Journal: iScience

Article Title: DAXX-dependent H3.3 deposition maintains myoblast cell identity independently of other histone chaperone complexes

doi: 10.1016/j.isci.2025.113119

Figure Lengend Snippet: Daxx KO impairs myogenic lineage progression (A) Immunostaining with DAXX (green) antibody and the nuclear marker DAPI (blue) in C2C12 (top) and Daxx KO (bottom) cell lines ( n = 3 C2C12 and n = 3 Daxx KO independent culture experiments). Scale bars, 40 μm. (B) Western blot for DAXX and GAPDH in C2C12 ( n = 2 independent protein samples) and Daxx KO (in 4 independent clones) ( n = 2 independent protein samples). (C) Co-immunostaining with PAX7 (red) and MYOD (green) antibodies and the nuclear marker DAPI (blue) in C2C12 (top) and Daxx KO (bottom) cell lines ( n = 5 C2C12 and n = 5 Daxx KO independent culture experiments). Scale bars, 100 μm. (D) Quantification of the number of PAX7-positive cells among DAPI-positive cells in (C) ( n = 5 C2C12 and n = 5 Daxx KO independent culture experiments). Error bars, mean ± SD, Mann-Whitney test. (E) Western blot or MYOD and H3 in C2C12 ( n = 2 independent protein samples) and Daxx KO (in 4 independent clones) ( n = 2 independent protein samples) cell lines. (F) RT-qPCR analyses of the mRNA levels of Pax7 , Myod1 , Daxx , and Hira in control (gray) ( n = 3 independent RNA samples) and Daxx KO (black) cells ( n = 3 independent RNA samples). For each gene, the mRNA levels of the control cells were normalized to 1. Error bars, mean ± SD, two-tailed unpaired t test. (G) Immunostaining with MF20 antibody (green) to visualize myosins and the nuclear marker DAPI (blue) in C2C12 (top) and Daxx KO (bottom) cell lines. Scale bars, 40 μm. (H) Quantification of the number of MF20-positive nuclei among DAPI-positive nuclei in (G) (C2C12 n = 6, Daxx KO n = 5, independent differentiation assays). Error bars, mean ± SD, Mann-Whitney test. (I) Immunostaining with MF20 antibody (green) to visualize myosins and the nuclear marker DAPI (blue) in C2C12 transfected with empty plasmid (left), and Daxx KO cell lines transfected with empty (middle) or Daxx cds-containing plasmid (right). Scale bars, 100 μm. (J) Quantification of the number of MF20-positive nuclei among DAPI-positive nuclei in (I) ( n = 3 independent differentiation assays for each condition). Error bars, mean ± SD, two-tailed unpaired t test. p values below 0.05 were considered significant. See also and .

Article Snippet: Mouse: C2C12 cell line , ATCC , CRL1772.

Techniques: Immunostaining, Marker, Western Blot, Clone Assay, MANN-WHITNEY, Quantitative RT-PCR, Control, Two Tailed Test, Transfection, Plasmid Preparation

Journal: iScience

Article Title: DAXX-dependent H3.3 deposition maintains myoblast cell identity independently of other histone chaperone complexes

doi: 10.1016/j.isci.2025.113119

Figure Lengend Snippet: Daxx KO prevents myogenic gene expression (A) MA plot of C2C12 over Daxx KO RNA-seq data. Significantly dysregulated genes are highlighted in blue (false discovery rate [FDR] < 0.05). (B) Number of up-regulated (2,364 genes, blue) and down-regulated (2,611 genes, red) genes in Daxx KO compared to C2C12. (C and D) Gene Ontology analysis for biological processes of the down-regulated (C) and up-regulated (D) genes in Daxx KO cells. Selected enriched terms are presented according to the fold enrichment. (E) Heatmap with the number of normalized reads of C2C12 and Daxx KO RNA-seq data for a selection of genes from the indicated cell lineages, in individual triplicates. (F) RT-qPCR analyses of the mRNA levels of selected genes down-regulated and up-regulated in Daxx KO cell lines. Control (gray) ( n = 3 independent RNA samples) and Daxx KO cells (black) ( n = 3 independent RNA samples). For each gene, the mRNA levels of the control cells were normalized to 1. Error bars, mean ± SD, two-tailed unpaired t test. (G) Total number of H3.3 called peaks (tag number) ( q value = 5e−2) in the ChIP-seq of C2C12 (gray, 38,439 peaks) ( n = 1) and Daxx KO cells (black, 12,779 peaks) ( n = 1). (H) Percentage of the total number of H3.3 peaks distributed in distinct genomic regions as indicated in C2C12 or Daxx KO cells. p values below 0.05 were considered significant. See also .

