Fig. 6: Screen for histone level modulators.

a Scheme of the siRNA screen for histone density modulators. b, c High-content imaging analysis of histone H3. MDA-MB-231 cells were transfected with the scrambled control siRNA and processed as in (a). Representative microscopy images are shown in (b). Examples for image-based cell cycle phase analysis and histone quantification are shown in (c). d Primary siRNA screen for histone modulators. MDA-MB-231 cells were transfected with the indicated siRNAs and processed as in (a). Three replicate plates (N = 3) with identical design were prepared, on which each siCtrl and siKIF11 were transfected in 4 wells, and all other siRNAs were transfected in one well. Quantifications are based on at least 1123 cells per well (excluding siKIF11). Histone H3 signal per DAPI signal was quantified separately for G1, S and G2/M phases, and the well-wise H3 signal was calculated as mean of these 3 values. The bars represent the mean across replicate wells. Significance against the scrambled contol siRNA was analyzed with two-tailed, unpaired Student’s t-test with Benjamini-Hochberg correction. e High-content imaging analysis of histones H3 and H4 in the secondary siRNA screen. MDA-MB-231 cells were transfected with the scrambled control siRNA and processed as in (a). Representative microscopy images are shown. f Secondary siRNA screen for histone modulators. MDA-MB-231 cells were transfected with the indicated siRNAs and processed as in (a). Two separate experiments were performed for the validations using either polyclonal anti-H3 antibody or the combination of monoclonal H3 and H4 antibodies. In each experiment, cells were transfected with siRNAs in 9 (siCtrl), 3 (siKIF11) or 4 (all other siRNAs) replicate wells. Quantifications are based on at least 1123 and 1174 cells per well for the pAb H3 and the mAb H3/H4 screens, respectively (excluding siKIF11). Histone H3 and H4 signals per DAPI signal were quantified separately for G1, S and G2/M phases, and the mean of these 3 values is represented. Significance against the scrambled contol siRNA was analyzed with two-tailed, unpaired Student’s t-test with Benjamini-Hochberg correction. g Western blot validation of histone modulators. MDA-MB-231 cells were transfected with the indicated siRNAs and protein lysates were produced after 3 days. Protein levels were analyzed by Western blot. The barplot represents histone levels normalized to DHX9. Significance against the scrambled contol siRNA was analyzed with two-tailed, unpaired Student’s t-test with Benjamini-Hochberg correction (N = 4 independent siRNA transfections). h–j Western blot and qPCR validation of candidate siRNAs. MDA-MB-231 cells were transfected with the indicated siRNAs. Protein lysates and RNA extracts were prepared after 3 days. Protein levels were analyzed by Western blot (h, i). The asterisk indicates that the apparent molecular weigth of DCAF6 was higher than expected, requiring an additional assay to control for DCAF6 depletion. The DCAF6 mRNA level was analyzed by qPCR using GAPDH mRNA as normalization control (j). Experiments were performed with three technical replicates. Data are represented as mean ± standard deviation. siCtrl is a scrambled siRNA. The scale bars correspond to 40 µm.