Journal: Viruses

Article Title: Coronaviral Main Protease Induces LPCAT3 Cleavage and Endoplasmic Reticulum (ER) Stress.

doi: 10.3390/v15081696

Figure Lengend Snippet: Figure 4. Mpro indirectly induced LPCAT3 cleavage. (A) SARS-CoV-2 Mpro inhibitor GC376 could not abolish Mpro-induced LPCAT3 cleavage. HEK293T cells were transfected with pcDNA3.1 (Vector) and pcDNA3.1-SCV2 Mpro-Flag (SCV2 Mpro). Overnight, cells were treated with GC376 at the final concentration as indicated in the figure for 12 h. Twenty micrograms of cellular lysates were loaded in each lane and submitted to Western blot analysis using anti-Flag, anti-LPCAT3, and anti- GAPDH antibodies. (B) Proteasome inhibitor MG132 could not abolish Mpro-induced LPCAT3 cleavage. HEK293T cells were transfected with pcDNA3.1 (Vector) and pcDNA3.1-SCV2 Mpro-Flag (SCV2 Mpro). Overnight, cells were treated with MG132 at the final concentration of 5 µM for 12 h. Twenty micrograms of cellular lysates were loaded in each lane and submitted to Western blot analysis using anti-Flag, anti-LPCAT3, and anti-GAPDH antibodies. (C) SARS-CoV-2 Mpro mutant could not abolish Mpro-induced LPCAT3 cleavage. HEK293T cells were transfected with either wild-type (WT) or protease-dead mutant (MUT) for 48 h. Twenty micrograms of cellular lysates were loaded in each lane and submitted to Western blot analysis using anti-Flag, anti-LPCAT3, and anti-GAPDH antibodies. The black solid arrows present uncleaved-LPCAT3, the red arrows present cleaved-LPCAT3, and the black hollow arrows present non-specific bands.

Article Snippet: Antibodies for β-actin (#4967), FLAG (#8146S), and MYC (#2276S) were purchased from Cell Signaling Technology.

Techniques: Transfection, Plasmid Preparation, Concentration Assay, Western Blot, Mutagenesis

Journal: bioRxiv

Article Title: Inducible and reversible inhibition of miRNA-mediated gene repression in vivo

doi: 10.1101/2021.06.01.445680

Figure Lengend Snippet: (a) Schematics of T6B action: T6B competes with TNRC6 for binding to AGO proteins preventing miRISC assembly. (b) Schematics of the size exclusion chromatography (SEC) assay for the fractionation of AGO-containing complexes according to their molecular weight. (c) SEC profiling of miRISC components upon T6B expression: Total lysates from HCT116 cells expressing no fusion protein (upper panel), T6B (middle panel) or T6B Mut (lower panel) were fractionated as described in (b) and immunoblotted to detect AGO2, TNRC6A, T6B and PABP1. (d) RNAseq analysis of total and small RNAs isolated from MEFs cell lines expressing either empty no fusion protein, T6B or T6B Mut . Upper panel: bubble plot of target derepression against miRNA abundance. The mean log2 Fold Change (T6B or T6B Mut vs control) of predicted targets for each conserved miRNA family was calculated, converted to a z-score and is plotted on the x-axis against the miRNA family abundance (log of the sum of read counts for each member of the family). The size of each circle is proportional to the number of predicted targets. A positive z score indicates that the targets for that family are preferentially upregulated upon T6B expression, while a negative score would indicate preferential downregulation. Expression of T6B, but not of T6B Mut , causes preferential upregulation of miRNA targets of the most miRNA families and the effect is roughly proportional to each miRNA family abundance. Lower panel: cumulative distribution plot of predicted let-7 targets compared to background in T6B-expressing MEFs. (e) Scatter plots of miRNA abundance as determined by small-RNAseq of total RNA extracted from MEFs expressing either T6B or T6B Mut . Each dot represents a miRNA in miRBase. (f) Effect of T6B expression on AGO2 slicing activity. MEFs expressing either T6B or T6B Mut were transfected with siRNAs targeting GAPDH mRNA (siGAPDH) or with scramble siRNA (siCTL). Levels of GAPDH, T6B and tubulin were assessed by immunoblot 72 hours post-transfection. T6B and T6B Mut have slightly different migration on PAGE, as previously observed by Hauptmann at al. .

