Fig 2: STAT5b (Tyr699 mutant) reversed the neuroprotection of neuron and activated the NF-κB pathway of microglia. (A) Vector map of STAT5b (Tyr-699 mutation). (B) Sequencing and matching results of STAT5b OV and STAT5b (Y699A). (C) The primary neurons were transfected with STAT5b OV and STAT5b (Y699A) plasmids for 48 h, and the neurons were deprived of oxygen and sugar for 30 min, then reperfusion for 3 h, and neurons were stained with calcein-AM and DAPI, and quantified (D). n = 5, (OGD + Vector) group vs. (OGD + Vector +dauricine) group, *** p < 0.001; (OGD + Vector + dauricine) group vs. (OGD + STAT5b (Y699A) + dauricine) group, # p < 0.05, unpaired Student’s t-test. (E) STAT5b OV and STAT5b (Y699A) plasmids were transfected into primary microglia for 48 h, and induced by LPS or LPS + dauricine for 3 h, then detected by the western blot and representative figure of NF-κB, P-NF-κB, and β-actin were shown, and was counted with ratio of P-NF-κB to NF-κB (F). n = 3, (Vector + LPS) group vs. (Vector + LPS + Dauricine) group, *** p < 0.001; (Vector + LPS + Dauricine) group vs. (STAT5b OV + LPS + Dauricine) group or (STAT5b (Y699A) + LPS + Dauricine) group, ### p < 0.001, unpaired Student’s t-test.

Fig 3: SENP1 knockdown suppresses MCL growth in vivo.A Images of the MCL xenografts in each mouse subcutaneously injected with shCon or shSENP1-3 transfected Jeko-1 cells (n = 5 per group). B Tumor volume in shCon and shSENP1-3 groups was recorded every two days for total 17 days. C Tumor growth rate was compared between shCon and shSENP1-3 groups. D Tumor weight was compared between shCon and shSENP1 groups. E Representative immunohistochemical images of SENP1 staining in MCL xenografts from shCon and shSENP1-3 groups. F The mean integrated optical density (IOD) of SENP1 protein in MCL xenografts from shCon and shSENP1-3 groups was calculated by Image-Pro Plus 6.0 software. G SENP1 protein level in each MCL xenograft from shCon and shSENP1-3 groups was measured by western blot. H Representative images of Ki67 staining, TUNEL assay and p-STAT5 in MCL xenografts from shCon and shSENP1-3 groups. I The mean integrated optical density (IOD) of Ki67, TUNEL and p-STAT5 in MCL xenografts from shCon and shSENP1-3 groups were calculated by Image-Pro Plus 6.0 software. Data were presented as mean ± SD and analyzed by two-sided Student’s t test. n = 3; *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 4: Pimozide abolishes B19V replication in primary CD36+ erythroid progenitor cells (EPCs).(A) Inhibition of phosphorylation of STAT5 by pimozide. CD36+ EPCs were treated with dimethyl sulfoxide (DMSO) vehicle or pimozide at various concentrations. At 48 h post-treatment, cells were collected, washed and lysed for Western blotting with detection of pSTAT5 by anti-pSTAT5(Y694) antibody. The blot was reprobed for total STAT5 using a mouse anti-STAT5 antibody and for ß-actin as a loading control. (B-D) Inhibition of B19V DNA replication by pimozide. CD36+ EPCs were pre-incubated with DMSO or pimozide at a final concentration of 15 µM or 25 µM 6 h prior to B19V infection. At 48 h post-infection, cells were subjected to (B) flow cytometry analysis for the B19V-infected cell population with an anti-B19V capsid antibody, (C) Hirt DNA extraction, followed by Southern blot analysis with a B19V M20 DNA probe, or (D) Western blotting with an anti-pSTAT5(Y694) antibody, and reprobing with an anti-ß-actin antibody. (E&F) Evaluation of the effect of pimozide on cell proliferation. CD36+ EPCs were treated with either DMSO or pimozide (15 µM or 25 µM). After 48 h, treated cells were incubated with bromodeoxyuridine (BrdU) for 1 h to analyze cell-cycle progression by a BrdU incorporation assay. (E) Results of a representative cell-cycle analysis experiment. (F) Relative fold change in the S phase cell population of each group is shown, with means and standard deviations of three independent experiments. P values are calculated using one-way and Tukey-Kramer post-test, compared with DMSO control. * denotes P<0.05; **** denotes P<0.0001; and n.s. (P>0.05) denotes no statistical significance.

Fig 5: Failure of B19V replicative form DNA clones with STAT5-binding element mutations in replication in transfected UT7/Epo-S1 cells.(A) Diagram of the B19V full-length M20 RF genome and various half ITR-deleted N8 RF genomes with mutations at STAT5-binding elements (STAT5BEs). Red squares indicate the position of the Ori sequences at both ITRs, and grey squares indicate mutated Ori (mOri). The sequence of the mOri is shown with mutated nucleotides in grey in the STAT5BE. (B&C) Southern blot analysis. (B) The N8 RF DNA, or derivatives with mutations in the STAT5BE of either the left ITR (N8mOriL), right ITR (N8mOriR), or both (N8mOri), were transfected into UT7/Epo-S1 cells. (C) M20, and M20mOri, a derivative of the M20 RF DNA with STAT5BEs of both ITRs mutated, were transfected into UT7/Epo-S1 cells. At 48 h post-transfection, cells were collected for Hirt DNA extraction. And Hirt DNA samples were analyzed by Southern blotting with an M20 DNA probe. RF DNA (RF), ssDNA (ss), and Dpn I-digested DNA (shown with a line) are indicated. Mitochondrial DNA (Mito DNA) was used as a loading control (lower panels). (D) Viral protein expression of B19V DNA mutants. M20 or M20mOri transfected UT7/Epo-S1 cells were stained with anti-NS1 or anti-capsid antibodies. Confocal images were taken with an Eclipse C1 Plus (Nikon) microscope at 100 × magnification.