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: Nature Communications

Article Title: Multimodal substrate recruitment enables CTLH MKLN1 E3 ligase to target acetylated N-, C-, and internal degrons

doi: 10.1038/s41467-026-78311-4

Figure Lengend Snippet: a Cartoon depicting the E3-MKLN1–Sub-MKLN1 tetrameric assembly, highlighting the discoidin and Kelch domains, and Asn447 within Kelch domain Loop-1 (starred), which is central to the E3-MKLN1–Sub-MKLN1 interface. b Cartoon depicting the Kelch domain central channel, which recognizes the C-terminal Arg of ZMYND19 and FAM72A, highlighting contact residues Gly273, Trp291, and Tyr346 (starred). c In vitro ubiquitylation assay comparing CTLH MKLN1 WT and the N447R mutant toward MKLN1, ZMYND19, and FAM72A-UNG2, n = 3 technical replicates. d Immunoblot analysis of MKLN1 levels in MKLN1-deficient cells transiently expressing WT, W291A/G273W, or N447R 3×Flag-MKLN1 , monitoring Torin1-dependent degradation after 16 h, Torin1 + MG132 (16 h) co-treatment, or matched DMSO controls. Actin serves as a loading control. e Quantification of MKLN1 immunoblots from ( d ). Data from each well were normalized to actin, and then each set expressing a particular 3×Flag-MKLN1 construct was normalized to the DMSO control for that construct. The graph shows results of mean ± s.d. of n = 4 biological replicates. P -values were calculated using two-way ANOVA with Dunnett’s multiple comparison tests ( p -value significance <0.05). f Top: sequence alignment of MKLN1, ZMYND19, and FAM72A C-termini. Bottom: superposition of cryo-EM structures of FAM72A (blue) and ZMYND19 (magenta) C-termini bound within the E3-MKLN1 Kelch domain central channel, highlighting their similar contacts between MKLN1 Gly273, Trp291, Tyr346, Arg407, and Arg517, and the terminal Arg residues of FAM72A (Arg149) and ZMYND19 (Arg227). g In vitro ubiquitylation assay examining effect of W291A/G273W Kelch central channel mutant on CTLH MKLN1 activity toward MKLN1, ZMYND19 and FAM72A-UNG2, n = 3 technical replicates. h Co-immunoprecipitation (using anti-RANBP9 antibody) in MKLN1- and ZMYND19-deficient HEK293 cells transiently expressing HA-ZMYND19 and 3×Flag-MKLN1 (WT or indicated N447R, or W291A/G273W mutants), assessing the role of Kelch channel surfaces on ZMYND19 association with the CTLH complex. Immunoprecipitates and inputs are shown. Actin serves as a loading control ( n = 2 biological replicates). i Immunoblot analysis of UNG and FAM72A levels in MKLN1 −/− knockout HEK293 cells transiently co-transfected with constructs expressing FAM72A and 3×Flag-MKLN1 WT or indicated mutant versions (W291A/G273W, or N447R) following mock (DMSO) treatment or treatment with proteasome inhibitor MG132. j Quantification of UNG2 immunoblots from ( i ). Data from each well were normalized to actin, and then to the DMSO control for cells transfected with a construct expressing WT 3×Flag-MKLN1 . The graph shows results of mean ± s.d. of n = 3 biological replicates. P -values were calculated using two-way ANOVA with Dunnett’s multiple comparison tests ( p -value significance <0.1). k Fluorescence polarization assay measuring binding of fluorescein-labeled peptides corresponding to the C-terminal 19 residues of ZMYND19 or FAM72A to the various versions of the MKLN1 Kelch domain (WT, W291A/G273W, or W291A/Y346A). K D for the ZMYND19 peptide–WT MKLN1 interaction is indicated. Data shown are the mean ± s.d. ( n = 3 technical replicates). l Co-immunoprecipitation via anti-RANBP9 antibody or rabbit IgG as a negative control in parental and ZMYND19 −/− knockout HEK293 cells transfected with constructs expressing HA-ZMYND19 (WT, ΔC-ter 3 res, or ΔC-ter Arg), assessing the role of the ZMYND19 C-terminal region in association with the CTLH complex. Immunoprecipitates and inputs are shown. Actin serves as a loading control ( n = 5 biological replicates). m Immunoblot analysis of ZMYND19 and MKLN1 levels in ZMYND19 −/− knockout HEK293 cells transiently expressing HA-ZMYND19 WT or ΔC-ter Arg mutant, and treated either with DMSO as control, Torin1, or Torin1 and proteasome inhibitor MG132. Actin serves as a loading control. n Quantification of ZMYND19 immunoblots from ( m ). Data from each well were normalized to actin, and then each set transfected with a particular HA-ZMYND19 expression construct was normalized to the DMSO control for that construct. The graph shows results of mean ± s.d. of n = 5 biological replicates. P -values were calculated using two-way ANOVA with Dunnett’s multiple comparison tests ( p -value significance <0.05). In vitro ubiquitylation assays examining the effect of C-terminal mutations in ZMYND19 ( o ) or FAM72A ( p ) on ubiquitylation by CTLH MKLN1 . n = 2 technical replicates. Source data are provided as a file.

