Display Settings:

Items per page
We are sorry, but NCBI web applications do not support your browser and may not function properly. More information

Results: 7

1.
Figure 7

Figure 7. SENP1 regulates of HIF1α stability in hypoxia. From: SUMO-specific Protease 1 is Essential for Stabilization of Hypoxia-inducible factor-1? During Hypoxia.

Hypoxia blocks the activity of PHD, preventing hydroxylation of HIF1α and its subsequent degradation in a VHL- and ubiquitin-dependent manner. On the other hand, hypoxia induces nuclear translocation and SUMOylation of HIF1α, which provides an alternative signal for VHL- and ubiquitin-dependent degradation. SENP1 stabilizes HIF1α by removing the alternative VHL-binding signal.

Jinke Cheng, et al. Cell. ;131(3):584-595.
2.
Figure 3

Figure 3. SENP1 regulates Epo production through HIF1α. From: SUMO-specific Protease 1 is Essential for Stabilization of Hypoxia-inducible factor-1? During Hypoxia.

(A) ELISA analysis of Epo production in Hep 3B cells. Data are presented as means ± SD of the results of three independent experiments for Figure 3A, B, C, and D. (NS = Non-specific siRNA).
(B) SENP1 enhanced Epo transcription induced by hypoxia. The indicated reporter gene, HIF1α, and SENP1 expression plasmids were co-transfected into Hep 3B cells. The cells were treated with hypoxia (1% O2) for 12 hr before luciferase assay. The Epo reporter gene with mutation of HIF1α binding sites on the Epo enhancer is indicated by mEpo-Luc. SENP1m is the catalytically inactive SENP1.
(C) SENP1 enhanced HIF1α-dependent Epo transcription. The indicated reporter gene, HIF1α, and SENP1 expression plasmids were co-transfected into Hep 3B cells.
(D) SENP1 was essential for HIF1α-dependent Epo transcription. Epo-Luc and the indicated siRNA expression plasmids were co-transfected into Hep 3B cells without or with HIF1α plasmids.

Jinke Cheng, et al. Cell. ;131(3):584-595.
3.
Figure 1

Figure 1. Severe anemia in the SENP1−/− embryos. From: SUMO-specific Protease 1 is Essential for Stabilization of Hypoxia-inducible factor-1? During Hypoxia.

(A) The ratios of the observed live and dead (in parentheses) SENP1−/− embryos to the total number of embryos analyzed at different stages of embryonic development.
(B) Appearance of SENP1+/+ and SENP1−/− embryos at E15.5. The SENP1−/− embryo and yolk sac were paler and smaller than those of the wild-type embryo and appeared to contain fewer red blood cells in major blood vessels.
(C) Relative numbers of red blood cells in peripheral blood in E15.5 wild-type (+/+, n = 5), heterozygote (+/−, n = 9), and SENP1 (−/−, n = 4) embryos. Results shown are means ± SD. A significant decrease in the number of red blood cells was found in SENP1 (−/−) embryos (p < 0.007), when compared with those in wild-type or heterozygote embryos.
(D) Total number of nucleated cells per fetal liver in E13.5 (+/+: n = 4; −/−: n = 3), E14.5 (+/+: n = 4; −/−: n = 4), and E15.5 embryos (+/+: n = 5; −/−: n = 3). Results shown are means ± SD. Significant differences between wildtype and mutant embryos were found at E14.5 (p < 0.015) and E15.5 (p < 0.004).
(E) Hematoxylin and Eosin-stained sections of fetal liver from E12.5 SENP1+/+ and −/− embryos. The SENP1−/− fetal liver showed a marked decrease in the number of erythropoietic foci and increase in apoptotic cells.

Jinke Cheng, et al. Cell. ;131(3):584-595.
4.
Figure 4

Figure 4. Defect in hypoxia-induced stabilization of HIF1α in SENP1−/− embryos. From: SUMO-specific Protease 1 is Essential for Stabilization of Hypoxia-inducible factor-1? During Hypoxia.

