Journal: Bioactive Materials

Article Title: Anisotropic mechanotransductive tissue constructs via brush-assisted bioprinting of microfiber-reinforced composite bioinks

doi: 10.1016/j.bioactmat.2025.12.017

Figure Lengend Snippet: Comparative analysis of conventional and brush-assisted bioprinting on cellular behavior. (a) Schematic illustration of shear stress distribution in normal versus brush-assisted printing. (b) Overview of the brush-assisted printing setup. (c) SEM images of collagen fibrils and fluorescence images showing cell viability (live/dead) and cytoskeletal organization (DAPI/phalloidin) of C2C12 myoblasts. Quantification of (d) cell viability post-printing (n = 4), (e) cell metabolic activity (MTT assay, in situ /day 3/day 7, n = 4), (f) nuclei aspect ratio (n = 180), (g) orientation factor (n = 3), and (h) F-actin–positive area at day 3 (n = 10). (i) Comparing normal and brush-assisted printing the mechanotransduction pathways activated by shear stress and collagen alignment. (j) Heatmap of relative gene expression ( YAP, TAZ, AKT1, PIEZO1, PI3K, and CAPN2 ) after 7 days of culture (n = 4). (k) Agarose gel electrophoresis of PCR products from cells cultured on normal versus brush-printed scaffolds for 7 days. (l) Schematic illustration of blocking mechano-sensing ion channel with GsMTx-4. (m) Relative gene expression levels associated with mechanosensing channel and ca 2+ pathway (n = 5), (n) Hippo pathway (n = 5), (o) PI3K-AKT pathway (n = 5). Student's t-test was applied for two-group comparisons, and one-way ANOVA with Tukey's HSD post-hoc test was used for multiple comparisons. Data are presented as mean ± standard deviation (SD). Statistical significance was set at p < 0.05 (∗), p < 0.01 (∗∗), and p < 0.001 (∗∗∗). Abbreviation: MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (cell metabolic activity assay); F-actin, Filamentous actin; PI3K , Phosphoinositide 3-kinase; CAPN2 , Calcium-activated neutral protease 2.

Article Snippet: H9C2 cardiomyoblasts (Korean Cell Line Bank, Seoul, Korea) and C2C12 murine skeletal muscle myoblasts (CRL-1772, ATCC, Manassas, USA) were cultured in high-glucose DMEM (Welgene, Korea) supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin–streptomycin (PS).

Techniques: Shear, Fluorescence, Activity Assay, MTT Assay, In Situ, Gene Expression, Agarose Gel Electrophoresis, Cell Culture, Blocking Assay, Standard Deviation, Metabolic Assay

Journal: Bioactive Materials

Article Title: Anisotropic mechanotransductive tissue constructs via brush-assisted bioprinting of microfiber-reinforced composite bioinks

doi: 10.1016/j.bioactmat.2025.12.017

Figure Lengend Snippet: Physical and biological evaluation of bioconstructs reinforced with straight (CSP) and coiled (CCP) PCL fibers. (a) Schematic illustration and optical images of flow tests with Col (collagen-only), CSP, and CCP bioinks, in which droplets were tilted at 90° for 10 min. (b) Quantification of droplet outflow (n = 10). Rheological measurements of bioinks: (c) storage modulus (G′) from frequency sweep (n = 3), (d) G′ and G″ from temperature sweep (n = 3), and (e) G′ and G″ under cyclic stress loading (10 and 200 Pa) showing viscoelastic recovery (n = 3). (f) SEM images of CCP constructs highlighting coiled PCL fiber and aligned collagen fibrils. (g) Optical image, live/dead staining, DAPI/phalloidin staining, and orientation factor analysis of C2C12 cells and PCL microfibers. Quantification of (h) cell viability (live/dead, n = 4), (i) F-actin–positive area (n = 20), and (j) metabolic activity (MTT assay, in situ /day 3/day 7, n = 4). Student's t-test was applied for two-group comparisons, and one-way ANOVA with Tukey's HSD post-hoc test was used for multiple comparisons. Data are presented as mean ± standard deviation (SD). Statistical significance was set at p < 0.05 (∗), p < 0.01 (∗∗), and p < 0.001 (∗∗∗).

