complementary fluorescent probes  (Macklin Inc)

Journal: International Journal of Molecular Sciences

Article Title: miR-193b-3p Promotes Proliferation of Goat Skeletal Muscle Satellite Cells through Activating IGF2BP1

doi: 10.3390/ijms232415760

Figure Lengend Snippet: miR-193b-3p induces IGF2BP through a seed-sites match. ( A ) miR-193b-3p promotes IGF2BP1 transcripts. ( B ) miR-193b-3p elevates IGF2BP1 protein. Western blotting (WB) assay was performed to detect IGF2BP1 protein. GAPDH works as a loading control. ( C ) The expression profile of IGF2BP1 in goat tissues and cells. ( D ) miR-193b-3p targets 3′ UTR of IGF2BP1. The potential seed match between miR-193b-3p within 3′ UTR regions of IGF2BP1 is predicted. The blue line marks the complementary nucleotide. Mfe indicates free energy between IGF2BP1-wt and miR-193b-3p. ( E ) The addition of miR-193b-3p activates luciferase activity of wild-type (R-Luc-IGF2BP1-wt) in MuSCs. ( F ) miR-193b-3p activates luciferase activity of R-Luc-IGF2BP1-wt in HeLa and mouse C2C12 myoblast. Data are shown as mean ± MSE. An unpaired two-tailed t -test is used to evaluate the means difference. *** p < 0.001, ** p < 0.01, * p < 0.05, and § p < 0.1.

Article Snippet: To detect the spatial localization of IGF2BP1 and miR-193b-3p RNA, the Cy5- labeled fluorescence oligonucleotide probes, complementary to goat IGF2BP1 mRNA (5′-GCCGGATTTGACCAAGAACTGGCCGCTGTAGGA-3′, red), and FAM-labeled chi-miR-193b-3p probes (5′-AGCGGGACTTTGTGGGCCAGTT-3′, green) were synthesized (Servicebio, Wuhan, China).

Techniques: Western Blot, Control, Expressing, Luciferase, Activity Assay, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: miR-193b-3p Promotes Proliferation of Goat Skeletal Muscle Satellite Cells through Activating IGF2BP1

doi: 10.3390/ijms232415760

Figure Lengend Snippet: IGF2BP1 promotes myoblast proliferation. ( A ) Levels of IGF2BP1 are successfully disturbed. mRNA (down panel) and protein (upper panel) levels of IGF2BP1 were measured in MuSCs 48 h post-transfection of pIGF2BP1 (3 μg/mL) or siIGF2BP1 (100 nM). ( B , C ) Effect of IGF2BP1 on myogenic gene RNA and protein levels. ( D ) IGF2BP1 elevates cell proliferation. Representative immunofluorescence images of EdU staining cells treated with pCtrl and pIGF2BP1 (3 μg/mL, red) or siCtrl and siIGF2BP1 (100 nM, green), cultured in GM consisting of 10 μM EdU (red or green) for 48 h, stained with anti-DAPI (blue). Scale bar = 100 μm. The fold change of EdU + cells is evaluated using randomly selected fields and normalized to control. ( E , F ) Cell cycle is affected by IGF2BP1 alteration. Flow cytometric assay is performed for cells treated with pIGF2BP1 or pCtrl ( n = 3). ( G ) Interfering IGF2BP1 retards miR-193b-3p-induced myogenic proliferation. Total RNA was extracted in cells cotransfected with miR-193b-3p mimic (mi-193b-3p, 50 nM) and interfering RNA against IGF2BP1 (siIGF2BP1, 100 nM) for 48 h in growth media (GM) or differentiated for 48 h (DM). RNA levels of target genes were quantified via RT-qPCR and calculated using the 2 −ΔΔCt . ( H ) Representative immunofluorescence images of EdU and DAPI (blue) staining cells post-transfection for 24, 48, and 72 h. Scale bar = 100 μm. The fold change of EdU + cells is evaluated using randomly selected fields and normalized to control. Data are shown as mean ± MSE. An unpaired two-tailed t -test is used to evaluate the means difference. *** p < 0.001, ** p < 0.01, and * p < 0.05.

Article Snippet: To detect the spatial localization of IGF2BP1 and miR-193b-3p RNA, the Cy5- labeled fluorescence oligonucleotide probes, complementary to goat IGF2BP1 mRNA (5′-GCCGGATTTGACCAAGAACTGGCCGCTGTAGGA-3′, red), and FAM-labeled chi-miR-193b-3p probes (5′-AGCGGGACTTTGTGGGCCAGTT-3′, green) were synthesized (Servicebio, Wuhan, China).

