Fig 2: YAP localization and the Hippo pathway are regulated by nucleus pulposus cell (NPC) density(A) NPCs were cultured at different densities, ranging from sparse density to confluence. (B and C) YAP localization is affected by cell density. YAP was stained with anti-YAP antibody (B) and cells in five random views were quantified for YAP subcellular localization (C). (D and E) Western blot analysis of Hippo pathway-related proteins using anti-LATS1, anti-LATS2, and anti-YAP antibodies (D). Histogram showing average relative protein levels normalized to GAPDH (* P<0.05).

Fig 3: AAV-delivery of SaCas9 for genome editing in the left thigh of aged C57BL/10 male mice.a T7EI assay of mice genes produced by intramuscular injections of rAAV–SaCas9. Numbers of mutant muscle tissues with bands of 168, 338 and 506 bp in AAV-sgMstn2, RAAV-sgMstn2 and only one band of 506 bp in AAV-CN. The red arrow indicates the band of enzyme digestion. b Stacked histogram showing the percentage distribution of indels at Mstn in muscle tissues of mice, as measured by sequencing analyses. Data are presented as mean ± SEM from five mice in each group. c The muscle tissues in AAV-sgMstn2, RAAV-sgMstn2 and AAV-CN groups analyzed by western blot using an anti-MSTN antibody. And anti-GAPDH was used as loading control. d Stacked histogram showing the percentage distribution of MSTN expression in different muscle tissues. Data represent means ± SEM (The number of samples was 3 per group, and each sample was repeated three times). *: 0.01 < P < 0.05, **P < 0.01 when AAV-sgMstn2, RAAV-sgMstn2-treated were compared with AAV-CN-treated. e Changes of left thigh muscle mass in the aged mice in AAV-CN, AAV-sgMstn2 and RAAV-sgMstn2 groups. Compared with the AAV-CN-treated group, the muscle mass of the MSTN gene knockout group was significantly increased by AAV-sgMstn2 and RAAV-sgMstn2-treated groups (in red cycle). f Changes in body weight from one to eight weeks in different experimental groups. Data represent means ± SEM, N = 10 per group. g, h The weight of the quadriceps and adductor muscle in the different groups at 8 week. Data represent means ± SEM, N = 10 per group. *: 0.01 < P < 0.05, **P < 0.01, when AAV-sgMstn2, RAAV-sgMstn2-treated were compared with AAV-CN-treated.

Fig 4: Effect of TS and APS on protein levels of ERS markers in transported-chicks. (A) The protein levels of ERS-UPR-related factors detected by Western blotting analysis; GAPDH was served as loading control for the total fractions respectively. (B) Luminance value analysis of XBP-1s/XBP-1u. (C) AFT4/GAPDH. (D) ATF6/GAPDH. (E) XBP-1s/XBP-1u. (F) Correlation analysis between ERS markers and damage score. Data are presented as the mean ± SD. *P < 0.05 and **P < 0.01. At the same time point, the same letters mean the difference is not significant (P > 0.05), different small letters mean a significant difference (P < 0.05) and different capital letters for the extremely significant difference (P < 0.01).

Fig 5: The rAAV-DJ/8-SaCas9 system efficiently targets the Mstn site in C2C12 cells. a The treatment groups infected into C2C12 cells with two dose rAAV-DJ/8-sgMstn1 and the control group was wild type cells cultured in the same batch. A 521 bp region of the sgMstn1 locus including the site targeted for editing was PCR amplified from the genomic DNA and analyzed for genome editing with a T7 endonuclease digestion identification method. The top band on the gel was uncut DNA at 521 bp in length, and the two smaller bands were edited DNA at 181 and 340 bp in length for sgMstn1 at 96 h, 120 h post infection (red arrow indicated). The nucleic acid marker was a 50 bp DNA ladder. b The percentage distribution of indels at sgMstn1 in C2C12 mutant cells, as measured by sequencing analyses. Data represent means ± SD from three technical replicates. c The treatment groups and the control group were analyzed by western blotting with an anti-MSTN antibody. Anti-GAPDH was used as a loading control. d Stacked histogram showing the percentage distribution of MSTN expression in different groups. The MSTN value of each group was divided by the corresponding GAPDH value, and then the data for each group were divided by the same multiple to normalize by the control group value. Data represent means ± SD from three technical replicates