socs2 plasmid  (Santa Cruz Biotechnology)

Journal: Frontiers in Neuroscience

Article Title: Suppressor of Cytokine Signaling 2 Regulates Retinal Pigment Epithelium Metabolism by Enhancing Autophagy

doi: 10.3389/fnins.2021.738022

Figure Lengend Snippet: RPE lacking the SOCS2 gene exhibited impaired autophagy. (A) In vivo , autophagic flux was detected by injecting 2 μl of 1 × 10 12 vg/ml adeno-associated virus 2 (AAV2)-mRFP-GFP-LC3 in the eyeball of adult WT and SOCS2 –/– mice subretinally. About 2-weeks later, mice were starved for 24 h before euthanasia, and RPE/choroid flat mounts were isolated from mice ( n = 8, 10-week old, including males and females). (B) The numbers of autophagosomes and autolysosomes were evaluated by counting yellow and RFP dots. Puncta dots per horizon are shown as the mean ± SEM. (C) LC3B puncta dots (green) and p62 puncta dots (green) were stained by immunofluorescence in mouse primary RPE cells that were starved and treated with or without 30 μM CQ for 24 h. One group of SOCS2 –/– RPE re-expressed SOCS2 by transient transfection. (D,E) Fluorescence intensity of LC3B and p62 were quantified by the ImageJ software. (F) Immunoblotting for LC3B and p62 was performed in mouse primary RPE with different treatments. (G) The ratio of LC3B-II/LC3B-I was quantified by ImageJ software and normalized to β-actin. (H) p62 was quantified by ImageJ software and normalized to β-actin. Values for the control were set to 1. Statistical significance was determined by using the two-tailed unpaired t -test.

Article Snippet: Plasmid cytomegalovirus 3 (pCMV3)-Human-SOCS2-orange fluorescent protein (OFP) expression plasmid (HG11285-ACR), control vector OFP expression plasmids (CV025), pCMV3-Human-SOCS2-green fluorescent protein (GFP) (HG11285-ACG), and control vectors GFP expression plasmids (CV026) were purchased from the Sino Biological Incorporation (Wayne, PA, United States).

Techniques: In Vivo, Isolation, Staining, Immunofluorescence, Transfection, Fluorescence, Software, Western Blot, Two Tailed Test

Journal: Frontiers in Neuroscience

Article Title: Suppressor of Cytokine Signaling 2 Regulates Retinal Pigment Epithelium Metabolism by Enhancing Autophagy

doi: 10.3389/fnins.2021.738022

Figure Lengend Snippet: SOCS2 promoted autophagy in ARPE-19 cells. (A) The mRNA expression of SOCS2 was detected by real-time quantitative PCR in ARPE-19 after transient transfection with pCMV3-Human-SOCS2-OFP expression plasmids or control vectors. The two-tailed unpaired t -test was used. (B,C) Western blot images of SOCS2-OFP and SOCS2-GFP were detected by the SOCS2 antibody to check the overexpression of SOCS2. (D) LC3B puncta dots (green) and p62 puncta dots (green) were stained by immunofluorescence in ARPE-19 cells transfected with pCMV3-Human-SOCS2-OFP expression plasmids or control vectors for 24 h, then cells were starved and treated with or without 30 μM CQ for 24 h. (E,F) Fluorescence intensity of LC3B and p62 was quantified by ImageJ software. (G) Western blot detected the expression of LC3B and p62 in SOCS2 overexpressing ARPE-19 cells that were treated with or without 30 μM CQ for 24 h. (H) The ratio of LC3B-II/LC3B-I was quantified by ImageJ software and normalized to β-actin. Values for the control were set to 1. (I) p62 was quantified by ImageJ software and normalized to β-actin. Values for the control were set to 1. Statistical significance was determined by using the two-tailed unpaired t -test.

Article Snippet: Plasmid cytomegalovirus 3 (pCMV3)-Human-SOCS2-orange fluorescent protein (OFP) expression plasmid (HG11285-ACR), control vector OFP expression plasmids (CV025), pCMV3-Human-SOCS2-green fluorescent protein (GFP) (HG11285-ACG), and control vectors GFP expression plasmids (CV026) were purchased from the Sino Biological Incorporation (Wayne, PA, United States).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Transfection, Two Tailed Test, Western Blot, Over Expression, Staining, Immunofluorescence, Fluorescence, Software

