Fig. 3: Acute degradation of SRBD1 results in anaphase bridges.
a Immunoblot analysis of SRBD1 (parental clone) and mAID-mClover-SRBD1 (mAC-SRBD1, clone 1) in asynchronous cells treated with DMSO (24 h) or 1 μM 5-Ph-IAA for the indicated amounts of time. Bio-Rad Stain-Free total protein was used as a loading control. A cross-reacting protein that migrates just below the untagged SRBD1 protein is denoted by an asterisk. b Asynchronously growing cells were treated with DMSO (24 h) or 1 μM 5-Ph-IAA for the indicated amounts of time and γH2AX was measured by immunofluorescence imaging. Each gray data point represents the nuclear intensity in one cell (arbitrary units x 107; total cells analyzed ≥19,388). The colored data points represent the mean of each biological replicate (n = 3) and black bars represent the mean of the three replicates. Significance was determined using a one-way ANOVA with Dunnett’s multiple comparisons test comparing the means of replicate experiments. c Asynchronously growing cells were treated with DMSO (24 h) or 1 μM 5 Ph-IAA for the indicated amounts of time and cell cycle distributions (from 25,000 gated cells) were analyzed by flow cytometry. pH3 staining was used to differentiate mitotic and G2 phase cells. d Asynchronously growing cells were treated with DMSO or 1 μM 5-Ph-IAA -/ + 8 mM caffeine for 16 h and cell cycle distributions were analyzed by flow cytometry on 25,000 gated cells. e–g G2 phase synchronized cells were treated with DMSO or 1 μM 5 Ph-IAA for 1 h, released into mitosis, and anaphase bridges were assessed by immunostaining. e Representative images of ultrafine anaphase bridges (detectable by PICH immunostaining, green), and anaphase chromatin bridges (identified with DAPI staining, blue). Scale bars represent 5 μm. The percentage of anaphases (total cells analyzed ≥360) with PICH or BLM-coated ultrafine bridges (f) and chromatin bridges (g) was quantified. Graphs display the mean +/− SD of biological replicates (n = 5 or 6 for UFBs and n = 8 for chromatin bridges). Significance was determined using a one-way ANOVA with Sidak’s multiple comparisons test. Source data are provided as a Source Data file.
