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. 2023 Jul 17;12(14):1868. doi: 10.3390/cells12141868

Figure 1.

Figure 1

Figure 1

CNOT1 depletion increases global transcription in HeLa and MCF7 cells. (A) Representative immunoblot shows CNOT1 depletion at different time points (0, 24 h, 48 h, 72 h, 96 h and 120 h) after transfection of HeLa cells with CNOT1 siRNA. (B) siRNA-transfected HeLa cells were lysed, and the lysates assessed by immunoblotting with the indicated antibodies against different CNOT subunits and Pan 3. (C) Representative immunoblot confirms depletion of CNOT1 via siRNA up to 72 h. (D) Representative IF images show the labelling of global nascent RNA using 5-ethynyluridine (EU) incorporation assay in control and CNOT1-depleted HeLa cells. The global transcription activity was measured after EU incorporation (1 h) shown after 24, 48, and 72 h in (E). To quantify EU incorporation, Hoechst was used to stain the nucleus of cells, generating a nuclear mask. Adobe Photoshop was used to adjust the levels of all panels equally. Quantification of EU signal per nucleus was performed using RStudio statistical software and the results shown (n = 3 independent experiments, 100 cells counted. Stats: Mann–Whitney Wilcoxon Rank Sum). (F) Representative Western blot showing the expression level of TBP and the phosphorylation of RNA Polymerase II C-terminal domain (S5) in each experimental condition. CDK2 was used as a loading control. (G) Representative Western blot showing the expression level of CNOT subunits in the presence or absence of CNOT1 in MCF7 TR/ Control and CNOT1 KD cells. (H) Representative Western blot confirms the CNOT1 (flag-tagged) pcDNA3 transfection in MCF7 TR/CNOT1 KD cells (Transfection efficiency was generally in the range 20–25%). (I,J) Representative images show transfection efficiency (22%). Images were collected using an EVOS fluorescence inverted digital microscope. MCF7 cells were fixed, permeabilized, and labelled with anti-FLAG antibody (green). Percentage transfection efficiency: (Fluorescent cells/Total number of cells) × 100. Bar 400 µm. (K,L) Transcription elongation was measured after EU incorporation (1 h) 3-day post +/− 2 μg/mL DOX treatment in MCF-7 TR control and MCF7 TR CNOT1KD cells (Representative cells are shown in (H)). To quantify EU incorporation, Hoechst was used to stain the nucleus of cells, generating a nuclear mask. Adobe Photoshop was used to adjust the levels of all panels equally. Quantification of EU signal per nucleus was performed using GraphPad Prism statistical software; (n = 3 independent experiments; >100 cells analysed per repeat; Stats: unpaired t-test, * p < 0.05). (M) Representative Western blot showing the expression level of TBP and the phosphorylation of RNA Polymerase II C-terminal domain (S5) in MCF-7 TR CNOT1KD cells up to 5 days post 2 μg/mL DOX induction. Scale bar in (C,H) = 50 µm. a.u, arbitrary units.