Journal: Cell Death and Differentiation

Article Title: Proteinase 3 depletion attenuates leukemia by promoting myeloid differentiation

doi: 10.1038/s41418-024-01288-4

Figure Lengend Snippet: a – d messenger RNA (mRNA), Protein level detected by WB and ELISA, and immunofluorescence staining of Prtn3 were determined in neutrophils and LSK cells from Prtn3 −/− and WT mice ( n = 3); e Representative flow cytometry plots for neutrophils, monocytes, T cells and B cells in bone marrow from WT and Prtn3 −/− mice. Numbers denote the frequency of each population among live singlets( n = 7). f Representative flow cytometry plots for neutrophils, monocytes, T cells, and B cells in bone marrow from the CD45.2 cells in CD45.1 recipient mice ( n = 7). Numbers denote the frequency of each population among live singlets. g , h Quantification of the frequency of CD45.1, CD45.2, the ratio of CD45.2/CD45.1, neutrophils, monocytes, T cells, and B cells in the CD45.2 recipient mice ( n = 7). scale bars, 50 μm; * p < 0.05, ** p < 0.01, *** p < 0.001. Data are the mean ± s.d.; n : biologically independent experiments. Statistical analysis was performed using an unpaired two-tailed Student’s t test.

Article Snippet: human primary CD34+ cells, HL-60 cell line, and NB4 cells line were transfected with non-targeting control siRNAs (sc-44230, Santa Cruz), or Prtn3 siRNA (sc-42968, Santa Cruz).

Techniques: Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Flow Cytometry, Two Tailed Test

Journal: Cell Death and Differentiation

Article Title: Proteinase 3 depletion attenuates leukemia by promoting myeloid differentiation

doi: 10.1038/s41418-024-01288-4

Figure Lengend Snippet: a The Co-immunoprecipitation (Co-IP) complex was subjected to silver-staining ( n = 3); b the peptide sequences of the STAT3 protein as detected in the Co-IP complex by mass spectrometry; c WB analysis of Co-IP complex confirmed that STAT3-Flag interacted with PRTN3-HA in HEK 293 T cells ( n = 3); d WB analysis of Co-IP complex confirmed that PRTN3-HA interacted with STAT3-Flag in HEK 293 T cells ( n = 3); e measurements of the binding affinity between STAT3 and PRTN3 by SPR method. f WB analysis of Co-IP complex confirmed that STAT3 interacted with PRTN3 in CD34+ cells ( n = 3); g WB analysis of Co-IP complex confirmed that PRTN3 interacted with STAT3 in CD34+ cells ( n = 3); h the immunofluorescence showed that STAT3 colocalized with PRTN3 and were expressed in both the cytoplasm of CD34+ cells ( n = 3). Data are the mean ± s.d.; n : biologically independent experiments. Statistical analysis was performed using an unpaired two-tailed Student’s t test.

Article Snippet: human primary CD34+ cells, HL-60 cell line, and NB4 cells line were transfected with non-targeting control siRNAs (sc-44230, Santa Cruz), or Prtn3 siRNA (sc-42968, Santa Cruz).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Silver Staining, Mass Spectrometry, Binding Assay, Immunofluorescence, Two Tailed Test

Journal: Cell Death and Differentiation

Article Title: Proteinase 3 depletion attenuates leukemia by promoting myeloid differentiation

doi: 10.1038/s41418-024-01288-4

Figure Lengend Snippet: a The schematic diagram of human STAT3 and its truncated mutants. b The schematic diagram of human PRTN3 and its truncated mutants. c Flag-tagged STAT3 or its mutants and HA-tagged PRTN3 were co-transfected into HEK293T cells. Cell lysates were immunoprecipitated with an anti-Flag antibody and then immunoblotted with the indicated antibodies ( n = 3). d HA-tagged PRTN3 or its mutants and Flag-tagged STAT3 were co-transfected into HEK293T cells. Cell lysates were immunoprecipitated with an anti-HA antibody and then immunoblotted with the indicated antibodies ( n = 3). e measurements of the binding affinity between the different domains of STAT3 and PRTN3 by SPR method. f SDS/PAGE analysis of hPR3-generated fragments of STAT3. At different times, the incubation mixtures were separated on a 10% SDS/PAGE gel denatured/reduced conditions and visualized by silver nitrate staining ( n = 3). g The solvent-accessible surface of the hPR3 active site was generated with Yasara ( http://www.yasara.org ). h Identification of the cleavage sites within the whole STAT3 after incubation with PRTN3 at pH 7.4 and 37 °C. Cleavage products were monitored by MALDI-TOF MS. Arrows indicate the cleavage sites of STAT3 that were identified. The width of the arrows indicates the intensity of the cleavage. Data are the mean ± s.d.; n : biologically independent experiments. Statistical analysis was performed using an unpaired two-tailed Student’s t test.

