Journal: Journal of Sport and Health Science

Article Title: Exercise training-induced extracellular miR-136-3p modulates glucose uptake and myogenesis through targeting of NRDC in human skeletal muscle

doi: 10.1016/j.jshs.2025.101091

Figure Lengend Snippet: MiR-136-3p content in cell culture medium and uptake of extracellular miR-136-3p into cultured myotubes. MiRNA content in (A) human myotubes and (B) human pancreatic islets culture media. Results were first normalized using RNU1A1 and then presented in relation to miR-23a-3p content for n = 4 different donors for myotubes cultures and n = 4 donors for human islets. Left panel (C) shows bright-field image of cultured human myotubes and right panel (C) shows a representative fluorescence image of cultured human myotubes with cells exposed to human serum-derived EVs loaded with Cy3-miR-136-3p. Cy3 fluorescence (red) is detected in the whole cytoplasm of the human myotubes. (D) Representative fluorescence image of human myotubes exposed to HEK293 culture medium with EVs loaded with Cy3-miR-136-3p (red). (E) Representative image of human myotubes exposed to EVs loaded with TexasRed-labeled with a control RNA (orange). Nuclear Hoechst staining is shown in blue. Scale bar = 100 μm. EVs = extracellular vesicles; HEK293 = human embryonic kidney; miR = microRNA; Rel = relative; RNU1A1 = U1 small nuclear RNA.

Article Snippet: Human embryonic kidney (HEK293) cells were obtained from American Type Culture Collection (ATCC) and cultured in high-glucose (4.5 g/L) Dulbecco's Modified Eagle Medium (DMEM; Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 10% (vol/vol) FBS.

Techniques: Cell Culture, Fluorescence, Derivative Assay, Labeling, Control, Staining

Journal: Journal of Sport and Health Science

Article Title: Exercise training-induced extracellular miR-136-3p modulates glucose uptake and myogenesis through targeting of NRDC in human skeletal muscle

doi: 10.1016/j.jshs.2025.101091

Figure Lengend Snippet: NRDC is a direct target of miR-136-3p in human myotubes. Skeletal muscle NRDC mRNA is responsive to training and inactivity. (A) Tissue mRNA expression of NRDC from the Human Protein Atlas database showing enriched expression of NRDC in human skeletal muscle. (B) The miR-136-3p target site in the NRDC gene is highly conserved in mammals. (C) Luciferase activity in HEK293 cells co-transfected the NRDC 3’UTR and miR-136-3p with or without anti-miR136-3p inhibitors. miR-136-3p transfection downregulates NRDC (D) mRNA and (E) representative image of protein abundance in human myotubes. (F) Publicly available data ( GSE14413 ) showing NRDC mRNA expression in human skeletal muscle of healthy young participants after 6 weeks of endurance training ( n = 8). (G) Publicly available data ( GSE120862 ) showing NRDC mRNA expression in human skeletal muscle of healthy young participants after 2 months of aerobic training ( n = 10). (H) Publicly available data ( GSE14901 ) showing NRDC mRNA expression in human skeletal muscle of healthy young participants after 14 days of immobilization ( n = 24). * p < 0.05, ** p < 0.005. GSE = gene set enrichment; HEK293 = human embryonic kidney; miR = microRNA; NC = negative control; NRDC = nardilysin convertase; nTPM = normalized transcripts per million; si NRDC = small interfering RNA of NRDC ; UTR = untranslated region.

Article Snippet: Human embryonic kidney (HEK293) cells were obtained from American Type Culture Collection (ATCC) and cultured in high-glucose (4.5 g/L) Dulbecco's Modified Eagle Medium (DMEM; Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 10% (vol/vol) FBS.

Techniques: Expressing, Luciferase, Activity Assay, Transfection, Quantitative Proteomics, Negative Control, Small Interfering RNA

Journal: Life Science Alliance

Article Title: The noncoding RNA GRAS1 interacts with NKAP and protects against DNA damage and cell death

doi: 10.26508/lsa.202403105

Figure Lengend Snippet: The cell line name is indicated on the left side of each expression track.

Article Snippet: Human lung adenocarcinoma A549 cell line (ATCC No. CCL-185), human colorectal carcinoma HCT116 cell line (ATCC No. CCL-247), and human kidney embryonic HEK293 cell line (ATCC No. CRL-1573) were purchased from ATCC.

Techniques: Expressing

Journal: Life Science Alliance

Article Title: The noncoding RNA GRAS1 interacts with NKAP and protects against DNA damage and cell death

doi: 10.26508/lsa.202403105

Figure Lengend Snippet: The cell line and replicate are indicated on the left side of each expression track.

Article Snippet: Human lung adenocarcinoma A549 cell line (ATCC No. CCL-185), human colorectal carcinoma HCT116 cell line (ATCC No. CCL-247), and human kidney embryonic HEK293 cell line (ATCC No. CRL-1573) were purchased from ATCC.

