Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Long Noncoding RNA Lnc-SEMT Modulates IGF2 Expression by Sponging miR-125b to Promote Sheep Muscle Development and Growth.

doi: 10.1159/000492979

Figure Lengend Snippet: Fig. 6. lnc-SEMT acts as a molecular sponge for miR-125b in vitro. (A) Potential binding sites in lnc-SEMT and miR-125b.Two seed sequence mutants of miR-125b (miR-125b-mut1 and miR-125b-mut2) are shown below. The underlined sequences represent the mutant site. (B) Luciferase reporter constructs: wild type lnc-SEMT and two lnc-SEMT mutants (lnc-SEMT-mut1 and lnc-SEMT-mut2) in the miR-125b binding site were inserted into psiCHECK-2 vector. The underlined sequences represent the mutant sites. (C) The relative luciferase activity of psiCHECK-2 containing WT lnc-SEMT co-transfected with AgomiR-125b or mutated AgomiR-125b. (D) The relative luciferase activity of psiCHECK-2 expressing WT or mutated lnc-SEMT co- transfected with AgomiR-125b. (E) Real-time PCR analysis of miR-125b and miR-125a expression in the different tissues of the sheep. (F) Real-time PCR analysis of miR-125b and miR-125a expression in muscle of the sheep.(G) The relative luciferase activity of psiCHECK-2 containing WT lnc-SEMT co-transfected with Antagomir-125b or mutated Antagomir-125b. (H) The relative luciferase activity of psiCHECK-2 expressing WT or mutated lnc-SEMT co-transfected with Antagomir-125b. (I) Ago2 immunoprecipitation was performed in sheep myoblasts transfected with control miRNA (miR-NC) or miR-125b, followed by real-time PCR to detect lnc-SEMT associated with Ago2. (J) Streptavidin capture was performed in sheep myoblasts transfected with biotin-miR-NC, biotin-miR-125b or mutated biotin-miR-125b, followed by real- time PCR to detect lnc-SEMT, IGF2 mRNA and control Gapdh mRNA abundances.

Article Snippet: Antibody signals were detected by Novex ® Chemiluminescent Substrates (Invitrogen, USA) Enzyme-linked immunosorbent assay for sheep Insulin-like growth factor 2 To quantify IGF2 in cell supernatants or serum, an enzyme-linked immunosorbent assay (ELISA) was performed in microtiter plates coated with antibody recognizing IGF2 (Cusabio, USA).

Techniques: In Vitro, Binding Assay, Sequencing, Mutagenesis, Luciferase, Construct, Plasmid Preparation, Activity Assay, Transfection, Expressing, Real-time Polymerase Chain Reaction, Immunoprecipitation, Control

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Long Noncoding RNA Lnc-SEMT Modulates IGF2 Expression by Sponging miR-125b to Promote Sheep Muscle Development and Growth.

doi: 10.1159/000492979

Figure Lengend Snippet: Fig. 7. (A) The relative luciferase activity of psiCHECK-2 expressing IGF2 3’UTR co-transfected with miR- 125b, miR-125b and lncRNA-SEMT, miR-125b and mutated lncRNA-SEMT respectively in sheep myoblasts. Mean values ± SEM, n=3, *P<0.05. (B) Streptavidin capture was performed in extracts of sheep myoblasts co-transfected with biotin-miR-NC or biotin-miR-125b and IGF2 3’UTR, IGF2 3’UTR together with lncRNA- SEMT or IGF2 3’UTR together with lncRNA-SEMT-mut, followed by Real-time PCR to detect IGF2 mRNA and control Gapdh mRNA abundances. Mean values ± SEM, n=3, *P<0.05.

Article Snippet: Antibody signals were detected by Novex ® Chemiluminescent Substrates (Invitrogen, USA) Enzyme-linked immunosorbent assay for sheep Insulin-like growth factor 2 To quantify IGF2 in cell supernatants or serum, an enzyme-linked immunosorbent assay (ELISA) was performed in microtiter plates coated with antibody recognizing IGF2 (Cusabio, USA).

Techniques: Luciferase, Activity Assay, Expressing, Transfection, Real-time Polymerase Chain Reaction, Control

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Long Noncoding RNA Lnc-SEMT Modulates IGF2 Expression by Sponging miR-125b to Promote Sheep Muscle Development and Growth.