Article Snippet: human primary CD34+ cells, HL-60 cell line, and NB4 cells line were transfected with non-targeting control siRNAs (sc-44230, Santa Cruz), or Prtn3 siRNA (sc-42968, Santa Cruz).

Techniques: Transfection, Immunoprecipitation, Binding Assay, SDS Page, Generated, Incubation, Staining, Solvent, Two Tailed Test

Journal: Cell Death and Differentiation

Article Title: Proteinase 3 depletion attenuates leukemia by promoting myeloid differentiation

doi: 10.1038/s41418-024-01288-4

Figure Lengend Snippet: a HEK293T transfected with empty vector or PRTN3-HA were treated with cycloheximide (100 ng/mL) for the indicated periods of time ( n = 3); b human CD34+ cells transfected with Si-Control or Si- Prtn3 were treated with cycloheximide (100 ng/mL) for the indicated periods of time ( n = 3); c HEK293T transfected with Empty vector or PRTN3-HA were treated with or without MG132 (50 μM) for 6 h ( n = 3). d human CD34+ cells transfected with Si-Control or Si- Prtn3 were treated with or without MG132 (50 μM) for 6 h ( n = 3). e In vitro ubiquitination assay of Empty vector, PRTN3-HA transfected to HEK293T, Si-Control or Si- Prtn3 transfected to human CD34+ cells. Cell lysates were immunoprecipitated with anti-FLAG antibody followed by immunoblotting analysis with anti-HA or anti-FLAG antibody ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001. Data are the mean ± s.d.; n : biologically independent experiments. Statistical analysis was performed using an unpaired two-tailed Student’s t test.

Article Snippet: human primary CD34+ cells, HL-60 cell line, and NB4 cells line were transfected with non-targeting control siRNAs (sc-44230, Santa Cruz), or Prtn3 siRNA (sc-42968, Santa Cruz).

Techniques: Transfection, Plasmid Preparation, Control, In Vitro, Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Two Tailed Test

Journal: Cell Death and Differentiation

Article Title: Proteinase 3 depletion attenuates leukemia by promoting myeloid differentiation

doi: 10.1038/s41418-024-01288-4

Figure Lengend Snippet: a protein expression of STAT3, P-STAT3, PRTN3, and β-Actin were analyzed in HEK293T cells with empty vector or PRTN3-HA transfection to overexpress PRTN3 and then treated with 10nM G-CSF for 24 h ( n = 3); (b)protein expression of STAT3, P-STAT3, PRTN3, and β-actin were analyzed in Prtn3 −/− primary Ckit+ cells with G-CSF treatment for 24 h, compared to WT cells ( n = 3); c immunofluorescence staining of STAT3 in Prtn3 −/− primary Ckit+ cells with G-CSF treatment for 2 h ( n = 3); d messenger RNA (mRNA) of P27 kpi1 and C/Ebpα were determined in Prtn3 −/− primary Ckit+ cells with G-CSF treatment for 24 hours, compared to WT cells ( n = 3); e Lineage- Sca-1+c-Kit+ (LSK) cells from WT and Prtn3 −/− mice were used for ex vivo myeloid culture ( n = 3). scale bars, 50 μm; * p < 0.05, ** p < 0.01, *** p < 0.001. Data are the mean ± s.d.; n : biologically independent experiments. Statistical analysis was performed using an unpaired two-tailed Student’s t test.

Article Snippet: human primary CD34+ cells, HL-60 cell line, and NB4 cells line were transfected with non-targeting control siRNAs (sc-44230, Santa Cruz), or Prtn3 siRNA (sc-42968, Santa Cruz).

Techniques: Expressing, Plasmid Preparation, Transfection, Immunofluorescence, Staining, Ex Vivo, Two Tailed Test

