Journal: Frontiers in Oncology

Article Title: YTHDF1 Aggravates the Progression of Cervical Cancer Through m 6 A-Mediated Up-Regulation of RANBP2

doi: 10.3389/fonc.2021.650383

Figure Lengend Snippet: RANBP2 is the key target of YTHDF1 in cervical cancer. (A) Western blot detecting the protein level of RANBP2 in Hela and Siha cells upon YTHDF1 knockdown. (B) RT-qPCR detecting relative RNA level of RANBP2 in Hela and Siha upon YTHDF1 knockdown. (C) RIP-PCR assays detecting the interactions between YTHDF1 and RANBP2 mRNA in Siha cells. IgG was used as an internal control. GAPDH was used as the negative control in western blot assays. (D) meRIP-PCR assays detecting the m 6 A modification of RANBP2 mRNA in Siha cells. (E) Schematic of wild-type (YTHDF1-wt) and mutant (YTHDF1-mut) YTHDF1 constructs. (F) RIP-derived RNA and protein of wild-type (YTHDF1-wt) group and mutant (YTHDF1-mut) group in Hela cells were measured by RT-qPCR and western blot after immunoprecipitation by using the antibody specific to Flag, respectively. GAPDH was used as the negative control in western blot assays. Data are shown as means ± S.D. **P < 0.01, ***P < 0.001.

Article Snippet: The slices were washed three times with PBS, and blocked with goat serum (ZSGB-BIO, ZLI-9021) at 37°C for 30 min. Rabbit anti-YTHDF1 antibody (ProteinTech, 1:100) or anti-RANBP2 antibody (ProteinTech, 1:100) was added to the slices and incubated overnight at 4°C.

Techniques: Western Blot, Knockdown, Quantitative RT-PCR, Control, Negative Control, Modification, Mutagenesis, Construct, Derivative Assay, Immunoprecipitation

Journal: Frontiers in Oncology

Article Title: YTHDF1 Aggravates the Progression of Cervical Cancer Through m 6 A-Mediated Up-Regulation of RANBP2

doi: 10.3389/fonc.2021.650383

Figure Lengend Snippet: RANBP2 plays an oncogenic role in cervical cancer cells. (A) Detection of RANBP2 knockdown in Hela and Siha cell lines by western blot. (B) The effect of RANBP2 knockdown on cell growth was determined by CCK-8 assays. (C) Colony formation assays were performed in RANBP2 knockdown and control cells. (D, E) Migration and invasion assays of Hela and Siha cells upon RANBP2 knockdown. Scale bar, 200 μm. Data are shown as means ± S.D. **P < 0.01, ***P < 0.001.

Article Snippet: The slices were washed three times with PBS, and blocked with goat serum (ZSGB-BIO, ZLI-9021) at 37°C for 30 min. Rabbit anti-YTHDF1 antibody (ProteinTech, 1:100) or anti-RANBP2 antibody (ProteinTech, 1:100) was added to the slices and incubated overnight at 4°C.

Techniques: Knockdown, Western Blot, CCK-8 Assay, Control, Migration

Journal: Frontiers in Oncology

Article Title: YTHDF1 Aggravates the Progression of Cervical Cancer Through m 6 A-Mediated Up-Regulation of RANBP2

doi: 10.3389/fonc.2021.650383

Figure Lengend Snippet: Knockdown of RANBP2 suppressed the proliferation, migration and invasion of YTHDF1-overexpressing Hela and Siha cells. (A) Colony formation assays were performed in YTHDF1-overexpressing Hela and Siha cells infected with the RANBP2 shRNA or controls. (B) The proliferation ability of YTHDF1-overexpressing Hela and Siha cells upon RANBP2 knockdown was assessed by CCK-8 assays. (C, D) Migration and invasion YTHDF1-overexpressing Hela (C) and Siha (D) cells upon RANBP2 knockdown was detected by transwell assays. Scale bar, 200 μm. *P < 0.05,**P < 0.01, ***P < 0.001.

Article Snippet: The slices were washed three times with PBS, and blocked with goat serum (ZSGB-BIO, ZLI-9021) at 37°C for 30 min. Rabbit anti-YTHDF1 antibody (ProteinTech, 1:100) or anti-RANBP2 antibody (ProteinTech, 1:100) was added to the slices and incubated overnight at 4°C.

Techniques: Knockdown, Migration, Infection, shRNA, CCK-8 Assay

Journal: Frontiers in Oncology

Article Title: YTHDF1 Aggravates the Progression of Cervical Cancer Through m 6 A-Mediated Up-Regulation of RANBP2

doi: 10.3389/fonc.2021.650383

Figure Lengend Snippet: The expression of RANBP2 is positively correlated with YTHDF1 in cervical cancer. (A) Representative immunohistochemical images of RANBP2 protein expression in cervical cancer tissues and cervical epithelium tissues. Scale bar, 100 μm. (B) The quantitative analysis of RANBP2 expression in cervical cancer tissues and cervical epithelium tissues assessed by immunohistochemistry. (C) Spearman’s correlation analysis of RANBP2 and YTHDF1 expression in cervical cancer tissues. (D) Representative immunohistochemical images of YTHDF1 and RANBP2 in the cervical cancer tissues. Scale bar, 100 μm. Data are shown as means ± S.D. *P < 0.05.

Article Snippet: The slices were washed three times with PBS, and blocked with goat serum (ZSGB-BIO, ZLI-9021) at 37°C for 30 min. Rabbit anti-YTHDF1 antibody (ProteinTech, 1:100) or anti-RANBP2 antibody (ProteinTech, 1:100) was added to the slices and incubated overnight at 4°C.

Techniques: Expressing, Immunohistochemical staining, Immunohistochemistry

Journal: Theranostics

Article Title: SUMO1 modification of methyltransferase-like 3 promotes tumor progression via regulating Snail mRNA homeostasis in hepatocellular carcinoma.

doi: 10.7150/thno.42539

Figure Lengend Snippet: Figure 1. Mitogen stimulates Mettl3 conjugated to SUMO-1. (A) Mettl3 was overexpressed in MHCC97H cells by transfection with Flag-tagged wild type Mettl3. Subsequently, the cells were transfected with His-SUMO1, -SUMO2, -SUMO3, and HA-Ubc9 or siUBC9. Immunoprecipitation (IP) and western blotting analyses were performed with the indicated antibodies for the SUMOylation assay. (B) Confocal immunofluorescence of endogenous Mettl3 and SUMO1 proteins in MHCC97H cells (Mettl3, green; SUMO1, red). Both Mettl3 and SUMO1 were observed in the nucleus and cytoplasm. Scale bars, 50 μm. (C-F) Representative IP immunoblot analysis was conducted with the anti-Mettl3 or anti-SUMO1 antibody and whole-cell extracts from HepG2 or MHCC97H cells incubated in serum-containing or serum-free medium for 24 h. (C and E) IP analysis was performed with anti-Mettl3 antibodies in HepG2 and MHCC97H cells. (D and F) IP analysis was performed with anti-SUMO1 antibodies in HepG2 and MHCC97H cells. (G) IP immunoblotting analysis examining SUMOylation of endogenous Mettl3 from whole-cell extracts after the addition of 5%, 10%, and 20% serum with anti-Mettl3 antibody or normal IgG, followed by western blotting with the indicated antibodies. (H) IP immunoblotting analysis examining SUMOylation of endogenous Mettl3 from whole-cell extracts after the addition of low (10 ng/ml) or high (20 ng/ml) concentration HGF with anti-Mettl3 antibody or normal IgG, followed by western blotting with the indicated antibodies.

Article Snippet: The primary antibodies, including anti-GAPDH, anti-LaminB1, anti-His, anti-HA, anti-Flag, anti-MMP2 (from proteintech), anti-Mettl3 (from abcam, Bethyl); anti-m6a (from Synaptic Systems); SUMO-1 and SUMO2/3 (from abcam); anti-MMP9, anti-E-cadherin and normal rabbit IgG (from CST); UBC9, Snail and normal mouse IgG (from santa cruz); anti-rabbit, Anti-mouse peroxidase-conjugated secondary antibodies (from proteintech). m6A dot blot assay mRNA was enriched using Dynabeads mRNA DIRECT Purification Kit (InvitrogenTM, 61012).

