Fig. 1

Identification and distribution of circLIFR. a Scheme illustrating the production of circLIFR. b, c The expression of circLIFR was detected by qRT-PCR in 79 pairs of bladder cancer and paired adjacent normal bladder tissues, SV-HUC-1, UROtsa, T24, and UMUC3 cells. GAPDH was used as internal control. Data were mean ± SD. ***P < 0.001 (Student’s t-test). d Kaplan-Meier curves of OS in bladder cancer patients. Patients were grouped by the median circLIFR expression. P-value was calculated using a log-rank test. e Sequencing analysis of head-to-tail splicing junction in circLIFR. f The existence of circLIFR was validated in T24 and UMUC3 bladder cancer cell lines by qRT-PCR. Divergent primers amplified circLIFR in cDNA but not genomic DNA (gDNA). GAPDH was used as negative control. Red arrows indicated divergent primers, and black arrows indicated convergent primers. g The relative RNA levels were analyzed by qRT-PCR in T24 and UMUC3 cells treated with or without RNase R. Data were mean ± SD, n = 3. ns, not significant, ***P < 0.001 (Student’s t-test). h The relative RNA levels of circLIFR and mLIFR were analyzed by qRT-PCR after treatment with actinomycin D at the indicated time points in T24 cells (n = 3). i Identification of circLIFR cytoplasmic and nuclear distribution by qRT-PCR analysis in T24 cells. GAPDH and U1 were applied as positive controls in the cytoplasm and nucleus, respectively (n = 3). Western blots of total cell lysates (T), cytosolic extracts (C) and nuclear extracts (N) with α-tubulin as a cytosolic marker, histone H3 as a nuclear marker. j Identification of circLIFR cytoplasmic and nuclear distribution by FISH in T24 cells. 18S and U6 were applied as positive controls in the cytoplasm and nucleus, respectively; circLIFR, 18S, and U6 probes were labeled with Cy3; nuclei were stained with DAPI