Journal: Cell death & disease

Article Title: SENP3-FIS1 axis promotes mitophagy and cell survival under hypoxia.

doi: 10.1038/s41419-024-07271-8

Figure Lengend Snippet: Fig. 1 deSUMO2/3-ylation induces mitophagy and promotes HIM. A Hypoxia induces mitophagy. HeLa cells were transfected with Mito- pHfluorin per 35mm-dish and exposed to normoxia (N) or hypoxia (H;1% O2 for 4, 8, 16, or 24 h) (Scale bar, 10 μm). Upper panel shows that hypoxia-induced mitophagy is detectable as early as 4 h after cells exposed to 1% O2. Histogram in the right panel shows the average number of puncta per cell under indicated time points (n = 19 ~ 51 cells; *p < 0.05; **p < 0.01; ***p < 0.001; ****; unpaired t-test). B, C Hypoxia causes decreased SUMO2/3-ylation (B, n = 5, biological replicates; *p < 0.05; paired t-test) but not SUMO1-ylation (C, n = 6, biological replicates; N.S., non-significant; paired t-test) in HeLa cells. HeLa cells were exposed to 1% O2 for 24 h. Whole cell lysate samples were prepared and blotted as indicated.

Article Snippet: The primary antibodies used were: β-actin (mouse monoclonal Ab; Sigma-Aldrich A2228; 1:20,000 dilution; rabbit polyclonal Ab; ProteinTech 20536-1-AP; 1:5,000 dilution), FEN1 (mouse monoclonal Ab; Santa Cruz biotechnology sc-28355, 1:500 dilution), FIS1 (mouse monoclonal Ab; ProteinTech 66635-1-Ig; 1:1,000 dilution), GAPDH (mouse monoclonal Ab; Santa Cruz biotechnology sc-365062; 1:1,000 dilution), GST (goat polyclonal Ab; GE Healthcare; 1:10,000 dilution), HA-tag (rabbit polyclonal Ab; ProteinTech 66006-2-Ig, 1:2,000 dilution), HIF1α (mouse monoclonal Ab, BD Bioscience 610959; 1:1,000 dilution), His (rabbit polyclonal Ab, Cell Signaling #2365; 1:1,000 dilution), LC3 (rabbit polyclonal Ab, Cell Signaling #4108; 1:2,000 dilution), SENP1 (recombinant monoclonal Ab, Abcam EPR3844, 1:2,000 dilution), SENP3 (rabbit monoclonal Ab, Cell Signaling #5591, 1:10,000 dilution), SUMO1 (rabbit polyclonal Ab, Cell Signaling #4930; 1:1,000 dilution), SUMO2/3 (rabbit monoclonal Ab, Cell Signaling #4971; 1:1,000 dilution), TBC1D17 (rabbit polyclonal Ab, ProteinTech 20482-1-AP; 1:1,000 dilution), and α-Tubulin (rabbit polyclonal Ab, ProteinTech 11224-1-AP; 1:2,000 dilution or mouse monoclonal Ab, ProteinTech 66031-1-Ig; 1:20,000 dilution).

Techniques: Transfection

Journal:

Article Title: Regulation of the SUMO pathway sensitizes differentiating human endometrial stromal cells to progesterone

doi: 10.1073/pnas.0603002103

Figure Lengend Snippet: Attenuated sumoylation in decidual HESCs enhances PR activity. (A) WT PR-A or K388R mutant (400 ng) were cotransfected with EGFP-SUMO-1 (200 ng) in primary HESCs. Cultures were treated with cAMP and MPA as indicated and harvested 72 h later. Whole-cell protein extracts were analyzed by Western blotting with anti-PR (Top), anti-SUMO-1 (Middle), and anti-β-actin (Bottom). ∗ indicates SUMO-1-modified PR-A, and arrowhead indicates free SUMO-1. (B) HESCs were transfected with PRE2-luciferase reporter (100 ng) and PR-A or PR-AK388R (400 ng) and treated with 8-br-cAMP and MPA in the indicated combinations. The cells were harvested after 72 h, and luciferase activity was assayed. The data are presented as fold induction in promoter activity when compared with untreated cells.

