Fig 2: ATF4 deficiency weakens apoptosis and protects human NP cell degeneration. NP cells of G2 tissues without or with the ATF4-siRNA transfection were cultured with 100 µM of H2O2 for 24 h. (A and B) The protein expression level of collagen II and caspase-9 were determined by (A) IF (magnification, x400) and (B) quantification analysis. (C) The mRNA expression levels of collagen II, ATF4, CHOP and caspase-9 were assayed by RT-PCR. (D and E) The protein expression levels of collagen II, ATF4, CHOP and caspase-9 were determined by (D) WB and (E) quantification analysis. (F and G) The ratio of apoptotic cells was analyzed by (F) flow cytometry and (G) quantification analysis. The values are mean ± SD of three independent experiments (n=3). (*P<0.05, **P<0.01, ***P<0.001, compared with 100 µM H2O2). ATF4, activating transcription factor 4; CHOP, C/EBP homologous protein; WB, western blot; NP, nucleus pulposus; RT-PCR, reverse transcription-polymerase chain reaction; IF, immunofluorescence.

Fig 3: CHOP deficiency weakens apoptosis and protects human NP cell degeneration. NP cells of G2 tissues without or with the CHOP-siRNA transfection were cultured with 100 µM of H2O2 for 24 h. (A and B) The protein expression level of collagen II and caspase-9 were determined by (A) IF (magnification, x400) and (B) quantification analysis. (C) The mRNA expression levels of collagen II, ATF4, CHOP and caspase-9 were assayed by RT-PCR. (D and E) The protein expression levels of collagen II, ATF4, CHOP, and caspase-9 were determined by (D) WB and (E) quantification analysis. (F and G) The ratio of apoptotic cells was analyzed by (F) flow cytometry and (G) quantification analysis. The values are mean ± SD of three independent experiments (n=3). (*P<0.05, **P<0.01, ***P<0.001, compared with 100 µM H2O2). ATF4, activating transcription factor 4; CHOP, C/EBP homologous protein; WB, western blot; NP, nucleus pulposus; RT-PCR, reverse transcription-polymerase chain reaction; IF, immunofluorescence.

Fig 4: ROS and apoptosis levels in human degenerated intervertebral disc tissues. Tissues were lysed and ROS measured with MDA methods. Total protein and mRNA were extracted from the tissues in mild (G2-G3) and severe (G4-G5) degeneration. (A) MDA level of different degenerated degrees of disc tissues. (B and C) The protein levels of collagen II, ATF4, CHOP and caspase-9 were determined by (B) Western blot and (C) quantification analysis. (D) mRNA levels of collagen II, ATF4, CHOP and caspase-9 were determined by RT-PCR. The values are mean ± SD of three independent experiments (n=3). (*P<0.05, **P<0.01, ***P<0.001, compared with G2-G3 group). ROS, reactive oxygen species; ATF4, activating transcription factor 4; CHOP, C/EBP homologous protein; RT-PCR, reverse transcription-polymerase chain reaction.

Fig 5: Apoptosis levels in H2O2 induce NP cells in vitro. NP cells of G2 tissues were cultured with a low or high concentration of H2O2 (50 or 100 µM) for 24 h. (A and B) The protein expression level of collagen II and caspase-9 were determined by (A) IF (magnification, x400) and (B) quantification analysis. (C) The mRNA expression levels of collagen II, ATF4, CHOP, and caspase-9 were assayed by RT-PCR. (D and E) The protein expression levels of collagen II, ATF4, CHOP and caspase-9 were determined by (D) WB and (E) quantification analysis. (F and G) The ratio of apoptotic cells was analyzed by (F) flow cytometry and (G) quantification analysis. The values are mean ± SD of three independent experiments (n=3). (*P<0.05, **P<0.01, ***P<0.001, compared with control; &P<0.05, &&P<0.01, compared with 50 µM H2O2). ATF4, activating transcription factor 4; CHOP, C/EBP homologous protein; WB, western blot; NP, nucleus pulposus; RT-PCR, reverse transcription-polymerase chain reaction; IF, immunofluorescence.