Article Snippet: Flp-In T-Rex HEK293 cells (ATCC CRL-1573) were purchased from Thermo Fisher Scientific (catalog no. R78007 ).

Techniques: In Vitro, Ubiquitin Assay, Mutagenesis, Western Blot, Analysis, Expressing, Control, Construct, Comparison, Sequencing, Cryo-Electron Microscopy, Activity Assay, Co-Immunoprecipitation Assay, Knock-Out, Transfection, Fluorescence, Binding Assay, Labeling, Negative Control

Journal: iScience

Article Title: A method for expressing two proteins at a tunable ratio allows for studying the kinetics of creatine transport by MCT12

doi: 10.1016/j.isci.2026.117719

Figure Lengend Snippet: Adaptable expression ratios of the creatine transporters CRT1 and MCT12 in HEK293 SLC6A8 −/− cells (A) Scheme of the CRT1 3xHA -P2A-K n -MCT12 2xmyc construct. (B) Immunoblot analysis of CRT1 3xHA -P2A-K n -MCT12 2xmyc . (C) Ratio of MCT12 2xmyc and CRT1 3xHA expression as determined by immunoblotting ( n = 5). Data are represented as mean ± SEM. (D) Immunoblot analysis of CRT1 3xHA -P2A-K n -MCT12 2xmyc . (E) Ratio of MCT12 2xmyc and CRT1 3xHA expression as determined by immunoblotting ( n = 4). Data are represented as mean ± SEM. See also .

Article Snippet: Human: HEK293 Lenti-X , Takara , Cat # 632180.

Techniques: Expressing, Construct, Western Blot, Analysis

Journal: iScience

Article Title: A method for expressing two proteins at a tunable ratio allows for studying the kinetics of creatine transport by MCT12

doi: 10.1016/j.isci.2026.117719

Figure Lengend Snippet: Relative cell surface expression of the creatine transporters CRT1 and MCT12 in HEK293 SLC6A8 −/− cells (A) Scheme of the CRT1 eGFP -P2A-K 0 -MCT12 mCherry (a) and MCT12 mCherry -P2A-K 0 -CRT1 eGFP construct (b). (B) Ratio of mCherry and eGFP expression in eGFP-P2A-K 0 -mCherry (K 0 , as a control), CRT1 eGFP -P2A-K 0 -MCT12 mCherry , and MCT12 mCherry -P2A-K 0 -CRT1 eGFP constructs as determined by flow cytometry ( n = 6). Data are represented as mean ± SEM. (C) Live-cell confocal microscopy of HEK293 SLC6A8 −/− cells expressing CRT1 eGFP -P2A-K 0 -MCT12 mCherry and MCT12 mCherry -P2A-K 0 -CRT1 eGFP . The plasma membrane was stained with CellMask Deep Red. Scale bars, 20 μm. (D) Analysis of cell surface expression of CRT1 eGFP and MCT12 mCherry . n = 30 (CRT1 eGFP -P2A-K 0 -MCT12 mCherry ); n = 28 (MCT12 mCherry -P2A-K 0 -CRT1 eGFP ).

Article Snippet: Human: HEK293 Lenti-X , Takara , Cat # 632180.

Techniques: Expressing, Construct, Control, Flow Cytometry, Confocal, Microscopy, Clinical Proteomics, Membrane, Staining, Analysis

Journal: iScience

Article Title: A method for expressing two proteins at a tunable ratio allows for studying the kinetics of creatine transport by MCT12

doi: 10.1016/j.isci.2026.117719

Figure Lengend Snippet:

Article Snippet: Human: HEK293 Lenti-X , Takara , Cat # 632180.

Techniques: Cell-Signaling, Virus, Recombinant, Clinical Proteomics, Membrane, Staining, Cloning, Software

Journal: Blood Advances

Article Title: Distinct epitope engagement confers differential activity of linvoseltamab vs teclistamab across BCMA mutations ∗

doi: 10.1182/bloodadvances.2026020381

Figure Lengend Snippet: Binding of linvoseltamab and teclistamab to WT or mutated BCMA expressed by engineered HEK293/hCD20 cells. (A) Histogram plots from cell staining with 100nM anti-BCMA clone 19F2 or mIgG isotype control. BCMA is expressed on all engineered cells. (B) Histogram plots from staining with 100nM linvoseltamab, teclistamab, or hIgG isotype control. Linvoseltamab binding to BCMA R27P, P33S, and S30del was comparable to WT BCMA. Teclistamab binding to R27P, S30del, and P34del was reduced. (C) EC 50 values (nM) derived from nonlinear regression based on the cell staining experiment. hIgG, human immunoglobulin G; mIgG, mouse immunoglobulin G; NC, not calculated (EC 50 cannot be calculated from the range of concentrations tested); ND, not determined (no concentration-dependent binding was observed).