Article Snippet: For IP of AGO-T6B complexes from human HCT116 cells, 500μg of lysates in 500 μL of SEC buffer were incubated for 3 hours with primary antibodies directed to either AGO proteins (WAKO anti-AGO2 #011-22033, EMD Millipore anti-panAGO #MABE56) or directed to T6B-fusion protein (Cell Signaling anti-FLAG #8146S, Cell Signaling anti-HA #2367S) or mouse IgG1 isotype control (Cell Signaling #5415).

Techniques: Binding Assay, Size-exclusion Chromatography, Fractionation, Molecular Weight, Expressing, Isolation, Control, Activity Assay, Transfection, Western Blot, Migration

Journal: bioRxiv

Article Title: Inducible and reversible inhibition of miRNA-mediated gene repression in vivo

doi: 10.1101/2021.06.01.445680

Figure Lengend Snippet: (a) HCTT116 cells transduced with retroviral vectors expressing a doxycycline-inducible T6B or T6B Mut transgene (FH-T6B-YFP) were cultured in the presence of doxycycline for 48. Whole cells lysates were probed with an anti-HA antibody. (b) Lysates from (a) were immunoprecipitated with the indicated antibodies and blotted against AGO2, FH-T6B-YFP (anti-HA) and GAPDH. Note that the T6B fusion protein, but not its mutant version (T6B Mut ), binds to AGO proteins. Lower panel. Aminoacid sequence of the T6B and T6B Mut fusion proteins. Both T6B versions have HA and FLAG tags at the N termini, and are fused to the yellow fluorescent protein (YFP) at the C-termini. In T6B Mut , all Tryptophan residues (red) are mutated to Alanine to prevent interaction with AGO proteins. Blue, FLAG-tag; light blue, HA-tag; bold black, T6B; green, YFP.

Article Snippet: For IP of AGO-T6B complexes from human HCT116 cells, 500μg of lysates in 500 μL of SEC buffer were incubated for 3 hours with primary antibodies directed to either AGO proteins (WAKO anti-AGO2 #011-22033, EMD Millipore anti-panAGO #MABE56) or directed to T6B-fusion protein (Cell Signaling anti-FLAG #8146S, Cell Signaling anti-HA #2367S) or mouse IgG1 isotype control (Cell Signaling #5415).

Techniques: Transduction, Retroviral, Expressing, Cell Culture, Immunoprecipitation, Mutagenesis, Sequencing, FLAG-tag

Journal: bioRxiv

Article Title: Inducible and reversible inhibition of miRNA-mediated gene repression in vivo

doi: 10.1101/2021.06.01.445680

Figure Lengend Snippet: Size exclusion chromatography was performed on whole cell lysates from MEFs transduced with retroviral vectors expressing a doxycycline-inducible T6B or T6B Mut transgene and cultured in presence of doxycycline for 48h. Eluted fractions were probed with the anti-AGO2 or anti-HA antibodies to determine the elution profile of AGO2 and T6B, respectively.

Article Snippet: For IP of AGO-T6B complexes from human HCT116 cells, 500μg of lysates in 500 μL of SEC buffer were incubated for 3 hours with primary antibodies directed to either AGO proteins (WAKO anti-AGO2 #011-22033, EMD Millipore anti-panAGO #MABE56) or directed to T6B-fusion protein (Cell Signaling anti-FLAG #8146S, Cell Signaling anti-HA #2367S) or mouse IgG1 isotype control (Cell Signaling #5415).

Techniques: Size-exclusion Chromatography, Transduction, Retroviral, Expressing, Cell Culture

Journal: bioRxiv

Article Title: Inducible and reversible inhibition of miRNA-mediated gene repression in vivo

