Journal: International Journal of Biological Sciences

Article Title: miR-487b-3p Suppresses the Proliferation and Differentiation of Myoblasts by Targeting IRS1 in Skeletal Muscle Myogenesis

doi: 10.7150/ijbs.25052

Figure Lengend Snippet: Primer information for miRNA and mRNA quantitative reverse transcription

Article Snippet: Then, the membranes were blocked with 5% skim milk and subsequently incubated overnight at 4°C with primary antibodies against Myf5 (MW: 28 kDa; Abscience; USA; 1:1000 dilution), Mef2c (MW: 51 kDa; Abscience; USA; 1:1000 dilution), MyoG (MW: 25 kDa; Abscience; USA; 1:1000 dilution), MyoD (MW: 35 kDa; Abscience; USA; 1:1000 dilution), Pax7 (MW: 57 kDa; EnoGene; China; 1:1000 dilution), PCNA (MW: 36 kDa; EnoGene; China; 1:1000 dilution), IRS1 (MW:130 kDa; Abscience; USA; 1:1000 dilution) and β-tubulin (MW: 55 kDa; Abscience; USA; 1:1000 dilution).

Techniques: Sequencing

Journal: International Journal of Biological Sciences

Article Title: miR-487b-3p Suppresses the Proliferation and Differentiation of Myoblasts by Targeting IRS1 in Skeletal Muscle Myogenesis

doi: 10.7150/ijbs.25052

Figure Lengend Snippet: miR-487b-3p suppresses C2C12 myoblast proliferation. (A, B) MyoD, Pax7, and PCNA mRNA expression after transfection with miR-487b-3p mimics (Mimics), negative control (NC), 2'-O-methylated oligonucleotides against miR-487b-3p (Inhibitors), and single-stranded negative control (Anti-NC) in GM at 24 h and 48 h. (C-F) MyoD (35 kDa; 1:1000 dilution), Pax7 (57 kDa; 1:1000 dilution), and PCNA (36 kDa; 1:1000 dilution) protein expression after transfection with miR-487b-3p mimics, NC, Inhibitors and Anti-NC in GM at 24 h (C, D) and 48 h (E, F). β-tubulin (55 kDa; 1:1000 dilution) was used as an internal control. (G) CCK-8 assay of C2C12 myoblasts at 24 h and 48 h after transfection with miR-487b-3p Mimics, NC, miR-487b-3p Inhibitors and Anti-NC. (H) Representative images of EdU assay of C2C12 myoblasts at 24 h after transfection with Mimics, NC, Inhibitors, Anti-NC in GM. Bars, 100 μm. (I) Quantification of EdU-positive cells(n=6). The results are shown as the mean ± SEM of three independent replicates. One-way ANOVA and t-tests were used for statistical analysis. Asterisks indicate significant differences. * P <0.05, ** P <0.01.

Article Snippet: Then, the membranes were blocked with 5% skim milk and subsequently incubated overnight at 4°C with primary antibodies against Myf5 (MW: 28 kDa; Abscience; USA; 1:1000 dilution), Mef2c (MW: 51 kDa; Abscience; USA; 1:1000 dilution), MyoG (MW: 25 kDa; Abscience; USA; 1:1000 dilution), MyoD (MW: 35 kDa; Abscience; USA; 1:1000 dilution), Pax7 (MW: 57 kDa; EnoGene; China; 1:1000 dilution), PCNA (MW: 36 kDa; EnoGene; China; 1:1000 dilution), IRS1 (MW:130 kDa; Abscience; USA; 1:1000 dilution) and β-tubulin (MW: 55 kDa; Abscience; USA; 1:1000 dilution).

Techniques: Expressing, Transfection, Negative Control, Methylation, CCK-8 Assay, EdU Assay

Journal: International Journal of Biological Sciences

Article Title: miR-487b-3p Suppresses the Proliferation and Differentiation of Myoblasts by Targeting IRS1 in Skeletal Muscle Myogenesis

