Fig 1: PIAS3 exerts an enhancing effect on UL44 SUMOylation. (a) The in vivo SUMOylation of UL44 in transfection assays, in the presence of exogenous PIAS3 using different SUMO peptides. pCMV-Myc-UL44 and pRK-Flag-SUMO-1/2/3, along with or without pcDNA-His-PIAS3, were co-introduced into HEK293T cells. At 48 h post transfection, Western blot with anti-Myc antibody was performed to detect UL44 and UL44-SUMO in total cell lysates. Free SUMO-1/2/3 peptides were probed with anti-Flag antibody. GAPDH was set as the internal control. (b) The in vivo SUMOylation of UL44 in transfection assays, in the presence of exogenous UBC9 and PIAS3 (WT or C334S mutant). pCMV-Myc-UL44, pRK-Flag-SUMO-1 and pcDNA-HA-UBC9, along with or without pcDNA-His-PIAS3 (WT or C334S mutant) were co-introduced into HEK293T cells. At 48 h post-transfection, Western blot with anti-Myc or anti-Flag antibody was performed to analyze UL44 and UL44-SUMO in total cell lysates. SUMO-1, UBC9 and PIAS3/PIAS3-C334S were probed with anti-Flag, anti-HA and anti-His antibodies, respectively. GAPDH served as the internal control. (c) The in vivo SUMOylation of UL44 during wt-HCMV infection, upon interference of endogenous PIAS3. U251 cells, after transfection with siControl (a negative control siRNA molecule) or siPIAS3 (a chemically synthesized siRNA that specially targets the CDS region of PIAS3 mRNA) for 24 h, were infected with wt-HCMV at an MOI of 1. At 48 hpi, the supernatant of total cell lysates, either immunoprecipitated with anti-UL44 antibody or not, was analyzed by Western blot using anti-UL44 or anti-SUMO1 antibody as indicated to detect UL44 and UL44-SUMO proteins. (d) The in vitro SUMOylation assays on UL44 protein. His-UL44, GST-PIAS3 and GST-PIAS3-C334S were individually expressed in E. coli BL21 (DE3), before further protein purification by affinity chromatography and SEC (size exclusion chromatography). Enzymatic reactions were set up with the SUMOylation-related components as indicated, and the production of SUMOylated UL44 was determined by Western blot with anti-His or anti-SUMO1 antibody. For all panels, each set of assays was repeated three times and a representative one was shown
Fig 2: UL44, HCMV DNA polymerase processivity factor, interacts with human E3 SUMO ligase PIAS3. (a) Co-IP analysis of the association between UL44 and PIAS3 in transfected cells. pCMV-Myc-UL44 and pRK-Flag-PIAS3 plasmids were co-introduced into 293 T cells. At 48 h post transfection, the supernatant of cell lysate was taken to IP (immuno-precipitate) with anti-Myc or anti-Flag mAbs. The immunoprecipitants, after heat denaturation, were analyzed by Western blot with anti-Myc or anti-Flag mAbs. Meanwhile, negative controls were set up by cell co-transfection with pCMV-Myc-UL44 and a plasmid expressing the Flag tag, or with pRK-Flag-PIAS3 and a plasmid expressing the Myc tag. (b) His and GST pull-down analyses of the direct interaction between UL44 and PIAS3. Expressed in E. coli BL21 (DE3), His-tagged UL44 and GST-tagged PIAS3 were individually purified by affinity chromatography using Ni2+-NTA or GST column. Then, the reciprocal GST and His pull-down assays were performed to verify the direct UL44-PIAS3 interaction in vitro. (c) Co-IP analysis of UL44 association with endogenous PIAS3 under HCMV infection. U251 cells were infected with wt-HCMV at an MOI of 1, before preparation of the total cell lysates at 48 hpi. The input proteins and IP samples were analyzed by Western blot, using as the indicated anti-UL84, anti-UL44 or anti-PIAS3 antibody, respectively. For all panels, results were representative of three independent experiments
Fig 3: Relative mRNA expression levels (RQ) of PIAS3, PIP, STAT5A, and STAT5B in T47D cell lines: control T47D CTRL vs. PIAS3-silenced T47D shPIAS3. The graph shows the level of mRNA isolated from cells cultured in medium (RPMI), after incubation with PRL (1 ng/mL, 20 min), DOX (0.5 µM, 48 h), and with both: PRL (1 ng/mL, 20 min) and DOX (0.5 µM, 48 h). Data represent the mean ± SD of three independent measurements. * p < 0.05, ** p < 0.01, *** p < 0.001; two-way MANOVA with Bonferroni post hoc tests. DOX, doxorubicin; PIAS3, protein inhibitor of activated STAT3; PIP, prolactin-induced protein; PRL, prolactin; PRLR, prolactin receptor; RQ, relative quantification, real-time PCR; SD, standard deviation; SOCS3, suppressor of cytokine signaling 3; STAT5A, signal transducer and activator of transcription 5A; STAT5B, signal transducer and activator of transcription 5B; STAT5-P, phosphorylated signal transducer and activator of transcription 5.
Fig 4: mRNA levels of (A) PIP and (B) PIAS3 according to the status of estrogen receptors (ER) (ER− n = 10, ER+ n = 32). * p < 0.01, Mann–Whitney U test. ER, estrogen receptor; PIAS3, protein inhibitor of activated STAT3; PIP, prolactin-induced protein; SD, standard deviation.
Fig 5: Immunohistochemical analysis of PIP, PIAS3, SOCS3, STAT5, STAT5-P, and PRLR expression. Magnification ×200. PIAS3, protein inhibitor of activated STAT3; PIP, prolactin-induced protein; PRLR, prolactin receptor; SOCS3, suppressor of cytokine signaling 3; STAT5, signal transducer and activator of transcription 5; STAT5-P, phosphorylated signal transducer and activator of transcription 5.
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