Figure 4(A) Nascent RNA imaging in control and Ncl depleted iKras PDAC cells, in presence or absence of KrasG12D. Cells were transfected with a non-targeting control siRNA, or two independent siRNAs against Ncl, grown for 48hrs in presence or absence of Dox, prior to pulse labeling with FUrd. Cells were then fixed and immunostained with anti-FUrd antibody (green) to visualize nascent RNA, along with anti-Fibrillarin (Fbl) antibody as a Nucleolar marker (red), and Hoechst (blue) as the Nuclear stain, followed by confocal microscopy analysis. (B) Quantification of Nucleolar FUrd levels in images from (A). FUrd fluorescence densities in single nucleoli were quantified from 182-220 individual cells per condition, combined from 2 independent biological replicate experiments (****: P < 0.0001; n.s.: not significant - calculated from one-way ANOVA with Šídák's multiple comparisons test).(C) RT-qPCR analysis of 5'ETS-containing pre-rRNA transcript levels in control and Ncl depleted iKras PDAC cells, in presence or absence of KrasG12D. Cells were transfected with a non-targeting control siRNA, or two independent siRNAs against Ncl, and grown for 48hrs in presence or absence of Dox, before RT-qPCR analysis with a specific probe against the mouse 5'ETS region. A probe against mouse Actb mRNA was used as loading control for normalization. A total of 4 biological replicate experiments were quantified (***: P < 0.001; n.s.: not significant - calculated from one-way ANOVA with Šídák's multiple comparisons test).(E) Assessment of the overall protein synthesis rates in control and Ncl depleted iKras PDAC cells, in presence or absence of KrasG12D, by puromycinylation. Cells were transfected with a non-targeting control siRNA, or two independent siRNAs against Ncl, and grown for 48hrs in presence or absence of Dox, before pulse labelling with Puromycin (10 μg/ml) for 15 min to label nascent proteins. Cells were subsequently lysed and analyzed by immunoblotting with the indicated antibodies. (F) Quantification of puromycin incorporation from (E), as an indicator of the overall protein synthesis rate. Total Erk1/2 levels were used as loading control. A total of 4 independent biological replicate experiments were quantified. Error bars depict SD. (**: P < 0.01; n.s.: not significant - calculated from one-way ANOVA with Šídák's multiple comparisons test). ((G) Nascent RNA imaging of WT, S4A, and S4D Myc-Ncl expressing iKras PDAC cells, in presence or absence of KrasG12D. Vectors encoding Myc-tagged WT, S4A, and S4D Ncl were transiently transfected into iKras PDAC cells, before reseeding and growing the cells for 48hrs in presence or absence of Dox. Cells were then subjected to pulse labeling with FUrd, fixation, and immunostaining with anti-FUrd antibody (green), anti-Myc-tag antibody (red), and Hoechst (blue), followed by confocal microscopy analysis. Scale bar = 10 µm. (H) Quantification of FUrd levels in Myc-positive nucleoli from (E). FUrd fluorescence densities in single nucleoli were quantified from 160-281 individual cells per condition, combined from three independent biological replicate experiments (****: P < 0.0001; n.s.: not significant - calculated from one-way ANOVA with Šídák's multiple comparisons test). ((I) RT-qPCR analysis of 5'ETS-containing pre-rRNA transcript levels in WT, S4A, and S4D Myc-Ncl expressing iKras PDAC cells, in presence or absence of KrasG12D. Vectors encoding Myc-tagged WT, S4A, and S4D Ncl were transiently transfected into iKras PDAC cells, before reseeding and growing the cells for 48hrs in presence or absence of Dox, followed by RT-qPCR analysis with a specific probe against the mouse 5'ETS region. A probe against mouse Actb mRNA was used as loading control for normalization. A total of 5 to 8 biological replicate experiments per condition were quantified (****: P < 0.0001; n.s.: not significant - calculated from one-way ANOVA with Šídák's multiple comparisons test).