Article Snippet: H9C2 cardiomyoblasts (Korean Cell Line Bank, Seoul, Korea) and C2C12 murine skeletal muscle myoblasts (CRL-1772, ATCC, Manassas, USA) were cultured in high-glucose DMEM (Welgene, Korea) supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin–streptomycin (PS).

Techniques: Construct, Staining, Activity Assay, MTT Assay, In Situ, Standard Deviation

Journal: Bioactive Materials

Article Title: Anisotropic mechanotransductive tissue constructs via brush-assisted bioprinting of microfiber-reinforced composite bioinks

doi: 10.1016/j.bioactmat.2025.12.017

Figure Lengend Snippet: In vitro myogenic differentiation of C2C12 cells cultured on Col, CSP and CCP scaffolds. (a) Live/dead staining at in situ , DAPI/phalloidin staining at day 3, and DAPI/ MHC staining at day 14. (b) Quantification of cell viability from live/dead assays (n = 4). (c) Nuclei orientation factor after 7 days of culture (n = 4). (d) Nuclei aspect ratio (n = 20). (e) F-actin positive area (n = 4). (f) Quantification of MHC fusion index (left, n = 5) and MHC maturation rate (right, n = 5). (g) Relative gene expression analysis and (h) agarose gel electrophoresis regarding mechanotransduction-related genes ( CAPN2, PIEZO1, RhoA, YAP, and TAZ ) (n = 4). (i) Western blot analysis of PIEZO1 . (j) Schematic illustrating differentiation progression and major genes involved at each stage. (k) Heatmap and (l) agarose gel electrophoresis of PCR products showing relative expression of myogenic markers ( MYF5, MYOD1, MYOG, MHC, MYH2, and MYH4 ) after 21 days of culture (n = 4). (m) Western blot analysis of MHC . Student's t-test was applied for two-group comparisons, and one-way ANOVA with Tukey's HSD post-hoc test was used for multiple comparisons. Data are presented as mean ± standard deviation (SD). Statistical significance was set at p < 0.05 (∗), p < 0.01 (∗∗), and p < 0.001 (∗∗∗). Abbreviation: MYF5 , Myogenic factor 5; MYOD1 , Myogenic differentiation 1; MYOG , Myogenin; MHC , Myosin heavy chain; MYH2 , Myosin heavy chain 2; MYH4 , Myosin heavy chain 4.

Article Snippet: H9C2 cardiomyoblasts (Korean Cell Line Bank, Seoul, Korea) and C2C12 murine skeletal muscle myoblasts (CRL-1772, ATCC, Manassas, USA) were cultured in high-glucose DMEM (Welgene, Korea) supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin–streptomycin (PS).

Techniques: In Vitro, Cell Characterization, Cell Culture, Staining, In Situ, Gene Expression, Agarose Gel Electrophoresis, Western Blot, Expressing, Standard Deviation

Journal: Journal of the Endocrine Society

Article Title: Insulin receptor trafficking and interactions in muscle cells

doi: 10.1210/jendso/bvag020

Figure Lengend Snippet: Biochemical and live-cell imaging characterization of the effects of insulin on INSR. (A) Internalized to total INSR ratio was quantified using surface biotinylation in undifferentiated C2C12 myoblasts. Cells were incubated in PBS (no insulin) or serum-free DMEM containing 0, 0.2, 2, or 20 nM insulin for 15 minutes. The ratios are normalized to the 0 nM group of each gel ( P > .05 when not specified). (B) Western blot showing SNAP-tagged INSR expressed from lentiviral vector in comparison to wild-type INSR in C2C12 myoblasts. (C, D) Representative images of INSR-A-SNAP-labeled using an Alexa Fluor 488 cell nonpermeable dye in 0, 0.2, or 20 nM insulin conditions in live undifferentiated C2C12 myoblasts from (C) 0 to 15 minutes or (D) after 20 minutes using a spinning disk confocal microscope. Time-lapse images of INSR vesicle interactions starting from the selected subregions (white squares) of snapshots are shown in the insets ( n = 3 cells).

