Fig. 5.

Upon exposure to PIC, Lnc13 translocates from the nucleus and facilitates the interaction between the PCBP2 and the 3′-UTR of STAT1 mRNA. (A) EndoC-βH1 cells were exposed to PIC (1 µg/mL) for 24 h, and relative Lnc13 expression was determined in nucleus and whole extracts. Expression of MALAT1 and RPLPO was used as a control for nucleus and whole cell fractions, respectively. Amounts of specific nuclear RNA were measured by Q-PCR and compared to the total amount of RNA in the whole cell. Results are represented as a logarithm (nucleus/whole) and are means ± SEM of three independent experiments; *P < 0.05; Student’s t test. (B) EndoC-βH1s were exposed to PIC (1 µg/mL) for 24 h and cytoplasmic (Cyt), nuclear (Nuc), and chromatin (Chro) fractions were purified. PCBP2 protein expression was determined in all cellular compartments by Western blot and protein expression of Hsp90, HDAC1, and H3 was used as a control for cytoplasmic, nuclear, and chromatin fractions, respectively. The results are representative of three independent experiments. (C) EndoC-βH1 cells were left nontransfected (NT) or transfected with PIC for 24 h. RNA immunoprecipitation was performed using a specific antibody for PCBP2 and Lnc13, and STAT1 expression was determined in PCBP2-bound RNA by Q-PCR. Results are means ± SEM of three independent experiments, and the amounts of Lnc13 and STAT1 are expressed as relative to the input. IgG was used as the negative control; ***P < 0.001 and **P < 0.01 as indicated; ANOVA followed by Student’s t test with Bonferroni correction. (D) In vitro transcribed biotinylated 3′-UTR region of STAT1 was incubated with cellular extracts overexpressing Lnc13-C+PCBP2, Lnc13-T+PCBP2, or PCBP2. Afterward, 3′-UTR-STAT1-bound proteins were purified using streptavidin beads, and PCBP2 and Hsp90 (as the negative control) were detected by Western blot. Incubation with streptavidin beads alone was used as the negative control. The results are representative of three independent experiments. (E) Densitometry results for purified PCBP2 amounts in RNA pull down experiments are represented as means ± SEM of three independent experiments. ANOVA followed by Student’s t test with Bonferroni correction; ***P < 0.001 and *P < 0.01 vs. negative control; ^†††P < 0.001 and ^†P < 0.05 as indicated. (F) Lnc13 antisense purification was performed in nontreated (NT) and PIC-treated nuclear and cytoplasmic fractions of EndoC-βH1 cells. Lnc13-bound 3′-UTR-STAT1 amounts were determined by Q-PCR using specific primers. A nonrelated similar lncRNA was used as the negative control (Ctrl). Results are represented as fold enrichment and are means ± SEM of four independent experiments. *P < 0.05 as indicated; ANOVA followed by Student’s t test with Bonferroni correction. (G) RNA-protein interaction assay. Cells were transfected with pPCBP2 alone, pPCBP2+pLnc13-C, pPCBP2+pLnc13-T, or pPCBP2+pLnc13-delSNP. Cell lysates were incubated with in vitro transcribed 3′-UTR-STAT1 molecules, and a native agarose gel electrophoretic mobility shift assay was performed (Lower). After electrophoresis, proteins were transferred to a nitrocellulose membrane for PCBP2 visualization (Upper). The 3′-UTR-STAT1 RNA molecule alone was loaded as the Ctrl. The results are representative of three independent experiments. (H) EndoC-βH1 cells were transfected with a plasmid encoding Lnc13-C or a mutant Lnc13 in which the region containing the T1D-associated SNP was deleted (pLnc13-delSNP). STAT1, CXCL10, and CCL5 expressions were determined by Q-PCR and normalized by the housekeeping gene β-actin and corrected by Lnc13 expression values. Results are means ± SEM of four independent experiments. P < 0.05 as indicated; Student’s t test.