Article Snippet: Primary HESCs were cultured on four-well multispot glass slides (CA Hendley-Essex, Loughton, U.K.), fixed in 1:1 methanol/acetone for 10 min, and permeabilized with 0.5% Triton X-100 in PBS for 30 min. Endogenous proteins were stained with mouse anti-SUMO-1 (Zymed), mouse anti-PR (NovoCastra), and rabbit anti-PIAS1 (sc-14016; Santa Cruz Biotechnology) followed by anti-mouse FITC (F0479; Dako, Glostrup, Denmark) anti-mouse Alexa Fluor 594 (Invitrogen), or anti-rabbit FITC (F0205; Dako).

Techniques: Activity Assay, Mutagenesis, Western Blot, Modification, Transfection, Luciferase

Journal:

Article Title: Regulation of the SUMO pathway sensitizes differentiating human endometrial stromal cells to progesterone

doi: 10.1073/pnas.0603002103

Figure Lengend Snippet: Regulated expression of the SUMO pathway enzymes during HESC decidualization. (A) Primary HESC cultures were kept untreated or stimulated with 8-br-cAMP and MPA for 2, 4, or 8 days. Whole-cell lysates were subjected to Western blot (Upper) and dot blot (Lower) analyses and immunoprobed with anti-SUMO-1 antibody. For dot blot analysis, protein lysates were serially diluted, and 10, 2, and 0.4 μg was spotted on the membrane. (B) HESCs transfected with WT SUMO-1 (SUMO-1-GG-HSTV) or matured SUMO-1 (SUMO-1-GG) were left untreated or treated with 8-br-cAMP and MPA for 3 days. Subsequently, whole-cell protein extracts were subjected to Western blotting together with in vitro-transcribed/translated products acting as positive controls for unprocessed and mature SUMO-1. Conjugated and free SUMO-1 was detected with an anti-SUMO-1 antibody, and an anti-β-actin antibody was used for loading control. The filled arrowhead indicates unprocessed full-length SUMO-1, and the open arrowhead indicates matured SUMO-1. (C) After treatment of HESCs with 8-br-cAMP and MPA in the indicated combinations for 2, 4, or 8 days, whole-cell protein extracts were subjected to Western blot analysis and probed for the expression of various E1, E2, and E3 enzymes and SUMO proteases.

Article Snippet: Primary HESCs were cultured on four-well multispot glass slides (CA Hendley-Essex, Loughton, U.K.), fixed in 1:1 methanol/acetone for 10 min, and permeabilized with 0.5% Triton X-100 in PBS for 30 min. Endogenous proteins were stained with mouse anti-SUMO-1 (Zymed), mouse anti-PR (NovoCastra), and rabbit anti-PIAS1 (sc-14016; Santa Cruz Biotechnology) followed by anti-mouse FITC (F0479; Dako, Glostrup, Denmark) anti-mouse Alexa Fluor 594 (Invitrogen), or anti-rabbit FITC (F0205; Dako).

Techniques: Expressing, Western Blot, Dot Blot, Transfection, In Vitro

Journal:

Article Title: Regulation of the SUMO pathway sensitizes differentiating human endometrial stromal cells to progesterone

doi: 10.1073/pnas.0603002103

Figure Lengend Snippet: Subcellular localization of SUMO-1 is altered upon HESC decidualization. Primary HESCs were treated with 8-br-cAMP, MPA, or both for 4 days. Methanol/acetone-fixed cells were immunostained with an antibody to SUMO-1, and the distribution of endogenous SUMO-1 (green channel) and DAPI nuclear staining (blue channel) was captured by confocal microscopy. (Magnifications: ×600.)

Article Snippet: Primary HESCs were cultured on four-well multispot glass slides (CA Hendley-Essex, Loughton, U.K.), fixed in 1:1 methanol/acetone for 10 min, and permeabilized with 0.5% Triton X-100 in PBS for 30 min. Endogenous proteins were stained with mouse anti-SUMO-1 (Zymed), mouse anti-PR (NovoCastra), and rabbit anti-PIAS1 (sc-14016; Santa Cruz Biotechnology) followed by anti-mouse FITC (F0479; Dako, Glostrup, Denmark) anti-mouse Alexa Fluor 594 (Invitrogen), or anti-rabbit FITC (F0205; Dako).