Techniques: Transfection, Immunoprecipitation, Western Blot, Incubation, Concentration Assay

Journal: Theranostics

Article Title: SUMO1 modification of methyltransferase-like 3 promotes tumor progression via regulating Snail mRNA homeostasis in hepatocellular carcinoma.

doi: 10.7150/thno.42539

Figure Lengend Snippet: Figure 2. Mettl3 SUMOylation correlates with UBC9 upregulation in response to mitogen in liver cancer cells. (A) qRT-PCR analysis of HEP3B or MHCC97H cells fed in the presence of serum or serum-starved for UBC9 expression. (B) Immunoblot analysis of whole-cell lysates (WCL) from HEP3B or MHCC97H cells stimulated in serum-containing or serum-free medium for Mettl3, UBC9, and SUMO1 expression. (C) MHCC97H cells were transfected with SUMO1 in combination with Ubc9, followed by IP and western blotting with the indicated antibodies. (D) IP immunoblot analysis was conducted with an anti-Mettl3 antibody and whole-cell extracts from MHCC97H cells stimulated with or without serum in the presence of scramble or Ubc9 siRNA. Twenty-four hours after transfection, cells were harvested and subjected to co-immunoprecipitation with the anti-Mettl3 antibody, followed by western blotting with the indicated antibodies. (E) UBC9 upregulation in HCC and its matched adjacent normal tissues of 26 patients was analyzed (mean ± s.d., * P < 0.05). (F and G) UBC9 expression level was elevated in 371 HCC tissues compared with 50 normal liver tissue samples (F) and positively correlated with metastatic HCC tissues (G) in the TCGA profile, based on the ualcan database (http://ualcan.path.uab.edu/index.html). (H and I) Overall survival analysis (H) and relapse-free survival (I) based on UBC9 expression in HCC, according to kmplot online database (http://kmplot.com/analysis/). (J-M) Effects of decreased UBC9 expression on cell proliferation (J), viability (K), apoptosis (L), and migration (M) in MHCC97H cells. Data are presented as mean ± s.d. * p < 0.05, ** p < 0.01, ***<0.001; Student’s t-test.

Article Snippet: The primary antibodies, including anti-GAPDH, anti-LaminB1, anti-His, anti-HA, anti-Flag, anti-MMP2 (from proteintech), anti-Mettl3 (from abcam, Bethyl); anti-m6a (from Synaptic Systems); SUMO-1 and SUMO2/3 (from abcam); anti-MMP9, anti-E-cadherin and normal rabbit IgG (from CST); UBC9, Snail and normal mouse IgG (from santa cruz); anti-rabbit, Anti-mouse peroxidase-conjugated secondary antibodies (from proteintech). m6A dot blot assay mRNA was enriched using Dynabeads mRNA DIRECT Purification Kit (InvitrogenTM, 61012).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Transfection, Immunoprecipitation, Migration

Journal: Theranostics

Article Title: SUMO1 modification of methyltransferase-like 3 promotes tumor progression via regulating Snail mRNA homeostasis in hepatocellular carcinoma.

doi: 10.7150/thno.42539

Figure Lengend Snippet: Figure 3. Mettl3-SUMO1 conjugates are essential for its oncogenic properties and correlates with Snail upregulation and is positively associated with high metastatic potential of liver cancer cells. (A) IP immunoblot analysis was conducted with an anti-Flag antibody and whole-cell extracts from Flag-tagged wild type Mettl3, or SUMOylation-defective Mettl3-K177/K211/K212/K215-Flag (KR)-expressing MHCC97H cells transfected with or without His-SUMO1 and HA-UBC9. (B-E) Effects of vector-, Mettl3-WT-, or Mettl3-KR-mutant-expressing cells on cell proliferation (B), viability and apoptosis (C), colony formation (D), migration and invasion (E) in MHCC97H cells. (F) IP immunoblot analysis was conducted with the anti-SUMO1 antibody and whole-cell extracts from HEP3B or MHCC97H cells stimulated with or without serum. Cells were harvested and subjected to co-immunoprecipitation with the anti-SUMO1 or control IgG antibody, followed by western blotting with the indicated antibodies. (G and H) qRT-PCR analysis of HEP3B or MHCC97H cells stimulated with serum or deprived of serum for Snail (G) and CDH1 (H) expression. (I) Immunoblot analysis of WCL from HEP3B or MHCC97H cells stimulated with serum or deprived of serum for Snail and CDH1 expression. (J) Immunoblot analysis of WCL from scramble or siMettl3-expressing cells stimulated with serum or deprived of serum for Mettl3, Snail and CDH1 expression. (K) Knock-down of endogenous Mettl3 impaired expression of EMT-related genes. Immunoblot analysis of WCL from scramble or siMettl3-expressing cells. (L) Effects of scramble or siMettl3-expressing cells on cell viability and apoptosis. Data are presented as mean ± s.d. * p < 0.05, ** p < 0.01, ***<0.001; Student’s t-test.

Article Snippet: The primary antibodies, including anti-GAPDH, anti-LaminB1, anti-His, anti-HA, anti-Flag, anti-MMP2 (from proteintech), anti-Mettl3 (from abcam, Bethyl); anti-m6a (from Synaptic Systems); SUMO-1 and SUMO2/3 (from abcam); anti-MMP9, anti-E-cadherin and normal rabbit IgG (from CST); UBC9, Snail and normal mouse IgG (from santa cruz); anti-rabbit, Anti-mouse peroxidase-conjugated secondary antibodies (from proteintech). m6A dot blot assay mRNA was enriched using Dynabeads mRNA DIRECT Purification Kit (InvitrogenTM, 61012).

Techniques: Western Blot, Expressing, Transfection, Plasmid Preparation, Mutagenesis, Migration, Immunoprecipitation, Control, Quantitative RT-PCR, Knockdown

Journal: Theranostics

Article Title: SUMO1 modification of methyltransferase-like 3 promotes tumor progression via regulating Snail mRNA homeostasis in hepatocellular carcinoma.

doi: 10.7150/thno.42539

Figure Lengend Snippet: Figure 5. SUMOylation of Mettl3 regulates Snail mRNA homeostasis via m6A methyltransferase. (A) Mettl3-WT or -KR was transiently transfected into MHCC97H cells and detected by the dot-blot assay with the anti-m6A antibody. Equal loading of mRNAs was confirmed by methylene blue staining. (B) Mettl3 with or without Ubc9 was transfected into MHCC97H cells and detected by the dot-blot assay with the anti-m6A antibody. Equal loading of mRNAs was confirmed by methylene blue staining. (C) Suppression or overexpression of Mettl3 in HCC was determined by RT-qPCR, and GAPDH was used as the normalized control. (D) mRNA of Snail in Mettl3 suppression or overexpression cells. (E) Mettl3-WT or Mettl3-KR-expressing cells were transfected with the pEZX-PL01-Snail promoter reporter plasmid and negative control plasmid for 36 h. Results were expressed as the ratios between F-luc and R-luc activities. (F) IP immunoblot analysis was performed in Mettl3-WT- and Mettl3-KR-expressing cells with the anti-Mettl3 antibody, followed by western blotting with Mettl3, anti-Eef2, anti-eIF4E, and anti-NCBP1 antibodies. One-tenth of lysates as the input was immunoblotted with indicated antibodies. (G) Mettl3-WT- and Mettl3-KR-expressing cells were pretreated with MG-132 for 6 h and stimulated with serum for indicated times. Subsequently, Mettl3 and Snail protein expression levels were analyzed by western blotting. (H) Scramble or siMettl3-expressing cells were fed with CHX for the indicated times, and protein expression of Mettl3 and Snail was analyzed by western blotting. (I) Mettl3-WT or Mettl3-KR-expressing cells transfected with different plasmids were treated with CHX for the indicated times, and protein expression of Mettl3 and Snail was detected by western blotting. (J and K) The decay rate of mRNA and qPCR analysis of Snail at the indicated times after exposure to the transcription inhibitor actinomycin D (5 μg/mL) in MHCC97H (J) and HepG2 (K) cells. The relative expression level was normalized to β-actin. Data are presented as mean ± s.d. * p < 0.05, ** p < 0.01; Student’s t-test.

Article Snippet: The primary antibodies, including anti-GAPDH, anti-LaminB1, anti-His, anti-HA, anti-Flag, anti-MMP2 (from proteintech), anti-Mettl3 (from abcam, Bethyl); anti-m6a (from Synaptic Systems); SUMO-1 and SUMO2/3 (from abcam); anti-MMP9, anti-E-cadherin and normal rabbit IgG (from CST); UBC9, Snail and normal mouse IgG (from santa cruz); anti-rabbit, Anti-mouse peroxidase-conjugated secondary antibodies (from proteintech). m6A dot blot assay mRNA was enriched using Dynabeads mRNA DIRECT Purification Kit (InvitrogenTM, 61012).

