Fig 1: cZYG11B regulates RGC function by acting as a miR-620 sponge. (A) Total cellular fractions were isolated from RGCs and immunoprecipitated using Ago2 or IgG antibody. The amount of cZYG11B in the IP was determined by RT-qPCR. *P<0.05 as indicated. (B) RGCs were transfected with pGL3-Basic (Ctrl) or Luc-cZYG11B with various miRNA mimics and pRL-TK vector (internal transfection control). Luciferase activity was detected at 24 h post-transfection using the Dual-Luciferase Reporter Assay kit. *P<0.05 vs. Ctrl. (C) Biotinylated miR-620 or miR-95 were transfected into RGCs. After streptavidin capture, the amount of cZYG11B and cZNF236 (negative control) in the input and bound fractions were detected by RT-qPCR. The relative IP/input ratios were plotted. *P<0.05 as indicated. (D) RGCs were transfected with Scr mimic, miR-620 mimic or were untreated. RT-qPCR was conducted to detect PTEN expression. *P<0.05 vs. Ctrl. (E and F) RGCs were left untreated or transfected with Scr mimic, miR-620 mimic, miR-620 mimic + pcDNA 3.1 (Vector) or PTEN-pcDNA 3.1 (PTEN) for 12 h. The medium was replaced and cells were cultured for an additional 12 h at 37°C. These groups were then exposed to 1% O2 to mimic hypoxic stress for 24 h at 37°C. (E) CCK-8 assays were conducted to detect the viability of RGCs. (F) TUNEL staining assays and semi-quantification analysis were conducted to detect the apoptosis of RGCs. Scale bar, 50 µm. *P<0.05 vs. hypoxia group; #P<0.05 as indicated; NS, no significant difference. All significant differences were evaluated by (A and C) Student's t-test or (B, D-F) one-way ANOVA followed by the post hoc Bonferroni test. n=4. Ctrl, control; cZYG11B, circZYG11B; IP, immunoprecipitated; miR, microRNA; RGC, retinal ganglion cell; RT-qPCR, reverse transcription-quantitative PCR; Scr, scrambled.
Fig 2: Age- dependent correlation of HOXA5, PTEN and p53 mRNA expression. (A) mRNA expression levels of HOXA5, PTEN and p53 are downregulated in the old subjects compared to the young cohort. (B) Moderate to strong linear correlations were observed between HOXA5 and PTEN mRNA expression (r = 0.7115) (C) between HOXA5 and p53 mRNA expression (r = 0.6294), and (D) between p53 with PTEN mRNA expression (r = 0.6685). Detection of mRNA expression was performed by qRT-PCR on total cellular RNA prepared from monocytes. Error bars denote SEM (n = 30, average age: 23.0 yrs and 81.1 yrs, respectively).
Fig 3: Clinical relevance of cZYG11B-mediated signaling in I/R-related ocular disease. Aqueous humor samples were collected from patients with glaucoma (n=20) and patients with cataracts (Ctrl; n=20). Reverse transcription-quantitative PCR was conducted to detect the expression levels of (A) cZYG11B and (B) miR-620. (C) ELISA was conducted to detect the concentration of PTEN. The significant differences were determined by unpaired Student's t-test. *P<0.05 vs. Ctrl group. Ctrl, control; cZYG11B, circZYG11B; miR, microRNA.
Supplier Page from Abcam for Human PTEN ELISA Kit