Techniques: Staining, Confocal Microscopy

Journal:

Article Title: Regulation of the SUMO pathway sensitizes differentiating human endometrial stromal cells to progesterone

doi: 10.1073/pnas.0603002103

Figure Lengend Snippet: PIAS1 interacts with and enhances PR-A sumoylation. (A) COS1 cells were transfected with PR-A WT or K388R mutant (500 ng), EGFP-SUMO-1 (50 ng), Ubc9 (500 ng), and Flag-PIAS1 (100, 500, and 1,000 ng) and treated with MPA for 36 h. Western blotting was performed on whole-cell protein extracts by using antibodies to PR, Flag tag, Ubc9, EGFP, and β-actin. (B) COS1 cells were transfected with PR-A and EYFP-tagged PIAS1 and left untreated or treated with MPA for 36 h. Cellular protein extracts were subsequently immunoprecipitated with an anti-PR antibody and analyzed by immunoblotting with an antibody against EYFP. Reprobing with the anti-PR antibody was used to check the amount of PR immunoprecipitated. Five percent of the amount of lysates used in immunoprecipitation (IP) was verified for equal expression of the transfected proteins. WB, Western blot. (C) HESCs were stained with antibodies to PR (red channel) and PIAS1 (green channel) and analyzed by confocal microscopy. Colocalization of PR and PIAS1 appears as yellow on the overlaid images. (Magnifications: ×600.)

Article Snippet: Primary HESCs were cultured on four-well multispot glass slides (CA Hendley-Essex, Loughton, U.K.), fixed in 1:1 methanol/acetone for 10 min, and permeabilized with 0.5% Triton X-100 in PBS for 30 min. Endogenous proteins were stained with mouse anti-SUMO-1 (Zymed), mouse anti-PR (NovoCastra), and rabbit anti-PIAS1 (sc-14016; Santa Cruz Biotechnology) followed by anti-mouse FITC (F0479; Dako, Glostrup, Denmark) anti-mouse Alexa Fluor 594 (Invitrogen), or anti-rabbit FITC (F0205; Dako).

Techniques: Transfection, Mutagenesis, Western Blot, FLAG-tag, Immunoprecipitation, Expressing, Staining, Confocal Microscopy

Journal:

Article Title: Regulation of the SUMO pathway sensitizes differentiating human endometrial stromal cells to progesterone

doi: 10.1073/pnas.0603002103

Figure Lengend Snippet: PIAS1 modulates PR-A activity. (A) Primary HESC cultures were transfected with 500 ng of WT PR-A or K388R mutant, 100 ng of EGFP-SUMO-1, and 400 ng of PRE2-luciferase in the presence or absence of 500 ng of WT PIAS1 or the E3 ligase-deficient mutant PIAS1 (PIAS1mt). The cultures were treated with MPA and harvested 24 h later for luciferase assay. The data are expressed as percentage PRE activity in the presence of cotransfected PIAS1 compared with the activity in the absence of overexpressed PIAS1. (B) HESCs were transfected with a nontargeting, control siRNA or PIAS1 siRNA and harvested 3 days later. Western blotting with an anti-PIAS1 antibody was used to determine the efficiency of the knockdown. (C) HESCs were first mock-transfected or transfected with PIAS1 siRNA. After 2 days, the cultures were transfected again with PR-A or PR-AK388R (500 ng) and PRE2-luciferase (400 ng) and treated with MPA. After 48 h cells were harvested and extracts were assayed for luciferase activity. The data are expressed as fold induction in PRE activity in the presence of PIAS1 siRNA when compared with mock-transfected cells. (D) HESCs transfected with or without PIAS1 siRNA were treated with MPA or 8-br-cAMP, as indicated, and RNA was harvested 3 days later. The abundance of decidual PRL (dPRL) transcripts was determined by quantitative RT-PCR and normalized to L19. The data represent the mean PRL mRNA induction (±SEM) of three independent experiments relative to the transcript levels in untreated cells.

