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Fig. S1. Depletion of lamin B1 correlates with a loss of nucleoplasmic transcription factories. HeLa cells were co-transfected with vectors expressing pSEChygro-lamin B1 and DsRed-lamin A/C as reporter. At different times after transfection, cells were fixed and nucleoplasmic sites containing the active form of RNA polymerase II (H5 epitope) visualised by indirect immunofluorescence (green). At different times, the fluorescent intensity of the active centres (n=100) was measured in cells expressing DsRed-lamin A/C reporter and the intensity of RNA polymerase II sites expressed as a percentage of levels in control cells transfected with empty RNAi vector (curve). A significant loss of activity was determined (48 hour time point) using Student’s t test (t=22.57; P<1.819e-32***). Scale bars:10 µm.
Fig. S2. Inhibition of RNA synthesis in HeLa cell with reduced lamin B1 expression. Fig. 2 shows that reduced lamin B1 expression (48 hours after transfection) correlated with inhibition of nascent RNA synthesis. The gallery of images presented here shows the range of transcription in cells with different degrees of lamin depletion. Typical examples from control, lamin B1 depleted (Kd LB1) and lamin A/C depleted (Kd LA/C) populations are shown. Immunostaining of lamin B1 in lamin-A/C-depleted cells shows that the lamin B1 expression in the residual lamin is not increased to compensate for loss of lamin A/C. Fluorescent images show merges of nascent transcripts (green) and lamin B1 (red). Scale bars: 5 µm.
Fig. S3. Reduced expression of lamin A/C does not affect levels of nascent RNA synthesis. BrUTP incorporation into nascent RNA was carried out in cells depleted of lamin A/C, at 48 and 72 hours after kd. This figure shows typical examples that were used to generate the data shown in Fig. 2F. BrdU-containing nascent transcripts and lamin A/C in the residual nuclear lamina were measured by quantitative image analysis and levels of transcription in cells with reduced lamin A/C expression were determined. Scale bars: 5 µm.
Fig. S4. Linearity of decay of active RNA polymerase II following depletion of lamin gene expression using siRNAs. Short duplex RNAs were used to deplete lamin gene expression and the concentration of active RNA polymerase engaged in transcription factories was monitored following the principle detailed in Fig. 2. The reduced lamin gene expression was monitored by quantitative immunofluorescence (A) as before (equivalent microscope settings were used to acquire the pictures except in the red channel after 48 hours, where contrast was increased 10% to reveal remaining lamin B1) and by immunoblotting (B) using whole cell extracts (10 µg whole cell protein/lane) from identical samples (24, 36 and 48 hours after transfecting the duplex RNAs in 24-well plates). In B, quantitative analysis of the blots shows the lamin protein expression, relative to untreated controls, to be 48, 25 and 9% for lamin A/C knockdown (1) and 30, 20 and 12% for lamin B1 knockdown (2) after 24, 36 and 48 hours, respectively. In this experiment, there was no significant difference in concentration of lamin B1 in lamin A/C kd cells quantitation of signal after blotting, in arbitrary units using ChemiImager software were 0.918 (CT), 0.905 (24 hours), 0.940 (36 hours) and 0.953 (48 hours), standardised values using actin. Using the same approach, a slight increase in lamin A/C expression was seen in lamin B kd cells arbitrary units were 2.065 (CT), 2.595 (24 hours), 1.675 (36 hours), 2.494 (48 hours). These data confirm the changes described using the vector-based RNAi shown in Fig. 2. The siRNA-induced reduction of lamin B1 expression correlated with a progressive loss of engaged RNA polymerase II (C) and nascent RNA (labelled with BrUTP in permeabilised cells; not shown). As before, siRNA-induced depletion of lamin A/C had no effect on the levels of engaged RNA polymerase II or nascent transcription (not shown). Scale bars: 20 µm.
Fig. S5. Inhibition of RNA synthesis in MRC5 cell with reduced lamin B1 expression. Lamin protein expression was reduced in MRC5 cells using siRNAs and sites of nascent transcription were labelled in permeabilised cells using BrUTP. Incorporation and lamin depletion were monitored by indirect immunofluorescence. (A) Alteration in lamin A/C or B1 expression in a panel of experiments in which different lamin depletions were performed. The anti-lamin A/C antibody blot shows protein level in MRC5 control cells, depleted in lamin A/C, lamin B1 (duplicates), lamin B2 and cells transfected after 48 hours with scrambled siRNA (A, 1a). Using this approach, lamin A/C in lamin A/C kd samples fell to 15-25% of controls in different experiments and expression of lamin A/C in lamin B kd samples and using scrambled siRNA (S) as control was essentially unaltered (95-110% of controls). The same experiment was performed using anti-lamin B1 (A, 1b). (A2) Cells with reduced lamin B1 expression were also analysed by blotting to assess any changes in global RNA polymerase II expression using the antibody CTD4H8, which recognises both phosphorylated (ser5) and unphosphorylated forms of the CTD. Quantitative image analysis shows that any overall decline in total polymerase II was less than 20%, which is probably not sufficient to alter transcription given that only ∼25% of RNA polymerase II is active at any time. (3A) Level of lamin B2 after knockdown using siRNA in MRC5 and HCT116 cells (p53+/+ and p53−/−). (B) Typical immunofluorescence images used to generate quantitative data shown. Fluorescent images show merges of nascent transcripts (green) and lamin A/C or B1 (red). The plots show the intensity of BrdU incorporation in arbitrary units (A.U.) for Pol I and Pol II transcripts in control, lamin A/C KD, lamin B1 kd and cells transfected with scrambled siRNA For each sample the nucleolar and nucleoplasmic signals in 50 cells were measured. Scale bars: 5 µm.
Fig. S6. Inhibition of RNA synthesis in HCT116 cell with reduced lamin B1 expression. HCT116 cells with or without normal p53 expression were transfected with siRNA and the structure and activity of transcription sites were monitored 48 hours later using immunofluorescence to recognise BrUTP incorporation in permeabilised cells (A). Nascent transcripts (BrdU, green) in either p53+/+ or p53−/− cells were analysed using siRNA against lamin B1 (LB1 kd, BrUTP, green, and anti-lamin B1, red), lamin B1 kd with apoptosis inhibitor (LB1 kd+V, BrUTP, green, and lamin B1, red), lamin A/C kd (LA/C kd, BrUTP, green, and anti-lamin A/C, red), lamin B2 kd (BrUTP, green, and anti-lamin B2, red), and cells transfected with scrambled siRNA (BrUTP, green, and anti-lamin B1, red). Quantitative image analysis of BrdU incorporation (B) in control (CT), lamin A/C kd, lamin B1 kd, lamin B2 kd, scrambled# siRNA and lamin B1 kd cells incubated with a inhibitor of apoptosis showed that transcription was unaffected in cells with lamin A/C or B2 depleted, whereas depletion of lamin B1 correlated with a clear decrease in transcription of both pre-mRNA (RNA pol II) and pre-rRNA (RNA pol I).
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