doi: 10.7150/ijbs.25052

Figure Lengend Snippet: Ectopic miR-487b-3p expression inhibits proliferation and differentiation in C2C12 myoblasts. (A) Myf5, MyoG, and Mef2c expression at D1, D3, D5, and D7 after transfection with pcDNA3.1 (+)-miR-487b-3p determined by RT-qPCR in DM. (B, C) Myf5 (28 kDa; 1:1000 dilution), MyoG (25 kDa; 1:1000 dilution) and Mef2c (51 kDa; 1:1000 dilution) protein levels were measured following overexpression of miR-487b-3p with pcDNA3.1(+)-miR-487b-3p in DM at D1, D3, D5 and D7. β-tubulin (55 kDa; 1:1000 dilution) was used as an internal control. (D) MyoD, Pax7, and PCNA mRNA levels after transfection with pcDNA3.1 (+)-miR-487b-3p in GM at 24 h and 48 h. (E) Relative MyoD, Pax7, and PCNA protein expression after transfection with pcDNA3.1 (+)-miR-487b-3p in GM at 24 h and 48 h. (F, G) MyoD (35 kDa; 1:1000 dilution), Pax7 (57 kDa; 1:1000 dilution), and PCNA (36 kDa; 1:1000 dilution) protein expression after transfection with pcDNA3.1 (+)-miR-487b-3p in GM at 24 h and 48 h. β-tubulin (55 kDa; 1:1000 dilution) was used as an internal control. (H) Representative images of EdU assays of C2C12 myoblasts at 24 h after transfection with pcDNA3.1 (+)-miR-487b-3p. Bars, 100 μm. (I) Quantification of EdU-positive cells (n=6). D1, 3, 5, and 7 indicate DM for 1, 3, 5, and 7 days, respectively. The results are shown as the mean ± SEM of three independent replicates. One-way ANOVA and t-tests were used for statistical analysis. Asterisks indicate significant differences. * P <0.05, ** P <0.01.

Article Snippet: Then, the membranes were blocked with 5% skim milk and subsequently incubated overnight at 4°C with primary antibodies against Myf5 (MW: 28 kDa; Abscience; USA; 1:1000 dilution), Mef2c (MW: 51 kDa; Abscience; USA; 1:1000 dilution), MyoG (MW: 25 kDa; Abscience; USA; 1:1000 dilution), MyoD (MW: 35 kDa; Abscience; USA; 1:1000 dilution), Pax7 (MW: 57 kDa; EnoGene; China; 1:1000 dilution), PCNA (MW: 36 kDa; EnoGene; China; 1:1000 dilution), IRS1 (MW:130 kDa; Abscience; USA; 1:1000 dilution) and β-tubulin (MW: 55 kDa; Abscience; USA; 1:1000 dilution).

Techniques: Expressing, Transfection, Quantitative RT-PCR, Over Expression

Journal: International Journal of Biological Sciences

Article Title: miR-487b-3p Suppresses the Proliferation and Differentiation of Myoblasts by Targeting IRS1 in Skeletal Muscle Myogenesis

doi: 10.7150/ijbs.25052

Figure Lengend Snippet: IRS1 knockdown inhibits proliferation and differentiation in cultured C2C12 myoblasts. (A) IRS1 expression was measured after transfection with siRNA control (si-NC), Lipo (lipofectamine 2000), si-IRS1-1, si-IRS1-2 and si-IRS1-3 in GM at 24 h. (B, C) IRS1 (130 kDa; 1:1000 dilution) protein expression was examined after transfection with siRNA control (si-NC), si-IRS1-3 in GM at 24 h, 48 h and 72 h. (D, E) MyoD (35 kDa; 1:1000 dilution), Pax7 (57 kDa; 1:1000 dilution), and PCNA (36 kDa; 1:1000 dilution) protein expression was examined after transfection with siRNA control (si-NC), si-IRS1-3 in GM at 24 h (D) and 48 h (E), respectively. (F, G) Myf5 (28 kDa; 1:1000 dilution), MyoG (25 kDa; 1:1000 dilution), and Mef2c (51 kDa; 1:1000 dilution) protein expression was examined after transfection with siRNA control (si-NC), si-IRS1-3 in DM at D2 (F) and D3 (G). β-tubulin (55 kDa; 1:1000 dilution) was used as an internal control. D2, 3 indicate DM for 2 and 3 days. (H) Representative images of EdU assays of C2C12 myoblasts at 24 h after transfection with siRNA control (si-NC), si-IRS1-3. Bars, 100 μm. (I) Quantification of EdU-positive cells (n=6). The results are shown as the mean ± SEM of three independent replicates. One-way ANOVA and t-tests were used for statistical analysis. Asterisks indicate significant differences. * P <0.05, ** P <0.01

Article Snippet: Then, the membranes were blocked with 5% skim milk and subsequently incubated overnight at 4°C with primary antibodies against Myf5 (MW: 28 kDa; Abscience; USA; 1:1000 dilution), Mef2c (MW: 51 kDa; Abscience; USA; 1:1000 dilution), MyoG (MW: 25 kDa; Abscience; USA; 1:1000 dilution), MyoD (MW: 35 kDa; Abscience; USA; 1:1000 dilution), Pax7 (MW: 57 kDa; EnoGene; China; 1:1000 dilution), PCNA (MW: 36 kDa; EnoGene; China; 1:1000 dilution), IRS1 (MW:130 kDa; Abscience; USA; 1:1000 dilution) and β-tubulin (MW: 55 kDa; Abscience; USA; 1:1000 dilution).