Article Snippet: Mouse: C2C12 cell line , ATCC , CRL1772.

Techniques: Gene Expression, RNA Sequencing, Selection, Quantitative RT-PCR, Control, Two Tailed Test, ChIP-sequencing

Journal: iScience

Article Title: DAXX-dependent H3.3 deposition maintains myoblast cell identity independently of other histone chaperone complexes

doi: 10.1016/j.isci.2025.113119

Figure Lengend Snippet: DAXX deposits H3.3 in myogenic gene loci (A) ChIP-seq average signal profiles (ratio to input) in the promoter region (±3 kb around the TSS), TSS, gene body, and TES for H3.3 in C2C12 (blue) and Daxx KO (red) cells are shown for all genes (left), up-regulated (middle) genes in Daxx KO, and down-regulated genes in Daxx KO (right). (B–E) ChIP-seq average signal profiles (ratio to input) in the promoter region (±3 kb around the TSS), TSS, gene body, and TES for H3K4me3 (B), H3K27ac (C), H3K9me3 (D), and H3K27me3 (E) in C2C12 (blue) and Daxx KO (red) cells shown for all genes. (F–K) ATAC-seq (green), ChIP-seq profiles for H3.3 (orange), H3K27ac (dark blue), H3K4me3 (red), H3K27me3 (purple), and H3K9me3 (light blue) in the genomic loci of Pax7 (F), Myod1 (G), Mymk (H), Dkk2 (I), Igtp (J), and Wnt5b (K) in control (WT, top lines) and Daxx KO (KO, bottom lines). See also and .

Article Snippet: Mouse: C2C12 cell line , ATCC , CRL1772.

Techniques: ChIP-sequencing, Control

Journal: iScience

Article Title: DAXX-dependent H3.3 deposition maintains myoblast cell identity independently of other histone chaperone complexes

doi: 10.1016/j.isci.2025.113119

Figure Lengend Snippet: Daxx and Hira dKO cell line has impaired myogenic identity Co-immunostaining with DAXX (green) and HIRA (red) antibodies and the nuclear marker DAPI (blue) in C2C12 (top) and dKO (bottom) cell lines ( n = 3 C2C12 and n = 3 dKO independent culture experiments). Scale bars, 40 μm. (B) Western blot for DAXX, HIRA, MYOD, and H3 in C2C12 ( n = 2 independent protein samples) and dKO (2 independent clones) ( n = 2 independent protein samples). (C) Co-immunostaining with PAX7 (green) and MYOD (red) antibodies and the nuclear marker DAPI (blue) in C2C12 (top) and dKO (bottom) cell lines ( n = 3 C2C12 and n = 3 dKO independent culture experiments). Scale bars, 40 μm. (D) Quantification of the number of PAX7-positive cells among DAPI-positive cells in (C) ( n = 3 C2C12 and n = 3 dKO independent culture experiments). Error bars, mean ± SD, two-tailed unpaired t test. (E) Quantification of the number of MYOD-positive cells among DAPI-positive cells in (C) ( n = 3 C2C12 and n = 3 dKO independent culture experiments). Error bars, mean ± SD, two-tailed unpaired t test. (F) Co-immunostaining with MYOG (red) and the nuclear marker DAPI (blue) in C2C12 (top) and dKO (bottom) cell lines ( n = 3 C2C12 and n = 3 dKO independent culture experiments). Scale bars, 40 μm. (G) Quantification of the number of MYOG-positive cells among DAPI-positive cells in (F) ( n = 3 C2C12 and n = 3 dKO independent culture experiments). Error bars, mean ± SD, two-tailed unpaired t test. (H) Immunostaining with MF20 antibody (green) to visualize myosins and the nuclear marker DAPI (blue) in C2C12 (top) and dKO (bottom) cell lines. Scale bars, 40 μm. (I) Quantification of the number of MF20-positive nuclei among DAPI-positive cells in (H) ( n = 3 C2C12 and n = 3 dKO independent culture experiments). Error bars, mean ± SD, two-tailed unpaired t test. (J) RT-qPCR analyses of the mRNA levels of Pax7 , Myf5 , Myod1 , Myog , and Tbp in the dKO cells normalized to control = 1 ( n = 3 independent RNA samples for control and dKO). For each gene, the mRNA levels of the control cells were normalized to 1. Error bars, mean ± SD, two-tailed unpaired t test. p values below 0.05 were considered significant. See also and .

