Fig 2: Hevin expression is altered at postnatal day (P) 7 and P21 in the hippocampus of Fmr1 KO mice. (A–C) Hevin expression, determined via Western blotting, in the hippocampus of WT (black; n = 8) and Fmr1 KO (white; n = 8) mice at P7, P14 and P21, respectively. Bands representing hevin were normalized against the total protein within the same lane on the membrane and cross gel controls, then expressed as a percent of the average level of hevin in the WT group. (D–F) Representative western blots show hevin (~130 kDa) in hippocampal samples (30 µg of protein per lane) from WT and Fmr1 KO mice at P7, P14 and P21, as well as the total protein within each lane. Statistical differences were denoted with either a single asterisk, P < 0.05, or a triple asterisks, P < 0.0005.

Fig 3: SPARCL1 regulates C2C12 cell differentiation through BMP/TGF-ß signaling pathway via BMP7.a shows the expression of proteins related to TGF-ß signaling pathway. b–h are greyscale scans of proteins in A. h shows Desmin expression in C2C12 cells when SPARCL1 was activated and BMP7 was inhibited at 72 h. i is the corresponding immunofluorescence blot of Desmin protein in A. In a, i, pSPgRNA-S-2 is the SPARCL1 activation group, while pSPgRNA is the SPARCL1 promotes C2C12 cell differentiation via BMP7-mediated BMP/TGF-ß blank control for SPARCL1 activation. SiRNA-B is the BMP7 interference group. NC is a negative control for BMP7 siRNA interference. The scale bar in I is 100 µm, green fluorescent signal is Desmin, and blue fluorescent signal is the nucleus. **P values < 0.01 and *P values < 0.05 were considered as significant

Fig 4: BMP7 influences C2C12 cells differentiation.a, e show the expression of BMP7 protein activated or inhibited in C2C12 cells when the cells were induced to differentiate at 72 h. pSPgRNA-B-3 was the BMP7 activation group, pSPgRNA was the blank control for BMP7 activation. NC was a negative control for BMP7 siRNA interference. b–d shows greyscale scans of the proteins in A. f–h shows greyscale scans of the proteins in e. i, k shows Desmin expression in C2C12 cells when SPARCL1 was activated or inhibited at 72 h. j, l shows quantification of myotubes according to Desmin staining in i, k, respectively. The SPARCL1 promotes C2C12 cell differentiation via BMP7-mediated BMP/TGF-ß scale bar in i, k is 100 µm, and the green fluorescent signal is Desmin, while the blue fluorescent signal is the nucleus. **P values < 0.01 and *P values < 0.05 were considered as significant

Fig 5: SPARCL1 influences C2C12 cell differentiation.a, e shows the expression of SPARCL1 protein activated or inhibited in C2C12 cells when the cells were induced to differentiate at 72 h. pSPgRNA-S-2 is the SPARCL1 activation group and pSPgRNA is the blank control for SPARCL1 activation. NC was the negative control for SPARCL1 siRNA interference. b–d are grayscale scans of the proteins shown in a. f–h are grayscale scans of the proteins shown in e. i, k show Desmin expression in C2C12 cells when SPARCL1 was activated or inhibited at 72 h. j, l shows the quantification of myotubes according to the Desmin staining of I and K. The scale bar in I and K is 100 µm; the green fluorescent signal is Desmin, while the blue fluorescent signal is the nucleus. **P < 0.01 were considered as significant