Fig. 3

circPINTexon2 encodes an 87-aa peptide. a Full-length or truncated circPINTexon2 IRES (478, 231, and 209 bp) were cloned between mCherry and GFP as indicated to construct several reporter plasmids. These plasmids were transfected into 293T cells as indicated, with or without 4EGI-1 treatment. IF was performed to determine mCherry and GFP signals. Scale bars, 50 μM. b Rluc and Luc were tandemly cloned into the luciferase reporter plasmid, with or without the indicated truncated IRES between them. Luc/Rluc activities were measured in each transfected plasmid. c RNC-RNA or total RNA from 293T cells was extracted and reverse-transcribed using oligo-dT or random primers as indicated. Specific primers for circPINTexon2 or LINC-PINT were analyzed by using q-PCR. d Upper, antibody recognition test for the predicted 87-aa peptide. Lanes 1 and 2, Coomassie blue staining of the GST-PINT87aa fusion protein; lanes 3 and 4, Western blot performed with GST antibody; lanes 5 and 6, Western blot performed with PINT87aa antibody. Lower, the predicted 87-aa peptide sequence and antibody generation region was shown as indicated. e Endogenous immunoprecipitation using anti-PINT87aa antibodies in 293T cells. LC-MS/MS analysis following SDS-PAGE was performed to identified peptide sequences of PINT87aa. f Upper panel, endogenous circPINTexon2: endogenous formation of circPINTexon2; CircPINTexon2 vector: the artificial circPINTexon2 overexpression plasmid. Note the junction was moved inside the 87-aa ORF. CicrPINTexon2 Del-IRES vector: negative control, in which the IRES sequence was deleted from the artificial circPINTexon2 plasmid. 87-aa overexpression vector: positive control, in which the 87-aa ORF was cloned downstream of a linear CMV promoter. Lower panel, PINT87aa expression was tested in 293T cells after transfection of the plasmids indicated above. g circPINTexon2 and PIN87aa expression were determined using junction-specific siRNA or shRNA specific for circPINTexon2-transfected 293T or hNSC. h Left, LINC-PINT, circPINTexon2, and PIN87aa expression were determined in two ASOs specific for LINC-PINT-transfected 293T. Right, LINC-PINT and PINT87aa were determined in LINC-PINT stably transduced 293T and hNSC. b, c, g, h Data are presented as mean ± s.e.m. from three independent experiments. **P < 0.01; ns, P > 0.05, determined by two-tailed Student’s t-tests