Techniques: Transfection, Dot Blot, Staining, Over Expression, Quantitative RT-PCR, Control, Expressing, Plasmid Preparation, Negative Control, Western Blot

Journal: Advanced Science

Article Title: FGF19‐Activated Hepatic Stellate Cells Release ANGPTL4 that Promotes Colorectal Cancer Liver Metastasis

doi: 10.1002/advs.202413525

Figure Lengend Snippet: FGF19 is associated with CRC liver metastasis (CRCLM) and poor prognosis in CRC patients. A) Seventeen cell subsets were identified by analysis of the scRNA‐Seq data from 6 patients with CRCLM. B) Analysis of the cellular communication in tumor samples from patients with CRCLM. C) The Venn diagram illustrating the overlap of interacting genes between CRC cells and CAFs, as well as the highly expressed genes in CRC from the TCGA database. D) Volcano plot showing the differential expression of genes, and nine of the interacting genes exhibited a marked increase in expression. E) Correlation analysis between FGF19 expression and CRC patient survival (n = 597). F) The FGF19 expression in colon adenocarcinoma (COAD) patients from the TCGA database (Normal, n = 41; Primary tumor, n = 286). G) Representative images of the luminance signals from the CRC (nonmetastasis), CRCLM, and CRC pulmanary metastasis (CRCPM) animal models. CT26‐luciference‐expressing (CT26‐luc) cells were injected into the caecum, spleen, and tail vein of mice to establish the CRC (CRC‐no metastasis), CRCLM, and CRCPM animal models, respectively. The luminance signals were detected by using the IVIS Lumina XRMS Series III instrument at the end of the experiment. H) Representative images and the luminance signals of the colon, lung, and liver tissues of the mice in CTL (n = 7), CRC (CRC‐no metastasis) (n = 6), CRCPM (n = 7), and CRCLM (n = 7) animal models. I) The levels of FGF15 in the serum of CRC, CRCLM, or CRCPM model mice were detected using the ELISA assay (CTL, n = 7; CRC, n = 6; CRCPM, n = 7; CRCLM, n = 7). J) IF staining showing the expression of FGF19 in the clinical samples using the microarray assay (n = 42). Data are shown as Mean ± SD. For I, ** p < 0.01, versus CTL.

Article Snippet: Infigratinib, human angiopoietin‐related 4 protein (ANGPTL4, HEK293, His), and mouse FGF15 Protein (His‐SUMO) were purchased from MedChemExpress (MCE, NJ, USA).

Techniques: Quantitative Proteomics, Expressing, Injection, Enzyme-linked Immunosorbent Assay, Staining, Microarray

Journal: Advanced Science

Article Title: FGF19‐Activated Hepatic Stellate Cells Release ANGPTL4 that Promotes Colorectal Cancer Liver Metastasis

doi: 10.1002/advs.202413525

Figure Lengend Snippet: CRC cells release FGF19 (FGF15) which activates HSCs and promotes HSCs‐to‐CAFs differentiation. A) The experimental setup for the control (upper panel) and experimental (lower panel) conditions for the conditioned medium (CM) systems. B) Experimental setup for the control (upper panel) and experimental (lower panel) coculture systems. C,D) The protein levels of α‐SMA and FAP in the CM system (C) and SW620‐LX‐2 coculture system (D) were determined by the Western blotting; and the quantitative results are shown in the right panel. E,F) The protein levels of α‐SMA and FAP in the CM system (E) and CT26‐JS1 coculture system (F) were determined by the Western blotting; and the quantitative results are shown in the right panel. G) Effects of the recombinant FGF19 protein on the protein levels of FAP and α‐SMA in LX‐2 cells. H) Effects of FGF15 recombinant protein on the protein levels of FAP and α‐SMA in JS1 cells. I,J) Effects of infigratinib, a FGFR inhibitor on the protein levels of FAP and α‐SMA in SW620 CM system (I) or CT26 CM system (J). Data are shown as Mean ± SD, n = 3. For C, E: * p < 0.05, ** p < 0.01 versus HSCs CM. For D, F‐H: * p < 0.05, ** p < 0.01 versus the corresponding CTL. For I‐J: * p < 0.05, ** p < 0.01 versus HSCs CM; # p < 0.05, ## p < 0.01 versus CRC CM.

Article Snippet: Infigratinib, human angiopoietin‐related 4 protein (ANGPTL4, HEK293, His), and mouse FGF15 Protein (His‐SUMO) were purchased from MedChemExpress (MCE, NJ, USA).

Techniques: Control, Western Blot, Recombinant

Journal: Advanced Science

Article Title: FGF19‐Activated Hepatic Stellate Cells Release ANGPTL4 that Promotes Colorectal Cancer Liver Metastasis

doi: 10.1002/advs.202413525

Figure Lengend Snippet: FGF‐activated HSCs increase CRC cells migration. A,B) Representative images of SW620 (A) and CT26 (B) cells migration in the CRC cells‐HSCs coculture system (left panel); and the quantitative data were analyzed using Image J software (right panel). Photographs were taken 24 h after treatment. C) Representative images of cell migration in the SW620, SW620‐LX‐2 coculture, and FGF19‐treated coculture systems (left panel); and the quantitative data were analyzed using Image J software (right panel). D) Representative images of cell migration in the CT26, CT26‐JS1 coculture, or FGF15 treated coculture system (left panel); and quantitative results were analyzed using Image J software (right panel). E) Representative images of cell migration in SW620, SW620‐LX‐2 coculture, and infigratinib‐treated coculture system (left panel); and quantitative results were analyzed using Image J software (right panel). F) Representative images of cell migration in the CT26, CT26‐JS1 coculture, and infigratinib‐treated coculture systems (left panel). All the quantitative results were analyzed using Image J software (right panel). Data are shown as Mean ± SD from three independent experiments, n = 3. For A‐D: * p < 0.05, ** p < 0.01 versus the corresponding CTL. For E‐F: * p < 0.05, ** p < 0.01 versus the corresponding CTL; # p < 0.05, ## p < 0.01 versus CRC cells‐HSCs coculture group.

Article Snippet: Infigratinib, human angiopoietin‐related 4 protein (ANGPTL4, HEK293, His), and mouse FGF15 Protein (His‐SUMO) were purchased from MedChemExpress (MCE, NJ, USA).

Techniques: Migration, Software

Journal: Advanced Science

Article Title: FGF19‐Activated Hepatic Stellate Cells Release ANGPTL4 that Promotes Colorectal Cancer Liver Metastasis

doi: 10.1002/advs.202413525

Figure Lengend Snippet: Effects of FGF15 and ANGPTL4 in mouse models of different degrees of CRCLM. A) Expression levels of MMP2 and MMP9 in the liver tissues of each group were detected by IHC staining. B,C) Protein levels of FGF15 in the liver tissues (B) and tumor sites (C) were determined by Western blotting. D) The serum level of FGF15 was determined by the ELISA assay. E) Expression of FGFR4, a FGF15 receptor in the liver tissues of each group was detected by IHC staining. F) Expression levels of α‐SMA and FAP in the liver tissues of each group were detected by IHC staining. G,H) Protein levels of α‐SMA in the liver tissues with tumors were determined by using Western blotting (G), and quantitative results were analyzed using Image J software (H). I) mRNA levels of FAP in the liver tissues with tumors were determined using RT‐qPCR analysis. J) Protein levels of ANGPTL4 in the liver tissues were determined by Western blotting (left panel), and the quantitative data were analyzed using Image J software (right panel). K,L) The liver tissue homogenates (K) and the serum (L) levels of ANGPTL4 were determined by the ELISA assay. M) Localization of FGF15 in CTCs within the liver of CRCLM mice by using the mIF analysis. N) Localization of ANGPTL4 in the liver tissue CAFs in CRCLM mouse model determined using the mIF analysis. Data are shown as Mean ± SD, n = 6. For B, D, H‐L, * p < 0.05, ** p < 0.01 versus CTL. For C, ** p < 0.01 versus 5.0 × 10 5 group.

Article Snippet: Infigratinib, human angiopoietin‐related 4 protein (ANGPTL4, HEK293, His), and mouse FGF15 Protein (His‐SUMO) were purchased from MedChemExpress (MCE, NJ, USA).