doi: 10.1101/2021.06.01.445680

Figure Lengend Snippet: (a) Schematic of the targeting strategy to generate the T6B mouse. The construct contains a flippase recognition target site (frt) that allows homing into the Col1A locus when electroporated together with a vector expressing the Flippase recombinase into KH2 (Col1A-frt/Rosa26-rtTA) murine embryonic stem cells. KH2 also express the rtTA trans-activator driven by the endogenous Rosa26 (R26) promoter. (b) Immunofluorescence imaging performed using an anti-YFP antibody, showing T6B expression in a panel of tissues of adult R26 T6B mice fed doxycycline for 7 days. Tissues from R26 CTL (carrying the rtTA allele but not the T6B allele) were used as negative controls. (c) Protein lysates from the liver of R26 T6B mice on or off doxycycline-containing chow for the indicated number of days were resolved by SDS-PAGE and Western blotting was performed with anti-HA antibody to detect expression of the T6B transgene. (d) Co-IP experiments using an anti-YFP antibody showing interaction between AGO and T6B in total liver extracts from T6B mice on doxycycline containing chow. (e) SEC elution profile of AGO2-containing complexes in liver lysates from T6B mice euthanized at the indicated time points after doxycycline administration. Notice the shift of AGO2 from the high molecular weight fractions to the low molecular weight fractions after 5 days of doxycycline treatment and the reconstitution of the full miRISC after removal of doxycycline from the diet. (f-g) Total RNA extracted from the large intestine (f) and the liver (g) of R26 CTL and R26 T6B mice was subjected to RNAseq. Left panel: scatter plot showing the effect of T6B expression on targets of all miRNA families were generated as described in . The abundance of each miRNA family was calculated using data set from Isakova et al. . Right panel: Representative cumulative distribution plot of log2 fold changes in expression of predicted targets of the indicated miRNA families.

Article Snippet: For IP of AGO-T6B complexes from human HCT116 cells, 500μg of lysates in 500 μL of SEC buffer were incubated for 3 hours with primary antibodies directed to either AGO proteins (WAKO anti-AGO2 #011-22033, EMD Millipore anti-panAGO #MABE56) or directed to T6B-fusion protein (Cell Signaling anti-FLAG #8146S, Cell Signaling anti-HA #2367S) or mouse IgG1 isotype control (Cell Signaling #5415).

Techniques: Construct, Plasmid Preparation, Expressing, Immunofluorescence, Imaging, SDS Page, Western Blot, Co-Immunoprecipitation Assay, High Molecular Weight, Molecular Weight, Generated

Journal: bioRxiv

Article Title: Inducible and reversible inhibition of miRNA-mediated gene repression in vivo

doi: 10.1101/2021.06.01.445680

Figure Lengend Snippet: (a) Two independent targeted ES clones were cultured in the presence or absence of doxycycline for 48h and examined by epifluorescence microscopy to detect FH-T6B-YFP expression. The same exposure was used for all images. Bright field images are also shown for each clone. (b) Whole cell lysates from the clones shown in (a) were reprobed with an anti-HA antibody to detect expression of the T6B fusion protein.

Article Snippet: For IP of AGO-T6B complexes from human HCT116 cells, 500μg of lysates in 500 μL of SEC buffer were incubated for 3 hours with primary antibodies directed to either AGO proteins (WAKO anti-AGO2 #011-22033, EMD Millipore anti-panAGO #MABE56) or directed to T6B-fusion protein (Cell Signaling anti-FLAG #8146S, Cell Signaling anti-HA #2367S) or mouse IgG1 isotype control (Cell Signaling #5415).

Techniques: Clone Assay, Cell Culture, Epifluorescence Microscopy, Expressing

Journal: bioRxiv

Article Title: Inducible and reversible inhibition of miRNA-mediated gene repression in vivo

doi: 10.1101/2021.06.01.445680

Figure Lengend Snippet: Immunofluorescence imaging using a YFP-specific antibody, showing T6B expression in a panel of tissues of adult R26 T6B mice (second column) and CAG T6B mice (third column) fed doxycycline-containing diet for 7 days. Tissues from R26 CTL (first column) mice fed doxycycline-containing diet for 7 days were included as negative controls.

Article Snippet: For IP of AGO-T6B complexes from human HCT116 cells, 500μg of lysates in 500 μL of SEC buffer were incubated for 3 hours with primary antibodies directed to either AGO proteins (WAKO anti-AGO2 #011-22033, EMD Millipore anti-panAGO #MABE56) or directed to T6B-fusion protein (Cell Signaling anti-FLAG #8146S, Cell Signaling anti-HA #2367S) or mouse IgG1 isotype control (Cell Signaling #5415).

