Additional file 1 of MYH1G-AS is a chromatin-associated lncRNA that regulates skeletal muscle development in chicken
Creators: name: Cai, Bolin
name: Ma, Manting
name: Yuan, Rongshuai
name: Zhou, Zhen
name: Zhang, Jing
name: Kong, Shaofen
name: Lin, Duo
name: Lian, Ling
name: Li, Juan
name: Zhang, Xiquan
name: Nie, Qinghua
Publisher: name: figshare
Year: 2024
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 1: Table S1. Information of primers. Table S2. Sequences of potential ORFs of MYH1G-AS. Table S3. Oligonucleotide sequences in this study. Fig. S1. Relative β-Tubulin and GAPDH protein expression in myoblast proliferation (myoblast cultured in growth medium [GM]) and differentiation (myoblast cultured in differentiation medium from 1 to 5 day [DM1 to DM5; DM indicate differentiation day]) periods. The numbers shown below the bands were folds of band intensities relative to control. Band intensities were quantified by ImageJ and normalized to β-Tubulin. Data are expressed as a fold-change relative to the control. Results are presented as mean ± SEM. Fig. S2. GO functions and KEGG pathways analysis of differentially expressed genes and ATAC-seq peaks between pectoralis major and soleus in 7-week-old Xinghua chicken. (A, B) GO functions (A) and KEGG pathways (B) analysis of differentially expressed genes between pectoralis major (PEM) and soleus (SOL) in 7-week-old Xinghua chicken. (C-D) GO functions (C) and KEGG pathways (D) analysis of differentially expressed ATAC-seq peaks between PEM and SOL in 7-week-old Xinghua chicken. Fig. S3. Heatmap of ATAC-seq signals at transcriptional start site. (A) Heatmap of ATAC-seq signals at transcriptional start site (TSS) in PEM samples. (B) Heatmap of ATAC-seq signals at TSS in SOL samples. Fig. S4. Characterization of MYH1G-AS. (A) Results of MYH1G-AS 3’ RACE and 5’ RACE. (B) The full-length sequence of MYH1G-AS. Coordinates are listed according to bGalGal1.mat.broiler.GRCg7b reference Annotation Release 106. (C) Conservative analysis of MYH1G-AS performed by using the NCBI’s BLAST. A total of eighteen species, including Anas platyrhynchos, Anser cygnoides, Apteryx mantelli mantelli, Aquila chrysaetos, Bos taurus, Coturnix japonica, Gallus gallus,Geospiza fortis, Homo sapiens, Meleagris gallopavo, Melopsittacus undulatus, Mus musculus, Numida meleagris, Ovis aries,Pan troglodytes, Rattus norvegicus, Sus scrofa and Zebra finch were used for Nucleotide BLAST. Top 5 most conservative results were listed above. Fig. S5. Prediction and identification of potential m6A modification sites on MYH1G-AS. (A) Potential m6A modification sites on MYH1G-AS were predict by using the SRAMP (http://www.cuilab.cn/sramp) software. (B–G) Relative single-base elongation and ligation-based PCR amplification method (SELECT) product at 250 (B), 436 (C), 495 (D), 970 (E), 1042 (F), and 1116 (G) sites of MYH1G-AS after ALKBH5 overexpression or interference. (H–M) Relative SELECT product at 250 (H), 436 (I), 495 (J), 970 (K), 1042 (L), and 1116 (M) sites of MYH1G-AS after ALKBH5 overexpression or interference. Results are presented as mean ± SEM. In panels B-M, the statistical significance of differences between means was assessed using an independent sample t-test. (NS, no significant difference). Fig. S6.GO functions and KEGG pathways analysis of differentially expressed genes between control group and MYH1G-AS interference. (A)GO functions analysis of differentially expressed genes between control group and MYH1G-AS interference. (B) KEGG pathways analysis of differentially expressed genes between control group and MYH1G-AS interference. Fig. S7. Overexpression of MYH1G-AS promotes myoblast proliferation but inhibits myoblast differentiation. (A–L) Relative MYH1G-AS expression (A), relative mRNA expressions of several cell cycle-inhibiting genes (B), EdU proliferation assays (C), proliferation rate of myoblasts (D), cell cycle analysis €, CCK-8 assays (F), MyHC immunostaining (G), myotube area (H), differentiation index (I), myoblast fusion index (J), and relative mRNA (K) and protein (L) expression levels of myoblast differentiation marker genes with MYH1G-AS overexpressionin vitro. In panel L, the numbers shown below the bands were folds of band intensities relative to control. Band intensities were quantified by ImageJ and normalized to β-Tubulin. Data are expressed as a fold-change relative to the control. Results are presented as mean ± SEM. In panels A, B, D–F, and H–K, statistical significance of differences between means was assessed using independent sample t-test. (* P &lt; 0.05; ** P &lt; 0.01). Fig. S8. Overexpression of MYH1G-AS represses mitochondria biogenesis to drive the transformation of slow-twitch to fast-twitch myofiber and induces muscle atrophy. (A-Q) Relative MYH1G-AS expression (A), relative mtDNA content (B), mitochondrial membrane potential (C), intracellular ROS ([ROS]i) (D), relative glycogen content (E), immunohistochemistry analysis of MYH1A/MYH7B (F), MYH1A/MYH7B protein content (G), frequency distribution of MYH1A+ (H) and MYH7B+ (I) myofiber CSA,  relative protein expression of MYH1A and MYH7B (J), relative mRNA expression of several