(A) HIF1α expression was decreased in SENP1−/− fetal liver. Fetal liver sections from E12.5 SENP1+/+ and SENP1−/− embryos were stained with anti-HIF1α antibody (brown).
(B) Hypoxia-induced HIF1α protein expression was decreased in SENP1−/− MEF cells. SENP1+/+ and −/− MEF cells were treated with hypoxia (1% O2) for 3 hrs before harvest. The whole cell lysate was analyzed by western blotting with anti-HIF1α and Actin antibodies.
(C) The half-life of HIF1α protein under hypoxia was decrease in SENP1−/− MEF cells.
(D) SENP1 was essential for hypoxia-induced HIF1α ODD (344–698) activity. 293 cells were transfected with pGal4-VP16 or pGal4-ODD (344–698)-VP16 plus other plasmids as indicated. Cells were incubated for 12 hrs in normoxia or hypoxia (1% O2) before harvest. The data are presented as the corrected (by internal control) pGal4-ODD-VP16 luciferase activity that normalized to the counts of pGal4-VP16.
(E) Hypoxia-induced HIF1α activity was significantly reduced in SENP1−/− MEF cells. Data are presented as means ± SD of three independent experiments
(F) Hypoxia-induced expression of VEGF and Glut-1 was reduced in SENP1−/− MEF cells. Expression of VEGF, Glut-1, and Actin-β was determined in SENP1+/+ or −/− MEF cells by RT-PCR.
(G) HIF1α restored hypoxia-induced expression of VEGF in SENP1−/− MEF cells. SENP1−/− MEF cells were transfected with HIF1α SM (SUMOylation mutant), or vector control plasmids and treated with hypoxia (1% O2) for different time as indicated. Expression of VEGF and Actin-β was determined by RT-PCR.

Jinke Cheng, et al. Cell. ;131(3):584-595.
5.
Figure 2

Figure 2. SENP1−/− erythroid progenitors undergo apoptosis due to Epo deficiency. From: SUMO-specific Protease 1 is Essential for Stabilization of Hypoxia-inducible factor-1? During Hypoxia.

(A) Analysis of CFU-e- and BFU-e-forming ability of fetal liver cells from E13.5 SENP1+/+ and SENP1−/− embryos. Results shown are means ± SD determined from three embryos. The number of CFU-e from liver cells of SENP1−/− embryos was significantly less than that from SENP1+/+ embryos (p<0.05). (B) Liver cells isolated from E13.5 SENP1+/+ and SENP1−/− embryos were stained with Ter-119 antibody and analyzed by the TUNEL procedure. Histograms show TUNEL staining profile of the Ter-119-positive population identified by flow cytometry. The percentages in the histograms are the percentages of TUNEL-positive cells.
(C) RT–PCR analysis of a variety of genes that are involved in erythroid differentiation in fetal livers of SENP1+/+ and SENP1−/− embryos at E11.5. Samples were amplified for 36, 39, and 42 cycles for Epo; 25, 28, and 32 cycles for EpoR, c-kit, SCF, Jak2, and STAT5. β-Actin mRNA levels (amplified for 18, 21, and 24 cycles) were measured as control.
(D) Fetal liver sections from E12.5 SENP1+/+ and SENP1−/− embryos were stained with anti-Epo antibody (brown).
(E and F) Fetal liver cells isolated from E13.5 SENP1+/+ and SENP1−/− embryos were cultured for 24 hours in the absence or presence of Epo (5 U/ml). Cell numbers were counted under microscope (E). Results shown in (E) are means ± SD determined from three embryos. TUNEL-positive cells were quantitated by flow cytometry. Percentages in histograms are the percentage of TUNEL-positive cells (F).

Jinke Cheng, et al. Cell. ;131(3):584-595.
6.
Figure 6

Figure 6. VHL is required for degradation of SUMOylated HIF1α. From: SUMO-specific Protease 1 is Essential for Stabilization of Hypoxia-inducible factor-1? During Hypoxia.

