Fig. 2

Identification of exon 2 of LINC-PINT as a circRNA. a Upper, visualization of the forward reads within the exon 2 region in the LINC-PINT junction site of NHA cell in RNA-seq and RNC-seq. These junction reads are specific for circular form of LINC-PINT exon 2. Lower, IGV plot of all reads located on exon 2 of LINC-PINT in RNA-seq and RNC-seq. The IGV plot also included the reads on exon 1 and 3 of LINC-PINT. b Illustration of the annotated genomic region of LINC-PINT (Ensembl number: ENSG00000231721), the putative different mRNA splicing forms (linear splicing and head-to-tail splicing) and the validation strategy for LINC-PINT circular exon 2 (circPINTexon2). Divergent primers detected the circular form of circPINTexon2 in cDNA but not in gDNA. Convergent primers spanning exon 1 and exon 2 of LINC-PINT (variants LINC-PINT-208, shown in a) specifically detected the linear splicing form. β-actin was used as a linear RNA control. c Sanger sequencing was performed following PCR using the indicated divergent flanking primers to confirm the head-to-tail splicing of circPINTexon2 in 293T cells. d Northern blots of 293T total RNA with the exon probe and the junction-specific circular probe for circPINTexon2. Lanes 1–4 detected circPINTexon2 with circular probes. Lanes 5–8 detected circPINTexon2 and LINC-PINT with exon probes. CircPINTexon2-overexpression plasmid was shown in Fig. 3f. e Q-PCR followed by with junction-specific primers was used to detect the expression of circPINTexon2 in vitro. Primers specific for linear LINC-PINT were also used to detect LINC-PINT expression. RNase R treatment was used to validate circPINTexon2. 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. f FISH with junction-specific probes specific to circPINTexon2, whereas linear specific probes specific to linear LINC-PINT indicated their cellular localization in vitro. Normal brain tissues and GBM samples were stained with indicated probes. Overexpressed or knocked-down circPINTexon2 or LINC-PINT using corresponding plasmids or siRNA/ASOs in 293T cells to indicate the specificity of these probes. Scale bars, 10 μM. EV empty vector, si-NC random scrambled siRNA, circPINTexon2 circPINTexon2 overexpression vector, ASO LINC-PINT anti-sense oligos