Figure 4. Triptolide perturbs condensation prior to its effects on condensin levels.

(A) Schematic of assay to test the timing of triptolide-induced defects in condensation and condensin levels. After a twenty-five minute incubation, either DMSO or triptolide (TPL) was added, and samples were taken at indicated times and processed for immunofluorescence. (B) Representative immunofluorescence images of DMSO or triptolide treated extracts at indicated timepoints. Chromatids were labeled with Hoechst and anti-CAP-E (condensin I and II). Note that condensation is already lost in in the first timepoint after triptolide addition, as in Figure 2A. Mean condensation parameters for each condition are indicated in lower left corner of each image. (C) Quantification of fluorescence intensity of CAP-E from experiment in (B), normalized to the 180 min DMSO-treated sample. n = 50 structures for each condition. Error bars represent SD, and asterisks indicate a statistically significant difference (*, p < 0.001). A.U., arbitrary units. Two biological replicates were performed, quantified structures are from a single experiment.

Figure 4—figure supplement 1. Condensation parameters of condensation kinetics after DMSO or Triptolide treatment.

(A) Scatter plots of the percentage of pixels below a threshold of 35% of image maximum fluorescence intensity (the condensation parameter), for each drug condition and indicated time point. Error bars represent SD, and the mean values are indicated. n = 20 structures for each condition. Two biological replicates were performed, quantified structures are from a single experiment.

Figure 4—figure supplement 2. Triptolide perturbs condensation prior to its effects on condensin levels.

(A) Left: Representative immunofluorescence images of DMSO or triptolide-treated extracts at indicated timepoints. Chromatids were labeled with DAPI and anti-CAP-E (condensin I & II). Right: Quantification of fluorescence intensity of CAP-E, normalized to the 180 min DMSO-treated sample. This data is also shown in Figure 4B in grayscale. (B) Left: Representative immunofluorescence images of DMSO or triptolide treated extracts at indicated timepoints. Chromatids were labeled with DAPI and anti-CAP-D3 (condensin II). Right: Quantification of fluorescence intensity of CAP-D3, normalized to the 180 min DMSO-treated sample. (C) Left: Representative immunofluorescence images of DMSO or triptolide treated extracts at indicated timepoints. Chromatids were labeled with DAPI and anti-XPB (TFIIH complex). Right: Quantification of fluorescence intensity of XPB, normalized to the 180 min DMSO-treated sample. n = 50 structures for each condition. Error bars represent SD, and asterisks indicate a statistically significant difference (*, p < 0.001). A.U., arbitrary units. Two biological replicates were performed, quantified structures are from a single experiment.