Techniques: Immunofluorescence, Imaging, Expressing

Journal: bioRxiv

Article Title: Inducible and reversible inhibition of miRNA-mediated gene repression in vivo

doi: 10.1101/2021.06.01.445680

Figure Lengend Snippet: Total extracts from the colon of R26 T6B mice kept on doxycycline-containing diet for 1 week were immunoprecipitated using an anti-YFP antibody and probed with the indicated antibodies to measure the interaction between the T6B fusion protein and Ago2 in vivo . An anti-HA antibody was used to detect T6B.

Article Snippet: For IP of AGO-T6B complexes from human HCT116 cells, 500μg of lysates in 500 μL of SEC buffer were incubated for 3 hours with primary antibodies directed to either AGO proteins (WAKO anti-AGO2 #011-22033, EMD Millipore anti-panAGO #MABE56) or directed to T6B-fusion protein (Cell Signaling anti-FLAG #8146S, Cell Signaling anti-HA #2367S) or mouse IgG1 isotype control (Cell Signaling #5415).

Techniques: Immunoprecipitation, In Vivo

Journal: bioRxiv

Article Title: Inducible and reversible inhibition of miRNA-mediated gene repression in vivo

doi: 10.1101/2021.06.01.445680

Figure Lengend Snippet: SEC fractionation followed by Western blotting of total extracts from the liver and large intestine of control and R26 T6B mice treated with doxycycline-containing chow for 7 days. The shift of AGO2 from high molecular weight to low molecular weight complexes confirms disruption of the miRISC.

Article Snippet: For IP of AGO-T6B complexes from human HCT116 cells, 500μg of lysates in 500 μL of SEC buffer were incubated for 3 hours with primary antibodies directed to either AGO proteins (WAKO anti-AGO2 #011-22033, EMD Millipore anti-panAGO #MABE56) or directed to T6B-fusion protein (Cell Signaling anti-FLAG #8146S, Cell Signaling anti-HA #2367S) or mouse IgG1 isotype control (Cell Signaling #5415).

Techniques: Fractionation, Western Blot, Control, High Molecular Weight, Molecular Weight, Disruption

Journal: bioRxiv

Article Title: Inducible and reversible inhibition of miRNA-mediated gene repression in vivo

doi: 10.1101/2021.06.01.445680

Figure Lengend Snippet: (a) R26 +/+ ; ColA1 T6B/T6B females were crossed with R26rt TA/+ ; ColA1 T6B/T6B males and doxycycline was administered by chow starting at 0.5 d.p.c. No viable pups positive for both the rtTA and T6B allele were observed (n = 15, p-value = 0.002, Fisher exact test). (b) Pregnant females were kept on doxycycline diet from E13.5 to E18.5 and the pups delivered on E18.5 by c-section. Note the significantly smaller size of R26 rtTA/rtTA ; ColA1 T6B/T6B embryos relative to R26rt TA/rtTA ; ColA1 +/+ control littermates. Lower row: YFP detection by epifluorescence in E18.5 pups of the indicated genotypes. (c) Comparison of intestine architecture in H&E sections from R26 T6B and R26 CTL mice (n = 3 for each genotype) maintained on doxycycline for 2 months. (d) Immunofluorescence imaging of the small intestine of R26 T6B and R26 CTL mice (n = 3-5 for each genotype) kept on doxycycline diet for a month (upper row), showing a reduction in lysozyme expression in Paneth cells in the crypts. Lysozyme expression in R26 T6B mice returned to normal levels upon removal of doxycycline from the diet (lower row). (e) Peripheral blood analysis conducted in R26 T6B and R26 CTL mice (R26 CTL n = 4; R26 T6B n = 5). (f) Flow cytometric analysis of bone marrow of R26 T6B and R26 CTL mice kept on doxycycline diet for 3 weeks showing developmental block at the Pro-B to Pre-B. p values (from left to right): *p = 0.0348, **p = 0.0023, *p = 0.0340, **p = 0.0004, unpaired t-test. R26 CTL n = 4; R26 T6B n = 5. (g) Flow cytometry analysis of the bone marrow of control and R26 T6B mice kept on doxycycline diet for 3 weeks. p values (from left to right): p = 0.0994, **p = 0.0092, **p = 0.0085, *p = 0.0312, unpaired t-test. R26 CTL n = 4; R26 T6B n = 5.

