t6b fusion protein  (Cell Signaling Technology Inc)

Journal: eLife

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

doi: 10.7554/elife.70948

Figure Lengend Snippet: Figure 1. T6B fusion protein prevents miRNA-induced silencing complex (miRISC) assembly and impairs microRNA (miRNA) activity in vitro. (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 T6BMut (lower panel) were fractionated as described in (B) and immunoblotted to detect AGO2, TNRC6A, T6B, and PABP1. For each blot, the relative signal intensity was assessed by densitometric analysis. (D) RNAseq analysis of total and small RNAs isolated from mouse embryo fibroblasts (MEFs) cell lines expressing either no fusion protein, T6B, or T6BMut (n = 3 for each cell line). Upper panel: bubble plot of target de-repression against miRNA abundance. The mean log2-fold change (T6B or T6BMut 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 T6BMut, 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 T6BMut (n = 3 for each cell line). Each dot represents a miRNA in miRbase. (F) Effect of T6B expression on AGO2 slicing activity. MEFs expressing either T6B or T6BMut were transfected with siRNAs targeting GAPDH mRNA (siGAPDH) or with scramble siRNA (siCTL). Levels of GAPDH, T6B, and tubulin were assessed by immunoblot 72 hr post-transfection. T6B and T6BMut have slightly different migration on PAGE, as previously observed by Hauptmann et al., 2015.

Article Snippet: For IP of AGO- T6B complexes from human HCT116 cells, 500 μg of lysates in 500 μl of SEC buffer were incubated for 3 hr 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, In Vitro, Binding Assay, Size-exclusion Chromatography, Fractionation, Molecular Weight, Expressing, Isolation, Control, Transfection, Western Blot, Migration

Journal: eLife

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

doi: 10.7554/elife.70948

Figure Lengend Snippet: Figure 2. Expression of T6B reversibly blocks miRNA-induced silencing complex (miRISC) assembly and inhibits microRNA (miRNA) function in vivo. (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 Col1a1 locus when electroporated together with a vector expressing the Flippase recombinase into KH2 (Col1a1-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 R26T6B mice fed doxycycline for 7 days. Tissues from R26CTL (carrying

Article Snippet: For IP of AGO- T6B complexes from human HCT116 cells, 500 μg of lysates in 500 μl of SEC buffer were incubated for 3 hr 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, In Vivo, Construct, Plasmid Preparation, Immunofluorescence, Imaging

Journal: eLife

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

doi: 10.7554/elife.70948

Figure Lengend Snippet: Figure 3. Phenotypic analysis of R26T6B mice during homeostasis. (A) Rosa26+/+; Col1a1T6B/T6B females were crossed with Rosa26rtTA/+; Col1a1T6B/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’s 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 Rosa26rtTA/rtTA; Col1a1T6B/T6B embryos relative to Rosa26rtTA/rtTA;Col1a1+/+ 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 R26T6B and R26CTL mice (n = 3 for each genotype) maintained on doxycycline for 2 months. (D) Immunofluorescence imaging of the small intestine of R26T6B and R26CTL 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 R26T6B mice returned to normal levels upon removal of doxycycline from the diet (lower row). (E) Peripheral blood analysis conducted in R26T6B and R26CTL mice (R26CTL n = 4; R26T6B n = 5). (F) Flow cytometric analysis of bone marrow of R26T6B and R26CTL 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. R26CTL n = 4; R26T6B n = 5. (G) Flow cytometry analysis of the bone marrow of control and R26T6B 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. R26CTL n = 4; R26T6B n = 5.

Article Snippet: For IP of AGO- T6B complexes from human HCT116 cells, 500 μg of lysates in 500 μl of SEC buffer were incubated for 3 hr 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: eLife

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

doi: 10.7554/elife.70948

Figure Lengend Snippet: Figure 4. T6B-induced block of miRNA-induced silencing complex (miRISC) assembly leads to impaired intestinal regeneration. (A) R26T6B and R26CTL mice (n = 6 for each genotype) kept on doxycycline diet were treated with dextran sulfate sodium (DSS) for 5 days to induce inflammatory colitis and their weight was monitored daily. Data are presented as mean ± SD. p-Values (from left to right): *p=0.034, *p=0.005, *p=0.029, *p=0.024, *p=0.011, from unpaired t-test. (B) Kaplan–Meier curves of animals treated with DSS as described in panel (A). p-Value from log-rank test (C) Representative hematoxylin-eosin-stained sections of intestine of R26T6B and R26CTL 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 R26T6B as compared to controls (n = 4 for each genotype).

Article Snippet: For IP of AGO- T6B complexes from human HCT116 cells, 500 μg of lysates in 500 μl of SEC buffer were incubated for 3 hr 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: Blocking Assay, Staining, Immunostaining, Control, RNA In Situ Hybridization

Journal: eLife

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

doi: 10.7554/elife.70948

Figure Lengend Snippet: Figure 6. The microRNA (miRNA) pathway is essential in heart and skeletal muscle during homeostasis. (A) Detection of T6B expression with an anti- YFP antibody in the heart and skeletal muscle of R26T6B, CAGT6B, and R26CTL 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 CAGCTL and CAGT6B mice (n = 3 for each strain) 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 was generated as described in Figure 1D. The abundance of each miRNA family was calculated using dataset from Isakova et al., 2020. 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 CAGT6B and CAGCTL mice (n = 8 for each genotype) maintained on doxycycline throughout the duration of the experiment. p-Value from log-rank test. (D) Upper row: representative H&E staining showing marked dilation of the four cardiac chambers in hearts of CAGT6B mice compared to controls (n = 9 for each genotype). Despite having thinner walls, the histomorphology of ventricular cardiomyofibers was within normal limits. Bottom row: representative H&E staining showing degenerative and regenerative changes in the skeletal muscle of the hind limbs of CAGT6B mice compared to controls (n = 9 for each genotype).

Article Snippet: For IP of AGO- T6B complexes from human HCT116 cells, 500 μg of lysates in 500 μl of SEC buffer were incubated for 3 hr 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: eLife

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

doi: 10.7554/elife.70948

Figure Lengend Snippet: Scheme 1. Cloning strategy for the generation of the targeting vector expressing the FH-T6B-YFP transgene.

Article Snippet: For IP of AGO- T6B complexes from human HCT116 cells, 500 μg of lysates in 500 μl of SEC buffer were incubated for 3 hr 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: Cloning, Plasmid Preparation, Expressing