Article Snippet: The C2C12 mouse myoblast (ATCC cell line provided by Dr Brian Rodrigues, University of British Columbia, Vancouver, Canada) was maintained in DMEM (Gibco) supplemented with 10% (v/v) fetal bovine serum (Gibco), and 1% penicillin–streptomycin (100 μg/mL; Gibco).

Techniques: Live Cell Imaging, Incubation, Western Blot, Plasmid Preparation, Comparison, Labeling, Microscopy

Journal: Journal of the Endocrine Society

Article Title: Insulin receptor trafficking and interactions in muscle cells

doi: 10.1210/jendso/bvag020

Figure Lengend Snippet: Interaction and colocalization between INSR, CAV3, CAV1, and CLTC under different insulin stimulations. (A) Western blot of mice skeletal muscle lysates after insulin injection. Phospho-AKT (Ser473) to total AKT ratio or phospho-ERK1/2 to total ERK ratio was quantified ( n = 6). (B) Co-immunoprecipitation of skeletal muscle INSR after PBS or insulin injection. CLTC to INSR ratio or CAV3 to INSR ratio were quantified ( n = 7-10). (C) Representative STED microscopy images of C2C12 myoblasts expressing INSR-A-SNAP (surface labeled) that were fixed after stimulation with 0, 0.2, or 20 nM insulin for 30 minutes and stained for CAV1 and CLTC (scale bar = 5 µm). (D, E) Colocalizations between INSR-A-SNAP, CAV1, and CLTC were quantified by Object Pearson Coefficient. Data are plotted to show differences between insulin concentrations (D) or between protein pairs (E) ( n = 7-9 images, 1-4 cells per image. * P < .05, Tukey's multiple comparison after 2-ANOVA. Box represents median and 25th to 75th percentiles).

Article Snippet: The C2C12 mouse myoblast (ATCC cell line provided by Dr Brian Rodrigues, University of British Columbia, Vancouver, Canada) was maintained in DMEM (Gibco) supplemented with 10% (v/v) fetal bovine serum (Gibco), and 1% penicillin–streptomycin (100 μg/mL; Gibco).

Techniques: Western Blot, Injection, Immunoprecipitation, Microscopy, Expressing, Labeling, Staining, Comparison

Journal: Journal of the Endocrine Society

Article Title: Insulin receptor trafficking and interactions in muscle cells

doi: 10.1210/jendso/bvag020

Figure Lengend Snippet: Colocalization and interaction between INSR and ANXA2 under different insulin concentrations. (A, B) Representative STED microscopy images of C2C12 myoblasts expressing INSR-A-SNAP (surface labeled) that were fixed after stimulation with 0, 0.2, or 20 nM insulin for (A) 15 or (B) 30 minutes and stained for ANXA2 (scale bar = 5 µm). (C, D) Colocalization between INSR-A-SNAP and ANXA2 at (C) 15 or (D) 30 minutes of insulin-stimulated were quantified by Object Pearson Coefficient ( n = 4-13 images, 1-3 cells per image. # P < .05, 1-ANOVA of all groups. Box represents median and 25th to 75th percentiles.).

Article Snippet: The C2C12 mouse myoblast (ATCC cell line provided by Dr Brian Rodrigues, University of British Columbia, Vancouver, Canada) was maintained in DMEM (Gibco) supplemented with 10% (v/v) fetal bovine serum (Gibco), and 1% penicillin–streptomycin (100 μg/mL; Gibco).