Article Snippet: For IP of AGO-T6B complexes from human HCT116 cells, 500μg of lysates in 500 μL of SEC buffer were incubated for 3 hours with primary antibodies directed to either AGO proteins (WAKO anti-AGO2 #011-22033, EMD Millipore anti-panAGO #MABE56) or directed to T6B-fusion protein (Cell Signaling anti-FLAG #8146S, Cell Signaling anti-HA #2367S) or mouse IgG1 isotype control (Cell Signaling #5415).

Techniques: Control, Comparison, Immunofluorescence, Imaging, Expressing, Blocking Assay, Flow Cytometry

Journal: bioRxiv

Article Title: Inducible and reversible inhibition of miRNA-mediated gene repression in vivo

doi: 10.1101/2021.06.01.445680

Figure Lengend Snippet: (a) Litter obtained by c-section from a pregnant R26 rtTA/rtTA ; ColA1 T6B/+ female crossed to a R26 rtTA/rtTA ; ColA1 T6B/+ male and maintained on doxycycline from d.p.c. 13.5 to d.p.c. 18.5. (b) Pups from (a) were weighted and genotyped and the results plotted. p-value: two-tailed unpaired t test.

Article Snippet: For IP of AGO-T6B complexes from human HCT116 cells, 500μg of lysates in 500 μL of SEC buffer were incubated for 3 hours with primary antibodies directed to either AGO proteins (WAKO anti-AGO2 #011-22033, EMD Millipore anti-panAGO #MABE56) or directed to T6B-fusion protein (Cell Signaling anti-FLAG #8146S, Cell Signaling anti-HA #2367S) or mouse IgG1 isotype control (Cell Signaling #5415).

Techniques: Two Tailed Test

Journal: bioRxiv

Article Title: Inducible and reversible inhibition of miRNA-mediated gene repression in vivo

doi: 10.1101/2021.06.01.445680

Figure Lengend Snippet: Immunofluorescence imaging of the small and large intestine of R26 T6B and R26 CTL mice kept on doxycycline diet for a month. An antibody against YFP was used to detect the T6B fusion protein.

Article Snippet: For IP of AGO-T6B complexes from human HCT116 cells, 500μg of lysates in 500 μL of SEC buffer were incubated for 3 hours with primary antibodies directed to either AGO proteins (WAKO anti-AGO2 #011-22033, EMD Millipore anti-panAGO #MABE56) or directed to T6B-fusion protein (Cell Signaling anti-FLAG #8146S, Cell Signaling anti-HA #2367S) or mouse IgG1 isotype control (Cell Signaling #5415).

Techniques: Immunofluorescence, Imaging

Journal: bioRxiv

Article Title: Inducible and reversible inhibition of miRNA-mediated gene repression in vivo

doi: 10.1101/2021.06.01.445680

Figure Lengend Snippet: Sections from the colon and small intestine sections of R26 T6B and control mice kept on doxycycline-containing diet for 2 months were probed by IHC with an anti-Ki67 antibody.

Article Snippet: For IP of AGO-T6B complexes from human HCT116 cells, 500μg of lysates in 500 μL of SEC buffer were incubated for 3 hours with primary antibodies directed to either AGO proteins (WAKO anti-AGO2 #011-22033, EMD Millipore anti-panAGO #MABE56) or directed to T6B-fusion protein (Cell Signaling anti-FLAG #8146S, Cell Signaling anti-HA #2367S) or mouse IgG1 isotype control (Cell Signaling #5415).

Techniques: Control

Journal: bioRxiv

Article Title: Inducible and reversible inhibition of miRNA-mediated gene repression in vivo

doi: 10.1101/2021.06.01.445680

Figure Lengend Snippet: Detection of goblet cells by staining of acidic and neutral mucins in intestine sections from R26 T6B and control mice kept on doxycycline diet for 2 months. Neutral mucins are stained with periodic acid-Shiff whereas acidic mucins are stained with Alcian blue.

Article Snippet: For IP of AGO-T6B complexes from human HCT116 cells, 500μg of lysates in 500 μL of SEC buffer were incubated for 3 hours with primary antibodies directed to either AGO proteins (WAKO anti-AGO2 #011-22033, EMD Millipore anti-panAGO #MABE56) or directed to T6B-fusion protein (Cell Signaling anti-FLAG #8146S, Cell Signaling anti-HA #2367S) or mouse IgG1 isotype control (Cell Signaling #5415).