Article Snippet: Human embryonic kidney 293 (HEK293) cells (ATCC CRL-1573) stably expressing human CD20 (hCD20) (MS4A1; M1-P297 of NP_690605.1 ) were generated by transfection with a mammalian expression vector and selected in media (Dulbecco's Modified Eagle Medium, 10% fetal bovine serum, and 1% penicillin-streptomycin; Thermo Fisher Scientific) supplemented with 0.5 mg/mL of G418.

Techniques: Binding Assay, Staining, Control, Derivative Assay, Concentration Assay

Journal: Blood Advances

Article Title: Distinct epitope engagement confers differential activity of linvoseltamab vs teclistamab across BCMA mutations ∗

doi: 10.1182/bloodadvances.2026020381

Figure Lengend Snippet: Effects of BCMA mutations on linvoseltamab- and teclistamab-mediated Jurkat-NFAT cell activation. Jurkat-NFAT reporter cells cocultured with HEK293/hCD20 cells with WT or mutated BCMA; reporter activity was detected as luminescence. A nontargeting CD3 antibody was used as a control. RLU, relative light units.

Article Snippet: Human embryonic kidney 293 (HEK293) cells (ATCC CRL-1573) stably expressing human CD20 (hCD20) (MS4A1; M1-P297 of NP_690605.1 ) were generated by transfection with a mammalian expression vector and selected in media (Dulbecco's Modified Eagle Medium, 10% fetal bovine serum, and 1% penicillin-streptomycin; Thermo Fisher Scientific) supplemented with 0.5 mg/mL of G418.

Techniques: Activation Assay, Activity Assay, Control

Journal: Blood Advances

Article Title: Distinct epitope engagement confers differential activity of linvoseltamab vs teclistamab across BCMA mutations ∗

doi: 10.1182/bloodadvances.2026020381

Figure Lengend Snippet: Effect of BCMA mutations on T-cell–mediated cytotoxicity against HEK293h/CD20 cells treated with linvoseltamab and teclistamab. HEK293 or OPM-2 myeloma cells were incubated for 3 days with isolated human T cells, with E:T of 12.5 (HEK293) and 5 (OPM-2). Target cell survival was determined by flow cytometry. Percentage survival was normalized to untreated cells. The OPM-2 cell line was used as a comparator because this is an MM cell line that endogenously expresses BCMA. E:T, effector-to-target ratio.

Article Snippet: Human embryonic kidney 293 (HEK293) cells (ATCC CRL-1573) stably expressing human CD20 (hCD20) (MS4A1; M1-P297 of NP_690605.1 ) were generated by transfection with a mammalian expression vector and selected in media (Dulbecco's Modified Eagle Medium, 10% fetal bovine serum, and 1% penicillin-streptomycin; Thermo Fisher Scientific) supplemented with 0.5 mg/mL of G418.

Techniques: Incubation, Isolation, Flow Cytometry

Journal: Blood Advances

Article Title: Distinct epitope engagement confers differential activity of linvoseltamab vs teclistamab across BCMA mutations ∗

doi: 10.1182/bloodadvances.2026020381

Figure Lengend Snippet: Effect of BCMA-binding interface mutations on primary T-cell activation by linvoseltamab and teclistamab. HEK293 or OPM-2 myeloma cells were incubated for 3 days with isolated human T cells, with E:T of 12.5 (HEK293) and 5 (OPM-2). T-cell activation was determined by flow cytometry based on the percentage of CD8 + T cells expressing CD25. The OPM-2 cell line was used as a comparator because this is an MM cell line that endogenously expresses BCMA.

Article Snippet: Human embryonic kidney 293 (HEK293) cells (ATCC CRL-1573) stably expressing human CD20 (hCD20) (MS4A1; M1-P297 of NP_690605.1 ) were generated by transfection with a mammalian expression vector and selected in media (Dulbecco's Modified Eagle Medium, 10% fetal bovine serum, and 1% penicillin-streptomycin; Thermo Fisher Scientific) supplemented with 0.5 mg/mL of G418.

Techniques: Binding Assay, Activation Assay, Incubation, Isolation, Flow Cytometry, Expressing