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. 2021 Jan 1;11(4):1732–1752. doi: 10.7150/thno.45302

Figure 4.

Figure 4

CircPGR is stably localized in the cytosol of cells. (A) MCF7 cells cultured in stripping medium and treated with or without estrogen (E2, 10-7 M) for duration as indicated were subjected to RNA extraction, reverse transcription and standard PCR by using primer sets specifically targeting circPGRs or PGR. The resultant PCR products were separated by DNA electrophoresis. Actin was served as a loading control. DNA marker (M) was shown as indicated. bp: base pair. (B) Prediction and visualization of full length circPGR expression in both control (CTL) and estrogen (E2)-treated conditions by CIRI-full and CIRI-vis, respectively, based on circRNA-seq as described in Figure 1C. (C, D) MCF7 cells cultured in stripping medium and treated with or without estrogen (E2, 10-7 M, 24 h) were subjected to polysome profiling and the resultant fractions were subjected to RNA exaction and RT-qPCR analysis to examine the expression of circPGR (C) and PGR (D). Fractions 1 to 3: free RNA (unbound with ribosome); Fraction 4: 40S (40S ribosomal subunit); Fractions 5 and 6: 60S (60S ribosomal subunit); Fractions 7 to 9: monosome; Fractions 10 to 15: polysome. (E) Total RNAs extracted from MCF7 cells cultured in stripping medium and treated with or without (E2, 10-7 M, 6 h) were incubated with or without RNase R (10 units/μg RNA) at 37 °C for 1 h, followed by RT-qPCR analysis to examine the expression of PGR or circPGR. (F) Estrogen-treated MCF7 cells were added with Actinomycin D (10 μg/ml) for duration as indicated, followed by RT-qPCR analysis to examine the expression of PGR or circPGR (± s.e.m., ***P < 0.001). (G) MCF7 cells cultured in stripping medium and treated with or without estrogen (E2, 10-7 M, 6 h) were subjected to cellular fractionation followed by RNA extraction and RT-qPCR analysis to quantify the amount of circRNA in both nucleus and cytosol of the cells. ACTIN and U6 snoRNA were served as purity control for cytosolic and nuclear fractions, respectively. (H) RNA-FISH analysis was performed in MCF7 cells transfected with si-CTL or si-circPGR and treated with estrogen (E2, 10-7 M, 6 h). Red: circPGR; Blue: DAPI.