Fig 2: Time-course of autophagy-related protein levels in the retinas of the rats after circumlimbal suture application. (A) Western blotting showing a decrease in the LC3-II/LC3-I ratio in the retinas of the rats 1 day after the surgery detected compared to that in control animals. 1 and 4 weeks after the circumlimbal suture placement, the LC3-II/LC3-I ratio significantly increased compared to control rats, indicating a late autophagy induction after the OHT insult. (B) Immunoblotting for p62, another important autophagic marker, showed increased protein levels 1 day after circumlimbal suturing, which decreased to baseline levels 1 and 4 weeks later. (C,D) Western blotting for ATG4 (C) and ATG7 (D) revealed a significant increase in their protein levels 1 and 4 weeks after circumlimbal suture placement, while no significant changes were observed one and 3 days after the surgery compared to contralateral control eyes. (E) Time-course of Beclin-1 expression in OHT retinas showed similar to ATG4 and ATG7 pattern, with no significant changes in the protein levels one and 3 days after the surgery and a profound increase 1 week after the surgery. Histograms represent the densitometric values of the protein bands shown in Western blotting. Data are normalized to β-actin and expressed as mean ± SEM. *p < 0.05 versus control (non-OHT) retina; statistical significance was determined using the Student’s t-test (see supplementary material).

Fig 3: HuR regulates Atg7 expression through binding to Atg7 AU-rich element and upregulating its mRNA level. The cells were transfected with LV-shHuR or LV-HuR before high-glucose treatment (100 mM). A, Multiple autophagy-related mRNA expressions in NP cells with high-glucose treatment, detected by qPCR. B-D, The protein expression of Atg7 in NP cells with or without high-glucose treatment (100 µM) for 6 hours, detected by Western blot and quantified by Image J software. E, Potential HuR-binding element at 3'UTR of Atg7 mRNA on multiple species. F, Schematic diagram showing the workflow of the RNA-binding protein immunoprecipitation (RIP) assay. G, The mRNA expression of Atg7 in rat NP cells, detected by qPCR, and normalized to IgG isotype controls. All data were shown as mean ± SD. *P < .05, **P < .01

Fig 4: Atg7 reverses HuR‐down‐regulated autophagy in high glucose‐treated NP cells. The cells were transfected with LV‐NC or LV‐Atg7 before high‐glucose treatment (100 mM) or LV‐shHuR. A, B, The protein expression of Atg7 in rat NP cells with LV‐Atg7 transfection, detected by Western blot and quantified by Image J software. C, TEM images of autophagic vesicles in rat NP cells, with LV‐NC or LV‐Atg7 (scale bar: 0.5 μm). D, LC3‐II was detected by immunofluorescence combined with DAPI staining for nuclei (scale bar: 20 μm) E, F, The protein expression of p62 and the ratio of LC3‐II/ LC3‐I were detected by Western blot and quantified by Image J software. All data were shown as mean ± SD. *P < .05, **P < .01, &P < .001

Fig 5: Overexpression of Atg7, instead of HuR, ameliorates the progress of DB-IVDD in vivo. A, T2 weighted MRI of a rat-tail disc at 3 weeks after disc puncture surgery, with or without lentivirus transfection (white arrows). B, The respective Pfirrmann grade scores of a rat-tail disc. C, X-ray of a rat-tail disc at 4 weeks after disc puncture surgery in normal and diabetic rats, with or without lentivirus transfection (white arrows). D, The disc height index (DHI) of a rat-tail disc. E, Representative H-E, S-O and Alcian Blue staining of NP tissues (scale bar: 800 µm). F, The histological grades evaluated in three groups. G-I, The immunohistochemical staining of p16 and LC3-II in intervertebral disc sections; the levels were determined using Image-Pro Plus software. All data were shown as mean ± SD. *P < .05, **P < .01