Article Snippet: Mouse: C2C12 cell line , ATCC , CRL1772.

Techniques: Immunostaining, Marker, Western Blot, Clone Assay, Two Tailed Test, Quantitative RT-PCR, Control

Journal: iScience

Article Title: DAXX-dependent H3.3 deposition maintains myoblast cell identity independently of other histone chaperone complexes

doi: 10.1016/j.isci.2025.113119

Figure Lengend Snippet: Transcriptomic analysis of dKO cells shows a distinct phenotype compared to single mutation myoblast lines (A) MA plot of C2C12 over dKO RNA-seq data. Significantly dysregulated genes are highlighted in blue (FDR < 0.05). (B) Number of up-regulated (6,816 genes, blue) and down-regulated (6,439 genes, red) genes in dKO compared to C2C12. (C and D) Gene Ontology analysis for biological processes of the down-regulated (C) and up-regulated (D) genes in dKO cells. Selected enriched terms are presented according to the fold enrichment. (E) Heatmap with the number of normalized reads of C2C12 and dKO RNA-seq data for a selection of genes from the indicated cell lineages, in individual triplicates. (F) RT-qPCR analyses of the mRNA levels of selected genes down-regulated and up-regulated in dKO cell lines. Control ( n = 3–6 independent RNA samples) and dKO cells ( n = 3–6 independent RNA samples). For each gene, the mRNA levels of the control cells were normalized to 1. Error bars, mean ± SD, two-tailed unpaired t test. (G) Venn diagram depicting the number of dysregulated genes in each single KO and in the dKO mutant lines. p values below 0.05 were considered significant. See also .

Article Snippet: Mouse: C2C12 cell line , ATCC , CRL1772.

Techniques: Mutagenesis, RNA Sequencing, Selection, Quantitative RT-PCR, Control, Two Tailed Test

Journal: iScience

Article Title: DAXX-dependent H3.3 deposition maintains myoblast cell identity independently of other histone chaperone complexes

doi: 10.1016/j.isci.2025.113119

Figure Lengend Snippet: DAXX and HIRA deposit H3.3 in both euchromatin and heterochromatin in C2C12 myoblasts (A) ChIP-seq average signal profiles (ratio to input) in enhancer regions for H3.3 in C2C12 (blue), Daxx KO (red), and Hira KO (green) cells. (B) ChIP-seq average signal profiles (ratio to input) for H3.3 plotted into the ATAC-seq peaks in C2C12 (blue), Daxx KO (red), and Hira KO (green) cells. (C and D) ChIP-seq average signal profiles (ratio to input) for H3.3 (C) and H3K9me3 (D) plotted into genomic repetitive regions in C2C12 (blue), Daxx KO (red), and Hira KO (green) cells. (E) UpSet plot depicting the H3.3 peak intersections between the different datasets, C2C12, Daxx KO, and Hira KO. (F) Percentage of the number of H3.3 shared peaks between Daxx KO and Hira KO datasets, distributed in distinct genomic loci. (G) Gene Ontology analysis for biological processes for the genes associated with peaks shared between Daxx KO and Hira KO datasets. Selected enriched terms are presented according to the fold enrichment. (H–J) ChIP-seq profiles for H3.3 (orange), in the genomic loci of Dlx4 (H), Cbx5 (I), Jmjd6 (J), in control (WT, top lines), Hira KO (middle lines), and Daxx KO (bottom lines). (K) ChIP-RT-qPCR for H3.3 on the regions selected from the ChIP-seq data for Myod1 , Tbx4 , Dll1 , and Notch1 genes in C2C12 (gray) and dKO (black) cell lines ( n = 3 independent biological samples per condition). Error bars, mean ± SD, two-tailed paired t test. p values below 0.05 were considered significant. See also .

