla) activity in Dgcr8-KO MEF of constructs carrying 30 UTR sequences of HMGA2-L (Rluc-30UTRwt_HMGA2-L) or a mutant derivative depleted for miRNA sites (Rluc-30UTRmt_HMGA2-L). Right: Normalized luciferase (Renilla) activity in Dgcr8-KO MEF of constructs carrying 30 UTR sequences of HMGA2-L (Rluc-30UTRwt_HMGA2-L) or HMGA2-S (Rluc-30UTRwt_HMGA2-S). Normalized luciferase activities were reported with respect to Rluc-30UTRmt_HMGA2-L (left) or Rluc-30UTRwt_HMGA2-S (right), set to 100%. Mean ± SEM values are shown. Unpaired t test was used; *p < 0.05, **p < 0.01, ***p < 0.005, borderline (०= 0.055). (B) Quantiﬁcation of miRNAs of interest in PC-3 and HPC-5F cells measured by qRT-PCR. U6 small nuclear RNA (snRNA) was used as control. (C) Relative quantiﬁcation of HMGA2 isoforms in PC-3 and HPC-5F cells transduced with lentiviral constructs carrying the HMGA2 ORFs equipped with their corresponding 30 UTRs (HMGA2-L+30UTRwt and HMGA2-S+30UTRwt) or a derivative HMGA2-L isoform mutated at its miRNA sites (HMGA2-L+30UTRmt). Infected cells were treated with actinomycin D (Act-D) and harvested at the indicated time points. Expression values were normalized to HPRT1 control and then reported with respect to HMGA2-S+30UTRwt, set to 1. Mean ± SEM values are shown. ANOVA was used; *p < 0.05, **p < 0.01, ***p < 0.005.

Journal: Cell stem cell

Article Title: A CLK3-HMGA2 Alternative Splicing Axis Impacts Human Hematopoietic Stem Cell Molecular Identity throughout Development.