Techniques: Cell Culture, Expressing, Transfection

Journal: Cellular and Molecular Life Sciences

Article Title: Inhibition of the membrane repair protein annexin-A2 prevents tumor invasion and metastasis

doi: 10.1007/s00018-023-05049-3

Figure Lengend Snippet: Highly expressed in MDA-MB-231 cells, ANXA2 promotes membrane repair. A Representative image of western-blot analysis showing the revelation of ANXA2 in MDA-MB-231 and MCF7 cells, as compared to GAPDH (loading control). B The histogram presents mean values (± SEM) of the ratio ANX/GAPDH from five independent experiments, analyzed by the gel analysis plugging of ImageJ. A representative membrane of the detection of ANXA1 is presented in supplementary Fig. . Student t test for independent samples. ** p < 0.01. C ANXA2-deficient MDA-MB-231 cells were generated by shRNA transduction strategy. The cellular content of ANXA2 in MDA-MB-231 cells transduced with lentiviral particles containing shRNA targeting ANXA2 (shA2) or a scrambled shRNA (ctl) was quantified by Western blotting. D Control and shANXA2 MDA-MB-231 cells, which expressed constitutively the tdTomato fluorescent protein were imaged by fluorescence microscopy. Right-hand histograms display mean cell area (in pixels 2 ) and nuclei circumference (in pixels) measured by the imageJ software using Tomato and DAPI images. The mean values (+ / − SEM) were calculated from at least 30 cells from three independent experiments. No statistical difference (student t test) was observed for the two parameters. Scale bar: 20 µm. E , G Sequences of representative images showing the response of a control ( E ) or shANXA2 ( G ) MDA-MB-231 cell to a membrane damage performed by 110-mW infrared laser irradiation, in the presence of FM1-43 (green). In all figures, the area of membrane irradiation is marked with a red arrow before irradiation and a white arrow after irradiation. Scale bars: 10 μm. F Kinetic data represent the FM1 − 43 fluorescence intensity for control (black filled circles) or shANXA2 (empty circles) MDA-MB-231 cells, integrated over whole cell sections, averaged for about 30 cells (+ / − SEM). H Recruitment of ANXA2 to the site of membrane injury. MDA-MB-231 cells transfected with the plasmid pA2-GFP were damaged by laser ablation. Red arrow, area before irradiation; white arrow, area after irradiation. I Subcellular localization of endogenous ANXA2 in damaged MDA-MB-231. MDA-MB-231 cells were irradiated with a 110-mW infrared laser (white arrow) in DPBS + Ca 2+ , then fixed and immunostained for ANXA2 and counterstained with DAPI (blue). After laser injury, MDA-MB-231 cells exhibited an accumulation of ANXA2 at the disruption site. The inset displays a magnified image of the disruption site where concentrates ANXA2. Scale bars: 10 µm on the images and 1 µm within the inset

Article Snippet: Semi-dry electrophoretic transfer (Bio-Rad) onto PVDF membrane was performed for 1 h at 100 V. The cellular content in ANXA1 (37 kDa), ANXA2 (36 kDa), ANXA4 (35 kDa), ANXA5 (35 kDa), ANXA6 (68 kDa), and actin (42 kDa) or GAPDH (37 kDa) was detected with rabbit anti-ANXA1 polyclonal antibody (PA1006, BosterBio, Pleasanton, CA, USA), mouse anti-ANXA2 monoclonal antibody (3E8-B6, Sigma-Aldrich), mouse anti-ANXA4 monoclonal antibody (SAB4200121, Sigma-Aldrich), mouse anti-ANXA5 monoclonal antibody (AN5, Sigma-Aldrich), mouse anti-ANXA6 monoclonal antibody (sc-271859, Santa cruz Biotechnology, Dallas, USA), rabbit anti-actin polyclonal antibody (A2066, Sigma-Aldrich), and rabbit anti-GAPDH polyclonal antibody (G9545, Sigma-Aldrich), respectively.

