Journal: Frontiers in Physiology

Article Title: Let-7b Regulates Myoblast Proliferation by Inhibiting IGF2BP3 Expression in Dwarf and Normal Chicken

doi: 10.3389/fphys.2017.00477

Figure Lengend Snippet: Let-7b has an enhanced inhibitory effect on IGF2BP3 expression in dwarf myoblast than in normal myoblast. (A) Desmin immunostaining of primary myoblast. (B) Relative let-7b expression in normal and dwarf myoblasts after transfection of let-7b mimic or NC mimic. (C) Relative mRNA expression after transfection of let-7b mimic or NC mimic in myoblast isolated from normal chicken. (D) Relative mRNA expression after transfection of let-7b mimic or NC mimic in myoblast isolated from dwarf chicken. (E) Relative IGF2BP3 protein expression in normal and dwarf myoblast after transfection of let-7b mimic or NC mimic. (F) ELISA analyzes of IGF2 protein expression in normal and dwarf myoblast after transfection of let-7b mimic or NC mimic. (G) Relative mRNA expression after transfection of let-7b inhibitor or NC inhibitor in myoblast isolated from normal chicken. (H) Relative mRNA expression after transfection of let-7b inhibitor or NC inhibitor in myoblast isolated from dwarf chicken. (I) Relative IGF2BP3 protein expression in normal and dwarf myoblast after transfection of let-7b inhibitor or NC inhibitor. (J) Relative luciferase activity of dwarf and normal myoblasts transfected with let-7b mimics and pmirGLO-IGF2BP3-3′UTR. Data were displayed as normalized fold change in relative luciferase activity (Firefly luciferase/Renilla luciferase, relative value of NC group in normal myoblast was set as 1). The data in (B–D,F–H,J) are mean ± S.E.M. with three cultures per group, and three wells per culture were assayed ( n = 9/treatment group). The data in (E,I) are mean ± S.E.M. from three independent experiments done in duplicate ( n = 6/treatment group). For (B–I) , independent sample t -test was used to analyze the statistical differences between groups. * p < 0.05; ** p < 0.01. For (F,J) , different letters above the bars indicate significant differences ( p < 0.05) by using the Duncan's Multiple Range Test, at the p < 0.05 significance level.

Article Snippet: The levels of IGF2 were determined using chicken IGF2 ELISA kit (CUSABIO BIOTECH Co. Ltd. China), following the manufacturer's instructions.

Techniques: Expressing, Immunostaining, Transfection, Isolation, Enzyme-linked Immunosorbent Assay, Luciferase, Activity Assay

Journal: Frontiers in Physiology

Article Title: Let-7b Regulates Myoblast Proliferation by Inhibiting IGF2BP3 Expression in Dwarf and Normal Chicken

doi: 10.3389/fphys.2017.00477

Figure Lengend Snippet: Schematic illustration for signaling pathways of skeletal muscle growth regulated by let-7b. let-7b can inhibit skeletal muscle development through let-7b-IGF2BP3-IGF2 signaling pathway and let-7b-GHR-GHR downstream genes pathway in normal chicken. However, the large deletion mutation at the exon 10 and 3′UTR of GHR gene in dwarf chicken lead to disruption of let-7b binding site in GHR 3′UTR. In this case, let-7b would enhance its inhibition on IGF2BP3 expression. Therefore, let-7b inhibits skeletal muscle development only through let-7b-IGF2BP3-IGF2 signaling pathway with enhancing effect in dwarf chicken.

Article Snippet: The levels of IGF2 were determined using chicken IGF2 ELISA kit (CUSABIO BIOTECH Co. Ltd. China), following the manufacturer's instructions.

Techniques: Protein-Protein interactions, Mutagenesis, Disruption, Binding Assay, Inhibition, Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Activation of Proneuronal Transcription Factor Ascl1 in Maternal Liver Ensures a Healthy Pregnancy

doi: 10.1016/j.jcmgh.2021.08.009

Figure Lengend Snippet: Igf2 activation in maternal livers deficient for Ascl1 . Maternal livers were collected and weighed from nonpregnant (NP) and GD15 and GD18 Ascl1 fl / fl and hep- Ascl1 -/- mice. ( A ) Hepatic Igf2 mRNA levels were measured using qRT-PCR and presented as the mean fold changes relative to NP controls ± SD (n = 4–5). ( B ) Western blot was performed using liver lysates with antibodies against the proteins indicated. ( C ) Igf2 in situ hybridization on liver sections. ( D ) IGF2 immunostaining. ( E ) Levels of hepatic Igf2 promoter-specific transcript variants were measured using qRT-PCR and presented as the mean fold changes relative to NP controls ± SD (n = 4–5). ( F ) Levels of IGF2 protein in serum were measured using enzyme-linked immunosorbent assay and presented as the mean fold changes ± SD (n = 3–5). ∗∗ P < .01, ∗∗∗ P < .001, and ∗∗∗∗ P < .0001. P0, placental-specific Igf2 promoter; P1-3, placental- and fetal liver-specific Igf2 promoter. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Serum IGF2 levels were quantified using a one-sixth dilution with the Mouse IGF2 ELISA Kit (EK0381; Boster Biological Technology, Pleasanton, CA) as per the manufacturer’s directions and read with the SpectraMax M2e spectrophotometer (Molecular Devices, San Jose, CA) using the SoftMax Pro 6 program (Molecular Devices).

