Fig 2: Effect of Adeno-siRNA-LIP on JCPyV-infected SVGA cells transiently transfected with C/EBPß-LIP expression plasmid in presence or absence of IFNa or IFNß treatment. Western blot on protein lysis from SVGA cells co-infected with JCPyV Mad-1 at MOI of 1 and Adeno-siRNA-LIP at MOI of 5 after co-transfected with pLIP and treatment with IFNa (A–D) or IFNß (E–H) for 5 days. On the 5th day post-infection (p.i.), the cells were harvested in order to evaluate the expression of LIP (B,F) and the late capsid protein VP1 (C,G) by Western blot and to measure the viral load in the supernatant culture media by quantitative PCR (Q-PCR) (D and H). Note: * p < 0.05; ** p < 0.01; *** p < 0.0001.

Fig 3: Endogenous expression of c/EBPß-LIP (LIP) after treatment with IFNa or IFNß. Western blot on protein lysis from SVGA cells collected 4, 8, 12, 24, 36 and 48 h after a single treatment with IFNa (A–C) or IFNß (D–F), respectively, in order to evaluate the endogenous expression of LIP (A,D). ?-tubulin was used as loading control and endogenous expression of STAT1 (B,E) and its phosphorylated form (C,F) (phosphoSTAT1 or pSTAT1) were also analyzed. For each panel, a densitometry assessment of LIP and STAT1 was also included, using untreated SVGA cells as negative control. At 36 and 24 h there was a significant increase in endogenous expression of LIP after treatment with IFNa (p < 0.01) and IFNß (p < 0.0001), respectively, whereas an important increasing of STAT1 phosphorylation was observed at 24 and 36 h after treatment with IFNa and IFNß, respectively. Standard deviation bars are depicted on the graph. p values < 0.05 were considered statistically significant. Note: * p < 0.05; ** p < 0.01; *** p < 0.0001.

Fig 4: Translocation of C/EBPß-LIP to the nucleus after IFNa or IFNß treatment. Immunocytochemistry (ICC) of SVGA cells transiently transfected with pLIP and treated with IFNa (A) or IFNß (B) for 24 h. After blocking, the slides were incubated overnight with mouse anti-CEBPß (H7) antibody (FITC) and then mounted with medium containing DAPI. The ICC images were acquired by BZ-X800 Fluorescence Microscope (KEYENCE Corporation) and analyzed using the dedicated software in order to calculate the percentage of FITC/DAPI merge. Treatment either with IFNa or IFNß showed a significant overlapping of FITC and DAPI signal (IFNa: p = 0.05 and IFNß: p < 0.01) which is an indication of C/EBPß-LIP enrichment in the nucleus. In order to confirm the nuclear localization of C/EBPß-LIP after treatment with IFNs, a cell fractionation assay was performed, cells were harvested at corresponding time points (4, 12 and 24 h) after one treatment with IFNa (C) or IFNß (D), and the nuclear and cytoplasmic proteins were isolated. Note: * p < 0.05; ** p < 0.01.

Fig 5: Silencing of CEBPB-AS1 results in epigenetic modifications at the CEBPB regulatory region. (a) RNA-IP (RIP) was performed in A375PR1 cells using antibodies against CTCF. The CEBPB-AS1 transcript was detected using semi-qRT-PCR. (b–d) The A375PR1 cells were transfected with either siControl or siCEBPB-AS1 and ChIP was performed using antibodies against CTCF (b), H3K27me3 (c) and EZH2 (d). Primers in the regulatory region of CEBPB were used for qPCR; independent experiments n = 3. Data represent mean ± SEM. ***p < 0.005. For EZH2, due to variations between the experiments, statistical significance was not reached, and results from individual experiments are shown. Each value was first adjusted to the IgG control by subtraction, calculated as a ratio to the input (normalization), and the data are presented as fold change to siControl. ChIP, chromatin immunoprecipitation; RIP, RNA immunoprecipitation; semi-qRT-PCR, semi-quantitative reverse transcription PCR.