Techniques: Microscopy, Expressing, Labeling, Staining

Journal: Nucleic Acids Research

Article Title: Repeat-rich RNA guides repetitive genomic elements into biomolecular condensates for heterochromatin organization and muscle integrity

doi: 10.1093/nar/gkag168

Figure Lengend Snippet: CU-rich RNA promotes heterochromatin condensate organization during differentiation. ( A ) Nuclei of C2C12 myotubes (MT) treated with 1.5% 1,6-hexanediol (1,6-HD) or 1.5% 2,5-hexanediol (2,5-HD). Left: Representative images of 4,6-diamidino-2-phenylindole (DAPI)-stained nuclei. Scale bar, 5 μm. Middle: Time-course quantification of the number of heterochromatin foci per nucleus. Right: Boxplots show foci area (μm 2 , y -axis) at 5, 10, and 15 min posttreatment ( x -axis). n = 55 nuclei, three biological replicates. ( B ) Representative live-cell images of Hoechst 33342-stained MT nuclei before and after 1,6-HD treatment (0 and 15 min, respectively), taken from . Arrows indicate changes in heterochromatin foci intensity (pink, increased; blue, decreased), and the red arrow highlights an alteration in chromocenter integrity. ( C ) Number of heterochromatin foci per nucleus in MT following recovery from 1.5% 1,6-hexanediol (1,6-HD) treatment for 15 min, measured at indicated time points. n = 68 nuclei, three biological replicates. ( D ) Representative images of nuclei of myoblast (MB) and MT with or without 1,6-HD treatment (1.5%, 15 min). Right: Quantification of the number of heterochromatin foci per nucleus. n = 40 (MB), n = 60 (MT), three biological replicates. ( E ) Quantification of colocalization between indicated proteins and DAPI foci in MT with or without 1,6-HD treatment (1.5%, 15 min), by Pearson’s correlation coefficients. n = 60, three biological replicates. ( F ) Boxplot showing the distribution of Z -score of the interchromosomal interaction frequencies in MB and MT. ( G ) RNA FISH using ChRO1 and LacZ biotinylated probes. Biotin signal was detected by Fluorescein-conjugated Avidin DCS and amplified with biotinylated anti-Avidin and additional Fluorescein Avidin DCS. Right: Quantification of colocalization between biotin signal and DAPI foci. n = 50 nuclei. ( H ) Number of heterochromatin foci per nucleus of mouse fibroblast cells (NIH3T3) with or without doxycyline (Dox)-induced ChRO1a expression and/or 1,6-HD treatment (1.5%, 15 min). (EV; empty vector). n = 50, three biological replicates. ( I ) Number of heterochromatin foci per nucleus in MB with or without Dox-induced ChRO1a fragment (1–413, CUR) expression and/or 1,6-HD treatment (1.5%, 15 min). n = 75, three biological replicates. Statistical analyses and data presentation details are described in the “Materials and methods” section.

Article Snippet: C2C12 murine myoblast cells and NIH3T3 mouse fibroblast cells were obtained from the American-type culture collection and grown in a growth medium (GM) consisting of Dulbecco’s modified Eagle medium (DMEM) with 10% (v/v) fetal bovine serum at 37°C and 5% CO 2 .

Techniques: Staining, Avidin-Biotin Assay, Amplification, Expressing, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: Repeat-rich RNA guides repetitive genomic elements into biomolecular condensates for heterochromatin organization and muscle integrity

