Fig 2: DUSP6 SUMOylation at K234 is protective to brain neurons in ischemic stroke.(A) Brain ischemia and brain damage in the tMCAO model. Left: Regional cerebral blood flow in the core of the middle cerebral artery territory of C57BL/6 mice before, during, and after ischemia as measured by LDF. Flow values were averaged at 5-min intervals and expressed as means ± SEM of nine experiments. Right: Representative photographs of TTC-stained brain sections (1 mm) showing brain infarction at 24 hours after the onset of ischemia. (B) Time-dependent decreases in DUSP6 SUMOylation and DUSP6 protein levels during reperfusion in mice subjected to tMCAO for 40 min. Cerebral cortices from C57BL/6 mice (wild type) that underwent 40 min tMCAO, followed by different periods of reperfusion as indicated, were lysed under denaturing conditions and subjected to IP with anti-DUSP6 (or the IgG control), which was followed by IB using anti-SUMO1 and anti-DUSP6. β-Actin was used as the loading control. Note also the time-dependent decrease in overall protein SUMOylation. (C to G) Transcriptional up-regulation of SENP1 during reperfusion of tMCAO injured mice. The original lysates as in (B) were analyzed by real-time quantitative RT-PCR for mRNA levels of DUSP6 (C), SAE1/SAE2 (D), UBC9 (E), and SENP1 (F), as well as by IB for protein levels of SAE1, SAE2, UBC9, and SENP1, with β-actin as the loading control (G). Only SENP1 showed time-dependent increases at both the mRNA (F) and protein levels (G). (H) Schematic representation of adeno-associated virus (AAV) infection into mouse primary somatosensory cortex, barrel field, and secondary somatosensory cortex (left) and representative brain images showing the expression of mNeonGreen in cerebral cortex (right). The boxed area is enhanced by high magnification to show mNeonGreen-positive neurons. Scale bar, 100 μm. (I to K) Overexpression of wild-type DUSP6 reduced, but the SUMOylation-deficient DUSP6K234R mutant exacerbated, cortical neuron apoptosis in response to brain I/R. AAV-DUSP6, AAV-DUSP6K234R, or a control AAV driven by the human synapsin promoter (hSyn) was injected into the primary somatosensory cortex, barrel field, and secondary somatosensory cortex of adult wild-type C57BL/6 mice as shown in (H). The animals were subjected to 40-min tMCAO, followed by 24-hour reperfusion at 4 weeks after stereotactic intracerebral injection. (I) The cerebral cortical tissues were lysed and subjected to IB using anti-cleaved caspase-3, anti–p–Drp1-S616, anti–p–Drp1-S637, anti-Drp1, anti-Flag, and anti-DUSP6. β-Actin was used as the loading control. Caspase-3 cleavage was reduced by AAV-DUSP6 but increased by AAV-DUSP6K234R. Note also the corresponding changes in p–Drp1-S616 levels; however, p–Drp1-S637 levels were affected by neither AAV-DUSP6 nor AAV-DUSP6K234R, despite the decrease caused by I/R. (J) The tissues were sectioned and stained by TUNEL (red) and DAPI (blue). The occurrence of TUNEL+ cells in areas with DUSP6 expression (green) was significantly less than that with DUSP6K234R expression. Representative confocal images in virus-infected cortical areas of brain sections are shown at the left. Scale bar, 100 μm. Quantification of TUNEL+ cells are shown at the right. Data represent means ± SEM of n = 3 independent experiments, with six to eight fields of each group used for statistics in each experiment. *P < 0.05 and ***P < 0.001, by one-way ANOVA with pairwise comparison using Tukey’s multiple comparisons test. (K) Brain infarction after I/R in mice injected with AAV-DUSP6, AAV-DUSP6K234R, or the control AAV. Left: Representative images of TTC-stained sequential brain sections (1 mm). Right: Quantification of infarct areas in ipsilateral hemisphere normalized to the total areas of the contralateral hemisphere in sequential coronal brain slices. Data represent means ± SEM of n = 5 mice in each group. *P < 0.05, **P < 0.01, ***P < 0.001, by one-way ANOVA with pairwise comparison using Tukey’s multiple comparisons test.

Fig 3: N-α-SUMOylation of CFL1 is reversible.The SUMOylation of CFL1 was inhibited by the knockdown of SAE1 (a), SAE2 (b), and Ubc9 (c), but enhanced by the knockdown of SENP1 (d). HEK-293T cells were transiently transfected as indicated. Cell lysates were used for De-IP by anti-CFL1 antibody followed by IB using anti-SUMO1 and anti-CFL1 antibodies. The original lysates were analyzed by IB using anti-CFL1, anti-SAE1 (a), anti-SAE2 (b), anti-Ubc9 (c), and anti-SENP1 (d) antibodies for input, as well as anti-GAPDH antibody for loading control. e CFL1 is physically associated with SAE1, SAE2, Ubc9 and SENP1. Lysates from HEK-293T cells transiently transfected with the vector control (−), CFL1WT-HA, CFL125KR-HA, HA-DUSP6, and His-SUMO1 as indicated for 24 h were subjected to IP with anti-HA antibody, followed by IB with anti-SAE1, anti-SAE2, anti-Ubc9, anti-SENP1 and anti-HA antibodies. The original lysates were analyzed by IB using anti-HA, anti-SAE1, anti-SAE2, anti-Ubc9 and anti-SENP1 antibodies for input, and anti-GAPDH antibody for loading control. f Similar to (e) but with expression of the vector control (−), CFL1WT-HA, CFL12KR-HA, CFL12KQ-HA, and His-SUMO1 as indicated. g–i Quantification of Western blots of (a)–(f). g Summary of SUMOylated CFL1 to total CFL1 presents the mean ± SEM of three independent experiments in (a)–(d); h Summary of coimmunoprecipitated (Co-IPed) SAE1, SAE2, Ubc9 and SENP1 normalized to total CFL1 or DUSP (HA) presents the mean ± SEM of three independent experiments in (e); i Summary of coimmunoprecipitated (Co-IPed) SAE1, SAE2, Ubc9 and SENP1 normalized to total CFL1 (HA) presents the mean ± SEM of three independent experiments in (f); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, by one-way ANOVA with Tukey’s multiple comparison test. All blots represent ≥3 independent experiments. The exact p value and source data are provided as Source Data file.