Techniques: Expressing

Journal: Life Science Alliance

Article Title: The noncoding RNA GRAS1 interacts with NKAP and protects against DNA damage and cell death

doi: 10.26508/lsa.202403105

Figure Lengend Snippet: (A) CCK8 assay for survival of A549, HCT116, and HEK293 cells transfected with control GapmeR compared with GRAS1 GapmeR1 or GRAS1 GapmeR2; N = 3. (B) CCK8 assay for survival of A549 and HCT116 cells transfected with empty vector compared with SNORA13 vector; N = 4. (C) CCK8 assay for survival of A549 and HCT116 cells transfected with empty vector compared with TIGA1 vector; N = 4. (D) Schematic gene structure of GRAS1 GapmeR–resistant construct. (E) CCK8 assay for survival of A549 and HCT116 GRAS1 knockdown cells complemented with GRAS1 GapmeR–resistant vector compared with empty vector; N = 4. (F) CCK8 assay for survival of A549 and HCT116 GRAS1 knockdown cells complemented with SNORA13 vector compared with empty vector; N = 4. (G) CCK8 assay for survival of A549 and HCT116 GRAS1 knockdown cells complemented with TIGA1 vector compared with empty vector; N = 4. Error bars represent SD in all panels; (ns) P > 0.05; (*) P ≤ 0.05; (**) P ≤ 0.01; (***) P < 0.001 by two-tailed t test.

Article Snippet: Human lung adenocarcinoma A549 cell line (ATCC No. CCL-185), human colorectal carcinoma HCT116 cell line (ATCC No. CCL-247), and human kidney embryonic HEK293 cell line (ATCC No. CRL-1573) were purchased from ATCC.

Techniques: CCK-8 Assay, Transfection, Control, Plasmid Preparation, Construct, Knockdown, Two Tailed Test

Journal: BMB Reports

Article Title: APC/C−CDH1 suppresses AROS-mediated protection against DNA damage-induced senescence by ubiquitination

doi: 10.5483/BMBRep.2025-0031

Figure Lengend Snippet: AROS interacts with APC2 of APC/C. (A) Schematic of human APC2. The Cullin homology domain is indicated. APC2 clone C1−41 (amino acids 468-822 of APC2) was isolated in a yeast two-hybrid screen. (B) Interaction of the AROS with APC2 analyzed by yeast two-hybrid assay using LexA DBD-fused AROS in pBTM116 and GAL4 AD-fused APC2 C1−41 in pGAD10. Error bars represent the mean ± standard deviation of three independent experiments. (C) HEK293 cells were lysed, and subjected to IP using anti-IgG or anti-AROS antibody. Western blotting was conducted using the indicated antibodies. (D) HEK293 cells were co-transfected with GFP−AROS and Flag-Empty or Flag–APC2. IP was performed using anti-Flag antibody, and bound proteins were analyzed by Western blotting with anti-GFP antibody. (E) For reverse IP, FLAG–APC2 was co-introduced with Myc–Empty or Myc–AROS into HEK293 cells. IP was performed using anti-Myc antibody, and bound proteins were detected by Western blotting with anti-Flag antibody.

Article Snippet: HEK293 cells (American Type Culture Collection [ATCC]) were cultured in Dulbecco’s modified Eagle’s medium.

Techniques: Isolation, Yeast, Two Hybrid Screening, Two Hybrid Assay, Standard Deviation, Western Blot, Transfection

Journal: BMB Reports

Article Title: APC/C−CDH1 suppresses AROS-mediated protection against DNA damage-induced senescence by ubiquitination

doi: 10.5483/BMBRep.2025-0031

Figure Lengend Snippet: AROS competes with APC11 for APC2 binding and inhibits APC/C−CDH1-mediated Cyclin B1 degradation. (A, B) AROS competes with APC11 for APC2 binding. (A) In vitro competition assays. GST or GST−APC11 and in vitro -translated Flag–APC2 were used in a GST pull-down assay with increasing amounts of in vitro -translated Myc–AROS. Bound APC2 was detected by Western blotting, using anti-Flag antibody. The decreasing APC2 binding to APC11 was quantified using ImageJ software. Error bars represent the mean ± SD of three independent experiments (*P < 0.05, **P < 0.01). (B) In vivo competition assays in which HEK293 cells were transfected with GFP−APC11, Flag–APC2, and increasing amounts of Myc–AROS. IP was performed using anti-Flag antibody, and bound APC11 was detected by Western blotting with anti-GFP antibody. The decreasing APC11 binding to APC2 was quantified using ImageJ software. Error bars represent the mean ± SD of three independent experiments (*P < 0.05). (C, D) AROS suppresses APC/C−CDH1-mediated Cyclin B1 degradation by competing with APC11. (C) Effects of AROS overexpression on APC/C−CDH1-mediated Cyclin Bl degradation. HEK293 cells co-expressing MyC−CDH1 with Flag-AROS were analyzed by Western blotting with the indicated antibodies. The level of Cyclin B1 was quantified using ImageJ software. Error bars represent the mean ± SD of three independent experiments (*P < 0.05, **P < 0.01). (D) Effect of AROS knockdown on APC/C−CDH1-mediated Cyclin Bl degradation. HEK293 cells were co-transfected with MyC−CDH1 and sh–AROS#1 or sh–AROS#2 (shRNA expression vector). Protein levels were assessed by Western blotting with the indicated antibodies. The level of Cyclin B1 was quantified using ImageJ software. Error bars represent the mean ± SD of three independent experiments (*P < 0.05).