Journal: Cell Death and Differentiation

Article Title: Proteinase 3 depletion attenuates leukemia by promoting myeloid differentiation

doi: 10.1038/s41418-024-01288-4

Figure Lengend Snippet: a Protein expression of STAT3, P-STAT3, PRTN3, and β-Actin were analyzed in LSK cells, which were isolated from Prtn3 −/− and WT mice, and treated with 10 nM Stattic for 24 h ( n = 3); b immunofluorescence staining of STAT3 and PRTN3 were analyzed in LSK cells, which were isolated from Prtn3 −/− and WT mice, and treated with 10 nM Stattic for 24 h ( n = 3); c Quantification of the number of cells with nuclear translocation from ( b ) ( n = 7). d messenger RNA (mRNA) of Prtn3 , C/EBPɑ , p27 kip1 , Bcl-X L , and C-myc were determined in LSK cells, which were isolated from Prtn3 −/− and WT mice, and treated with 10 nM Stattic for 24 h ( n = 3); e Representative flow cytometry plots for neutrophils and monocytes in LSK cells from WT and Prtn3 −/− mice. Numbers denote the frequency of each population among live singlets ( n = 3). f The schematic diagram of WT and Prtn3 −/− mice with fed STAT3 inhibitor. g Representative flow cytometry plots for neutrophils and monocytes in LSK cells from WT and Prtn3 −/− mice. Numbers denote the frequency of each population among live singlets ( n = 7). scale bars, 50 μm; * p < 0.05, ** p < 0.01, *** p < 0.001. Data are the mean ± s.d.; n : biologically independent experiments. Statistical analysis was performed using an unpaired two-tailed Student’s t test.

Article Snippet: human primary CD34+ cells, HL-60 cell line, and NB4 cells line were transfected with non-targeting control siRNAs (sc-44230, Santa Cruz), or Prtn3 siRNA (sc-42968, Santa Cruz).

Techniques: Expressing, Isolation, Immunofluorescence, Staining, Translocation Assay, Flow Cytometry, Two Tailed Test

Journal: Cell Death and Differentiation

Article Title: Proteinase 3 depletion attenuates leukemia by promoting myeloid differentiation

doi: 10.1038/s41418-024-01288-4

Figure Lengend Snippet: a The experimental design. b Survival curves of AML recipients with WT or Prtn3 −/− AML cells ( n = 10 per group). c histogram analysis of the percentage of GFP+ leukemia cells in the peripheral blood (PB) of recipients 30 days after transplantation ( n = 6). d immunofluorescence staining assay shows GFP+ leukemia cells in the spleen of recipients 30 days after transplantation ( n = 6). e The bone marrow color (left) and histogram (right) of recipient on 30 days after transplantation ( n = 6). f Flow cytometry (left) and histogram (right) analysis show the percentages of neutrophils from AML cells (GFP+) in the BM of recipients ( n = 6). g Histogram analysis shows the percentages of GFP+ neutrophils with PE-E.coli uptake from BM of AML Prtn3KO recipients at different time points ( n = 3). h Histogram analysis shows the percentages of GFP+ neutrophils capable of chemotaxis migration in the abdominal cavity of AML Prtn3KO recipients with E. coli injection for 6 h ( n = 3). i messenger RNA of Prtn3 expression in healthy peoples and patients ( n = 6). j , k messenger RNA and protein expression of PRTN3 and β-Actin were analyzed in HL60 cells with Si-control or Si- Prtn3 treatment for 24 h ( n = 3). l Flow cytometry (left) and histogram (right) analysis of the percentage of CD11b+ leukemia cells in the HL60 cells with Si-control or Si- Prtn3 treatment for 7 days ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001. Data are the mean ± s.d.; n : biologically independent experiments. Statistical analysis was performed using an unpaired two-tailed Student’s t test.

Article Snippet: human primary CD34+ cells, HL-60 cell line, and NB4 cells line were transfected with non-targeting control siRNAs (sc-44230, Santa Cruz), or Prtn3 siRNA (sc-42968, Santa Cruz).

Techniques: Transplantation Assay, Immunofluorescence, Staining, Flow Cytometry, Chemotaxis Assay, Migration, Injection, Expressing, Control, Two Tailed Test

Journal: Cell Death and Differentiation

Article Title: Proteinase 3 depletion attenuates leukemia by promoting myeloid differentiation

doi: 10.1038/s41418-024-01288-4

Figure Lengend Snippet: In WT mice, the level of G-CSF-induced STAT3 in LSK cells is counterbalanced by the PRTN3-dependent ubiquitin-mediated degradation of STAT3. In prtn3 knockout (KO) mice, G-CSF-induced a sustained STAT3 phosphorylation and nuclear translocation that promotes robust expression of differentiation genes, including C/Ebpα and P27 kpi1 , that stimulates myeloid differentiation to promote an increased.

Article Snippet: human primary CD34+ cells, HL-60 cell line, and NB4 cells line were transfected with non-targeting control siRNAs (sc-44230, Santa Cruz), or Prtn3 siRNA (sc-42968, Santa Cruz).

Techniques: Ubiquitin Proteomics, Knock-Out, Phospho-proteomics, Translocation Assay, Expressing