Techniques: Transfection, Immunofluorescence, Staining, Cell Culture, Control, Flow Cytometry, Quantitative RT-PCR, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: miR-193b-3p Promotes Proliferation of Goat Skeletal Muscle Satellite Cells through Activating IGF2BP1

doi: 10.3390/ijms232415760

Figure Lengend Snippet: miR-193b-3p activates IGF2BP1 transcriptionally as an enhancer-related miRNA. ( A ) The overlapped signals of miR-193b-3p and IGF2BP1 mRNA. The Cy5-labeled probes target goat IGF2BP1 mRNA (red), and FAM-labeled chi-miR-193b-3p probes (green) are synthesized to localize them in MuSCs. The nucleus is stained with anti-DAPI (blue). Scale bar = 50 μm. ( B ) miR-193b-3p destabilizes IGF2BP1 mRNA. ( C ) miR-193b-3p distributes in both cytoplasm and nucleus. N, Nucleus; C, Cytoplasm. ( D ) miR-193b-3p promotes levels of IGF2BP1 hnRNA. ( E ) Human miR-193b (hsa-mir-193b) is located in the enhancer region. The upper panel, hsa-mir-193b sits in the peak and valley of H3K4Me1/H3K4me3/ H3K27ac derived from 7 cell lines in ENCODE UCSC. Lower panel, the sequence of goat pri-miR-193b and pre-miR-193b-5p and 3p. ( F ) Pre-miR-193b-3p induces gene expression. Promoter vectors including pre-193b-3p, pre-193b-5p as well as the blank (pBasic) and control (pCtrl) (1.28 μg/mL) were transfected into MuSC and HeLa, and F-Luc/R-Luc levels were measured 48 h post transection. ( G ) Neighboring genes of miR-193b-3p, including MKL2, BFAR, and PARN, are the same in the goat and mouse genome. ( H ) Goat miR-193b induces the expression of its neighboring genes. ( I ) Pri-miR-193b activates the abundance of its neighboring genes and IGF2BP1. ( J ) Goat miR-193b-3p is unable to induce miR-365-3p. Left panel, Disturbing miR-193b-3p in goat MuSCs fails to elevate transcripts of miR-365 (right panel). Right panel, miR193b (red box) and miR365a (green box) in humans, mouse, and bovine are neighboring, while in goats they are distributed on different chromosomes. An arrow marks the transcriptional direction. Data are shown as mean ± MSE. An unpaired two-tailed t -test is used to evaluate the means difference. *** p < 0.001, ** p < 0.01, * p < 0.05, and § p < 0.1.

Article Snippet: To detect the spatial localization of IGF2BP1 and miR-193b-3p RNA, the Cy5- labeled fluorescence oligonucleotide probes, complementary to goat IGF2BP1 mRNA (5′-GCCGGATTTGACCAAGAACTGGCCGCTGTAGGA-3′, red), and FAM-labeled chi-miR-193b-3p probes (5′-AGCGGGACTTTGTGGGCCAGTT-3′, green) were synthesized (Servicebio, Wuhan, China).

Techniques: Labeling, Synthesized, Staining, Derivative Assay, Sequencing, Gene Expression, Control, Transfection, Expressing, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: miR-193b-3p Promotes Proliferation of Goat Skeletal Muscle Satellite Cells through Activating IGF2BP1

doi: 10.3390/ijms232415760

Figure Lengend Snippet: miR-193b-3p negatively regulates mouse IGF2BP1 and suppresses C2C12 proliferation. ( A , B ) miR-193b-3p decreases transcripts of IGF2BP1 ( A ) but barely affects the abundance of its neighboring genes in C2C12 ( B ). ( C ) miR-193b-3p deduces C2C12 proliferation. Left panel, Representative immunofluorescence images of EdU staining cells in miR-193b-3p-disturbed C2C12 (mi-Ctrl and mi-193b-3p, in-Ctrl and in-193b-3p, 50 nM). Scale bar = 100 μm. Right panel, Fold change of EdU + cells (ratio of EdU + myoblasts to all) are evaluated using randomly selected fields and normalized to control. Each treatment is repeated ten times. ( D ) Cell cycle is affected by miR-193b-3p mimic. Flow cytometric assay is performed for cells treated with mi-193b-3p or mi-Ctrl ( n = 3). Data are shown as mean ± MSE. ** p < 0.01, * p < 0.05, and § p < 0.1.

Article Snippet: To detect the spatial localization of IGF2BP1 and miR-193b-3p RNA, the Cy5- labeled fluorescence oligonucleotide probes, complementary to goat IGF2BP1 mRNA (5′-GCCGGATTTGACCAAGAACTGGCCGCTGTAGGA-3′, red), and FAM-labeled chi-miR-193b-3p probes (5′-AGCGGGACTTTGTGGGCCAGTT-3′, green) were synthesized (Servicebio, Wuhan, China).

Techniques: Immunofluorescence, Staining, Control, Flow Cytometry