(A) HIF1α ubiquitination was regulated by SUMO-1 and SENP1. COS-7 cells were co-transfected with indicated plasmids and treated with MG132 (10 μM) for 9 hours before harvesting. HIF1α was immunoprecipitated with anti-HIF1α (IP) and bound proteins were detected by anti-HA (top panel), anti-Myc (second panel), and anti-HIF1α immunoblotting (third panel) (IB). Whole-cell lysates (WCL) were immunoblotted (IB) with anti-HA (fourth panel) or anti-Flag (bottom panel) antibodies. Asterisk indicates IgG band.
(B) RCC4 or RCC4/VHL cells were treated by hypoxia (1% O2) and/or MG132 (10 μM) for 4 hrs as indicated. HIF1α was immunoprecipitated with anti-HIF1α antibody from cell lysates. The precipitates were immunoblotted (IB) with anti-SUMO-1 or HIF1α antibodies. Asterisk indicates IgG band.
(C) VHL bound to SUMOylated HIF1α proline mutant (HIF1α PM) in vivo. COS-7 cells were co-transfected with indicated plasmids and treated without or with MG132 (10 μM) for 4 hrs before harvesting. VHL was immunoprecipitated with anti-Flag (IP) from the nuclear fraction of the transfected cells and bound proteins were detected by anti-HIF1α (top panel), anti-HA (second panel), or anti-Flag immunoblotting (third panel) (IB). HIF1α was also immunoprecipitated with anti-HIF1α (IP) from the nuclear fraction of transfected cells and bound proteins were detected by anti-HA antibody (IB) (fourth panel). Whole-cell lysates (WCL) were immunoblotted (IB) with anti-HIF1α (bottom panel). Asterisk indicates IgG band.
(D) VHL specifically bound to SUMOylated HIF1α ODD with proline mutation (GST-ODD (344–698) PM) in vitro. GST-ODD (344–698) PM recombinant protein and SUMOylated GST-ODD (344–698) PM produced by in vitro SUMOylation was incubated with Flag-VHL produced by in vitro translation. After washing and eluting with Flag peptide, the precipitates were immunoblotted with ant-HIF1α (middle panel) or anti-SUMO-1(right panel) antibodies.
(E) Mapping of VHL domain that bound to SUMO-1-fused ODD. GST-ODD (344–698) PM and SUMO-1-fused GST-ODD (344–698) PM recombinant proteins were incubated with HA-VHL and its mutant produced by in vitro translation for two hr. The precipitates with glutathione-agarose beads were detected by immunoblotting with ant-HA (top and right panel) or anti-HIF1α antibodies (bottom panel).
(F) HIF1α degradation induced by SENP1 silencing was VHL-dependent. RCC4 or RCC4/VHL cells and SENP1-siRNA stable transfected RCC4 or RCC4/VHL cells were treated by hypoxia (1% O2) for 4 hrs as indicated. Cell lysates were detected by immunoblotted with anti-HIF1α or anti-actin antibodies.

Jinke Cheng, et al. Cell. ;131(3):584-595.
7.
Figure 5

Figure 5. SUMOylated HIF1α accumulated in SENP1−/− MEF cells and underwent proteasomal-dependent degradation in the hypoxic condition. From: SUMO-specific Protease 1 is Essential for Stabilization of Hypoxia-inducible factor-1? During Hypoxia.

(A) SENP1 de-conjugated SUMOylated HIF1α in vitro. SUMOylated GST-ODD (344–698) PM recombinant protein was produced by in vitro SUMOylation. Flag-SENP1 or SENP1m was generated by in vitro translation. SUMOylated GST-ODDPM and SENP1 (or SENP1m) were incubated for 1 hr at 37 °C. The reaction mixtures were detected by western blot with anti-HIF1α (left panel), anti-SUMO-1 (right panel), and anti-Flag antibodies (bottom panel).
(B) Mutation of SUMOylation sites increased HIF1α-dependent Epo transcription and reduced HIF1α response to SENP1. Epo-Luc and the indicated plasmids were co-transfected into Hep-3B cells. Data are presented as means ± SD of three independent experiments.
(C) SUMOylated HIF1α accumulated in SENP1−/− MEF cells, but not in wildtype or SENP2−/−MEF cells after exposure to hypoxia. Wildtype (+/+), SENP1−/−, or SENP2−/− MEF cells were treated with or without hypoxia (1% O2) for 4 hrs as indicated. HIF1α was immunoprecipitated with anti-HIF1α antibody from these cell lysates. The precipitates were immunoblotted (IB) with anti-SUMO-1 antibody (top panel). Asterisk indicates IgG band.
(D) SUMOylated HIF1α level was controlled by SENP1 and proteasome-dependent degradation. COS-7 cells were transfected with indicated plasmids and then treated without or with MG132 (10 μM) for 4 hours before harvesting and immunoprecipitated with anti-HIF1α (IP). Bound proteins were detected by anti-HA and anti-HIF1α immunoblotting (IB). Whole-cell lysates (WCL) were immunoblotted (IB) with anti-HA. Asterisk indicates IgG band.
(E) SUMOylated HIF1α accumulated in SENP1−/− MEF cells after exposure to hypoxia and undergoes proteasome-dependent degradation. SENP1+/+ or −/− MEF cells were treated by hypoxia (1% O2) and MG132 (10 μM) for 4 hrs as indicated. HIF1α was immunoprecipitated with anti-HIF1α antibody from cell lysates. The precipitates were immunoblotted (IB) with anti-SUMO-1 and HIF1α antibodies. Asterisk indicates IgG band.

Jinke Cheng, et al. Cell. ;131(3):584-595.

Display Settings:

Items per page

Supplemental Content

Recent activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...
Write to the Help Desk