Fig. 5: PPIA regulates protein phase separation of its substrates.
a, Stress-granule formation was visualized and quantified with G3BP1 staining after stress induction with sodium arsenite in HeLa control or PPIA Kd cells. DAPI, blue; G3BP1, green. Scale bars, 50 µm. PPIA knockdown was partially rescued by the reintroduction of knockdown-resistant PPIA. Cell viability was measured on an automated cell counter with acridine orange/propidium iodide staining solution using n = 6 independently treated replicates per group. Data are means ± s.d.; **P < 0.01, ****P < 0.0001; two-sided Wilcoxon rank-sum test; n = 616 (control), n = 656 (control + PPIA), n = 254 (PPIA knockdown) and n = 293 (PPIA knockdown + PPIA) cells were analysed following blinding. Data are representative of three independent experiments. b, Staining for DDX6 revealed significantly fewer P-bodies in HeLa cells following PPIA knockdown. Scale bars, 20 μm. The arrowhead indicates a representative P-body. ****P < 0.0001; two-sided Wilcoxon rank-sum test; n = 457 (control) and n = 284 (PPIA knockdown) cells were analysed following blinding. Data are representative of three independent experiments. c, OCI-AML3 cells that express a stable, gene-edited NPM1-mCherry fusion, exhibited smaller, more fragmented nucleoli following PPIA knockdown. Cell designation was blinded for the analyst. Scale bars, 20 μm. ***P < 0.001, ****P < 0.0001; two-sided Wilcoxon rank-sum test; n = 564 (control) and n = 617 (PPIA knockdown) cells were analysed following blinding. Data are representative of three independent experiments. d, PLAs in primary haematopoietic stem cells (lin−/c-Kit+/Sca1+/CD34−/CD135−) between PPIA and PABPC1, DDX6 and NPM1. Shown is the signal quantification using single-antibody staining as a control. Scale bar, 5 μm. ****P < 0.0001; two-sided Wilcoxon rank-sum test. Cells were analysed following blinding. Box plots indicate minima, maxima and quartiles; n = 20 randomly chosen cells per group. Data are representative of two independent experiments.