Techniques: Expressing, Immunohistochemistry, Western Blot, Enzyme-linked Immunosorbent Assay, Software, Quantitative RT-PCR

Journal: Advanced Science

Article Title: FGF19‐Activated Hepatic Stellate Cells Release ANGPTL4 that Promotes Colorectal Cancer Liver Metastasis

doi: 10.1002/advs.202413525

Figure Lengend Snippet: The effects of FGF15 and ANGPTL4 in CRCLM mouse models with different time points. A) Timeline for the establishment of the mouse CRCLM model. CT26‐luc cells were injected into the spleens of BALB/c mice, then the mice were sacrificed at different time points. B) Representative images of live tumor‐bearing mice with tumors. C) Representative fluorescence signal imaging of the liver tissues. D,E) Quantitative results of the mice fluorescence intensity (D) and the liver fluorescence intensity (E) were analyzed using the Living Image software 4.4. F–H) Representative IHC staining of FGF15 and FGFR4 (F) MMP2 and MMP9 (G), α‐SMA, and FAP (H) in the liver tissues of mice in each group. I) Localization of ANGPTL4 in CAFs of liver tissues in CRCLM mouse model by using the mIF analysis. J) Protein levels of ANGPTL4 in the liver tissues with tumors were determined by using Western blotting (upper panel); and quantitative results were quantified and analyzed using Image J software (lower panel). K,L) Liver tissue homogenates (K) and the serum (L) levels of ANGPTL4 were determined by the ELISA assay. Data are shown as Mean ± SD. n = 6. * p < 0.05, ** p < 0.01 versus CTL.

Article Snippet: Infigratinib, human angiopoietin‐related 4 protein (ANGPTL4, HEK293, His), and mouse FGF15 Protein (His‐SUMO) were purchased from MedChemExpress (MCE, NJ, USA).

Techniques: Injection, Fluorescence, Imaging, Software, Immunohistochemistry, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Advanced Science

Article Title: FGF19‐Activated Hepatic Stellate Cells Release ANGPTL4 that Promotes Colorectal Cancer Liver Metastasis

doi: 10.1002/advs.202413525

Figure Lengend Snippet: The FGF15/ANGPTL4 axis is involved in the progression of CRCLM. A,B) Representative images of IHC staining for FGF15 and FGFR4 (A), and for α‐SMA and FAP (B) in the liver tissues of mice in each group. C) The protein levels of ANGPTL4 in tumor‐bearing liver tissues were determined by using Western blotting (upper panel); and quantitative results were analyzed using Image J software (lower panel). D) Representative live‐animal imaging images of mice with tumors. CT26‐luc‐shNC cells and CT26‐luc‐shFGF15 cells were inoculated into the hepatic portal vein of BALB/c mice, respectively. Mice were randomly divided into 3 groups, including the sham, shNC, and shFGF15 groups. E) Quantitative results of the fluorescence intensity were analyzed using Living Image software 4.4. F) Representative H&E‐stained images of liver tissues in each group of mice. G) Representative images of IHC for MMP2 and MMP9 in mouse liver tissues from each group. H) Diagram showing that the FGF19/ANGPTL4 axis mediates the interaction between CRC cells and HSCs, and promotes CRC liver metastasis. Data are shown as Mean ± SD, n = 6. ** p < 0.01 versus Sham; # p < 0.05, ## p < 0.01 versus shNC.

Article Snippet: Infigratinib, human angiopoietin‐related 4 protein (ANGPTL4, HEK293, His), and mouse FGF15 Protein (His‐SUMO) were purchased from MedChemExpress (MCE, NJ, USA).

Techniques: Immunohistochemistry, Western Blot, Software, Imaging, Fluorescence, Staining

Journal: Journal of Cell Science

Article Title: Coilin and SUMOylation influence PARP1 dynamics and the DNA damage response

doi: 10.1242/jcs.263953

Figure Lengend Snippet: Coilin and PIAS4 promote the SUMOylation of PARP1 in cells treated with MMS plus Talazoparib. (A) HeLa cells were transfected with control siRNA 72 h. At the 48 h mark, cells were left untransfected, transfected with His–SUMO1 or co-transfected with His–SUMO1 and Myc–coilin. At the 71 h mark, cells were either untreated or treated with MMS+Talazoparib for 1 h. Lysate was generated and subjected to Ni-NTA pulldown (PD), SDS-PAGE, western blotting and probing with antibodies to PARP1 (upper panel) or SUMO1 (lower panel). SUMOylated PARP1 is indicated by a bracket. (B) HeLa cells were treated as in A, except cells were transfected with PIAS4 siRNA instead of control siRNA. The blot was probed with anti-PARP1 (top panel), anti-Myc (middle panel) and anti-SUMO1 (bottom panel) antibodies. SUMOylated PARP1 and SUMOylated Myc–coilin are indicated by a bracket. 20 µl of total lysate was used for input in A and B. (C) Quantification of A and other blots showing that PIAS4 KD decreases the amount of SUMOylated PARP1 in the presence of MMS+Talazoparib. Results are mean±s.e.m., n =3. * P <0.05 (unpaired two-tailed Student's t -test). (D) Quantification of B and other blots showing that the expression of Myc–coilin in the presence of PAIS4 KD and MMS+Talazoparib treatment increases the amount of SUMOylated PARP1 compared to reactions lacking Myc–coilin. Quantification was done as described in Fig. 1C , with the amount of SUMOylated PARP1 present in PIAS4 and MMS+Talazoparib set to 1. Results are mean±s.e.m., n =3. * P <0.05 (unpaired two-tailed Student's t -test).

Article Snippet: The primary antibodies used were anti-β-actin mouse monoclonal antibody (1:15,000; 8H10D10, Cell Signaling, Danvers, MA), anti-Myc monoclonal antibody (1:1000; 9E10, Santa Cruz Biotechnology Inc., Dallas, TX, USA), anti-SUMO-1 polyclonal antibody (1:1000; 4930, Cell Signaling, Danvers, MA), anti-coilin rabbit polyclonal antibody (1:1000; sc-32860, Santa Cruz Biotechnology Inc., Dallas, TX, USA), anti-PARP1 rabbit polyclonal antibody (1:1000; 13371, Proteintech, Rosemont, IL, USA), anti-Ubc9 rabbit monoclonal antibody (1:1000; 4918, Cell Signaling Technology, Danvers, MA, USA), and anti-PIAS4 rabbit polyclonal antibody (Proteintech, Rosemont, IL, USA).

Techniques: Transfection, Control, Generated, SDS Page, Western Blot, Two Tailed Test, Expressing

Journal: Endocrinology

Article Title: miR-26a Plays an Important Role in Cell Cycle Regulation in ACTH-Secreting Pituitary Adenomas by Modulating Protein Kinase Cδ

doi: 10.1210/en.2012-2070

Figure Lengend Snippet: PRKCD and SENP5 mRNA expression. A and B, PRKCD basal mRNA expression levels were assessed in AtT20/D16v-F2 cells (A) and human ACTH-secreting pituitary adenoma tissues (B) by RQ-PCR. C and D, SENP5 basal mRNA expression levels were assessed in AtT20/D16v-F2 cells (C) and human ACTH-secreting pituitary adenoma tissues (D) by RQ-PCR. Results are expressed as mean fold of induction ± SE percent mRNA expression vs normal pituitary from at least 5 independent experiments in 5 replicates. ** P < .01 vs normal pituitary.

Article Snippet: Relative expression ratios of SUMO1/sentrin-specific peptidase 5 ( SENP5 ) (Mm00769150_m1 and Hs01090047_m1; Applied Biosystems) and protein kinase Cδ ( PRKCD ) (Mm00440891_m1 and Hs01090047_m1; Applied Biosystems) mRNAs were calculated by applying the method described by Pfaffl et al ( ).

Techniques: Expressing

Journal: Endocrinology

Article Title: miR-26a Plays an Important Role in Cell Cycle Regulation in ACTH-Secreting Pituitary Adenomas by Modulating Protein Kinase Cδ

doi: 10.1210/en.2012-2070

Figure Lengend Snippet: PRKCD is a direct target of miR-26a. Panel A, AtT20/D16v-F2 cells were transfected with a luciferase gene linked to the 3′-UTR of PRKCD (PRKCD pmirGLO) or the antisense sequence (antisense PRKCD pmirGLO) and cotransfected with pre–miR-26a, in the presence or absence of anti–miR-26a. Panel B, The same experiments were performed for SENP5 by using SENP5 pmirGLO or antisense SENP5 pmirGLO vectors. Results are expressed as mean value ± SE percent RLU vs vector only (pmirGLO) from 3 independent experiments in 6 replicates. ** P < .01 vs pmirGLO vector. Panel C, AtT20/D16v-F2 cells were transfected with pre–miR-26a (P), anti–miR-26a (A), or specific control oligos (cP and cA). Cell extracts were harvested 48 hours after transfection and assayed for PRKCD and SENP5 protein expression by Western blot. Data presented were confirmed in 3 independent experiments. Panel D, PRKCD basal protein expression levels were assessed by Western blot, and miR-26a expression levels were assessed by RQ-PCR in human ACTH-secreting pituitary adenoma tissues. Results are expressed as mean percent expression levels ± SE vs normal pituitary from at least 3 replicates. ** P < .01 vs normal pituitary. In the lower panel are representative Western blots showing PRKCD protein levels in human normal pituitary and in human ACTH-secreting pituitary adenomas. Abbreviation: C, control cells.

