Fig 1: Nucleolar alterations can be detected by DHM.Diverse nucleolar alterations were induced in HeLa-FBL-GFP cells, observed by correlative DHM-fluorescence microscopy, and quantified. (A) Drug-induced nucleolar alterations. Cells were treated for 2 h with DRB (0.1 mM), roscovitine (ROS, 50 µM), actinomycin-D (ActD, 160 nM), CX-5461 (5 µM), or the vehicle control (DMSO 0.1%). White arrows point towards nucleolar caps (ActD and CX-5461 pictures). Scale bar, 20 µm. (B) Quantification of the nucleolar optical thickness reveals a significant reduction in cells treated with DRB or ROS and an increase upon treatment with ActD or CX-5461, by comparison with a DMSO control. Number of independent experiments, n = 10. The mean of each independent experiment is represented by a black circle, the mean of the means by a black line, and the individual nucleoli are counted by colored dots. The total numbers of nucleoli counted were 290, 256, 234, 301, and 287 for DRB, ROS, ActD, CX-5461, and DMSO, respectively. Data were analyzed by one-way ANOVA (p < 0.0001) and by uncorrected Fisher’s LSD (DMSO vs. DRB, p < 0.0001; DMSO vs. ROS, p < 0.0001; DMSO vs. ActD, p = 0.038; DMSO vs. CX-5461, p = 0.030). *p ≤ 0.05; ****p ≤ 0.0001. (C) Nucleolar alterations induced by ribosomal protein knockdown. HeLa-FBL-GFP cells were treated for three days with a siRNA (10 nM) specific to the mRNA encoding ribosomal protein uL5 or uL18 or with a non-targeting scramble control (SCR). Scale bar, 20 µm. (D) Western blot assessment of uL5 and uL18 depletion after siRNA-mediated knockdown. ß-actin was used as a loading control. Note that uL18 is reduced upon uL5 knockdown and vice versa. (E) Knockdown of ribosomal protein uL5 or uL18 leads to a significant reduction in nucleolar optical thickness as compared to the SCR control. Number of independent experiments, n = 11. The mean of each independent experiment is represented by a black circle, the mean of the means by a black line, and the individual nucleoli are counted by colored dots. The total numbers of nucleoli counted were 311, 235, and 332 for uL5, uL18, and the SCR control, respectively. Data were analyzed by one-way ANOVA (p < 0.0001) and by uncorrected Fisher’s LSD (SCR vs. uL5, p < 0.0001; SCR vs. uL18, p < 0.0001). ****p ≤ 0.0001. Source data are available online for this figure.