Article Snippet: Mouse: C2C12 cell line , ATCC , CRL1772.

Techniques: ChIP-sequencing, Control, Quantitative RT-PCR, Two Tailed Test

Journal: Agronomy

Article Title: Effects of Sweet Potato Leaf Extracts and Chlorogenic Acid on Glucose Uptake in C2C12 Cells

doi: 10.3390/agronomy14122855

Figure Lengend Snippet: Figure 1. Percentage viability of C2C12 cells treated with different concentrations of (A) SPLEs and (B) CGA. Treatment groups in (A) were P, purple SPLE; G, green SPLE; Y, yellow SPLE, at 200 and 400 µg/mL concentrations; in (B) were CGA at 0, 10, 20, 40, and 80 µg/mL concentrations. Different letters denote statistically significant differences between results (p < 0.05). Bars indicate standard deviation with three repetitions.

Article Snippet: Mouse muscle myoblast C2C12 cell line (derived from ATCC; CRL-1772) was purchased from Agronomy 2024, 14, 2855 3 of 12 the Bioresource Collection and Research Center (BCRC) of the Institute of Food Industry, Hsinchu City, Taiwan.

Techniques: Standard Deviation

Journal: Agronomy

Article Title: Effects of Sweet Potato Leaf Extracts and Chlorogenic Acid on Glucose Uptake in C2C12 Cells

doi: 10.3390/agronomy14122855

Figure Lengend Snippet: Figure 2. Effects of SPLEs, CGA, and the addition of 150 nM insulin on the uptake of 2-NBDG by C2C12. SPLEs (400 µg/mL) and CGA (10 and 20 µg/mL) were treatments. A fluorescence inverted microscope was used for an exposure time of 57 µs/750 ms to obtain a fluorescent image (A) (SAGE vision SGHD-3600 HDMI high-pixel microscope system), followed by analysis of fluorescence inten- sity (B). More detailed explanations of cellular glucose uptake are described in Section 3.2. C, control, cells without any treatment; I, containing 150 nM insulin; P, purple SPLE containing 150 nM insulin; G, green SPLE containing 150 nM insulin; Y, yellow SPLE containing 150 nM insulin; CGA10, 10 µg/mL CGA containing 150 nM insulin; CGA20, 20 µg/mL CGA containing 150 nM insulin. Data are expressed as mean ± SD with three repetitions. Different small letters in (B) indicate significant differences among treatments and the control (p < 0.05).

Article Snippet: Mouse muscle myoblast C2C12 cell line (derived from ATCC; CRL-1772) was purchased from Agronomy 2024, 14, 2855 3 of 12 the Bioresource Collection and Research Center (BCRC) of the Institute of Food Industry, Hsinchu City, Taiwan.

Techniques: Fluorescence, Inverted Microscopy, Microscopy, Control

Journal: Agronomy

Article Title: Effects of Sweet Potato Leaf Extracts and Chlorogenic Acid on Glucose Uptake in C2C12 Cells

doi: 10.3390/agronomy14122855

Figure Lengend Snippet: Figure 4. Analysis of (A) IR and (B) IRS1 abundances of SPLEs and CGA on C2C12 cells. SPLEs (400 µg/mL) and CGA (10 and 20 µg/mL). C, control, cells without any treatment; I, 150 nM insulin treated only; P, purple SPLE containing 150 nM insulin; G, green SPLE containing 150 nM insulin; Y, yellow SPLE containing 150 nM insulin; CGA10, 10 µg/mL CGA containing 150 nM insulin; CGA20, 20 µg/mL CGA containing 150 nM insulin. Data are expressed as mean ± SD with three repetitions. Different small letters indicate significant differences among treatments and the control (p < 0.05).

Article Snippet: Mouse muscle myoblast C2C12 cell line (derived from ATCC; CRL-1772) was purchased from Agronomy 2024, 14, 2855 3 of 12 the Bioresource Collection and Research Center (BCRC) of the Institute of Food Industry, Hsinchu City, Taiwan.

Techniques: Control