Article Snippet: Relative expression ratios of SUMO1/sentrin-specific peptidase 5 ( SENP5 ) (Mm00769150_m1 and Hs01090047_m1; Applied Biosystems) and protein kinase Cδ ( PRKCD ) (Mm00440891_m1 and Hs01090047_m1; Applied Biosystems) mRNAs were calculated by applying the method described by Pfaffl et al ( ).

Techniques: Transfection, Luciferase, Sequencing, Plasmid Preparation, Control, Expressing, Western Blot

Journal: The EMBO Journal

Article Title: SENP3-mediated deSUMOylation of dynamin-related protein 1 promotes cell death following ischaemia

doi: 10.1038/emboj.2013.65

Figure Lengend Snippet: OGD increases global SUMO-2/3 conjugation and decreases SENP3 in neurons. ( A ) Lysates of primary cortical neurons were blotted for SUMO-2/3 and β-tubulin after OGD (1 h). ( B ) sumo-2 mRNA levels are decreased and sumo-3 mRNA levels remain unchanged after OGD (1 h) in primary cortical neurons. ( C ) Levels of SENP3 are reduced in neurons after OGD (30 min). ( D ) OGD (30 min) does not alter SENP1, SENP2 or SENP5 levels in neurons. ( E ) senp3 mRNA levels are not reduced by OGD (1 h). For ( B ) and ( E ) significance was determined using a two-tailed Paired t -test; for sumo-2 mRNA ( n =9; ** P <0.01), for sumo-3 mRNA ( n =7) and for senp3 mRNA ( n =8). Source data for this figure is available on the online page.

Article Snippet: The probes used were Rn00821719_g1* for Rat SUMO2 , Rn01429214_g1 for Rat SUMO3 and Rn01496196_g1 for Rat SENP3 (Applied Biosystems).

Techniques: Conjugation Assay, Two Tailed Test

Journal: The EMBO Journal

Article Title: SENP3-mediated deSUMOylation of dynamin-related protein 1 promotes cell death following ischaemia

doi: 10.1038/emboj.2013.65

Figure Lengend Snippet: PERK activation is required for decreased SENP3. ( A ) PERK is phosphorylated (p-PERK) in neurons by OGD (30 min). Blots were probed with phospho-PERK, PERK and β-tubulin antibodies. ( B ) Overexpression of HA-PERK causes loss of Flag-SENP3 in HEK293 cells, which is blocked by overexpression of the PERK inhibitor p58IPK. Lysates were blotted with Flag, phospho-PERK and HA antibodies. ( C ) The kinase activity of PERK is required for SENP3 removal in HEK293 cells. HA-PERK, but not kinase-dead HA-PERK K618A decreases SENP3. The top panel shows the effect of PERK on overexpressed Flag-SENP3, while the lower panel shows endogenous SENP3. ( D ) PERK is required for OGD-induced loss of SENP3. Confluent wild-type and PERK −/− MEFs were subjected to OGD (2 h), harvested and the lysates probed as indicated. ( E ) Overexpression of HA-PERK in HEK293 cells reduces levels of SENP3 but not of SENP1, SENP2 or SENP5. Source data for this figure is available on the online page.

Article Snippet: The probes used were Rn00821719_g1* for Rat SUMO2 , Rn01429214_g1 for Rat SUMO3 and Rn01496196_g1 for Rat SENP3 (Applied Biosystems).

Techniques: Activation Assay, Over Expression, Activity Assay

Journal: The EMBO Journal

Article Title: SENP3-mediated deSUMOylation of dynamin-related protein 1 promotes cell death following ischaemia

doi: 10.1038/emboj.2013.65

Figure Lengend Snippet: SENP3 regulates cytochrome c release via deSUMOylation of Drp1. ( A ) Overexpression of Flag-SENP3, but not inactive C532A mutant causes cytochrome c release in HEK293 cells. The cytosol fraction or whole cell lysate was blotted for cytochrome c , Flag, and the cytosolic marker GAPDH, or β-actin. ( B ) Overexpressing SENP3 decreases mitochondrial cytochrome c in HeLa cells. Fixed cells were immunostained to assess the localization of cytochrome c in mitochondria labelled with MitoTracker. Representative images showing GFP-SENP3 (top panel) and GFP-SENP3 C532A expressing cells (lower panel, Green: SENP3; Red: MitoTracker; Blue: cytochrome c ; scale bar: 10 μm). White arrows indicate transfected cells with percentages denoting the relative proportions of transfected cells with significantly reduced cytochrome c in mitochondria for wild-type versus inactive mutant overexpression ( n =75 cells for GFP-SENP and n =113 for GFP-SENP3 C532A). ( C ) SENP3-mediated cytochrome c release is Drp1 dependent. HEK293 cells expressing Flag-SENP3 were treated with Mdivi-1 (50 μM) for 4 h. The cytosol fraction or whole cell lysate was blotted for cytochrome c , Smac/Diablo, Flag, and GAPDH, or β-actin. ( D ) Drp1 is SUMO-2-ylated in HEK293 cells expressing Flag-Ubc9, His-SUMO-2 and HA-Drp1. Lysate was incubated with Ni 2+ beads to precipitate His-SUMO-2-ylated proteins (His PD). ( E ) Overexpression of SENP3 decreases Drp1 SUMOylation. Constructs expressing HA-Drp1, Flag-Ubc9, His-SUMO-2 and either GFP or GFP-SENP3 were transfected into HEK293 cells. ( F ) SENP3 knockdown enhances Drp1 SUMOylation. Either non-specific siRNA (Nsi) or SENP3 siRNA (SENP3i) together with constructs expressing HA-Drp1, Flag-Ubc9 and His-SUMO-2 were co-expressed in HEK293 cells. ( G ) OGD enhances Drp1 SUMOylation. Two days post transfection, HEK293 cells were exposed to OGD (2 h). In ( C – F ), His-pulldown and lysate samples were blotted as indicated with antibodies against HA, Flag, His, GFP, SENP3 and β-actin. ( H ) OGD (2 h) for primary cortical neurons increases SUMOylation of endogenous Drp1. ( I ) Preventing Drp1 SUMOylation results in cytochrome c release. YFP-Drp1 R WT or Drp1 R 4KR were expressed in HEK293 cells after knockdown of endogenous Drp1. Source data for this figure is available on the online page.

Article Snippet: The probes used were Rn00821719_g1* for Rat SUMO2 , Rn01429214_g1 for Rat SUMO3 and Rn01496196_g1 for Rat SENP3 (Applied Biosystems).

Techniques: Over Expression, Mutagenesis, Marker, Expressing, Transfection, Incubation, Construct, Knockdown