Techniques: Staining, Control

Journal: bioRxiv

Article Title: Inducible and reversible inhibition of miRNA-mediated gene repression in vivo

doi: 10.1101/2021.06.01.445680

Figure Lengend Snippet: Body weight of R26 T6B (n = 5) and control (n = 8) female mice was assessed after 2 month-administration of doxycycline-containing chow. ns, not significant (p = 0. 6264, unpaired t test).

Article Snippet: For IP of AGO-T6B complexes from human HCT116 cells, 500μg of lysates in 500 μL of SEC buffer were incubated for 3 hours with primary antibodies directed to either AGO proteins (WAKO anti-AGO2 #011-22033, EMD Millipore anti-panAGO #MABE56) or directed to T6B-fusion protein (Cell Signaling anti-FLAG #8146S, Cell Signaling anti-HA #2367S) or mouse IgG1 isotype control (Cell Signaling #5415).

Techniques: Control

Journal: bioRxiv

Article Title: Inducible and reversible inhibition of miRNA-mediated gene repression in vivo

doi: 10.1101/2021.06.01.445680

Figure Lengend Snippet: Representative flow cytometry plots showing the gating strategy for the identification of hematopoietic stem and progenitor cells from whole bone marrow harvested from R26 T6B and R26 CTL mice maintained on doxycycline diet for 3 weeks. LT-HSC: Lin- Kit+ Sca1+ CD150+ CD48-, ST-HSC: Lin- Kit+ Sca1+ CD150- CD48-, MPP2: Lin- Kit+ Sca1+ CD150+ CD48+, MPP3/4: Lin- Kit+ Sca1+ CD150- CD48+.

Article Snippet: For IP of AGO-T6B complexes from human HCT116 cells, 500μg of lysates in 500 μL of SEC buffer were incubated for 3 hours with primary antibodies directed to either AGO proteins (WAKO anti-AGO2 #011-22033, EMD Millipore anti-panAGO #MABE56) or directed to T6B-fusion protein (Cell Signaling anti-FLAG #8146S, Cell Signaling anti-HA #2367S) or mouse IgG1 isotype control (Cell Signaling #5415).

Techniques: Flow Cytometry

Journal: bioRxiv

Article Title: Inducible and reversible inhibition of miRNA-mediated gene repression in vivo

doi: 10.1101/2021.06.01.445680

Figure Lengend Snippet: Representative flow cytometry plots showing the gating strategy for the identification of B cell lineage populations from whole bone marrow harvested from R26 T6B and R26 CTL mice maintained on doxycycline diet for 3 weeks. Pro-B: B220+CD19+IgD-IgM-CD25-Kit+, Pre-B: B220+CD19+IgD-IgM-CD25+, Imm B: B220+CD19+IgD-IgM+, Mat B: B220+CD19+IgD+IgM+/lo.

Article Snippet: For IP of AGO-T6B complexes from human HCT116 cells, 500μg of lysates in 500 μL of SEC buffer were incubated for 3 hours with primary antibodies directed to either AGO proteins (WAKO anti-AGO2 #011-22033, EMD Millipore anti-panAGO #MABE56) or directed to T6B-fusion protein (Cell Signaling anti-FLAG #8146S, Cell Signaling anti-HA #2367S) or mouse IgG1 isotype control (Cell Signaling #5415).

Techniques: Flow Cytometry

Journal: bioRxiv

Article Title: Inducible and reversible inhibition of miRNA-mediated gene repression in vivo

doi: 10.1101/2021.06.01.445680

Figure Lengend Snippet: (a) R26 T6B and R26 CTL mice (n = 6 for each genotype) kept on doxycycline diet were treated with Dextran Sodium Sulphate (DSS) for 5 days to induce inflammatory colitis and their weight was monitored daily. (b) Kaplan-Meier curves of animals described in panel (a). (c) Representative hematoxylin-eosin-stained sections of intestine of R26 T6B and R26 CTL mice (n = 3 for each genotype) at different time points pre- and post-DSS treatment. (d) Ki67 immunostaining of section of intestine at the indicated time points. (e) Sections from the large intestine of control and T6B mice euthanized at day 13 were subjected to RNA in situ hybridization with a probe against the Igfbp5 transcript. The results show increased levels of IGFBP5 mRNA in ulcerated areas of R26 T6B as compared to controls (n = 4 for each genotype).