Article Snippet: HEK293 cells (American Type Culture Collection [ATCC]) were cultured in Dulbecco’s modified Eagle’s medium.

Techniques: Binding Assay, In Vitro, GST Pulldown Assay, Western Blot, Software, In Vivo, Transfection, Over Expression, Expressing, Knockdown, shRNA, Plasmid Preparation

Journal: BMB Reports

Article Title: APC/C−CDH1 suppresses AROS-mediated protection against DNA damage-induced senescence by ubiquitination

doi: 10.5483/BMBRep.2025-0031

Figure Lengend Snippet: AROS is ubiquitinated and degraded by APC/C−CDH1. (A) CDH1 promotes AROS degradation. HEK293 cells were co-transfected with MyC−CDH1 or Myc–CDC20 with Flag–AROS. Cell lysates were analyzed by Western blotting with the indicated antibodies. (B) CDH1-mediated degradation of AROS was restored by treatment with the proteasome inhibitor MG132. (C) CHX chase assays. HEK293 cells were co-transfected with Flag–AROS and Myc–Empty or MyC−CDH1 for 12 h. Cells were then treated with CHX (50 μg/ml) for the indicated times, and Western blotting was performed with the indicated antibodies. Degradation of AROS was quantified using ImageJ software. Error bars represent the mean ± SD of three independent experiments (**P < 0.01, ***P < 0.001). (D) CDH1 knockdown increases AROS levels. HEK293 cells were transfected with sh–CDH1 or sh–Luciferase (Luc) as a control. After 48 h, cell lysates were subjected to Western blotting with the indicated antibodies. (E) AROS is ubiquitinated by CDH1. HEK293 cells were co-transfected under the indicated conditions for 24 h, followed by treatment with 10 μM MG132 for 12 h, then lysed, and subjected to IP using anti-Flag antibody. AROS ubiquitination was assessed by Western blotting with anti-Ub antibody. (F) In vitro ubiquitination of AROS by CDH1. In vitro -translated Flag–AROS and MyC−CDH1 were incubated with APC/C immunoprecipitated with anti-APC2 antibody using a ubiquitination kit. Ubiquitinated AROS was visualized by Western blotting with anti-poly–Ub antibody.

Article Snippet: HEK293 cells (American Type Culture Collection [ATCC]) were cultured in Dulbecco’s modified Eagle’s medium.

Techniques: Transfection, Western Blot, Software, Knockdown, Luciferase, Control, Ubiquitin Proteomics, In Vitro, Incubation, Immunoprecipitation

Journal: BMB Reports

Article Title: APC/C−CDH1 suppresses AROS-mediated protection against DNA damage-induced senescence by ubiquitination

doi: 10.5483/BMBRep.2025-0031

Figure Lengend Snippet: APC/C−CDH1-mediated ubiquitination of AROS requires the AROS D–box. (A) Alignment of the amino acid sequence of the D–box motifs of AROS. The regions corresponding to the putative D–boxes of AROS in various species are indicated. (B) Mutation of the M1 D–box of AROS results in a loss of CDH1-mediated degradation. HEK293 cells were co-transfected with the indicated constructs, and Western blotting was performed with the indicated antibodies. (C) CHX chase assays with AROS M1. HEK293 cells were co-transfected with individual constructs, and treated with CHX (50 μg/ml) for the indicated times. AROS turnover was analyzed by Western blotting with the indicated antibodies. Error bars represent the mean ± SD of three independent experiments (*P < 0.05). (D) Flag–AROS WT or Flag–AROS M1 was used in ubiquitination assay. AROS ubiquitination was measured by Western blotting with the indicated antibodies.

Article Snippet: HEK293 cells (American Type Culture Collection [ATCC]) were cultured in Dulbecco’s modified Eagle’s medium.

Techniques: Ubiquitin Proteomics, Sequencing, Mutagenesis, Transfection, Construct, Western Blot