doi: 10.1159/000492979

Figure Lengend Snippet: Fig. 8. lnc-SEMT regulates the expression of IGF2. (A) Western blot analysis of IGF2 protein abundances in sheep myoblasts transfected with control siRNA or lnc-SEMT siRNA. (B) Western blot analysis of IGF2 protein abundances in sheep myoblasts transfected with control vector or lnc-SEMT vector. (C) ELISA analysis of secreted IGF2 protein abundances in supernatant from sheep myoblasts transfected with control siRNA or lnc-SEMT siRNA. (D) ELISA analysis of secreted IGF2 protein levels in supernatant from sheep myoblasts transfected with control vector or lnc-SEMT vector. Mean values ± SEM, n=3, * P<0.05.(E)Real- time PCR analysis of IGF2 expression levels in sheep myoblasts transfected with control siRNA or IGF2 siRNA.Mean values ± SEM, n=5, * P<0.05. (F) Western blot analysis of IGF2 protein levels in sheep myoblasts transfected with control siRNA or IGF2 siRNA. (G)Real-time PCR analysis of expression of myogenic gene in sheep myoblasts transfected with control vector or lnc-SEMT vector or control vector with IGF2 siRNA or lnc-SEMT vector with IGF2 siRNA. (H) Real-time PCR analysis of miR-125b expression in longissimus dorsi musclefrom WT and TG sheep. Mean values ± SEM, n=5, * P<0.05. (I) Western blot analysis of IGF2 protein abundances in longissimus dorsimuscle from WT and TG sheep. (J) ELISA analysis of IGF2 protein abundances in serum from WT and TG sheep. Mean values ± SEM, n=5, * P<0.05.

Article Snippet: Antibody signals were detected by Novex ® Chemiluminescent Substrates (Invitrogen, USA) Enzyme-linked immunosorbent assay for sheep Insulin-like growth factor 2 To quantify IGF2 in cell supernatants or serum, an enzyme-linked immunosorbent assay (ELISA) was performed in microtiter plates coated with antibody recognizing IGF2 (Cusabio, USA).

Techniques: Expressing, Western Blot, Transfection, Control, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Reply to the Comment on "The m6A Reader IGF2BP2 Regulates Macrophage Phenotypic Activation and Inflammatory Diseases by Stabilizing TSC1 and PPARγ".

doi: 10.1002/advs.202201452

Figure Lengend Snippet: Figure 1. IGF2BP2 expression in LPS activated human macrophages. A) IGF2BP2 mRNA level in HMDMs treated by saline (Control) or LPS (100 ng mL−1) at indicated time. B) IGF2BP2 protein expression in HMDMs treated by normal saline or LPS (100 ng mL−1) for 4 and 8 h. C) Statistically analysis of (B). D) IGF2BP2 protein expression in HMDMs treated by saline or LPS (100 ng mL−1) for 24, 36, and 48 h. E) Statistically analysis of (D). F–I) Inflammatory cytokine and TSC1 mRNA level in HMDMs determined by RT-qPCR after normal saline or LPS treatment at indicated time points; qPCR biological replicates come from six different donors; western blot biological replicates come from three different donors; anti-IGF2BP2 antibody was purchased from Proteintech (11601-1-AP). The mRNA levels in the Control group were set to 1 and levels in others experimental conditions were relative to that after normalization with GAPDH. The dates were shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, versus the control group. p-values were determined by using two-way analysis of variance (ANOVA).

Article Snippet: F–I) Inflammatory cytokine and TSC1 mRNA level in HMDMs determined by RT-qPCR after normal saline or LPS treatment at indicated time points; qPCR biological replicates come from six different donors; western blot biological replicates come from three different donors; anti-IGF2BP2 antibody was purchased from Proteintech (11601-1-AP).

Techniques: Expressing, Saline, Control, Quantitative RT-PCR, Western Blot

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Reply to the Comment on "The m6A Reader IGF2BP2 Regulates Macrophage Phenotypic Activation and Inflammatory Diseases by Stabilizing TSC1 and PPARγ".

doi: 10.1002/advs.202201452

Figure Lengend Snippet: Figure 2. IGF2BP2 expression in LPS activated mouse macrophages. A) IGF2BP2 mRNA level in BMDMs treated by saline (Control) or LPS (100 ng mL−1) at the indicated time. B) IGF2BP2 protein expression in BMDMs treated by normal saline or LPS (100 ng mL−1) at the indicated time points, anti-IGF2BP2 antibody was purchased from Proteintech (11601- 1-AP); qPCR and western blot biological replicates from three different donors; IGF2BP2 mRNA levels in Control group were set to 1 and levels in others experimental conditions were relative to that after normalization with GAPDH. The dates were shown as mean ± SEM. *p < 0.05, **p < 0.01, versus the Control group. p-values were determined by using a two- way analysis of variance (ANOVA).

Article Snippet: F–I) Inflammatory cytokine and TSC1 mRNA level in HMDMs determined by RT-qPCR after normal saline or LPS treatment at indicated time points; qPCR biological replicates come from six different donors; western blot biological replicates come from three different donors; anti-IGF2BP2 antibody was purchased from Proteintech (11601-1-AP).

Techniques: Expressing, Saline, Control, Western Blot