Article Snippet: For IP of AGO-T6B complexes from human HCT116 cells, 500μg of lysates in 500 μL of SEC buffer were incubated for 3 hours with primary antibodies directed to either AGO proteins (WAKO anti-AGO2 #011-22033, EMD Millipore anti-panAGO #MABE56) or directed to T6B-fusion protein (Cell Signaling anti-FLAG #8146S, Cell Signaling anti-HA #2367S) or mouse IgG1 isotype control (Cell Signaling #5415).

Techniques: Staining, Immunostaining, Control, RNA In Situ Hybridization

Journal: bioRxiv

Article Title: Inducible and reversible inhibition of miRNA-mediated gene repression in vivo

doi: 10.1101/2021.06.01.445680

Figure Lengend Snippet: Bar plots showing measurement of colon length, aggregated length of ulcers, percentage of colon with ulcers, area of ulcers, number of immune nodules and the area of immune nodules performed on H&E longitudinal sections of colon from R26 CTL and R26 T6B mice 5 days post-DSS treatment. Measurements of these parameters were obtained using OMERO ( https://www.openmicroscopy.org/omero/ ) and used to estimate the extent of damage and colitis induced by DSS treatment. Plots show that no significant differences between R26 CTL and R26 T6B mice were observed, suggesting that both groups experienced similar level of DSS-induced colitis.

Article Snippet: For IP of AGO-T6B complexes from human HCT116 cells, 500μg of lysates in 500 μL of SEC buffer were incubated for 3 hours with primary antibodies directed to either AGO proteins (WAKO anti-AGO2 #011-22033, EMD Millipore anti-panAGO #MABE56) or directed to T6B-fusion protein (Cell Signaling anti-FLAG #8146S, Cell Signaling anti-HA #2367S) or mouse IgG1 isotype control (Cell Signaling #5415).

Techniques:

Journal: bioRxiv

Article Title: Inducible and reversible inhibition of miRNA-mediated gene repression in vivo

doi: 10.1101/2021.06.01.445680

Figure Lengend Snippet: (a) Long term HSC in the bone marrow of R26 T6B and T6B CTL mice treated with 5-FU or subjected to repeated bleeding (n = 5 for each genotype). Mice were maintained on doxycycline containing diet throughout the experiment. (b) Kaplan-Meier plots of R26 T6B (n = 5) and R26 CTL (n = 5) mice treated weekly with 5-FU for seven weeks. (c) Schematic of the bone marrow transplantation experiments: T6B was induced at different time points post-transplantation, and multi line age reconstitution was assessed at the indicated time points by FACS. (d) FACS analysis conducted on the peripheral blood of irradiated recipients transplanted 1:1 with T6B-expressing and wild-type bone marrow, and maintained on doxycycline diet according to scheme shown in panel c. Data are presented as mean ± s.d. *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA. off > off, n = 9; off > on, n = 10; on > off, n = 8; on>on, n = 8. (e) FACS analysis showing the frequency of T6B-extressing HSCs in the bone marrow of transplanted recipient mice kept on doxycycline diet according to scheme shown in panel c. off > off, n = 5; off > on, n = 5; on > off, n = 4; on > on, n = 5, one-way ANOVA.

Article Snippet: For IP of AGO-T6B complexes from human HCT116 cells, 500μg of lysates in 500 μL of SEC buffer were incubated for 3 hours with primary antibodies directed to either AGO proteins (WAKO anti-AGO2 #011-22033, EMD Millipore anti-panAGO #MABE56) or directed to T6B-fusion protein (Cell Signaling anti-FLAG #8146S, Cell Signaling anti-HA #2367S) or mouse IgG1 isotype control (Cell Signaling #5415).

Techniques: Transplantation Assay, Irradiation, Expressing

Journal: bioRxiv

Article Title: Inducible and reversible inhibition of miRNA-mediated gene repression in vivo