doi: 10.1016/j.stem.2018.03.012

Figure Lengend Snippet: Figure 4. Post-transcriptional Regulation of HMGA2 Isoforms (A) Left: Normalized luciferase (Renilla) activity in Dgcr8-KO MEF of constructs carrying 30 UTR sequences of HMGA2-L (Rluc-30UTRwt_HMGA2-L) or a mutant derivative depleted for miRNA sites (Rluc-30UTRmt_HMGA2-L). Right: Normalized luciferase (Renilla) activity in Dgcr8-KO MEF of constructs carrying 30 UTR sequences of HMGA2-L (Rluc-30UTRwt_HMGA2-L) or HMGA2-S (Rluc-30UTRwt_HMGA2-S). Normalized luciferase activities were reported with respect to Rluc-30UTRmt_HMGA2-L (left) or Rluc-30UTRwt_HMGA2-S (right), set to 100%. Mean ± SEM values are shown. Unpaired t test was used; *p < 0.05, **p < 0.01, ***p < 0.005, borderline (०= 0.055). (B) Quantiﬁcation of miRNAs of interest in PC-3 and HPC-5F cells measured by qRT-PCR. U6 small nuclear RNA (snRNA) was used as control. (C) Relative quantiﬁcation of HMGA2 isoforms in PC-3 and HPC-5F cells transduced with lentiviral constructs carrying the HMGA2 ORFs equipped with their corresponding 30 UTRs (HMGA2-L+30UTRwt and HMGA2-S+30UTRwt) or a derivative HMGA2-L isoform mutated at its miRNA sites (HMGA2-L+30UTRmt). Infected cells were treated with actinomycin D (Act-D) and harvested at the indicated time points. Expression values were normalized to HPRT1 control and then reported with respect to HMGA2-S+30UTRwt, set to 1. Mean ± SEM values are shown. ANOVA was used; *p < 0.05, **p < 0.01, ***p < 0.005.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies CD34 PE-Cy7 BD Biosciences Cat. 348791; CLONE 8G12 CD38 PE-Cy5 BD Biosciences Cat. 555461; CLONE HIT2 CD90 PE BD Biosciences Cat. 555596; CLONE 5E10 CD45RA FITC BioLegend Cat. 304105; CLONE HI100 DAPI solution BD Biosciences Cat. 564907 CD45RA V450 BD Biosciences Cat. 560362; CLONE HI100 CD133/1 APC Miltenyi Biotec Cat. 130-090-826; CLONE AC133 CD19 PE BD Biosciences Cat. 349209; CLONE 4G7 CD45 PE-Cy5 Coulter CLONE Immu19.2 CD33 APC BD Biosciences Cat. 340474; CLONE P67.6 CD45 APC-Cy7 BD Biosciences Cat. 561863; CLONE 2D1 V5 MBL Cat. M167-3 H3K4me2 Diagenode Cat. 035-050 Biological Samples Cord Blood CD34+ Cells Lonza Cat. 2C-101 Cord Blood CD34+ Cells AllCells Cat. CB008F Fetal Liver CD34+ Cells AllCells Cat. FL-CD34-002F Bone Marrow CD34+ Cells AllCells Cat. ABM017F Bone Marrow CD34+ Cells Lonza Cat. 2M-101C Chemicals, Peptides, and Recombinant Proteins Actinomycin D Sigma-Aldrich Cat. A1410 Doxycycline Hyclate Sigma-Aldrich Cat. D9891 Protamine Sulfate Sigma-Aldrich Cat. P4020 Recombinant Human SCF Peprotech Cat. 300-07 Recombinant Human FLT3L Peprotech Cat. 300-19 Recombinant Human TPO Peprotech Cat. 300-18 Recombinant Human IL6 Peprotech Cat. 200-06 Retronectin Clontech Cat. T100A DOTAP Liposomal Transfection Reagent Sigma-Aldrich Cat. 11202375001 DharmaFECT Duo Transfection Reagent Dharmacon Cat. T-2010-03 Critical Commercial Assays Dual-Luciferase Reporter Assay System Promega Cat. E1910 TruSeq RNA Library Prep Kit v2 Illumina Cat. RS-122-2001 SMART-Seq v4 Ultra Low Input RNA Kit Clontech Cat. 634888 Nextera XT DNA Library Preparation Kit Illumina Cat. FC-131-1024 nCounter Human v2 miRNA Expression Assay NanoString Technologies Cat. GXA-MIR2-24 ChiP-Seq Assay Broad Institute Epigenomics Program N/A Experimental Models: Cell Lines PC-3 ATCC Cat. CRL-1435 K562 ATCC Cat. CCL-243 HPC and HPC-5F Doulatov et al., 2013 N/A DGCR8 knockout MEF Novus Biologicals Cat. NBP2-25171 (Continued on next page) Cell Stem Cell 22, 575–588.e1–e7, April 5, 2018 e1

Techniques: Luciferase, Activity Assay, Construct, Mutagenesis, Quantitative RT-PCR, Control, Transduction, Infection, Expressing

( a ) (left) Susceptibility (TCID 50 /ml) of miRNA deficient cells ( Dgcr8 -/- , Dicer -/- ) and wild-type parental cells ( Dgcr8 +/+ (ESC1), Dicer +/+ (ESC2)) to TMEV infection, higher values represent higher susceptibility (n = 4, p-value<0.05, t-test). (right) Quantification of Influenza A replication after infection of the same cell lines, data show the average (n = 3)±s.d. (*) p-value<0.05 by t-test. ( b ) Quantification of Ifnb1 expression of ESCs lacking Dgcr8 or Dicer to stimulation with poly(I:C) and G 3 -YSD. Data show average (n = 3)±s.d., normalized to mock, (*) p-value<0.05 by t-test. ( c ) Quantification of TMEV vRNA upon JAK1/2 inhibition by Ruxolitinib treatment. Data show the average (n = 3)±s.d. ( d ) qRT-PCR analyses of ISGs expression after stimulation of wild-type and miRNA-deficient ESCs with IFN-β. Data show average (n = 3)±s.d., normalized to mock treated cells ( e, f ) Quantification of TMEV replication after infection in Dgcr8 ( e ) and Dicer ( f ) parental (+/+), deficient (-/-) and rescued (resc) cell lines. Data are normalized to miRNA-deficient cell lines susceptibility. Data show the average (n = 3)±s.d (*) p-value<0.05 by t-test. Northern blots for three stem-cell specific miRNAs, as control for knock-out and rescue of Dgcr8 and Dicer , are shown at the right of each panel.