Journal: The EMBO Journal

Article Title: SENP3-mediated deSUMOylation of dynamin-related protein 1 promotes cell death following ischaemia

doi: 10.1038/emboj.2013.65

Figure Lengend Snippet: SENP3 regulates mitochondrial association of Drp1. ( A ) Knockdown of Drp1 decreases cytosolic cytochrome c . ( B ) Knockdown of SENP3 decreases the mitochondrial association of endogenous Drp1 and reduces levels of cytosolic cytochrome c in HEK293 cells ( n =3; ** P <0.01). ( C ) SENP3 knockdown decreases mitochondrial localization of Drp1 in HeLa cells. Fixed cells were immunostained to assess the co-localization of endogenous Drp1 with mitochondria. Representative images showing control (top panel, Nsi) and SENP3 knockdown cells (lower panel, SENP3i; Green: Drp1; Red: MitoTracker; Yellow: co-localization of Drp1 and MitoTracker; Magenta: SENP3; Blue: Hoechst; scale bar: 10 μm). The region of interest defined by the white box is enlarged in the middle panels (scale bar: 5 μm). Right hand panels illustrate the individual channel data. The histogram shows the comparative levels of Drp1 localization at mitochondria ( n =36 cells for Nsi and n =27 cells for SENP3i; ** P <0.01). ( D ) SENP3 overexpression increases localization of Drp1 at mitochondria in HEK293 cells ( n =3; * P <0.05). ( E ) SUMOylation affects Drp1 dynamics. YFP-Drp1 R WT or non-SUMOylatable Drp1 R 4KR was expressed in HeLa cells after knockdown of endogenous Drp1 and the cells subjected to FRAP analysis. The left hand images show the representative cells sampled and the subsequent image panels are enlargements of frames at the specified time points (Green: YFP-DRP1 R ; Red: MitoTracker; Yellow: co-localization. Scale bar: 10 μm for first frame, 5 μm for enlarged frames). The area defined by the white box is the photobleached region of interest. Recovery curves for Drp1 R WT (black) and Drp1 R 4KR (white) are shown and the values presented in the table. Values=mean±s.e.m. ( n =12 cells per condition). ( F ) Non-SUMOylatable Drp1 shows enhanced mitochondrial association. YFP-Drp1 R WT or Drp1 R 4KR was expressed in HeLa cells after knockdown of endogenous Drp1, and cytosolic fraction, mitochondrial fraction, or whole cell lysates were blotted as indicated. Source data for this figure is available on the online page.

Article Snippet: The probes used were Rn00821719_g1* for Rat SUMO2 , Rn01429214_g1 for Rat SUMO3 and Rn01496196_g1 for Rat SENP3 (Applied Biosystems).

Techniques: Knockdown, Control, Over Expression

Journal: The EMBO Journal

Article Title: SENP3-mediated deSUMOylation of dynamin-related protein 1 promotes cell death following ischaemia

doi: 10.1038/emboj.2013.65

Figure Lengend Snippet: SENP3 regulates Drp1-mediated mitochondrial fission. ( A ) Overexpression of SENP3 does not cause Bax/Bak activation in HeLa cells whereas staurosporine (STS; 1 μM; 2 h) potently activates Bax/Bak. Representative images show immunostaining for active Bax (left panels) or active Bak (right panels) in GFP-SENP3 or GFP-SENP C532A transfected cells or untransfected staurosporine-treated cells (Green: GFP-SENP3, Red: active Bax/Bak; Blue: Hoechst; Scale bar: 10 μm). ( B ) Overexpression of SENP3 promotes mitochondrial fission in HeLa cells. Cells overexpressing GFP-SENP3 or inactive C532A mutant were stained with MitoTracker and mitochondrial morphology classified into one of three categories: (I) Elongated/Tubular, (II) Intermediate or (III) Fragmented. There was a significantly increased proportion of cells with fragmented mitochondria in GFP-SENP3 compared to C532A overexpressing cells ( P <0.0001, chi square test; n =99 cells for GFP-SENP3 and n =86 cells for GFP-SENP3 C532A). ( C ) Representative images showing GFP-SENP3 expressing (upper panels) and GFP-SENP3 C532A expressing neurons (lower panels) co-transfected with Mito-DsRed (scale: 10 μm, white box ROI magnified below). Data were quantified and expressed as a dendritic mitochondrial index (total mitochondrial length/dendrite length). Values bar charts are shown as mean±s.e.m. n =29 cells for GFP-SENP3 and n =27 for C532A, *** P <0.0001). ( D ) Representative images showing knockdown of endogenous Drp1 and rescue with GFP-Drp1 R WT or non-SUMOylatable Drp1 R 4KR in neurons. Non-SUMOylatable Drp1 R 4KR increases mitochondrial fission visualized using Mito-DsRed (scale bar: 10 μm, white box ROI magnified below). Mitochondria morphology analysis was performed as in ( C ). Values in bar charts are shown as mean±s.e.m. ( n =8 cells for GFP-Drp1 R WT and n =7 cells for GFP-Drp1 R 4KR), * P <0.05.

Article Snippet: The probes used were Rn00821719_g1* for Rat SUMO2 , Rn01429214_g1 for Rat SUMO3 and Rn01496196_g1 for Rat SENP3 (Applied Biosystems).

Techniques: Over Expression, Activation Assay, Immunostaining, Transfection, Mutagenesis, Staining, Expressing, Knockdown

Journal: The EMBO Journal

Article Title: SENP3-mediated deSUMOylation of dynamin-related protein 1 promotes cell death following ischaemia

doi: 10.1038/emboj.2013.65

Figure Lengend Snippet: SENP3 regulation of Drp1 SUMOylation plays a critical role in cell death following reoxygenation. ( A ) SENP3 knockdown decreases OGD plus reoxygenation-induced caspase 3 cleavage in HEK293 cells. Two days post transfection with Nsi or SENP3i HEK293 cells were subjected to OGD (2 h) and then reoxygenation (24 h). Lysates were blotted as indicated. ( B ) SENP3 knockdown decreases LDH release from HEK293 cells after OGD plus reoxygenation. Cells were treated as in ( A ) and culture media was sampled after OGD (2 h) and after 24 h reoxygenation (2 h+24 h). ( C ) SENP3 knockdown decreases LDH release in neurons following OGD plus reoxygenation. Neurons were infected with retrovirus containing either scrambled shRNA (Scr) or SENP3 shRNA (SENP3 sh), subjected to OGD (2 h) and then reoxygenation (24 h), and media sampled for LDH as in ( B ). Inset panels in ( B ) and ( C ) show immunoblots of cell lysates confirming SENP3 knockdown. ( D ) Knockdown of both SENP3 and Drp1 is not additive on OGD plus reoxygenation-evoked LDH release from HEK293 cells. Immunoblots (right panel) confirm knockdown of SENP3 and/or Drp1. ( E ) Drp1 knockdown and rescue with non-SUMOylatable Drp1 R 4KR increases LDH release in response to OGD plus reoxygenation in HEK293 cells (upper panel). Immunoblots (lower panel) confirm knockdown and rescue of Drp1. ( F ) SENP3 knockdown does not reduce LDH release induced by OGD plus reoxygenation in Drp1 R 4KR-rescued cells. Immunoblots (lower panels) confirm knockdown of Drp1/SENP3 and rescue of Drp1. In ( B – F ), values are presented as mean±s.e.m. ( n ?5 replicates for each group; * P <0.05; ** P <0.01; *** P <0.001). Source data for this figure is available on the online page.

Article Snippet: The probes used were Rn00821719_g1* for Rat SUMO2 , Rn01429214_g1 for Rat SUMO3 and Rn01496196_g1 for Rat SENP3 (Applied Biosystems).

Techniques: Knockdown, Transfection, Infection, shRNA, Western Blot

Journal: The EMBO Journal

Article Title: SENP3-mediated deSUMOylation of dynamin-related protein 1 promotes cell death following ischaemia

doi: 10.1038/emboj.2013.65

Figure Lengend Snippet: Schematic of proposed cell death/survival pathway. During ischaemic stress, the UPR kinase PERK is activated, which leads to lysosome-mediated degradation of the SUMO-2/3-specific deSUMOylating enzyme SENP3. The absence of SENP3 prolongs Drp1 SUMOylation, favouring localization in the cytosol and reducing Drp1-mediated cytochrome c release. Following reoxygenation, however, SENP3 levels recover, promoting mitochondrial association of Drp1 and cell death.

Article Snippet: The probes used were Rn00821719_g1* for Rat SUMO2 , Rn01429214_g1 for Rat SUMO3 and Rn01496196_g1 for Rat SENP3 (Applied Biosystems).

Techniques:

Journal: mBio

Article Title: EBNA1 SUMOylation by PIAS1 suppresses EBV lytic replication and enhances episome maintenance

doi: 10.1128/mbio.02639-25

Figure Lengend Snippet: PIAS1 enhances EBNA1 SUMOylation both in vivo and in vitro . ( A ) HEK-293T cells were transfected with plasmids encoding Halo-V5-EBNA1, Halo-PIAS1, and His-SUMO2. Whole-cell lysates (input) were analyzed by WB using antibodies against SUMO2/3, PIAS1, V5, and β-actin. SUMOylated proteins are indicated by brackets. EBNA1 was immunoprecipitated using anti-V5 magnetic beads, followed by WB analysis with antibodies as indicated. Arrows denote SUMOylated EBNA1. ( B ) In vitro SUMOylation assay was conducted using a combination of purified proteins, including E1, E2, SUMO2, PIAS1, and the substrate V5-EBNA1, as specified. The reaction was stopped by adding 2× SDS-PAGE loading buffer, followed by WB analysis using anti-V5-HRP antibody. SUMOylated EBNA1 is indicated by brackets. ( C ) WB analysis of PIAS1 and β-actin expression in non-targeting control (NC) and PIAS1-depleted (sg-PIAS1) Akata (EBV+) cells. ( D ) Control (NC) and PIAS1-depleted (sg-PIAS1) Akata (EBV+) cells were blocked with 3% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) for 1 h at room temperature, followed by incubation with mouse anti-EBNA1 and rabbit anti-SUMO2/3 antibodies. PLA probes were subsequently added for ligation and amplification. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) and visualized using a Nikon AXR confocal microscope. The close proximity between EBNA1 and SUMO2/3 is indicated by red fluorescent PLA signals.