Article Snippet: Primary HESCs were cultured on four-well multispot glass slides (CA Hendley-Essex, Loughton, U.K.), fixed in 1:1 methanol/acetone for 10 min, and permeabilized with 0.5% Triton X-100 in PBS for 30 min. Endogenous proteins were stained with mouse anti-SUMO-1 (Zymed), mouse anti-PR (NovoCastra), and rabbit anti-PIAS1 (sc-14016; Santa Cruz Biotechnology) followed by anti-mouse FITC (F0479; Dako, Glostrup, Denmark) anti-mouse Alexa Fluor 594 (Invitrogen), or anti-rabbit FITC (F0205; Dako).

Techniques: Activity Assay, Transfection, Mutagenesis, Luciferase, Western Blot, Quantitative RT-PCR

Journal: Cell reports

Article Title: The polySUMOylation axis promotes nucleolar release of Tof2 for mitotic exit

doi: 10.1016/j.celrep.2024.114492

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse monoclonal anti-Smt3 , Santa Cruz , Cat# SC-137158; RRID:AB_201891.

Techniques: Recombinant, Protease Inhibitor, Gel Extraction, Plasmid Preparation, Software

Journal: PLoS ONE

Article Title: Stanniocalcin-1 Regulates Re-Epithelialization in Human Keratinocytes

doi: 10.1371/journal.pone.0027094

Figure Lengend Snippet: (A) In the cell spreading assay, e-lam formation on fibronectin-coated plate was induced by increasing doses of STS treatment (0, 2.5, 5, 10 nM) for 24 h. The cell images were captured under magnification of 200x ( top ). (B) Cell migration through the transwell was induced by 5 nM STS for 24 h and the cell images were captured under 100x magnification ( top ). (C) Western blot analysis demonstrated the increase of FAK phosphorylation (pY397) upon 5 nM STS treatment from 10–360 s. (D) Cell migration was increased upon STS treatment at 24 h, but was blocked by 2 µM PF573228. Cell images were captured under 100x magnification ( top ). (E) STC1 mRNA and protein expressions were induced by increasing doses of STS treatment (0, 2.5, 5, 10 nM). (F) STS-induced STC1 mRNA expression was not significantly abolished by PF573228 at 24 h. Asterisks (***) denote p <0.0001, (**) denote p <0.005 and (*) denote p <0.01 as compared to the respective control treatment.

Article Snippet: The blotting was conducted using mouse anti-V5 (Invitrogen), anti-human STC1 (R&D), human p-FAK (pY397) (BD Biosciences), rabbit anti-human p-GSK3β (Ser9), anti-human total GSK3β, anti-human p-Akt (Ser473), anti-human total Akt (Cell Signaling), and anti-human actin serum (Sigma).

Techniques: Migration, Western Blot, Expressing

Journal: PLoS ONE

Article Title: Stanniocalcin-1 Regulates Re-Epithelialization in Human Keratinocytes

doi: 10.1371/journal.pone.0027094

Figure Lengend Snippet: (A) STS treatment increased [Ca 2+ ]i as measured using Ca 2+ detection dye, Fura 2-AM. (B) The number of migrated cells was significantly reduced by 10 µM BAPTA/AM at 24 h (vs Ctrl) and the cell images were captured under 100x magnification ( top ). (C) STS-induced STC1 mRNA was further induced by 1 µM ionomycin but was downregulated by 10 µM BAPTA/AM at 24 h of the cotreatments. Asterisks (*) denote p <0.05 as compared to the Ctrl.

Article Snippet: The blotting was conducted using mouse anti-V5 (Invitrogen), anti-human STC1 (R&D), human p-FAK (pY397) (BD Biosciences), rabbit anti-human p-GSK3β (Ser9), anti-human total GSK3β, anti-human p-Akt (Ser473), anti-human total Akt (Cell Signaling), and anti-human actin serum (Sigma).