Journal: eLife

Article Title: MicroRNA-deficient mouse embryonic stem cells acquire a functional interferon response

doi: 10.7554/eLife.44171

Figure Lengend Snippet: ( a ) (left) Susceptibility (TCID 50 /ml) of miRNA deficient cells ( Dgcr8 -/- , Dicer -/- ) and wild-type parental cells ( Dgcr8 +/+ (ESC1), Dicer +/+ (ESC2)) to TMEV infection, higher values represent higher susceptibility (n = 4, p-value<0.05, t-test). (right) Quantification of Influenza A replication after infection of the same cell lines, data show the average (n = 3)±s.d. (*) p-value<0.05 by t-test. ( b ) Quantification of Ifnb1 expression of ESCs lacking Dgcr8 or Dicer to stimulation with poly(I:C) and G 3 -YSD. Data show average (n = 3)±s.d., normalized to mock, (*) p-value<0.05 by t-test. ( c ) Quantification of TMEV vRNA upon JAK1/2 inhibition by Ruxolitinib treatment. Data show the average (n = 3)±s.d. ( d ) qRT-PCR analyses of ISGs expression after stimulation of wild-type and miRNA-deficient ESCs with IFN-β. Data show average (n = 3)±s.d., normalized to mock treated cells ( e, f ) Quantification of TMEV replication after infection in Dgcr8 ( e ) and Dicer ( f ) parental (+/+), deficient (-/-) and rescued (resc) cell lines. Data are normalized to miRNA-deficient cell lines susceptibility. Data show the average (n = 3)±s.d (*) p-value<0.05 by t-test. Northern blots for three stem-cell specific miRNAs, as control for knock-out and rescue of Dgcr8 and Dicer , are shown at the right of each panel.

Article Snippet: Dgcr8 knockout ( Dgcr8 -/- ) mouse ESCs were purchased from Novus Biologicals (NBA1-19349) and the parental strain, v6.5 ( Dgcr8 +/+ , also named in the text ESC1) from ThermoFisher (MES1402).

Techniques: Infection, Expressing, Inhibition, Quantitative RT-PCR, Northern Blot, Control, Knock-Out

( a ) RT-PCR of poly(I:C) activated ESCs. Only Dgcr8 -/- (lane 6, left panel) and Dicer -/- ( lane 5 and 6, right panel) express detectable Ifnb1 transcript (top panels). Actb amplification serves as a loading control. ( b ) Ifnb mRNA quantification by qRT-PCR after increasing amounts of poly(I:C) in Dgcr8 -/- and Dicer -/- mESCs. Data show the average (n = 3)±s.d., normalized to mock, (*) p-value<0.05 by t-test. ( c ) IFN-β ELISA analyses from mock and poly(I:C) stimulated wild-type and miRNA-deficient ESCs. Data show the average (n = 3)±s.d., (*) p-value<0.05 by two-tailed T test ( d ) TMEV vRNA quantification in wild-type and miRNA-deficient cell lines after mock or Ruxolitinib treatment (JAK1/2 inhibitor). Data show the average (n = 3)±s.d., (*) p value < 0.05 by Student’s t-test, n.s. non-significant ( e ) TCID50 quantification after TMEV infection of Dgcr8 (left) and Dicer (right) rescued cell lines.

Journal: eLife

Article Title: MicroRNA-deficient mouse embryonic stem cells acquire a functional interferon response

doi: 10.7554/eLife.44171

Figure Lengend Snippet: ( a ) RT-PCR of poly(I:C) activated ESCs. Only Dgcr8 -/- (lane 6, left panel) and Dicer -/- ( lane 5 and 6, right panel) express detectable Ifnb1 transcript (top panels). Actb amplification serves as a loading control. ( b ) Ifnb mRNA quantification by qRT-PCR after increasing amounts of poly(I:C) in Dgcr8 -/- and Dicer -/- mESCs. Data show the average (n = 3)±s.d., normalized to mock, (*) p-value<0.05 by t-test. ( c ) IFN-β ELISA analyses from mock and poly(I:C) stimulated wild-type and miRNA-deficient ESCs. Data show the average (n = 3)±s.d., (*) p-value<0.05 by two-tailed T test ( d ) TMEV vRNA quantification in wild-type and miRNA-deficient cell lines after mock or Ruxolitinib treatment (JAK1/2 inhibitor). Data show the average (n = 3)±s.d., (*) p value < 0.05 by Student’s t-test, n.s. non-significant ( e ) TCID50 quantification after TMEV infection of Dgcr8 (left) and Dicer (right) rescued cell lines.

Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Control, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Infection

( a, b ) Susceptibility of Dgcr8 -/- , Dicer -/- and parental cells to TMEV infection after inhibition of IRF3 (BX795) ( a ) and Nf-κB (BMS345541) ( b ), normalized to mock-treated cells. ( c ) Heat map of significantly differentially expressed proteins (p<0.05) in the absence ( Dgcr8 -/- ) or presence ( Dgcr8 resc ) of miRNAs identified by STRING analysis. ( d ) Western blot analysis of MAVS expression in miRNA-deficient cells ( Dgcr8 -/- and Dicer -/- , lanes 2 and 5), wild-type counterparts ( Dgcr8 +/+ and Dicer +/+ , lanes 1 and 4) and respective rescued ESCs lines ( Dgcr8 resc and Dicer resc , lanes 3 and 6). MAVS quantification normalized to Tubulin and relative to wild-type levels is shown at the top of the panel. 10.7554/eLife.44171.010 Figure 3—source data 1. Source data of mass spectrometry results.

Journal: eLife

Article Title: MicroRNA-deficient mouse embryonic stem cells acquire a functional interferon response

doi: 10.7554/eLife.44171

Figure Lengend Snippet: ( a, b ) Susceptibility of Dgcr8 -/- , Dicer -/- and parental cells to TMEV infection after inhibition of IRF3 (BX795) ( a ) and Nf-κB (BMS345541) ( b ), normalized to mock-treated cells. ( c ) Heat map of significantly differentially expressed proteins (p<0.05) in the absence ( Dgcr8 -/- ) or presence ( Dgcr8 resc ) of miRNAs identified by STRING analysis. ( d ) Western blot analysis of MAVS expression in miRNA-deficient cells ( Dgcr8 -/- and Dicer -/- , lanes 2 and 5), wild-type counterparts ( Dgcr8 +/+ and Dicer +/+ , lanes 1 and 4) and respective rescued ESCs lines ( Dgcr8 resc and Dicer resc , lanes 3 and 6). MAVS quantification normalized to Tubulin and relative to wild-type levels is shown at the top of the panel. 10.7554/eLife.44171.010 Figure 3—source data 1. Source data of mass spectrometry results.

Techniques: Infection, Inhibition, Western Blot, Expressing, Mass Spectrometry

( a ) Treatment of mESCs with inhibitors of IRF3 and NF-κB activation improves viral replication to a much greater extend in the Dgcr8 -/- and Dicer -/- cells compared to the parental cells, suggesting that the inhibitory effect that miRNAs have on the IFN response is upstream of these two transcription factors. ( b ) Western blot analyses of phospho-IRF3 upon stimulation of Dgcr8 -/- ESCs with poly(I:C) in the presence or absence of BX759 (inhibitor IRF3 phosphorylation). Tubulin serves as a loading control ( c ) Quantification of Tnf mRNA upon poly(I:C) stimulation in Dgcr8 +/+ and Dgcr8 -/- in the presence or absence of BMS345541 (inhibitor of NFkB activation). Data show the average (n = 2) normalized to Gapdh ( d ) Quantification of oxidative phosphorylation activity by Rhodamine 123 uptake assay in Dgcr8 (left) and Dicer (right) cell lines. ( e ) qRT-PCR quantification of Mavs mRNA. Mavs expression is higher in the absence of miRNAs ( Dgcr8 -/- and Dicer -/- ), when compared to parental cell lines ( Dgcr8 +/+ and Dicer +/+ ). Data show the average (n = 3)±s.d, (*) denotes p<0.05, t-test. ( f ) Western blot analyses for MDA5, RIG-I and PKR for wild-type cell lines (+/+), Dgcr8 and Dicer KO (-/-), and the respective rescued cell lines (resc). Quantification of the signal normalized to Tubulin and relative to wild-type is shown at the top of each panel.