Article Snippet: Briefly, cells were blocked with 3% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) at room temperature for 1 h, then incubated with PBS control or a mixture of mouse anti-EBNA1 (Cat. #sc-81581, Santa Cruz) and rabbit anti-PIAS1 (Cat. #ab77231, Abcam) or rabbit anti-SUMO2/3 (Cat. # 11251-1-AP, Proteintech) antibodies (1:50 dilution in 3% BSA) at 4°C overnight.

Techniques: In Vivo, In Vitro, Transfection, Immunoprecipitation, Magnetic Beads, Purification, SDS Page, Expressing, Control, Saline, Incubation, Ligation, Amplification, Staining, Microscopy

Journal: Journal of cell science

Article Title: Cajal body formation is regulated by coilin SUMOylation.

doi: 10.1242/jcs.263447

Figure Lengend Snippet: Fig. 1. NSMCE2 KD decreases SUMOylated coilin and increases CB number in HeLa cells. (A) HeLa cells either remained untransfected or were transfected with His–SUMO-1 for 48 h, as indicated. Ni-NTA pulldown and input (20 μl of total lysate saved and run for inputs) samples were visualized on western blots probed for coilin and SUMO-1. SUMOylated coilin is denoted by a bracket. Blots shown are representative of three experiments. (B) HeLa cells were transfected with control or NSMCE2 A siRNA for 96 h and either untransfected or transfected with His–SUMO-1 (as indicated) at 48 h after KD. After an additional 48 h, protein was collected and subject to Ni-NTA pulldown. Pulldown and input samples (20 μl of total lysate saved and run for inputs) were visualized on western blots probed for coilin and SUMO-1. SUMOylated coilin is denoted by a bracket. A transformed image of the coilin probing is also shown (middle panel) but was not used for quantification. Adjustments to this image were made using the transformation settings on QuantityOne software and applied across the entire image. (C) Quantification of B and replicate experiments, showing the level of SUMOylated coilin normalized to coilin input levels relative to control (which is set to 1) when NSMCE2 is knocked down. Data represent three biological replicates (N=3). Bars show the mean, error bars represent s.d. and points represent individual data points. **P<0.01 (two-tailed unpaired Student’s t-test). (D) SuperPlot of the number of CBs in HeLa cells following control siRNA treatment and two types of NSMCE2 KD for 72 h. ***P<0.001, ****P<0.0001 (two-tailed unpaired Student’s t-test). CBs were counted by two people who were not aware of the sample identities. Data are from five biological repeats for control siRNA (n=389 cells), six biological repeats for NSMCE2 A siRNA (n=488 cells) and four biological repeats for NSMCE2 B siRNA (n=297 cells). Horizontal line, mean; error bars, s.d.; larger points show the mean for each biological repeat and points represent individual data points. (E) Immunofluorescence of HeLa cells with control siRNA treatment and two types of NSMCE2 KD for 72 h, as indicated. Coilin is shown in red and DAPI (blue) is used to visualize the nucleus. Scale bars: 20 μm. Images are representative of six experiments.

Article Snippet: The primary antibodies usedwere anti-β-actinmouse monoclonal antibody (1:5000; 8H10D10; Cell Signaling Technology, Danvers, MA, USA); anti-NSMCE2 polyclonal antibody (1:1000; 13627-1- AP; Proteintech, Rosemont, IL, USA), anti-GFP monoclonal antibody (1:500; 11814460001; Roche, Germany), anti-SUMO-1 polyclonal antibody (1:1000; 10329-1-AP; Cell Signaling Technology, Danvers, MA, USA), antiSUMO-2/3 polyclonal antibody (1:1000; 11251-1-AP; Cell Signaling Technology, Danvers, MA, USA), anti-coilin polyclonal antibody (1:1000; sc-32860; Santa Cruz Biotechnology, Dallas, TX, USA), anti-NOCL1 (Nopp140) polyclonal antibody (1:1000; 11815-1-AP; Proteintech) and Jo u rn al o f Ce ll Sc ie n ce anti-myc (1:1000; sc-40; Santa Cruz Biotechnology).

Techniques: Transfection, Western Blot, Control, Transformation Assay, Software, Two Tailed Test, Immunofluorescence

Journal: Frontiers in Immunology

Article Title: Foot-and-Mouth Disease Virus Counteracts on Internal Ribosome Entry Site Suppression by G3BP1 and Inhibits G3BP1-Mediated Stress Granule Assembly via Post-Translational Mechanisms

doi: 10.3389/fimmu.2018.01142

Figure Lengend Snippet: Confirmation of differentially expressed proteins and phosphoproteins by western blotting and Phos-tag western Blotting. (A) Analysis of ubiquitin conjugating enzyme E2 I, ubiquitin conjugating enzyme E2 L3, glyceraldehyde-3-phosphate dehydrogenase, β-actin expression levels in foot-and-mouth disease virus (FMDV)-infected and control cells by western blotting. SILAC-ratios and immunoblotting ratios (infection/control) were shown on the right side. (B) Analysis of the dynamic phosphorylation alterations of the three differentially phosphoproteins (ribosomal protein L15, chromosome 5 open reading frame 24, and FOS-like 2) in FMDV-infected and control cells by Phos-tag western blotting.

Article Snippet: To confirm the expression levels of GAPDH, ubiquitin conjugating enzyme E2 I (UBE2I), ubiquitin conjugating enzyme E2 L3 (UBE2L3), ribosomal protein L15 (RPL15), chromosome 5 open reading frame 24 (C5ORF24) and FOS-like 2 (FOSL2), anti-GAPDH antibody (Beyotime, China), anti-UBE2I antibody (Proteintech, China), anti-UBE2L3 antibody (Proteintech, China), anti-RPL15 antibody (Proteintech, China), anti-C5ORF24 antibody (Proteintech, China), and anti-FOSL2 antibody (Proteintech, China) were used for immunoblotting.

Techniques: Western Blot, Ubiquitin Proteomics, Expressing, Virus, Infection, Control, Multiplex sample analysis, Phospho-proteomics

Journal: Disease Markers

Article Title: PIAS1 Alleviates Hepatic Ischemia-Reperfusion Injury in Mice through a Mechanism Involving NFATc1 SUMOylation

doi: 10.1155/2022/4988539

Figure Lengend Snippet: Bioinformatics analysis of the potential mechanism involved in the pathogenesis of HIRI. (a) The heat map of the top 20 DEGs with the smallest p value in the microarray GSE10657, the color scale from green to red indicated the gene expression value from low to high. (b) The correlation between DEGs and ischemia reperfusion injury, the X-ray indicated the correlation score. (c) The expression of PIAS1 in HIRI ( n = 24) and sham ( n = 6). (d) The Venn diagram of interaction factor obtained from STRING and reperfusion injury-related genes obtained from the CTD database. (e) KEGG enrichment analysis of the candidate genes. (f) The interaction network between NFATc1 and other candidate genes. (g) The interaction between NFATc1 and HDAC1 in liver tissue GTEX liver ( n = 110) (Pearson's r = 0.5578, p value = 2.44 e -10).

Article Snippet: The antibodies used are as follows: rabbit anti PIAS1 (1: 2000, 23395-1-AP, Proteintech), NFATc1 (1: 1000, #8032S, CST), HDAC1 (1 μ g/mL, PA1-860, Thermo Fisher Scientific), IRF-1 (1: 1000, #8478S, CST), tumor necrosis factor alpha (TNF- α , 1: 1000, # 11948S, CST), IL-1 β (1: 1000, ab254360, Abcam), IL-6 (1: 1000, P620, Thermo Fisher Scientific), Bcl2 (1: 2000, ab196495, Abcam), Bax (1: 2000, ab182733, Abcam), cleaved caspase-3 (1: 1000, #9664, CST), p38 (1: 1000, #8690S, CST), phosphorylated-p38 (p-p38, 1: 1000, #4511S, CST), JNK (1: 1000, ab179461, Abcam), p-JNK (1: 1000, ab124956, Abcam), ERK1/2 (1: 10000, ab184699, Abcam), p-ERK1/2 (1: 1000, ab278538, Abcam), GAPDH (1: 2500, ab9485, Abcam), and goat anti-rabbit secondary antibody (ab97051, 1: 2000, Abcam).