Techniques:

Journal: PLoS ONE

Article Title: Stanniocalcin-1 Regulates Re-Epithelialization in Human Keratinocytes

doi: 10.1371/journal.pone.0027094

Figure Lengend Snippet: (A) The inhibitory effects of 30 mM LiCl and 10 µM SH6 on GSK3β and Akt-signaling respectively, were determined by probing the phosphorylated and total forms of GSK3β and Akt. The band intensities were measured and the ratios of phosphorylated/total form were plotted. Bars with the same letter are not significantly different according to the results of one-way ANOVA followed by Duncan’s multiple range tests ( p <0.05). STS-induced (B) e-lam formation and (C) cell migration were suppressed by LiCl but were induced by SH6. The cell images were captured under 200x and 100x magnification for e-lam formation and cell migration, respectively ( top ). (D) STS-induced STC1 mRNA expression was inhibited by LiCl, but was increased by SH6 (vs STS alone). Asterisks (*) denote p <0.05 as compared to their corresponding control.

Article Snippet: The blotting was conducted using mouse anti-V5 (Invitrogen), anti-human STC1 (R&D), human p-FAK (pY397) (BD Biosciences), rabbit anti-human p-GSK3β (Ser9), anti-human total GSK3β, anti-human p-Akt (Ser473), anti-human total Akt (Cell Signaling), and anti-human actin serum (Sigma).

Techniques: Migration, Expressing

Journal: PLoS ONE

Article Title: Stanniocalcin-1 Regulates Re-Epithelialization in Human Keratinocytes

doi: 10.1371/journal.pone.0027094

Figure Lengend Snippet: (A) After the transfection of STC1 siRNA for 48 h, the knockdown efficiency was examined by real-time PCR. In STC1 siRNA-transfected cells, STS-induced (B) cell migration and (C) e-lam formation on the fibronectin-coated plates were significantly reduced as compared to the NS siRNA-transfected cells. The cell images were captured for e-lam formation (200x magnification) and cell migration (100x magnification) ( top ). Under the STS+LiCl or STS+SH6 cotreatment, (D) the number of migrated cells and (E) e-lam formation on fibronectin-coated plate were compared between the STC1 siRNA-transfected cells and the NS siRNA-transfected cells. The knockdown of STC1 was found to inhibit cell migration induced by the STS + LiCl/SH6 cotreatments. The synergistic effect of STS/SH6 on the increase of e-lam formation was significantly reduced in the STC1 siRNA transfected cells. Asterisks (**) denote p <0.01 and (*) denote p <0.05 as compared to their corresponding NS siRNA-transfected cells.

Article Snippet: The blotting was conducted using mouse anti-V5 (Invitrogen), anti-human STC1 (R&D), human p-FAK (pY397) (BD Biosciences), rabbit anti-human p-GSK3β (Ser9), anti-human total GSK3β, anti-human p-Akt (Ser473), anti-human total Akt (Cell Signaling), and anti-human actin serum (Sigma).

Techniques: Transfection, Real-time Polymerase Chain Reaction, Migration

Journal: PLoS ONE

Article Title: Stanniocalcin-1 Regulates Re-Epithelialization in Human Keratinocytes

doi: 10.1371/journal.pone.0027094

Figure Lengend Snippet: (A) After transient transfection of V5-tagged STC1/pLenti (STC1/pLenti) and empty vector control (pLenti) into the HaCaT for 24 h, the cells were treated with 5 nM STS for 24 h and the protein expression levels of V5, GSK3β and Akt were determined using western blotting. The band intensities were measured and the ratios of the respective phosphorylated/total proteins were plotted. STC1 overexpression was found to have synergistic effect on STS-inhibited phosphorylation of Akt. STS-induced (B) e-lam formation on fibronectin-coated plate and (C) cell migration were synergistically induced by the transient overexpression of STC1 (vs STS-treated pLenti). The cell images were captured for e-lam formation (200x magnification) and cell migration (100x magnification) ( top ). (D) In the scratched wound healing assay, the scratched wound was closed significantly more rapidly in the cells maintained in the conditioned medium (CM) containing overexpressed STC1 protein (CM-STC1) than the control medium (CM Ctrl). The cell images were captured under 100x magnification. The levels of STC1 proteins in the conditioned media (CM-Ctrl and CM-STC1) were determined from day1 to day3 using western blotting. ( top ); CM-Ctrl depicts as C and CM-STC1 depicts as T. Asterisks (**) denote p <0.01 and (*) denote p <0.05 as compared to STS-treated pLenti-transfected cells.