Journal: eLife

Article Title: MicroRNA-deficient mouse embryonic stem cells acquire a functional interferon response

doi: 10.7554/eLife.44171

Figure Lengend Snippet: ( a ) Treatment of mESCs with inhibitors of IRF3 and NF-κB activation improves viral replication to a much greater extend in the Dgcr8 -/- and Dicer -/- cells compared to the parental cells, suggesting that the inhibitory effect that miRNAs have on the IFN response is upstream of these two transcription factors. ( b ) Western blot analyses of phospho-IRF3 upon stimulation of Dgcr8 -/- ESCs with poly(I:C) in the presence or absence of BX759 (inhibitor IRF3 phosphorylation). Tubulin serves as a loading control ( c ) Quantification of Tnf mRNA upon poly(I:C) stimulation in Dgcr8 +/+ and Dgcr8 -/- in the presence or absence of BMS345541 (inhibitor of NFkB activation). Data show the average (n = 2) normalized to Gapdh ( d ) Quantification of oxidative phosphorylation activity by Rhodamine 123 uptake assay in Dgcr8 (left) and Dicer (right) cell lines. ( e ) qRT-PCR quantification of Mavs mRNA. Mavs expression is higher in the absence of miRNAs ( Dgcr8 -/- and Dicer -/- ), when compared to parental cell lines ( Dgcr8 +/+ and Dicer +/+ ). Data show the average (n = 3)±s.d, (*) denotes p<0.05, t-test. ( f ) Western blot analyses for MDA5, RIG-I and PKR for wild-type cell lines (+/+), Dgcr8 and Dicer KO (-/-), and the respective rescued cell lines (resc). Quantification of the signal normalized to Tubulin and relative to wild-type is shown at the top of each panel.

Techniques: Activation Assay, Western Blot, Phospho-proteomics, Control, Activity Assay, Quantitative RT-PCR, Expressing

( a ) Dual luciferase assay with Mavs , Rig-I and Mda5 3’UTRs in miRNA-deficient cells lines ( Dgcr8 -/- and Dicer -/- ). Data show the average (n = 3)±s.d normalized to Renilla and relative to the parental lines, (*) p-value<0.05 by t-test ( b ) Western blot of cell line overexpressing MAVS lacking the 3’UTR in Dgcr8 +/+ cells (lane 3). MAVS quantification normalized to Tubulin and relative to wild-type is shown at the top ( c ) Susceptibility (TCID 50 /ml) of same cells lines as in ( b ) to TMEV infection (left panel) and quantification of viral RNA after TMEV infections in the same cell lines (right panel). Data show the average (n = 5)±s.d. (*) p-value<0.05 by t-test ( d ) Ifnb mRNA expression after poly(I:C) transfection of the same cell lines as in ( b ), average is represented (n = 3)±s.d, normalized to Dgcr8 +/+ cell line, (*) p-value<0.05 by t-test.

Journal: eLife

Article Title: MicroRNA-deficient mouse embryonic stem cells acquire a functional interferon response

doi: 10.7554/eLife.44171

Figure Lengend Snippet: ( a ) Dual luciferase assay with Mavs , Rig-I and Mda5 3’UTRs in miRNA-deficient cells lines ( Dgcr8 -/- and Dicer -/- ). Data show the average (n = 3)±s.d normalized to Renilla and relative to the parental lines, (*) p-value<0.05 by t-test ( b ) Western blot of cell line overexpressing MAVS lacking the 3’UTR in Dgcr8 +/+ cells (lane 3). MAVS quantification normalized to Tubulin and relative to wild-type is shown at the top ( c ) Susceptibility (TCID 50 /ml) of same cells lines as in ( b ) to TMEV infection (left panel) and quantification of viral RNA after TMEV infections in the same cell lines (right panel). Data show the average (n = 5)±s.d. (*) p-value<0.05 by t-test ( d ) Ifnb mRNA expression after poly(I:C) transfection of the same cell lines as in ( b ), average is represented (n = 3)±s.d, normalized to Dgcr8 +/+ cell line, (*) p-value<0.05 by t-test.

