Extended Data Fig. 2. Dynamics of cytoplasmic hybrid production.

(a) cytoDRIP blot showing cytoplasmic hybrids in mock or PlaB treated (500 nM, 3 h) BAX^−/−BAK^−/− HeLa cells with or without in vitro RH treatment.

(b) Flow cytometry analysis of asynchronous or serum-starved MCF10A cells following incubation with BrdU. Cells were segmented based on DNA content (propidium iodide staining) and BrdU intensity. The percentage of cells in G1, S and G2/M are indicated. At least 50,000 cells were quantified per condition.

(c) Western blot showing fractionation of asynchronous and serum-starved MCF10A cells into soluble nuclear and cytoplasmic compartments with LaminB1 and GAPDH as markers, respectively.

(d) RT-qPCR from asynchronous or serum-starved MCF10A cells showing increased unspliced mRNA following PlaB treatment (500 nM, 3 h). Shown is the mean ± s.d. from three independent biological replicates (n = 3), p-values are indicated in the figure; unpaired, two-tailed t-test.

(e) As in (c) but for foreskin fibroblasts.

(f) Cell cycle quantification from high-content imaging of foreskin fibroblasts after EdU incorporation, using DAPI staining for DNA content. Shown is the mean ± s.d. from three independent biological replicates (n = 3).

(g) As in (d) but for foreskin fibroblasts.

(h) cytoDRIP blot showing cytoplasmic hybrids extracted from equal numbers of asynchronous or serum-starved foreskin fibroblasts following DMSO or PlaB treatment (500 nM, 3 h), with mock and RH treatment prior to pull-down.

(i) As in (b) but for BAX^−/−BAK^−/− MCF10A cells.

(j) Western blot showing fractionation of asynchronous and serum-starved BAX^−/−BAK^−/− MCF10A cells into soluble nuclear and cytoplasmic compartments with LaminB1 and GAPDH as markers, respectively.

(k) cytoDRIP blot showing cytoplasmic hybrids in asynchronous and serum-starved BAX^−/−BAK^−/− MCF10A cells with DMSO or PlaB treatment (500 nM, 3 h). Each sample was treated with RH in vitro to confirm the specificity of the hybrid IP.

(l) Left: experimental workflow. Right: blots showing hybrids isolated from the cytoplasm or nucleoplasm of siCon or siSETX-treated HeLa cells, with mock or LMB treatment (3 h, 5 nM) prior to harvest.

(m) Left: experimental workflow. Middle: western blot as in (c) from HeLa cells treated with vehicle control (DMSO) or PlaB (500 nM, 3 h). Right: blot showing hybrids as in (i) but in HeLa cells treated with LMB (2 h, 5 nM) followed by PlaB + LMB for a further 3 h.

(n) Representative images showing cyclin B1 localization in fixed HeLa cells treated with LMB (5 h, 5 nM) or vehicle control (EtOH). Scale bar is 10 μm.

(o) RT-qPCR from HeLa cells showing increased unspliced mRNA following treatment with PlaB (500 nM) for the times indicated. Shown is the mean ± s.d. from three independent biological replicates (n = 3).

(p) As in (o) but cells were treated with PlaB (500 nM, 3 h) and then fresh media was added following PlaB withdrawal for the times indicated. Shown is the mean ± s.d. from four independent biological replicates (n = 4).