Techniques: Activation Assay, Quantitative RT-PCR, Western Blot, In Situ Hybridization, Immunostaining, Enzyme-linked Immunosorbent Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Activation of Proneuronal Transcription Factor Ascl1 in Maternal Liver Ensures a Healthy Pregnancy

doi: 10.1016/j.jcmgh.2021.08.009

Figure Lengend Snippet: Primers for Promoter-Specific Igf2 Transcripts

Article Snippet: Serum IGF2 levels were quantified using a one-sixth dilution with the Mouse IGF2 ELISA Kit (EK0381; Boster Biological Technology, Pleasanton, CA) as per the manufacturer’s directions and read with the SpectraMax M2e spectrophotometer (Molecular Devices, San Jose, CA) using the SoftMax Pro 6 program (Molecular Devices).

Techniques: Sequencing

Journal: International journal of molecular sciences

Article Title: SNHG15-Mediated Localization of Nucleolin at the Cell Protrusions Regulates CDH2 mRNA Expression and Cell Invasion.

doi: 10.3390/ijms242115600

Figure Lengend Snippet: Figure 2. IMP1 regulates the localization of SNHG15 at the cell protrusions. (A) FISH was per- formed to detect the subcellular localization of SNHG15 in WT BT-549 cells (upper panel) and SNHG15-expressing BT-549 cells (lower panel). The arrowheads indicate localized SNHG15 at the cell protrusions. Scale bar: 10 µm. (B) FISH indicated that, in comparison with WT MDA-MB-231 cells (upper panel), IMP1 expression greatly increased the localization of SHNG15 at cell protrusions (lower panel). Scale bar: 10 µm. (C) A bar graph indicates the percentage of protrusion-localized SNHG15 in tested cells. Localization was increased to 70% from 40% when ectopic IMP1 was ex- pressed. About 80–100 cells were counted in each group. ** p < 0.01. WT: cell origin. IMP1: cells expressing ectopic IMP1. Control: cells transfected with an empty plasmid. (D) RT-qPCR showed that IMP1 expression does not affect cellular levels of SNHG15.

Article Snippet: Primary antibodies against IMP1, GAPDH and nucleolin (NCL) were purchased from Cell Signaling Co. (Danvers, MA, USA); CDH2 and CDH1 antibodies were purchased from Boster Biological Technology Co. (Wuhan, China).

Techniques: Expressing, Comparison, Control, Transfection, Plasmid Preparation, Quantitative RT-PCR

Journal: International journal of molecular sciences

Article Title: SNHG15-Mediated Localization of Nucleolin at the Cell Protrusions Regulates CDH2 mRNA Expression and Cell Invasion.

doi: 10.3390/ijms242115600

Figure Lengend Snippet: Figure 3. Localization of SNHG15 at the cell protrusions increases cell invasive potential. (A) The full-length SNHG15 and three dissected fragments of SNHG15 are separately tagged with six MS2 repeats. The relative position of the truncated SNHG15 is shown. The black box indicates the “ACACCC” motif for IMP1 binding. (B) FISH experiments showed that deletion of the “ACACCC” motif (Mut SNHG15) prevented SNHG15 from localizing at the cell protrusions. The arrowhead indicates cell-protrusion-localized SNHG15. Scale bar: 10 µm. (C) A bar graph shows that the cell population with protrusion-localized SNHG15 was largely decreased when the “ACACCC” motif was absent. ** p < 0.01. (D) Localization of the full-length and three truncated fragments of SNHG15 was determined by FISH. Results indicated that T1 and T3, but not T2, were still able to localize at the cell protrusions. About 80–100 cells were counted in each group. ** p < 0.01. (E) Transwell assays showed that, compared to the cells expressing full-length SNHG15, all three stable cell lines expressing truncated SNHG15 displayed lower invasive potentials. ** p < 0.01.

Article Snippet: Primary antibodies against IMP1, GAPDH and nucleolin (NCL) were purchased from Cell Signaling Co. (Danvers, MA, USA); CDH2 and CDH1 antibodies were purchased from Boster Biological Technology Co. (Wuhan, China).

Techniques: Binding Assay, Expressing, Stable Transfection