Techniques: Luciferase, Western Blot, Infection, Expressing, Transfection

( a ) Transfection of miRNA mimics miR-125a-5p, miR-125b-5p, miR-185–5p and miR-673–5p in Dgcr8 -/- cells followed by MAVS western blot. MAVS quantification normalized to Tubulin and relative to wild-type is shown at the top ( b ) Quantification of TMEV replication by qRT-PCR in the same cell lines as in ( a ) (n = 3) ( c ) Western blot analysis of MAVS expression in Dgcr8 +/+ cells transfected with antagomirs against miR-125a-5p, miR-125b-5p and miR-673–5p. MAVS quantification normalized to Tubulin and relative to wild-type is shown at the top ( d ) Western blot analysis of MAVS expression in miR-673 -/- cell lines. MAVS quantification normalized to Tubulin and relative to wild-type is shown at the top ( e ) Quantification of TMEV vRNA in miR-673 CRISPR knock-out cell lines in a Dgcr8 +/+ background. Data show the average (n = 3)±s.d (*) p-value<0.05 by t-test. ( f ) Quantification of TMEV vRNA shown as fold-change compared to mock upon JAK1/2 inhibition by Ruxolitinib treatment. Data show the average (n = 3)±s.d ( g ) qRT-PCR quantification of mmu-miR-673–5p expression after retinoic acid (RA) differentiation of ESC. Data show the average (n = 2)±s.e.m, normalized to U6 snRNA, (*) p-val <0.05 by t-test.

Journal: eLife

Article Title: MicroRNA-deficient mouse embryonic stem cells acquire a functional interferon response

doi: 10.7554/eLife.44171

Figure Lengend Snippet: ( a ) Transfection of miRNA mimics miR-125a-5p, miR-125b-5p, miR-185–5p and miR-673–5p in Dgcr8 -/- cells followed by MAVS western blot. MAVS quantification normalized to Tubulin and relative to wild-type is shown at the top ( b ) Quantification of TMEV replication by qRT-PCR in the same cell lines as in ( a ) (n = 3) ( c ) Western blot analysis of MAVS expression in Dgcr8 +/+ cells transfected with antagomirs against miR-125a-5p, miR-125b-5p and miR-673–5p. MAVS quantification normalized to Tubulin and relative to wild-type is shown at the top ( d ) Western blot analysis of MAVS expression in miR-673 -/- cell lines. MAVS quantification normalized to Tubulin and relative to wild-type is shown at the top ( e ) Quantification of TMEV vRNA in miR-673 CRISPR knock-out cell lines in a Dgcr8 +/+ background. Data show the average (n = 3)±s.d (*) p-value<0.05 by t-test. ( f ) Quantification of TMEV vRNA shown as fold-change compared to mock upon JAK1/2 inhibition by Ruxolitinib treatment. Data show the average (n = 3)±s.d ( g ) qRT-PCR quantification of mmu-miR-673–5p expression after retinoic acid (RA) differentiation of ESC. Data show the average (n = 2)±s.e.m, normalized to U6 snRNA, (*) p-val <0.05 by t-test.

Techniques: Transfection, Western Blot, Quantitative RT-PCR, Expressing, CRISPR, Knock-Out, Inhibition

( a ) Dgcr8 -/- cells were transfected with miRNA mimics for 24 hr, followed by RNA extraction and Mavs mRNA quantification by qRT-PCR. Transfection of all miRNA mimics result in (non-significant) decrease of Mavs levels. ( b ) Dgcr8 -/- mESCs were first transfected with mimics, as in ( a ) followed by TMEV infections 36 hr later, and measurement of the TCID50. All mimics except for mir-185-5p resulted in higher TCID50 values compared to mock transfected cells, suggesting that in mESC cells targeting Mavs with these miRNAs leads to a higher susceptibility to viral infection.

Journal: eLife

Article Title: MicroRNA-deficient mouse embryonic stem cells acquire a functional interferon response

doi: 10.7554/eLife.44171

Figure Lengend Snippet: ( a ) Dgcr8 -/- cells were transfected with miRNA mimics for 24 hr, followed by RNA extraction and Mavs mRNA quantification by qRT-PCR. Transfection of all miRNA mimics result in (non-significant) decrease of Mavs levels. ( b ) Dgcr8 -/- mESCs were first transfected with mimics, as in ( a ) followed by TMEV infections 36 hr later, and measurement of the TCID50. All mimics except for mir-185-5p resulted in higher TCID50 values compared to mock transfected cells, suggesting that in mESC cells targeting Mavs with these miRNAs leads to a higher susceptibility to viral infection.

Techniques: Transfection, RNA Extraction, Quantitative RT-PCR, Infection

( a ) Genomic sequencing of miR-673 locus contained in chromosome 12. In WT, the position of the two miRNAs encoded by the locus, miR-673–5p and miR-673–3p are indicated by yellow boxes. For miR-673 -/- clones (1,2 and 3) both sequenced alleles are shown with the indicated deletions induced by CRISPR. ( b ) Quantification of mmu-miR-673–5p abundance by qRT-PCR. Data show the average (n = 3)±s.d, normalized to U6 snRNA. (n.d, not detected) ( c ) Quantification of Mavs mRNA levels by RT-qPCR. Data show the average of at least (n = 3)±s.d, normalized to Actb , and relative to Dgcr8 +/+ .