doi: 10.1093/nar/gkag168

Figure Lengend Snippet: ChRO1 deficiency and condensate disruption induce a muscle atrophic phenotype. ( A ) Strategy for generating ChRO1 KO (ChRO1−/−) mice using CRISPR–Cas9 genome editing targeting two gRNA sites to delete ChRO1 promoter, exon1, and part of intron1 region. ( B ) qRT-RCR quantification of ChRO1a and ChRO1b expression in gastrocnemius from 13-months-old-male mice WT and KO mice (ChRO1+/+ and ChRO1−/−). ( C ) qRT-PCR analysis of atrophic genes and satellite RNAs in gastrocnemius muscle from WT and ChRO1 KO mice. ( D ) Representative wheat germ agglutinin staining (left) and myofiber CSA (μm 2 ) quantification (right) of indicated skeletal muscle tissues from WT and ChRO1 KO mice. n ≥ 500 fibers analyzed per tissue. Scale bars, 40 μm. ( E ) Representative images (left) and quantification (right) of heterochromatin foci in gastrocnemius muscle from WT and ChRO1 KO mice. Scale bars, 1 μm. n = 50 nuclei. ( F ) Immunostaining for DAXX and H3K9me3 in gastrocnemius muscle of WT and ChRO1 KO mice. n = 50 nuclei. Scale bars, 2 μm. ( G ) qRT-PCR analysis of Atrogin1 (left) and Murf1 (right) in C2C12 MT differentiated for 5 days followed by 1,6-HD treatment at indicated concentration (%) for varying durations (1.5, 5.5, or 24 h). Expression normalized to Gapdh and shown as fold change relative to untreated control for each time point. ( H ) Western blot analysis of MyHC protein levels in C2C12 MT treated with 1,6-HD at indicated concentration (%) for varying durations (1.5, 5.5, or 24 h). Immunofluorescence staining of MyHC (AF488, green) and nuclei (DAPI, blue) ( I ) and quantification of myotube diameters [( J ), n = 100 cells] of C2C12 MT treated with 1,6-HD at indicated concentrations. ( K ) Quantification of myotube diameters of C2C12 MT treated with 2,5-HD or 1,6-HD at indicated concentration for 24 h. n = 45 cells. Statistical analyses, box plot elements, and data normalization procedures are detailed in the “Materials and methods” section.

Article Snippet: C2C12 murine myoblast cells and NIH3T3 mouse fibroblast cells were obtained from the American-type culture collection and grown in a growth medium (GM) consisting of Dulbecco’s modified Eagle medium (DMEM) with 10% (v/v) fetal bovine serum at 37°C and 5% CO 2 .

Techniques: Disruption, CRISPR, Expressing, Quantitative RT-PCR, Staining, Immunostaining, Concentration Assay, Control, Western Blot, Immunofluorescence

Journal: eLife

Article Title: Competition for the conserved branch point sequence influences physiological outcomes in pre-mRNA splicing

doi: 10.7554/eLife.103167

Figure Lengend Snippet: ( A ) UCSC Genome Browser screenshot (top panel) with RNA-seq reads mapping to RAI14 for shQKI (top), shNT (middle), or shSF1 (bottom); percent spliced in (PSI) values are measured by Vast-tools; the inset shows boxed regions of (from top) two ACUAAC elements with Cola-seq branchpoints mapping to one nucleotide downstream of a branchpoint adenine, ACUAA elements by oligomatch, then Quaking (QKI) eCLIP peaks from K562 (top) or HepG2 (bottom) cells that overlap with these; conservation of 100 vertebrates is shown at the bottom. ( B ) Western blot of protein extracted from HEK293 QKI KO cells (top) probed with anti-tubulin (magenta) or anti-panQKI (green) antibodies (MW in kD is shown on right); RT-PCR of RAI14 exon 11 and BioAnalyzer gel-like image (bottom) of RNA extracted from the cells above showing mean percent included ± standard deviation below (n=3 biological replicates; **** p <0.001 by Student’s t-test compared to ctrl; size of amplicon in bp is shown on right). ( C ) Western blot (top) of proteins extracted from HEK293 cells transfected with siNT, siSF1_2, or siSF1_1+2, probed with anti-SF1 (green) or anti-Gapdh (magenta) antibodies; the protein abundance (fold change relative to the siNT control ± standard deviation) is shown below and MW in kD is shown on right; RT-PCR of RAI14 exon 11 and BioAnalyzer gel-like image (bottom) of RNA extracted from the cells described above with mean percent included ± standard deviation below (n=3 biological replicates; ** p <0.01 or *** p <0.001 by Student’s t-test compared to siNT; size of amplicon in bp is shown on right). ( D ) Western blot (top) of proteins extracted from C2C12 myoblasts transfected with siNT, siQki or siSf1_1+2, probed with anti-SF1 (magenta; top), anti-tubulin (magenta; middle) or anti-panQki (green; bottom); MW in kD is shown on right. The protein abundance (fold change relative to the siNT control ± standard deviation) is shown below (n=3 biological replicates; ** p <0.01 or *** p <0.001 by Student’s t-test); RT-PCR of Rai14 exon 11 and BioAnalyzer gel-like image (bottom) of RNA extracted from the C2C12 cells described above with mean percent included ± standard deviation indicated below (**** p <0.0001 by Student’s t-test; size of amplicon in bp is shown on right). Figure 2—source data 1. Original membranes and BioAnalyzer gel-like images corresponding to (left), (middle), or (right). See text under each set of images for additional details. Figure 2—source data 2. Source data containing original uncropped western blots and BioAnalyzer gel-like images/files.