Techniques: Membrane, Western Blot, Control, Generated, shRNA, Transduction, Fluorescence, Microscopy, Software, Irradiation, Transfection, Plasmid Preparation, Disruption

Journal: Molecular medicine reports

Article Title: Therapeutic delivery of cyclin-A2 via recombinant adeno-associated virus serotype 9 restarts the myocardial cell cycle: an in vitro study.

doi: 10.3892/mmr.2015.3147

Figure Lengend Snippet: Figure 1. Expression of cyclin‑A2 in the myocardium of the experimental and control groups. (A) Representative western blot analysis of cyclin‑A2 expression in the myocardium of the control group and the experi mental group at Tg-2w and Tg-4w. Expression levels were normalized to GAPDH. (B) Cyclin-A2 expression levels were significantly higher in the AAV9‑cyclin-A2 group than those of the control (saline) group and two and four weeks following injection. Values are expressed as the mean ± standard deviation. Saline, n=5 and AAV9‑cyclin‑A2, n=7. *P<0.0001 vs. saline group at two weeks. #P<0.001 vs. saline group at four weeks. Tg‑2w, two weeks following injection; Tg‑4w, four weeks following injection; AAV9, adeno‑associated virus serotype 9.

Article Snippet: Rabbit anti-human GAPDH (Cell Signaling Technology, Inc.; 1:1,000; 36 KDa) was used as reference.

Techniques: Expressing, Control, Western Blot, Saline, Injection, Standard Deviation, Virus

Journal: Molecular medicine reports

Article Title: Therapeutic delivery of cyclin-A2 via recombinant adeno-associated virus serotype 9 restarts the myocardial cell cycle: an in vitro study.

doi: 10.3892/mmr.2015.3147

Figure Lengend Snippet: Figure 5. Expression levels of PCNA and H3P detected by western blot analysis. (A) PCNA expression levels displayed a significant difference between the control and experimental groups, two and four weeks following injection. *P<0.0001, #P<0.0001, AAV9‑cyclin‑A2 vs. saline group. (B) There was no significant difference in H3P expression levels between the two groups, P>0.05. (control group, n=5; experimental group, n=7). Values are expressed as the mean ± standard deviation. (C) Representative western blot of control group and experimental group at Tg-2w and Tg-4w. Size of proteins: PCNA, 36KD; H3P, 17 KDa; GAPDH, 36 KDa. Tg-2w, two weeks following injection; Tg-4w, four weeks following injection. PCNA, proliferating cell nuclear antigen; H3P, phospho‑histone H3; AAV9, adeno‑associated virus serotype 9.

Article Snippet: Rabbit anti-human GAPDH (Cell Signaling Technology, Inc.; 1:1,000; 36 KDa) was used as reference.

Techniques: Expressing, Western Blot, Control, Injection, Saline, Standard Deviation, Virus

Journal: Acta Pharmaceutica Sinica. B

Article Title: The substitution of SERCA2 redox cysteine 674 promotes pulmonary vascular remodeling by activating IRE1 α /XBP1s pathway

doi: 10.1016/j.apsb.2021.12.025

Figure Lengend Snippet: Upregulation of XBP1s promotes SKI PASMC proliferation. (A) Xbp1 mRNA in PASMCs. n = 5–9. (B) Representative images of XBP1s immunofluorescence staining (red) in PASMCs and quantitative analysis of fluorescence density in graph. Nuclei are indicated by DAPI (blue). n = 5. Scale bar: 50 μm. (C) Representative images of XBP1s immunofluorescence staining (green) in pulmonary vessels indicated by α -SMA staining (red), and quantitative analysis of fluorescence density in graph. n = 5. The white arrow indicates that XBP1s is mainly expressed in the remodeled pulmonary vessels. Scale bar: 50 μm. (A)‒(C), ∗ P < 0.05, SKI vs. WT, unpaired t -test. (D) Representative Western blot of WT PASMCs overexpressed Xbp1s plasmid or vector control (Ctrl), and quantification of band intensities in graph. ∗ P < 0.05, Xbp1s vs. Ctrl, unpaired t -test, n = 3–7. (E) The relative percentage of XBP1s (red) or Ki67-positive (green) cells in WT PASMCs overexpressed Xbp1s plasmid or vector control (Ctrl). ∗ P < 0.05, Xbp1s vs. Ctrl, unpaired t -test, n = 5. Scale bar: 100 μm. (F) Cell proliferation in WT PASMCs. ∗ P < 0.05, Xbp1s vs. Ctrl, unpaired t -test, n = 5. Data were presented as mean ± SEM.