Journal: eLife

Article Title: MicroRNA-deficient mouse embryonic stem cells acquire a functional interferon response

doi: 10.7554/eLife.44171

Figure Lengend Snippet: ( a ) Genomic sequencing of miR-673 locus contained in chromosome 12. In WT, the position of the two miRNAs encoded by the locus, miR-673–5p and miR-673–3p are indicated by yellow boxes. For miR-673 -/- clones (1,2 and 3) both sequenced alleles are shown with the indicated deletions induced by CRISPR. ( b ) Quantification of mmu-miR-673–5p abundance by qRT-PCR. Data show the average (n = 3)±s.d, normalized to U6 snRNA. (n.d, not detected) ( c ) Quantification of Mavs mRNA levels by RT-qPCR. Data show the average of at least (n = 3)±s.d, normalized to Actb , and relative to Dgcr8 +/+ .

Techniques: Genomic Sequencing, Clone Assay, CRISPR, Quantitative RT-PCR

( a ) TMEV vRNA quantification in wild-type and miRNA-deficient cell lines after mock or Ruxolitinib treatment (JAK1/2 inhibitor). Data show the average (n = 3)±s.d., (*) p value < 0.05 by Student’s t-test, n.s. non-significant ( b ) Growth curves of ESCs. Data show the average (n = 3)±s.d. ( c ) No obvious morphological changes were observed between wild-type ( Dgcr8 +/+ ) and miR-673-deficient ESCs ( d ) Quantification of Nanog and Pouf5f1 pluripotent markers upon knocking out miR-673 by CRISPR in comparison to wild-type ESCs, data show the average (n = 3)±s.e.m, (n.s.) non-significant by t-test. NIH3T3 serves as a negative control for pluripotent maker expression ( e ) Quantification of Nanog and Pouf51 expression after reintroduction of miR-673 mimics and controls for 24 or 48 hr. Data show the average (n = 3)±s.e.m. (n.s.) non-significant by t-test.

Journal: eLife

Article Title: MicroRNA-deficient mouse embryonic stem cells acquire a functional interferon response

doi: 10.7554/eLife.44171

Figure Lengend Snippet: ( a ) TMEV vRNA quantification in wild-type and miRNA-deficient cell lines after mock or Ruxolitinib treatment (JAK1/2 inhibitor). Data show the average (n = 3)±s.d., (*) p value < 0.05 by Student’s t-test, n.s. non-significant ( b ) Growth curves of ESCs. Data show the average (n = 3)±s.d. ( c ) No obvious morphological changes were observed between wild-type ( Dgcr8 +/+ ) and miR-673-deficient ESCs ( d ) Quantification of Nanog and Pouf5f1 pluripotent markers upon knocking out miR-673 by CRISPR in comparison to wild-type ESCs, data show the average (n = 3)±s.e.m, (n.s.) non-significant by t-test. NIH3T3 serves as a negative control for pluripotent maker expression ( e ) Quantification of Nanog and Pouf51 expression after reintroduction of miR-673 mimics and controls for 24 or 48 hr. Data show the average (n = 3)±s.e.m. (n.s.) non-significant by t-test.

Techniques: CRISPR, Comparison, Negative Control, Expressing

Journal: Cell stem cell

Article Title: A CLK3-HMGA2 alternative splicing axis impacts human hematopoietic stem cell molecular identity throughout development

doi: 10.1016/j.stem.2018.03.012

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: DGCR8 knockout MEF , Novus Biologicals , Cat. NBP2-25171.

Techniques: Recombinant, Transfection, Reporter Assay, DNA Library Preparation, Expressing, Knock-Out, Negative Control, Software

Journal: Cell stem cell

Article Title: A CLK3-HMGA2 alternative splicing axis impacts human hematopoietic stem cell molecular identity throughout development

doi: 10.1016/j.stem.2018.03.012

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: DGCR8 knockout MEF , Novus Biologicals , Cat. NBP2-25171.

Techniques: Recombinant, Transfection, Reporter Assay, DNA Library Preparation, Expressing, Knock-Out, Negative Control, Software

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