Skip to main content
. 2023 Nov 15;24(12):e57585. doi: 10.15252/embr.202357585

Figure 5. Accumulation of GNL3 into the nucleolus limits origin firing.

Figure 5

  1. Schematic representation of human GNL3 protein with its associated domains (B: basic domain; C: coiled‐coil domain; G1: GTP‐binding motif 1; G4: GTP‐binding motif 4; I: intermediate domain; A: acidic domain). GNL3‐WT and GNL3‐dB are fused with FLAG.
  2. Western‐blot analysis of Flp‐In T‐Rex HeLa cells expressing exogenous GNL3‐WT or GNL3‐dB. Cells were transfected with siControl or siGNL3 for 48 h then expression of exogenous GNL3‐FLAG (resistant to the siRNA against GNL3) was induced using 10 μg/ml of doxycycline for 16 h.
  3. Immunofluorescence analysis of Flp‐in T‐Rex HeLa cells expressing exogenous GNL3‐WT or GNL3‐dB.
  4. PLA (proximity ligation assay) analyzing the proximity between ORC2 and GNL3‐FLAG or GNL3‐dB‐FLAG in HeLa Flp‐In cells upon doxycycline induction.
  5. Graphic representation of the average number of PLA ORC2‐FLAG foci in three biological replicates. For statistical analysis paired t‐test was used; *P < 0.05.
  6. Immunofluorescence experiment of HeLa Flp‐In cells expressing GNL3‐dB with or without pre‐extraction with cytoskeletal buffer (CSK).
  7. Analysis of GIFD (Global Instant Fork Density) by DNA combing in HeLa cells (n = 1; a biological replicate is shown in Fig EV5C). GIFD value is indicated in red.

Source data are available online for this figure.