fast- and slow-twitch myofiber genes (K), relative enzymes activity of LDH and SDH (L), relative gastrocnemius muscle weight (M), H&amp;E staining (N), frequency distribution of fiber CSA (O), and relative mRNA (P) and protein (Q) expression of FBXO25 in gastrocnemius with MYH1G-AS overexpression. In panel J and Q, the numbers shown below the bands were folds of band intensities relative to control. Band intensities were quantified by ImageJ and normalized to β-Tubulin. Data are expressed as a fold-change relative to the control. Results are showed as mean ± SEM. In panels A–E, G, K–M, and P, statistical significance of differences between means was assessed using independent sample t-test. (* P &lt; 0.05; ** P &lt; 0.01). Fig. S9. The mRNA level of MYH1G and FGF18 didn’t change with MYH1G-AS overexpression and knockdown both in vitro and in vivo. (A-B) Relative mRNA expression of MYH1G with MYH1G-AS overexpression and knockdown in vitro (A) and in vivo (B). (C-D)Relative mRNA expression of FGF18 with MYH1G-AS overexpression and knockdown in vitro (C) and in vivo (D). Results are shown as mean ± SEM. In all panels, statistical significance of differences between means was assessed using independent sample t-test. (NS, no significant difference). Fig. S10. The location and expression analysis of FGF18.(A) Subcellular location of FGF18 protein annotated by UniProt Knowledgebase (https://www.uniprot.org/). (B) Tissue expression profiles of FGF18. The horizontal axis and vertical axis indicate different tissues and their relative expression values, respectively. (C) Relative FGF18 expression during CPM proliferation and differentiation. Results are presented as mean ± SEM. In panels B-C, statistical significance of differences between means was assessed using independent sample t-test. (* P &lt; 0.05; ** P &lt; 0.01). Fig. S11. FGF18 inhibits myoblast proliferation, promotes myoblast differentiation, and facilitates mitochondria biogenesis. (A-R) Relative mRNA (A) and protein (B) expression levels of FGF18, relative mRNA expressions of several cell cycle-inhibiting genes (C), EdU proliferation assays (D), proliferation rate of myoblasts (E), cell cycle analysis (F), CCK-8 assays (G-H), MyHC immunostaining (I), myotube area (J), differentiation index (K), myoblast fusion index (L), relative mRNA (M) and protein (N) expression levels of myoblast differentiation marker genes, relative mtDNA content (O), mitochondrial membrane potential (P), intracellular ROS ([ROS]i) (Q), and relative FBXO25 expression (R) with FGF18 overexpression or interference in vitro. In panels B and N, the numbers shown below the bands were folds of band intensities relative to control. Band intensities were quantified by ImageJ and normalized to β-Tubulin. Data are expressed as a fold-change relative to the control. Results are showed as mean ± SEM. In panels A, C, E-H, J-M, and O-R, statistical significance of differences between means was assessed using independent sample t-test. (* P &lt; 0.05; ** P &lt; 0.01). Fig. S12. Neither MYH1G-AS nor FGF18 regulate the mRNA and protein expression of SMARCA5. (A-D) Relative mRNA (A and C) and protein (B and D) expression levels of SMARCA5 after MYH1G-AS overexpression or interference in vitro (A-B) orin vivo (C-D). (E-F) Relative mRNA (E) and protein (F) expression levels of SMARCA5 after FGF18 overexpression or interference. In panels B, D, and F, the numbers shown below the bands were folds of band intensities relative to control. Band intensities were quantified by ImageJ and normalized to β-Tubulin. Data are expressed as a fold-change relative to the control. Results are presented as mean ± SEM. In panels A, C,and E, statistical significance of differences between means was assessed using independent sample t-test. (NS, no significant difference). Fig. S13. FGF18 and SMARCA5 promotes the expression and transcription of SMAD4. (A-B) Protein expression levels of phosphorylated SMAD2 and phosphorylated SMAD3 after MYH1G-AS overexpression or interference in vitro (A) or in vivo (B). (C-F) Relative SMAD4 mRNA expression (C), protein expression levels of SMAD4, phosphorylated SMAD2 and phosphorylated SMAD3 (D), the transcriptional activity of SMAD4 core promoter region (E), and ChIP analysis of the binding capacity of POU2F1 to SMAD4 promoter (F) with FGF18 overexpression or knockdown. (G-L) Relative mRNA (G) and protein (H) expression levels of SMARCA5, relative SMAD4 mRNA expression (I), protein expression levels of SMAD4, phosphorylated SMAD2 and phosphorylated SMAD3 (J), the transcriptional activity of SMAD4 core promoter region (K), and ChIP analysis of the binding capacity of POU2F1 to SMAD4 promoter (L) with SMARCA5 overexpression or knockdown. In panels A-B, D, H, and J, the numbers shown below the bands were folds of band intensities relative to control. Band intensities were quantified by ImageJ and normalized to β-Tubulin. Data are expressed as a fold-change relative to the control. Results are presented as mean ± SEM. In panels C, E-G, I, and K-L, statistical significance of differences between means was assessed using independent sample t-test. (* P &lt; 0.05; ** P&lt; 0.01).