Fig 2: Biological effects of thioredoxin deletion on murine hematopoietic stem/progenitor cells. TXN1fl/fl (control) and ROSA-CreER-TXN1.fl/fl mice were treated with tamoxifen (75 mg/kg) i.p. daily for 5 days. Mice were sacrificed at day 10. Bone marrow cells were harvested and enriched for hematopoietic stem/progenitor cells using lineage depletion kit. The hematopoietic stem/progenitor- enriched cells were then measured for (A) beta-gal expression by flow cytometry staining, and Annexin (B). ROS measurement by carboxy-H2DCFDA staining. C JC probe to measure mitochondrial membrane polarization (D). E, F Seahorse measurement of oxygen consumption and cellular ATP concentration. G Relative mitochondrial DNA content (mtDNA:nDNA), The results were normalized to the ratio of mtDNA:nDNA of control cells. (n = 10 Mean ± SEM or SD). H–J PARK2, PINK1 and LC3B mRNA expression in murine HSPCs were quantified using qPCR (n = 3–5 Mean ± SEM or SD)

Fig 3: TXN1 regulates P53 at both post transcriptional and transcriptional levels. A TXN1 deletion prevented p53 and MDM2 from degradation. EML/TXN1KO and EML control cells were incubated with 50uM cycloheximide (CHX). At various time-points (2, 4, 6, 8, 10, 12, 14 and 24 h), the cells were harvested and p53, MDM2 and ß-actin were measured by Western Blot analysis. B Expression levels of p53 and MDM2 were determined by image J software of the immunoblots in A (errors bars indicate mean ± sd; n = 3). C EML cells were transfected with flag-tag plasmid encoding TXN1 as indicated. Then the binding of TXN1 to the P53 promotor was analyzed by chromatin immunoprecipitation (CHIP-qPCR). D CHIP-qPCR representing the significant enrichment of the total input of the TP53 promotor region in a CHIP experiment using the TXN1 flag-tag antibody relative to the enrichment by nonspecific IgG. E) Tp53 promoter region (- 1600 to - 100) was cloned into pGL3 firefly/renilla luciferase reporter system. The p53 promoter-pGL3 reporter was transduced into EML cells and the EML cells were then treated with PX12 (thioredoxin inhibitor, recombinant TXN1 or NAC). Relative luciferase activity Luc (fold change) was calculated from the ratio of P53PGL3 Luc activity after normalization to the pRenilla

Fig 4: p53 plays a critical role in TXN1 mediated effects. A EML cells were transduced with Cas9 control, TXN1 specific CRISPR/Cas9, p53 specific CRISPR/cas9 or both. p53 and P53 downstream signaling pathway were assessed by immunoblot. ß-actin was used to assess equal loading. B Cells proliferation rate was assessed using BrdU proliferation assay. C JC-I mitochondrial membrane depolarization was measured D ROS measurement by carboxy-H2DCFDA staining. EML/TXN1KO cells were treated with 250uM NAC and ROS levels were measured. E Cell proliferation. EML/TXN1KO cells were treated with 250uM NAC and cells proliferation rate was assessed using BrdU proliferation assay as described. F In vitro cultured colony forming unit in EML control cells, EML Cas9 control cells, EML TXN1 KO cells and EML TXN1 KO/NAC cells. EML/TXN1KO cells were treated with 250uM NAC, and plated at 2X104/dish in Meth3434 semi-solid medium and incubated for 10-12 days. G JC-1 mitochondrial membrane depolarization was not affected by NAC treatment. EML/TXN1KO cells were treated with 250uM NAC and JC-1 levels were measured. (n = 4 Mean ± SEM or SD)

Fig 5: In-vivo thioredoxin deletion is lethal and is associated with reduced splenocytes and bone marrow cells in a murine model. A Mice with floxed TXN1 were crossed with mice carrying the ROSA-CreER transgene to develop an inducible model for thioredoxin deletion. TXN1fl/fl (control) and ROSA-CreER-TXN1fl/fl mice were given TAM (75 mg/kg) i.p. daily for 5 days. Mice were sacrificed at day 10. Thioredoxin was measured by Western blot in bone marrow cells. B Kaplan–Meier survival curve. TXN1fl/fl mice and ROSA-Cre-TXN1fl/fl mice were treated with TAM (75 mg/kg) i.p. daily for 5 days. Animal survival was monitored daily. C The weight of the mice as described in B was measured, TXN1 deletion resulted in drastic weight loss (humane endpoints required euthanasia following a > 20% reduction in body weight). D Total BM cells (one femur and one tibia) and total thymic cells following harvesting at day 10 of TAM injection. E Number of spleenocytes following harvesting at day 10 of TAM treatment. F Spleen weight (g) at day 10 of TAM treatment (n = 10 mice for each group each experiment, one of 3 separate experiments was shown). G Histology of bone marrow. Bone was harvested at day 10 from the mice as described in F. H Histology of spleen: Spleen was harvested at day 10 from the mice as described in F