Fig. 8

G4 promotes NFATC2 transcription by changing promoter mCpG. A The left panel is a schematic diagram of PSA. Simply put, after G4 is formed, it is not easy or cannot be unlocked to initiate the PCR reaction. The G4 and mutant G4 sequences were amplified with a specific FAM labelled primer, under the G4 induction or not. PCR product couldn’t be obtained with a G4 induction, while PCR product could be obtained without G4 induction. The PSA on mutant G4 sequence was used as a negative control (n = 3). B The upper panel is the principle schematic diagram that NFATC2 G4 specific ASO regulates G4 formation. During G4 induction, ASO and unspecific single-strand DNA were separately added. The specific primer was used to PCR amplification. Double-strand DNA (PCR product) could be obtained with ASO treatment, while double-strand DNA could not be obtained with ASO control (n = 3). C Total RNA was extracted from myoblasts that treated with NFATC2 G4 specific ASO or 5-AZA and mRNA level of NFATC2 was quantified by qPCR (n = 6). Student’s t-test was used to analyze the difference among groups and *P < 0.05. D DNA was extracted from myoblasts that treated with NFATC2 G4 specific ASO or 5-AZA and mCpG level was quantified by MSP (n = 6 for NC, n = 11 for ASO control, n = 12 for ASO and n = 12 for ASO + 5-AZA). Student’s t-test was used to analyze the difference among groups. *P < 0.05 and ****P < 0.0001. E Total RNA was extracted from the myoblasts treated with NFATC2 overexpression or knockdown and mRNA levels of MyoD1, MyoG and MyHC were quantified by qPCR (n = 6). Student’s t-test was used to analyze the difference among groups. *P < 0.05 and **P < 0.01. F Myoblasts treated with NFATC2 overexpression or knockdown for 48 h were collect for MyoG-immunofluorescence staining and quantified in a flow cytometry. The fluorescence value ≥ 10⁴ was considered as the threshold for MyoG-stained cells. Student’s t-test was used to analyze the difference among groups. **P < 0.01, ****P < 0.0001. G Myoblasts treated with NFATC2 overexpression and knockdown in inducing myotube fusion for 72 h (n = 3). Myotubes were labelled by MyHC immunofluorescence and captured in a fluorescence microscope. ImageJ was used to measure myotube fusion index. Student’s t-test was used to analyze the difference among groups. **P < 0.01, ****P < 0.0001