Fig 2: Onvansertib effects on sensitive and resistant CAL33 cell lines. (A) Clonogenicity assay of sensitive and resistant CAL33 cell lines after onvansertib (10 nM and 25 nM) or cisplatin (3 µM) treatment (n = 2). (B) Hematoxylin eosin saffron staining of sensitive and resistant CAL33 cell lines treated with onvansertib (25 nM) for 24 h. Abnormal mitosis was indicated with an arrow. (C-D) Cells were treated with onvansertib (25 nM, 50 nM or 100 nM) for 24 h. Cell cycle was measured by flow cytometry (n = 3) (C), G2/M arrest was evaluated by phospho-NPM and, phospho-H3 immunoblotting. HSP90 served as a loading control (n = 2) (D). (E-F) Polyploidy (number of nuclei 8N and 16N) was assessed by flow cytometry (n = 3) (E). Representation of polyploidy in CAL33 RR (F). (G) Cells were treated with onvansertib (25 nM and 50 nM) for 48 h. Cell death was evaluated by flow cytometry. Cells were stained with propidium iodure (PI) and Annexin V (AV). Histograms show AV+/PI- cells (early apoptosis) and AV+/PI+ cells (late-apoptosis or another cell death) (n = 3). (H) Immunostaining of PARP expression on CAL33 cell lines after exposure to onvansertib (100 nM) for 24 h. HSP60 served as loading control (n = 2). (I-J) Invasion of sensitive and resistant CAL33 cells using 3D cell culture assay (spheroids) after 6 days of onvansertib treatment (100 nM) grown in (I). Results are represented as arbitrary units (the no-treatment condition was used as a control) (n = 2) (J). Statistics were analyzed using ANOVA tests: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Fig 3: LSD1 is involved in breast cancer growth and is enriched in chemoresistant cells. (a) Western blots of indicated breast cancer cell line total cell extracts probed for LSD1. NPM and histone H3 were loading controls. Copped images are from samples and antibodies processed and run on the same gel. Full-length blot is shown in Supplementary Fig. 6. (b) LSD1 mRNA levels as measured by qPCR in breast cancer cell lines characterised as ER positive and ER negative. Data are expressed as arbitrary copy numbers normalised to PPIA (n = 2). LSD1 expression in: (c) local recurrent and non-recurrent breast carcinomas from GEO dataset GSE4913 (n = 19); (d) primary and secondary locally recurrent breast carcinomas from GEO dataset GSE4913 (n = 9); (e) Immunofluorescence microscopy was performed on matched parental and docetaxel-resistant MCF-7, T47D and MDA-MB-231 cells fixed and probed with anti-ALDH1A1, anti-PKC-? and anti-LSD1-s111p. Graphs indicate ALDH1A, PKC- ? and LSD1-s111p TNFI and PKC and LSD1-s111p PCC (n = 20 individual cells). -1 = inverse of colocalization; 0 = no colocalization; +1 = perfect colocalization. (f) Tumour growth (mm3) up to 5 weeks post-treatment with indicated treatments (n = 5). Coloured asterisk (*) denotes significance at that time point. (g) Tumours sizes 5 weeks post-treatment from left to right: control, nab-paclitaxel 60 mg/kg, nab-paclitaxel 30 mg/kg, nab-paclitaxel 10 mg/kg, docetaxel 10 mg/kg, and docetaxel 4 mg/kg. Tumours were excised and digested into single cell suspension and then subjected to immunofluorescence microscopy where TNFI was determined for control, nab-paclitaxel 60 mg/kg, and docetaxel 10 mg/kg treated tumours after probing with: (h) anti-EGFR, anti-Snail; or (i) anti-LSD1, or anti-LSD1-s111p (n > 10). All data represents the mean ± SE. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, Mann-Whitney test.

Fig 4: Quantitative analysis of B23 and fibrillarin nucleolar localization. The abundance of proteins B23 and fibrillarin in nucleoli is regulated by stress. HeLa cells were grown under normal conditions or heat-shocked for 1 hour at 45.5°C as shown in Figure 1. Nucleolar and nuclear fluorescence was quantified and pixel values/area were calculated. For B23, 21 control cells (38 nucleoli) and 22 heat-shocked cells (36 nucleoli) were measured; for fibrillarin 20 control (28 nucleoli) and 20 heat-treated cells (31 nucleoli) were analyzed. Results obtained for control conditions were defined as 1; significant differences were identified with two-tailed Student's t-test. Raw data were further analyzed in part a-e. (a) Fluorescence intensities were measured for all nucleoli. Individual nucleoli were given the same weight in the analysis, independent of the number of nucleoli in each nucleus. (b) Fluorescence intensities were measured for all nucleoli, average values were calculated for nucleoli if multiple nucleoli were in the same nucleus. Based on these average values, it was determined whether nucleolar fluorescence is different in control and stressed cells. (c) Changes in nucleolar fluorescence/area are shown as histograms. The same binning was applied to control and stressed samples for each protein. (d) Single cell analysis depicting nucleolar fluorescence intensities/area for individual nuclei. Results are shown for individual nuclei; each line represents the measurement for the sum of nucleoli of one nucleus of control (blue lines) or heat-shocked cells (red lines). Data were not normalized. (e) The values obtained for nucleolar fluorescence intensities in part b were used to determine the ratio of nucleolar/nuclear fluorescence intensities. Note that there is no drastic change for B23 upon heat shock, whereas a significant change is observed for fibrillarin.

Fig 5: The dynamic organization of nucleoli requires new tools for quantification of nucleolar fluorescence. The nucleolar accumulation of B23 and fibrillarin, frequently used as marker proteins for the nucleolus, is sensitive to changes in growth conditions. HeLa cells were grown under normal conditions or exposed to a 1-hour heat shock at 45.5°C. Cells were fixed and processed for indirect immunofluorescence. Size bar is 20 µm. Note that a portion of either protein relocates from the nucleolus upon heat stress.