Fig 2: Proliferation assay in LNCap cells using PCAT92 and ABCC4 knockdown experiments(A) LNCaP cells transfected with shRNA constructs against ABCC4, PCAT92 and scrambled. Based on the number of fluorescent cells, it was observed that transfection efficiency was about 75%. (B) MTT assay showing the effect of ABCC4 and PCAT92 knockdown on RPWE-1 cell viability. As we have shown that RPWE-1 cells does not express ABCC4 and PCAT92, we observed that there no reduction in cell viability under both ABCC4 and PCAT92 knockdown when compared to control indicating that there is no off-target effect due to shRNA. (C) MTT assay showing the effect of ABCC4 and PCAT92 knockdown on LNCaP cell viability. It was observed that there a reduction in cell viability under both ABCC4 and PCAT92 knockdown when compared to control. This indicates that both ABCC4 and PCAT92 are required for cancer cell growth. (D) FACS analysis showing an increase in number of dead cell population under the knockdown condition indicating that ABCC4 and PCAT92 has a role in cell proliferation. No cell arrest and any specific cycle stage was observed. (E) qRT-PCR results from ABCC4, PCAT92 and ZIC2 post knockdown of ABCC4 and PCAT92: A significant reduction in the transcript level of ABCC4 and PCAT92 was observed in their respective knockdown LnCAP cells. Interestingly, upon PCAT92 knockdown, a significant reduction in the transcript level of ABCC4 was also observed, indicating a possible regulatory role of PCAT92 in ABCC4 gene regulation. No significant change in the PCAT92 transcript level was observed upon ABCC4 knockdown. No significant change in the level of ZIC2 was observed under both ABCC4 and PCAT92 knockdown condition. Actin was used as a housekeeping gene to calculate ΔΔCt. Both the knockdowns were performed in three independent batches. (F) Western blot analysis, showing a significant reduction in ABCC4 protein level in both ABCC4 and PCAT92 knockdown condition, thereby strengthening our prediction of PCAT92 playing a role in ABCC4 gene regulation. The bars in the graph are plotted based on the band intensity observed in the western blot. Actin was used as a control. (*** P<=0.001, ** P<=0.01 and ns P>0.05).

Fig 3: Pulldown/binding assays for ZIC2-ABCC4(A) ChIP RTPCR to show the enrichment of ABCC4 promoter region using ZIC2 pulldown. H3 pulldown for enrichment of RPL30 exon 3 was used as a positive control and anti-rabbit IgG was used as a negative control. Three independent set of experiment was performed. (B) Mobility shift assay, confirming the binding of ZIC2 at ABCC4 promoter region. (C) ChIP-PCR under PCAT92 knockdown a reduction in the enrichment of ABCC4 promoter region was observed indicating a possible interaction between PCAT92 and ZIC2 to help the later in forming a stable complex at the promoter region. H3 pulldown for enrichment of RPL30 exon 3 was used as a control. The bars in the graph are plotted based on the band intensity observed in the immunoblot gel. Three independent set of experiment was performed. (*** P<=0.001, ** P<=0.01 and ns P>0.05).

Fig 4: PCAT92-ABCC4 Triplex(A) Triplex forming oligo (TFO) on PCAT92 strand and Triplex Target Site (TTS) on ABCC4. (B) NanoITC result showed a sequencing specific interaction between PCAT92 transcript and ABCC4. Red indicate the interaction between sense PCAT92 with ABCC4 and green indicate random RNA with ABCC4. (C) EMSA showing a sequence specific interaction between ABCC4 and sense PCAT92 RNA forming triplex. Random RNA was used as a negative control. (D) The model of the predicted PCAT92-ABCC4 Triplex.

Fig 5: Cellular localization of PCAT92 and ZIC2 and hypothesis for the mechanism of PCAT92 involvement in PC(A) Co-localization of PCAT92 RNA and ZIC2 protein to the nucleus and cytoplasm has been shown. The yellow color in the nucleus (blue) indicates that both PCAT92 RNA and ZIC2 protein are interacting in the nucleus of the LNCaP cells. (B) Hypothesis for how PCAT92 might be regulating ABCC4 via protein ZIC2.