Article Snippet: C2C12 mouse myoblasts (obtained from ATCC) and HEK293 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with high glucose (Invitrogen) and 10% (v/v) heat-inactivated fetal bovine serum (Thermo Fisher).

Techniques: RNA Sequencing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Standard Deviation, Amplification, Transfection, Quantitative Proteomics, Control

Journal: eLife

Article Title: Competition for the conserved branch point sequence influences physiological outcomes in pre-mRNA splicing

doi: 10.7554/eLife.103167

Figure Lengend Snippet: ( A ) Schematic of the beta globin pDUP-RAI14 ex 11 splicing reporters indicating the region of intron 10, exon 11, and intron 11 included; the inset below shows the different constructs (wild-type or mutant) tested. ( B ) RT-PCR and BioAnalyzer gel-like image from RNA extracted from C2C12 cells transfected with RAI14 ex 11 wild-type or deletion mutant reporters; mean percent included values are shown below (+/- the standard deviation; *indicates an unidentified/spurious product). ( C ) RT-qPCR measuring total reporter RNA level, normalized to Eef1a1 , from RNA described in B., and shown as fold-change relative to WT (** p <0.01 or **** p <0.001 by Student’s t-test). ( D ) RT-PCR and BioAnalyzer gel-like image from RNA extracted from C2C12 cells transfected with RAI14 ex 11 wild-type or substitution mutant reporters, analyzed as described in B. ( E ) RT-qPCR measuring total reporter RNA level, normalized to Eef1a1 , from RNA described in D., and shown as fold-change relative to WT (* p <0.05 or **** p <0.001 by Student’s t-test). ( F ) RT-PCR and BioAnalyzer gel-like image from RNA extracted from C2C12 cells transfected with RAI14 ex 11 wild-type or half-site mutant reporters, analyzed as described in B (*** p <0.001, **** p <0.0001). G. RT-qPCR measuring total reporter RNA level, normalized to Eef1a1 , from RNA described in F., and shown as fold-change relative to WT (**p<0.01 or ****p<0.001 by Student’s t-test). Each experiment was conducted in biological triplicate, and for RT-PCR with BioAnalyzer measurement, each comparison compared to WT reporter showed p <0.0001 by Student’s t-test. Figure 3—source data 1. Original BioAnalyzer gel-like images corresponding to (left), (middle), or (right). Figure 3—source data 2. Original BioAnalyzer gel-like images and data files corresponding to (left), (middle), or (right).

Article Snippet: C2C12 mouse myoblasts (obtained from ATCC) and HEK293 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with high glucose (Invitrogen) and 10% (v/v) heat-inactivated fetal bovine serum (Thermo Fisher).

Techniques: Construct, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Transfection, Standard Deviation, Quantitative RT-PCR, Comparison

Journal: eLife

Article Title: Competition for the conserved branch point sequence influences physiological outcomes in pre-mRNA splicing

doi: 10.7554/eLife.103167

Figure Lengend Snippet: PCR was performed with RNA from C2C12 cells transfected with RAI14 reporter plasmids. Figure 3—figure supplement 1—source data 1. Original uncropped agarose gel images from . Figure 3—figure supplement 1—source data 2. Original uncropped agarose gel images from .