Techniques: Microarray, Gene Expression, Expressing

Journal: Disease Markers

Article Title: PIAS1 Alleviates Hepatic Ischemia-Reperfusion Injury in Mice through a Mechanism Involving NFATc1 SUMOylation

doi: 10.1155/2022/4988539

Figure Lengend Snippet: The effect of PIAS1 on NFATc1 SUMOylation and the inflammatory response and apoptosis of hepatocytes. (a) The mRNA expression of PIAS1 and NFATc1 in the liver tissues of sham-operated and HIRI mice determined by RT-qPCR ( ∗ p < 0.05). (b) The protein expression of PIAS1 and NFATc1 in the liver tissues of sham-operated and HIRI mice determined by western blot analysis ( ∗ p < 0.05). (c) The SUMOylation of NFATc1 in 293 T cells transduced with Flag-NFATc1 and HA-SUMO1 detected by Co-IP assay. (d) The SUMOylation of endogenous NFATc1 in AML12 cells detected by Co-IP assay. (e) The SUMOylation of NFATc1 in H/R-exposed AML12 cells treated with oe-PIAS1 or combined with ML-792 detected by Co-IP assay. (f) Apoptosis of H/R-exposed AML12 cells detected by TUNEL staining ( ∗ p < 0.05 vs. control, # p < 0.05 vs. H/R, & p < 0.05 vs. H/R + oe-PIAS1). (g) RT-qPCR detection of PIAS1 and NFATc1 expression in H/R-exposed AML12 cells treated with oe-PIAS1 or combined with oe-NFATc1. (h) The mRNA expression of TNF- α , IL-1 β , IL-6, Bax, Bcl2, and caspase-3 in H/R-exposed AML12 cells treated with oe-PIAS1 or combined with oe-NFATc1 measured by RT-qPCR. (i) The protein expression of TNF- α , IL-1 β , IL-6, Bax, Bcl2, and cleaved caspase-3 in H/R-exposed AML12 cells treated with oe-PIAS1 or combined with oe-NFATc1 measured by Western blot analysis. (j) Apoptosis of H/R-exposed AML12 cells following treatment with oe-PIAS1 or combined with oe-NFATc1 detected by TUNEL staining. In (g)–(j), ∗ p < 0.05 vs. oe-NC + sh-NC, # p < 0.05 vs. oe-PIAS1 + oe-NC. The cell experiment was repeated 3 times.

Article Snippet: The antibodies used are as follows: rabbit anti PIAS1 (1: 2000, 23395-1-AP, Proteintech), NFATc1 (1: 1000, #8032S, CST), HDAC1 (1 μ g/mL, PA1-860, Thermo Fisher Scientific), IRF-1 (1: 1000, #8478S, CST), tumor necrosis factor alpha (TNF- α , 1: 1000, # 11948S, CST), IL-1 β (1: 1000, ab254360, Abcam), IL-6 (1: 1000, P620, Thermo Fisher Scientific), Bcl2 (1: 2000, ab196495, Abcam), Bax (1: 2000, ab182733, Abcam), cleaved caspase-3 (1: 1000, #9664, CST), p38 (1: 1000, #8690S, CST), phosphorylated-p38 (p-p38, 1: 1000, #4511S, CST), JNK (1: 1000, ab179461, Abcam), p-JNK (1: 1000, ab124956, Abcam), ERK1/2 (1: 10000, ab184699, Abcam), p-ERK1/2 (1: 1000, ab278538, Abcam), GAPDH (1: 2500, ab9485, Abcam), and goat anti-rabbit secondary antibody (ab97051, 1: 2000, Abcam).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Transduction, Co-Immunoprecipitation Assay, TUNEL Assay, Staining, Control

Journal: Disease Markers

Article Title: PIAS1 Alleviates Hepatic Ischemia-Reperfusion Injury in Mice through a Mechanism Involving NFATc1 SUMOylation

doi: 10.1155/2022/4988539

Figure Lengend Snippet: Overexpression of PIAS1 inactivated the NFATc1/HDAC1/IRF-1/p38 MAPK pathway to alleviate HIRI in mice. HIRI mice were treated with oe-PIAS1 or combined with anisomycin (p38 MAPK activator). (a) The protein expression of PIAS1, NFATc1, HDAC1, IRF-1, and phosphorylation levels of p38 in the liver tissue of mice determined by western blot analysis. (b) The serum ALT activity in mice determined by ELISA. (c) The serum AST activity in mice determined by ELISA. (d) The degree of liver injury in mice determined by H&E staining. (e) The degree of liver injury in mice determined by MDA detection. (f) The neutrophil activity in the liver tissue of mice determined by MPO detection. (g) The expression of TNF- α , IL-1 β , and IL-6 levels in the liver tissue of mice determined by ELISA. (h) The levels of Gr-1 and CD68 in liver tissues of mice determined by immunofluorescence staining. (i) Hepatocyte apoptosis in liver tissues of mice determined by TUNEL assay. n = 8, ∗ p < 0.05 vs. sham-operated mice, # p < 0.05 vs. HIRI + oe-NC, & p < 0.05 vs. HIRI + oe-PIAS1.

Article Snippet: The antibodies used are as follows: rabbit anti PIAS1 (1: 2000, 23395-1-AP, Proteintech), NFATc1 (1: 1000, #8032S, CST), HDAC1 (1 μ g/mL, PA1-860, Thermo Fisher Scientific), IRF-1 (1: 1000, #8478S, CST), tumor necrosis factor alpha (TNF- α , 1: 1000, # 11948S, CST), IL-1 β (1: 1000, ab254360, Abcam), IL-6 (1: 1000, P620, Thermo Fisher Scientific), Bcl2 (1: 2000, ab196495, Abcam), Bax (1: 2000, ab182733, Abcam), cleaved caspase-3 (1: 1000, #9664, CST), p38 (1: 1000, #8690S, CST), phosphorylated-p38 (p-p38, 1: 1000, #4511S, CST), JNK (1: 1000, ab179461, Abcam), p-JNK (1: 1000, ab124956, Abcam), ERK1/2 (1: 10000, ab184699, Abcam), p-ERK1/2 (1: 1000, ab278538, Abcam), GAPDH (1: 2500, ab9485, Abcam), and goat anti-rabbit secondary antibody (ab97051, 1: 2000, Abcam).

Techniques: Over Expression, Expressing, Phospho-proteomics, Western Blot, Activity Assay, Enzyme-linked Immunosorbent Assay, Staining, Immunofluorescence, TUNEL Assay

Journal: Disease Markers

Article Title: PIAS1 Alleviates Hepatic Ischemia-Reperfusion Injury in Mice through a Mechanism Involving NFATc1 SUMOylation

doi: 10.1155/2022/4988539

Figure Lengend Snippet: The molecular mechanism of SUMO E3 ligase PIAS1 in HIRI through regulating NFATc1/HDAC1/IRF-1/p38 MAPK signaling pathway.

Article Snippet: The antibodies used are as follows: rabbit anti PIAS1 (1: 2000, 23395-1-AP, Proteintech), NFATc1 (1: 1000, #8032S, CST), HDAC1 (1 μ g/mL, PA1-860, Thermo Fisher Scientific), IRF-1 (1: 1000, #8478S, CST), tumor necrosis factor alpha (TNF- α , 1: 1000, # 11948S, CST), IL-1 β (1: 1000, ab254360, Abcam), IL-6 (1: 1000, P620, Thermo Fisher Scientific), Bcl2 (1: 2000, ab196495, Abcam), Bax (1: 2000, ab182733, Abcam), cleaved caspase-3 (1: 1000, #9664, CST), p38 (1: 1000, #8690S, CST), phosphorylated-p38 (p-p38, 1: 1000, #4511S, CST), JNK (1: 1000, ab179461, Abcam), p-JNK (1: 1000, ab124956, Abcam), ERK1/2 (1: 10000, ab184699, Abcam), p-ERK1/2 (1: 1000, ab278538, Abcam), GAPDH (1: 2500, ab9485, Abcam), and goat anti-rabbit secondary antibody (ab97051, 1: 2000, Abcam).

Techniques: