Extended Data Fig. 3: PPIA’s effect on transcription and translation in haematopoietic cells.
a, Impaired translation in haematopoietic stem and progenitor cells following pharmacological PPIA inhibition. Haematopoietic stem cells (lin−/cKit+/Sca1+/CD34−/CD135−) were isolated and expanded in vitro as previously published75. Translation rates were measured through bio-orthogonal labeling with a fluorescently labeled puromycin analog for two hours in cells that were pre-treated with DMSO or PPIA inhibitor TMN355 (10 μM, 24 h). Shown is a representative of three independent biological replicates, in which n = 1,880 (DMSO) and n = 1,303 (TMN355) cells were analysed and fluorescence was measured per cell. Scale bar, 100 μm (****P < 0.0001, two-sided Wilcoxon rank-sum test). Bottom panel depicts DAPI counterstain. b, Reduced expression of PPIA substrates in OCI-AML3 cells following PPIA knockdown. Western blot analyses to detect protein expression of PPIA and PPIA protein partners PABPC1, DDX6, and NPM1 in the OCI-AML3 cell line. GAPDH was used as a loading control for protein normalization and densitometry was measured relative to GAPDH expression. The images represent results of two independent experiments. c, Protein expression of PPIA client proteins is decreased in Ppia−/− HSPCs. Western blot analyses to detect protein expression of PPIA and PPIA protein partners PABPC1, DDX6, G3BP1, and NPM1 in mouse lineage-depleted bone marrow cells. The images represent results of Ppia+/− and Ppia−/− animals (n = 1 each). β-tubulin was used as a loading control for protein normalization. This experiment was performed once. d, Ppia knockout versus heterozygous haematopoietic stem and progenitor cells (lin−/c-Kit+) upregulate genes involved in translation. Volcano plot shows comparable up- and down−regulation of genes. Gene set enrichment analysis of haematopoietic stem and progenitor cells of Ppia knockout or heterozygous animals. N = 3 independent animals were analysed per group. Genes encoding the entire mouse chaperome or the ubiquitin-proteasome system (UPS) were not significantly increased in knockout cells. However, the gene ontology ‘cytoplasmic translation’ was significantly upregulated in knockout cells. e, Ppia knockout cells show the transcriptional signature of aging. Haematopoietic stem and progenitor cells (lin−/cKit+) of three Ppia knockout animals compared to three heterozygous animals show significant upregulation of the aging marker gene P-selectin45. In addition, we observed a strong gene set enrichment resembling aged haematopoietic stem cells44. Statistics derived using two-sided Wilcoxon rank-sum test (n = 3 mice per group). f, Genes encoding PPIA substrates are upregulated in Ppia knockout haematopoietic stem and progenitor cells. Relative FPKM changes shown in cumulative violin plots representing three animals per genotype (Ppia knockout versus heterozygous); y-axis represented in log2. Gene set enrichment analysis of haematopoietic stem and progenitor cells (lin−/c-Kit+) of Ppia knockout or heterozygous animals shows significant upregulation of PPIA substrates (n = 307) compared to the overall proteome (n = 6,114) in knockout animals. Statistics derived using two-sided Wilcoxon rank-sum test. g, No increased spontaneous aggregation of misfolded proteins in absence of PPIA. Left panel: Protein misfolding was quantified using the molecular rotor ProteoStat with affinity for aggregated proteins. Increased protein aggregation causes the dye to stop spinning and emit fluorescence. We analysed misfolding in HeLa cells transduced with either scramble lentivirus or following PPIA knockdown, and used proteasome inhibition as positive control (MG132, 10 μM, 16 h). Scale bar, 80 μm. Right panel: Relative mean intensity per cell was plotted and calculated after blinding. A total of 8 independently treated replicates were analysed per group in this assessment. Statistics calculated using two-sided Wilcoxon rank-sum test (n = 154 control cells, n = 147 PPIA knockdown cells for, n = 75 for MG132-treated control cells, and n = 86 for MG132-treated PPIA knockdown cells; the experiment was performed once).