Article Snippet: C2C12 mouse myoblasts (obtained from ATCC) and HEK293 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with high glucose (Invitrogen) and 10% (v/v) heat-inactivated fetal bovine serum (Thermo Fisher).

Techniques: Transfection, Agarose Gel Electrophoresis

Journal: eLife

Article Title: Competition for the conserved branch point sequence influences physiological outcomes in pre-mRNA splicing

doi: 10.7554/eLife.103167

Figure Lengend Snippet: RNA affinity chromatography (RAC) with liquid chromatography and tandem mass spec or western blot analysis. ( A ) Schematic representation of substrates used for RNA affinity chromatography (RAC) which included 64 nt of RAI14 intron 10, 6 nt of exon sequence, and the tobramycin aptamer (tobra): wild-type, upDEL, dnDEL, 2xDEL, and aptamer only; C2C12 nuclear extract (NE) was incubated with these, and RAC-associated eluates were analyzed by liquid chromatography with tandem mass spectrometry (LC-MS/MS) or western blot. ( B ) Heatmap showing hierarchical clustering of early spliceosome and 17S U2 snRNP protein (E/U2) abundance detected in the RAC-LC-MS/MS datasets for each substate shown and in the presence of ATP; these represent background-corrected levels relative to NE (see Methods) and the scale bar shows row Z-score values. ( C ) Numbers observed for relative levels of E/U2 proteins (background corrected relative to NE) observed associating with each RAC substate with either a positive (n pos ) or negative (n neg ) value. ( D ) Western blot of NE (input) or WT, dnDEL (dn), or 2xDEL (2 x) RAC time-course (+ATP as described in B and C) for 7.5 min (left), 15 min (middle), or 30 min (right) probed with anti-panQki (top) or anti-SF1 (bottom) antibodies; MW shown in kD to the right. ( E ) Heatmap showing hierarchical clustering of early spliceosome and 17S U2 snRNP protein (E/U2) abundance detected in the RAC-LC-MS/MS datasets for each substate shown and in the absence of ATP; these represent data-independent acquisition (DIA; see methods) values normalized to NE and each mutant is shown as log2 fold change relative to the WT substate and passed cutoff of log 2 fold change > |0.2| and p <0.01. ( F ) Volcano plot comparing LC-MS/MS log 2 protein abundance (log 2 fold-change (log2FC); x-axis) of E/U2 (circles) and other RNA-binding proteins (RBPs) (squares) observed associating with RAC substates dnDEL compared to WT (y-axis shows -log 10 p -value) of enriched proteins (cutoff: L2FC > |0.7| and p <0.01); inset shows the number observed for those enriched in dnDEL (yellow) or WT (blue); schematic below shows model of RAC substates recruitment to distinct protein-associated species. ( G ) Western blot of NE (input) or WT, dnDEL (dn), or 2xDEL (2 x) RAC time-course (-ATP as described in E and F) for 7.5 min (left), 15 min (middle), or 30 min (right) probed with anti-panQki (top), anti-SF1 (middle), or anti-TATSF1 (bottom); MW shown in kD to the right. Figure 4—source data 1. Original membranes corresponding to western blots depicted in (left), or . Figure 4—source data 2. Original membranes corresponding to western blots depicted in (left), or .

Article Snippet: C2C12 mouse myoblasts (obtained from ATCC) and HEK293 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with high glucose (Invitrogen) and 10% (v/v) heat-inactivated fetal bovine serum (Thermo Fisher).

Techniques: Affinity Chromatography, Liquid Chromatography, Mass Spectrometry, Western Blot, Sequencing, Incubation, Liquid Chromatography with Mass Spectroscopy, Data-independent acquisition, Mutagenesis, Quantitative Proteomics, RNA Binding Assay