doi: 10.1101/2021.06.01.445680

Figure Lengend Snippet: (a) Detection of T6B expression with an anti-YFP antibody in the heart and skeletal muscle of R26 T6B , CAG T6B , and R26 CTL mice maintained on doxycycline containing diet for 7 days. (b) Total RNA extracted from the heart (upper panel) and the skeletal muscle (lower panel) of CAG CTL and CAG T6B mice maintained on dox for 7 days was analyzed by RNAseq. Left panels: Scatter plot showing the effect of T6B expression on targets of conserved miRNA families were generated as described in . The abundance of each miRNA family was calculated using dataset from Isakova et al. . Right panels: Representative cumulative distribution plot of log2 fold changes in expression of predicted targets of the indicated miRNA families. (c) Kaplan-Meier curves of CAG T6B and CAG CTL mice (n = 8 for each genotype) maintained on doxycycline throughout the duration of the experiment. (d) Upper row: representative H&E staining showing marked dilation of the four cardiac chambers in hearts of CAG T6B mice compared to controls (n = 9 for each genotype). Despite having thinner walls, the histomorphology of ventricular cardiomyofibers were within normal limits. Bottom row: representative H&E staining showing degenerative and regenerative changes in the skeletal muscle of the hind limbs of CAG T6B mice compared to controls (n = 9 for each genotype).

Article Snippet: For IP of AGO-T6B complexes from human HCT116 cells, 500μg of lysates in 500 μL of SEC buffer were incubated for 3 hours with primary antibodies directed to either AGO proteins (WAKO anti-AGO2 #011-22033, EMD Millipore anti-panAGO #MABE56) or directed to T6B-fusion protein (Cell Signaling anti-FLAG #8146S, Cell Signaling anti-HA #2367S) or mouse IgG1 isotype control (Cell Signaling #5415).

Techniques: Expressing, Generated, Staining

Journal: bioRxiv

Article Title: Inducible and reversible inhibition of miRNA-mediated gene repression in vivo

doi: 10.1101/2021.06.01.445680

Figure Lengend Snippet: Body weight of CAG T6B and control mice maintained on doxycycline for up to 45 days was assessed the day on which euthanasia was performed. n = 8 (4 females and 4 males) for each genotype (age and sex matched). Mice were kept on doxycycline diet throughout the duration of the experiment and control mice were euthanized at day 45. P-values: unpaired t-test.

Article Snippet: For IP of AGO-T6B complexes from human HCT116 cells, 500μg of lysates in 500 μL of SEC buffer were incubated for 3 hours with primary antibodies directed to either AGO proteins (WAKO anti-AGO2 #011-22033, EMD Millipore anti-panAGO #MABE56) or directed to T6B-fusion protein (Cell Signaling anti-FLAG #8146S, Cell Signaling anti-HA #2367S) or mouse IgG1 isotype control (Cell Signaling #5415).

Techniques: Control

Journal: bioRxiv

Article Title: Inducible and reversible inhibition of miRNA-mediated gene repression in vivo

doi: 10.1101/2021.06.01.445680

Figure Lengend Snippet: T6B blocks miRNA activity in sea urchins and zebrafish. (a) Left panel: Representative examples of Mediterranean sea urchin ( P. lividus ) zygotes injected with 1 pg of in vitro-transcribed mRNA coding for either T6B or T6B Mut proteins and observed under DIC optics at 48 hours post-fertilization. Both embryos are oriented in a vegetal view. T6B-expressing embryos displayed severe developmental aberrations ranging from the failure to form a proper archenteron and skeletal structures, to overall delay in development and embryonic lethality. By contrast, control T6B Mut -expressing embryos observed at the same developmental stage went through embryogenesis normally and exhibited the characteristic easel-like shape of the echinoid pluteus larva. Right panel: quantitative PCR showing dysregulation of genes involved in the developmental gene regulatory network of the sea urchin upon T6B expression. (b) Zebrafish ( Danio rerio ) fertilized eggs were injected with 75 pg of in vitro-transcribed mRNA coding for either T6B or T6BMut fusion proteins. While T6B Mut -expressing embryos developed normally, the majority of T6B-expressing embryos under went severe developmental defects.

Article Snippet: For IP of AGO-T6B complexes from human HCT116 cells, 500μg of lysates in 500 μL of SEC buffer were incubated for 3 hours with primary antibodies directed to either AGO proteins (WAKO anti-AGO2 #011-22033, EMD Millipore anti-panAGO #MABE56) or directed to T6B-fusion protein (Cell Signaling anti-FLAG #8146S, Cell Signaling anti-HA #2367S) or mouse IgG1 isotype control (Cell Signaling #5415).

Techniques: Activity Assay, Injection, In Vitro, Expressing, Control, Real-time Polymerase Chain Reaction