Article Snippet: The blotting was conducted using mouse anti-V5 (Invitrogen), anti-human STC1 (R&D), human p-FAK (pY397) (BD Biosciences), rabbit anti-human p-GSK3β (Ser9), anti-human total GSK3β, anti-human p-Akt (Ser473), anti-human total Akt (Cell Signaling), and anti-human actin serum (Sigma).

Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot, Over Expression, Migration, Wound Healing Assay

Journal: PLoS ONE

Article Title: SUMO regulates p21 Cip1 intracellular distribution and with p21 Cip1 facilitates multiprotein complex formation in the nucleolus upon DNA damage

doi: 10.1371/journal.pone.0178925

Figure Lengend Snippet: A) Immunodetection of endogenous SUMO-1 (green) using anti-SUMO-1 mouse antibody and p21 (red) using anti-p21 rabbit antibody in control (CTL) or treated with Adr for 48 hours (Adr) HCT116 cells. Scale bar: 5μm. B) Immunogold electronic microscopy of SUMO-1 showing the presence of SUMO-1 in the INoB HCT116 cells treated with Adr for 24 hours. Scale Bar: 0.5μm. C) Immunostaining of endogenous p21 (red) in GFP-SUMO-1 (two representative cells are shown) or GFP-SUMO-1ΔGly-Gly transfected cells treated 24h with Adr. Scale bar: 5μm. D) Immunostaining of p21 (red) and UBC9 (green) (rabbit polyclonal antibody) in 24-h Adr-treated HCT116 cells. Scale bar: 5μm. E) Immunostaining of SUMO-1 (green) and UBC9 (red) (rabbit monoclonal antibody) of 24-h Adr-treated HCT116 cells transfected with non-targeting (siNT) or UBC9 (siUBC9). Scale bar: 5 μm.

Article Snippet: The antibodies used were the following: anti-p21Waf-1 mouse monoclonal antibody (diluted 1:200; Ab-1, OP64, Calbiochem); anti-p21 rabbit polyclonal antibody (diluted 1:200; C-19, sc-397, Santa Cruz Biotechnology); anti-UBF mouse monoclonal antibody (diluted 1:100; F-9, sc-13125, Santa Cruz Biotechnology); anti-fibrillarin rabbit polyclonal antibody (diluted 1:50; H-140, sc-25397, Santa Cruz Biotechnology); anti-p53 mouse monoclonal antibody (diluted 1:25; Pab 240, sc-99, Santa Cruz Biotechnology); anti-Mdm2 mouse monoclonal antibody (diluted 1:100; Ab-1, OP46, Calbiochem); anti-Cyclin E mouse monoclonal antibody (diluted 1:50; HE-12, sc-247, Santa Cruz Biotechnology); anti-SUMO-1 mouse monoclonal antibody (anti-GMP-1, diluted 1:100; 33–2400, Zymed), anti-SUMO-1 rabbit polyclonal antibody (FL-101, sc-9060, Santa Cruz Biotechnology); anti-UBC9 rabbit polyclonal antibody (diluted 1:50; ab30502, Abcam); anti-UBC9 rabbit monoclonal (diluted 1:50; D26F2 Cell Signaling); anti-HA mouse monoclonal antibody (diluted 1:200; 12CA5, 11 583816 001, Roche); anti-PML rabbit polyclonal antibody (diluted 1:200; H-238, sc-5621, Santa Cruz Biotechnology); anti-CRM1 (exportin-1) mouse monoclonal antibody (diluted 1:100; 611832, BD Biosciences); anti-Cdk2 rabbit polyclonal antibody (diluted 1:50; sc-163, Santa Cruz Biotechnology); and anti-PCNA mouse monoclonal antibody (diluted 1:50; Ab-1, NA03, Oncogene).

Techniques: Immunodetection, Microscopy, Immunostaining, Transfection