Article Snippet: Immunoblotted PVDF membrane at 4 °C overnight with specific antibodies against the following proteins: SERCA2 (C498 antibody was a customized polyclonal antibody from Bethyl Laboratories, Inc.; 110 kDa), SERCA2 C674-SO 3 H (Cat# A300-BL2103; Bethyl Laboratories, Inc.; 110 kDa), phosphorylated IRE1 α (p-IRE1 α ; Cat# GTX132808; Genetex, TX, USA; 133 kDa), IRE1 α (Cat# 3294; CST, Danvers, MA, USA; 130 kDa), binding immunoglobulin protein (BIP; Cat# E90019; EnoGene; 78 kDa), activating transcription factor 6 (ATF6; Cat# E90009A; EnoGene; 75 kDa), unspliced XBP1 (XBP1u; 25997-1-AP; Proteintech; 36 kDa), XBP1s (Cat# 658802; Biolegend, San Diego, CA, USA; 55 kDa), cyclin A1 (55 kDa), cyclin A2 (50 kDa), cyclin B1 (60 kDa), cyclin D1 (Cat# 60186-1-Ig; Proteintech; 34 kDa), cyclin dependent kinase 1 (CDK1; Cat# AP16160b; abcepta; 35 kDa), CDK2 (Cat# AP7518d; abcepta; 34 kDa), β -actin (Cat# E12-051-3; EnoGene; 42 kDa), followed by incubation with HRP-conjugated goat-anti-rabbit secondary antibody (Cat# SSA003; Sino Biological Inc., Beijing, China) 1 h at room temperature.

Techniques: Immunofluorescence, Staining, Fluorescence, Western Blot, Plasmid Preparation, Control

Journal: Acta Pharmaceutica Sinica. B

Article Title: The substitution of SERCA2 redox cysteine 674 promotes pulmonary vascular remodeling by activating IRE1 α /XBP1s pathway

doi: 10.1016/j.apsb.2021.12.025

Figure Lengend Snippet: Inhibition of IRE1 α /XBP1s pathway prevents SKI pulmonary vascular remodeling. (A) Summary of the severity of vascular remodeling in PAs and veins. (B) The protein expression of XBP1 and cell cycle related proteins in lungs. ∗ P < 0.05, SKI treated with solvent control (SKI/Ctrl) vs. WT treated with solvent control (WT/Ctrl), # P < 0.05, SKI treated with 4 μ 8C (SKI/4 μ 8C) vs. SKI/Ctrl. ANOVA with Bonferroni correction, n = 9. The WT/Ctrl group ( n = 9) includes the data from Fig. S8 ( n = 5). Data were presented as mean ± SEM.

Article Snippet: Immunoblotted PVDF membrane at 4 °C overnight with specific antibodies against the following proteins: SERCA2 (C498 antibody was a customized polyclonal antibody from Bethyl Laboratories, Inc.; 110 kDa), SERCA2 C674-SO 3 H (Cat# A300-BL2103; Bethyl Laboratories, Inc.; 110 kDa), phosphorylated IRE1 α (p-IRE1 α ; Cat# GTX132808; Genetex, TX, USA; 133 kDa), IRE1 α (Cat# 3294; CST, Danvers, MA, USA; 130 kDa), binding immunoglobulin protein (BIP; Cat# E90019; EnoGene; 78 kDa), activating transcription factor 6 (ATF6; Cat# E90009A; EnoGene; 75 kDa), unspliced XBP1 (XBP1u; 25997-1-AP; Proteintech; 36 kDa), XBP1s (Cat# 658802; Biolegend, San Diego, CA, USA; 55 kDa), cyclin A1 (55 kDa), cyclin A2 (50 kDa), cyclin B1 (60 kDa), cyclin D1 (Cat# 60186-1-Ig; Proteintech; 34 kDa), cyclin dependent kinase 1 (CDK1; Cat# AP16160b; abcepta; 35 kDa), CDK2 (Cat# AP7518d; abcepta; 34 kDa), β -actin (Cat# E12-051-3; EnoGene; 42 kDa), followed by incubation with HRP-conjugated goat-anti-rabbit secondary antibody (Cat# SSA003; Sino Biological Inc., Beijing, China) 1 h at room temperature.

Techniques: Inhibition, Expressing, Solvent, Control