Fig 2: Verification of miRNA target sites. (A) miR-3163 could be overexpressed by Mimics (Mim) as measured by qRT-PCR. Scramble (Scr) was used as the negative control. (B) Western blot analysis showing the protein levels of ABCG2 in RCSCs treated with Scramble or miR-3163 Mimics. GAPDH was used as a loading control. (C) Luciferase activity was measured in HeLa cells co-transfected with miR-3163 and either wide-type Reporter ABCG2 3'UTR (grey) or mutated Reporter ABCG2 3’UTR (black). Renilla activity was used as an internal control. * P < 0.05 compared with the control. The data represent the results of three independent experiments.

Fig 3: Identification of RCSCs in the WERI-Rb1 retinoblastoma cell line. (A) ABCG2 positive (ABCG2+) and negative (ABCG2-) cells were sorted by flow cytometry based on expression of the RCSC surface marker ABCG2. For FACS sorting, the blank group was incubated with IgG2a antibody, whereas the ABCG2+ group was incubated with ABCG2 antibody. The area of R3 was the ABCG2+ cells sorted for further research, the rest cells was ABCG2- cells. (B) Expression levels of ABCG2, OCT4 and NANOG were detected in ABCG2+ and ABCG2- cells by Western blot.

Fig 4: Representative photomicrographs of putative progenitor cell markers and associated proteins in the human and rat LGs. Expression of nestin appeared to be restricted to blood vessels (arrowheads) in both the human (A) and rat (B) LGs. Occasional ABCG2-positive cells were present within both the human (C) and rat (D) LGs (arrowheads). In the human LG, expression of CD34 was observed in blood vessels plus other cells surrounding acini (E). CD34 was non-reactive in rat tissue (F). Ki-67-positive cells were infrequently encountered in both the human (G) and rat (H) LGs, being typically located within acini (arrows) when observed. Smaller ki-67 positive cells were evident in rat LG in close proximity to ducts (arrowheads). Comparison of numbers of Ki-67-positive cells in the human and rat LGs revealed that there was no statistically significant difference between the two species (non-paired Student's t-test; n = 12 for each; I). Scale bar = 60 µm.

Fig 5: miRNA prediction and expression of miR-3163 in RCSCs. (A) Putative miR-3163-binding sites on the ABCG2 3'UTR with potential complementary residues shown in black. (B) Expression levels of miR-3163 were detected in ABCG2+ and ABCG2- cells by qRT-PCR. * P < 0.05 compared with the control. The data represent the results of three independent experiments.