Fig. 2. Reduced lamin B1 expression correlates with inhibition of RNA synthesis. HeLa cells were co-transfected with RNAi vectors and a vector expressing YFP-tubulin and the structure and activity of transcription sites monitored 48 hours later using either indirect immunofluorescence of the active form of RNA polymerase II (A) or BrUTP incorporation in permeabilised cells (B). (A) RNA polymerase (red) in transfected (expressing YFP-tubulin – green) cells after transfection with: lamin B1 RNAi vector (Kd LB1); RNAi vector co-transfected with lamin B1 rescue vector (Kb LB1 + Rescue); rescue vector alone (Rescue vector) and lamin A/C RNAi vector (Kd LA/C). (B) Nascent transcripts (BrU – red) in control cells and cells transfected with lamin A/C (Kd LA/C) and lamin B1 (Kd LB1) RNAi vectors. Lamin B1 depletion was assessed by indirect immunofluorescence (Anti-LB1, red). As a control for possible labelling artefacts, the order of antibody labelling was also reversed (Anti-LB1/BrU – merged image of lamin B1 labelled green and transcripts labelled red). Quantitative image analysis of active RNA polymerase II – H5 epitope (C) and nascent transcripts (D-F) in control and lamin-depleted cells showed that transcription was unaffected in cells with lamin A/C depleted (D,F) whereas depletion of lamin B1 (C-E) correlated with a clear decrease in transcription of both pre-mRNA and pre-rRNA (see supplementary material Fig. S2). For pre-mRNA synthesis, statistical analysis using two-tailed Student's t test showed the lamin-B1-depleted and control cell populations (E) to be unrelated. Scale bars: 10 µm (A); 5 µm (B).