Fig 2: Ssu72 depletion in BAT affects translation of OXPHOS subunits.a Western blots of ClpP and phosphorylated eIF2α (P-eIF2αSer51) in BAT from Ssu72 WT and aKO mice housed at RT (n = 3 mice per group). b Relative mRNA expression levels of OXPHOS complex subunits encoded by nuclear DNA in BAT from Ssu72 WT and aKO mice housed at RT (n = 5 mice per group). Data are presented as mean ± SEM. Statistical comparisons were made using two-tailed Student’s t test. c Western blots of mitochondrial OXPHOS complex subunits (CI-V) encoded by nuclear DNA in BAT from Ssu72 WT and aKO mice housed at RT (n = 3 mice per group). d Relative mRNA expression levels of OXPHOS complex subunits encoded by mitochondrial DNA (Complex I, IV, and V) in BATs from Ssu72 WT and aKO mice housed at RT (n = 5 mice per group). Data are presented as mean ± SEM. Statistical comparisons were made using two-tailed Student’s t test. e Western blots of MT-ND1 and MT-CO1 in BAT from Ssu72 WT and aKO mice housed at RT (n = 3 mice per group). f Western blots of OXPHOS complex subunits (CI-V) encoded by nuclear DNA in BAT from 12-week-old Ssu72 WT, aKO, and aKO;cTg female mice housed at RT (n = 3 mice per group). g Western blots of MT-ND1 and MT-CO1 in BAT from 12-week-old Ssu72 WT, aKO and aKO;cTg female mice housed at RT (n = 3 mice per group). For a–e, 11-week-old male Ssu72 WT and aKO mice were used. Source data are provided as a Source Data file.

Fig 3: Ssu72 phosphatase is potentially involved in physiological regulation of thermogenic adipose tissues, especially BAT.a Various mouse tissues including epididymal white adipose tissue (eWAT), inguinal white adipose tissue (iWAT), and brown adipose tissue (BAT) were extracted from 7–8 weeks old C57BL/6 (WT) male mice, and lysates from tissues were immunoblotted with antibodies against several representative phosphatases, which were classified into four phosphatase groups (n = 5, biologically independent samples). b Relative mRNA expression levels of Ssu72 in eWAT, iWAT, and BAT from 9-week-old WT mice (n = 5 mice per group). Data are presented as mean ± SEM. Statistical comparisons were made using one-way ANOVA. c Relative mRNA expression levels of Ssu72 in BAT of 7 to 8-week-old WT mice after mild cold exposure (15 °C) (n = 5 mice per group). Data are presented as mean ± SEM. Statistical comparisons were made using one-way ANOVA. d Western blots of lysates from BAT of WT mice housed at RT (23 °C) or exposed to acute cold (4 °C) for 4 h (left) (n = 3 mice per group), with quantification of Ssu72 and UCP1 protein levels (right). Graph shows quantification of Ssu72 protein levels normalized to β-actin protein level using ImageJ. Data are presented as mean ± SEM. Statistical comparisons were made using two-tailed Student’s t test. e Animated images of Ssu72flox/flox (Ssu72 WT), Adiponectin-Cre, and Ssu72flox/flox; Adiponectin-Cre mice (Ssu72 aKO). f Representative images of BAT from 11-week-old Ssu72 WT and aKO male mice (scale bar, 5 mm; n = 10 per group, biologically independent samples from 8–11 weeks old mice). g Hematoxylin and eosin (H&E) staining of representative sections of BAT from 6- and 22-weeks-old Ssu72 WT and aKO male mice (n = 5 per group, biologically independent samples). For (f) and (g), both male and female mice were used, and no sex-specific phenotype was observed. Source data are provided as a Source Data file.

Fig 4: The characteristic feature of the fission yeast Pin1 identified is conserved in humans. (A) Coimmunoprecipitation was performed with extracts prepared from human HeLa cells expressing p38 tagged with flag. Immunoprecipitates were analyzed by Western immunoblotting analysis using antibodies specific for Rpb1 and flag. The relative level of Rpb1 pull down by p38 was shown. Data were expressed as means±SD in triplicate. Asterisks indicated statistical significance (P< 0.05, t-test unpaired) verse control of no treatment. (B) Purified GST-CTD from Escherichia coli and p38-flag precipitated from HeLa cells were subjected to GST pulldown assay followed by Western immunoblotting analysis with antibodies against GST and flag. (C) In vitro kinase assay using p38 extracted and immunoprecipitated from activated cells. Following incubation with GST-CTD, proteins were resolved by SDS-PAGE and probed with antibodies indicated. (D) Coimmunoprecipitation was performed with extracts prepared from human HeLa cells using anti-Ssu72 antibody and IgG as control. Immunoprecipitates were analyzed by Western immunoblotting analysis with antibodies specific for human Pin1 and Ssu72. (E) Total lysates of human cell indicated were subjected to Western immunoblotting analysis with indicated antibodies. The relative level of protein of interest was indicated beneath each lane. Data were expressed as means±SD in triplicate. Asterisks indicated statistical significance (P< 0.05, t-test unpaired) verse RWPE-1 cells. (F) (G) Human cells indicated were subjected to measurement of intracellular ROS Levels in triplicate using CM-H2DCFDA as indicator. Data were expressed as means±SD in triplicate. Asterisks indicated statistical significance (P< 0.05, t-test unpaired) verse normal RWPE-1 cells.

Fig 5: TRT is reduced and lung injury is ameliorated in SSU72 transgenic mice infected with the lethal H5N1 virus. A Western blot analysis of mouse SSU72 expression in mouse lung tissues at 3 days after treatment with AF/H1N1/H5N1. β-Actin expression served as an internal control. B Numbers of TRT genes (expression of the TRT region upregulated by more than 5 compared with the AF-treated condition) in lung tissues from control (n = 5) and SSU72 transgenic mice (n = 5) at 3 days after intratracheal infection with H5N1 (106 TCID50). The relative mRNA expression ratios of C mouse STAT1 and D STAT2 in lung tissues from control (n = 8) and SSU72 transgenic mice (n = 4) at 3 days after intratracheal infection with H5N1 virus (106 TCID50). Mouse β-actin expression served as the reference control. E Kaplan–Meier survival curves for control (n = 8) and SSU72 transgenic mice (n = 10) after intratracheal infection with H5N1 (106 TCID50). F–H Control and SSU72 transgenic mice were infected with AF or H5N1 (106 TCID50) via intratracheal instillation. F Viral titers in the lungs were assessed 4 days after infection with H5N1 in control (n = 7) and SSU72 transgenic mice (n = 3). G Wet-to-dry weight ratios of the lungs of control (n = 4) and SSU72 transgenic mice (n = 4) at 3 days after infection with H5N1. H Representative images of lung pathology in control and SSU72 transgenic mice at 3 days after H5N1 infection. The lung injury scores (means ± SEMs) and numbers of infiltrating cells per microscopic field (means ± SEMs) are shown in the bar graphs. N = 100 fields for control (n = 15) and SSU72 transgenic (n = 6) mice. Bar = 100 μm. *P < 0.05 and **P < 0.01. Each experiment except for RNA-seq analysis of lungs from mice with or without H5N1 infection was repeated at least three times