Journal: Frontiers in Neuroscience

Article Title: Suppressor of Cytokine Signaling 2 Regulates Retinal Pigment Epithelium Metabolism by Enhancing Autophagy

doi: 10.3389/fnins.2021.738022

Figure Lengend Snippet: Ubiquitylated proteins recognized by SOCS2 were transferred into autophagosomes and autolysosomes. (A) The endogenous detection of protein interaction between SOCS2 and ubiquitin was detected in starved ARPE-19 by Duolink in situ fluorescence. The red fluorescence indicated the interaction of SOCS2 and ubiquitin. (B) Immunofluorescence staining with ubiquitin (red) and DAPI (blue) was performed in starved ARPE-19 with transient transfection of GFP control vectors or SOCS2-GFP plasmids. The yellow dots indicated the colocalization of SOCS2 and ubiquitin in merged pictures. (C) Immunofluorescence staining with the antibody p62 (red) and DAPI (blue) in ARPE-19 with the same treatment of B. The yellow dots indicated the colocalization of SOCS2 and p62 in merged pictures. (D) The immunoprecipitation of p62 and SOCS2 was detected in ARPE-19 with the same treatment of B. (E) Immunofluorescence staining was performed with the antibodies against p62 (green) and ubiquitin (red) in starved ARPE-19. (F) Immunofluorescence staining with the antibody LC3B (red) in ARPE-19 with the same treatment of B. (G) The immunoprecipitation of LC3B and SOCS2 was detected in ARPE-19 with the same treatment of B. (H) The endogenous detection of protein interaction between SOCS2 and LAMP2 was detected in starved ARPE-19 by Duolink in situ fluorescence. The red fluorescence indicated the interaction of SOCS2 and LAMP2. (I) Immunofluorescence staining with the antibody LAMP2 in ARPE-19 after transient transfection of SOCS2-OFP plasmids or OFP control vectors. (J,K) The immunoprecipitation of p62 and phosphorylated mammalian target of rapamycin (p-mTOR), SOCS2, and p-mTOR were detected in ARPE-19 with the same treatment of B.

Article Snippet: Plasmid cytomegalovirus 3 (pCMV3)-Human-SOCS2-orange fluorescent protein (OFP) expression plasmid (HG11285-ACR), control vector OFP expression plasmids (CV025), pCMV3-Human-SOCS2-green fluorescent protein (GFP) (HG11285-ACG), and control vectors GFP expression plasmids (CV026) were purchased from the Sino Biological Incorporation (Wayne, PA, United States).

Techniques: In Situ, Fluorescence, Immunofluorescence, Staining, Transfection, Immunoprecipitation

Journal: BMC Cancer

Article Title: Favorable prognostic value of SOCS2 and IGF-I in breast cancer

doi: 10.1186/1471-2407-7-136

Figure Lengend Snippet: Kaplan-Meier curve assessment of risk of death in a cohort of 89 patients with invasive breast cancer. Overall survival is shown for patients with high and low SOCS2 and IGF-I expression, respectively. The median was taken as a cut-off. P values were determined using the log-rank test.

Article Snippet: To test for antibody specificity, lysates from COS7 cells transiently transfected with a myc-tagged SOCS2 plasmid were probed with an anti-c-myc antibody (9E10, Santa Cruz Biotechnology, Santa Cruz) and anti-SOCS2 antibodies.

Techniques: Expressing

Journal: BMC Cancer

Article Title: Favorable prognostic value of SOCS2 and IGF-I in breast cancer

doi: 10.1186/1471-2407-7-136

Figure Lengend Snippet: Comparison of SOCS2 and IGF-I expression. (A) Box blots with data from a set of seven tumors comparing the relative mRNA expression levels in tumor (CA) and normal adjacent tissue (TA). (B) SOCS2 protein and corresponding SOCS2 mRNA expression in normal and cancerous tissue of two tumor specimens. SOCS2 protein expression was assessed by immuno-blot with an antibody directed against SOCS2. Actin was used as a loading control.

Article Snippet: To test for antibody specificity, lysates from COS7 cells transiently transfected with a myc-tagged SOCS2 plasmid were probed with an anti-c-myc antibody (9E10, Santa Cruz Biotechnology, Santa Cruz) and anti-SOCS2 antibodies.

Techniques: Expressing

Journal: BMC Cancer

Article Title: Favorable prognostic value of SOCS2 and IGF-I in breast cancer

doi: 10.1186/1471-2407-7-136

Figure Lengend Snippet: Detection of SOCS2 in primary mammary carcinoma samples by immunohistochemistry. Representative immunohistochemical stainings of samples with low SOCS2 (A) and high SOCS2 (B) mRNA expression are shown. Note a strong cytoplasmatic epithelial staining for SOCS2 in tumors with high SOCS2 mRNA levels (B).

Article Snippet: To test for antibody specificity, lysates from COS7 cells transiently transfected with a myc-tagged SOCS2 plasmid were probed with an anti-c-myc antibody (9E10, Santa Cruz Biotechnology, Santa Cruz) and anti-SOCS2 antibodies.

Techniques: Immunohistochemistry, Immunohistochemical staining, Expressing, Staining

Journal: Cell Death and Differentiation

Article Title: SOCS2-enhanced ubiquitination of SLC7A11 promotes ferroptosis and radiosensitization in hepatocellular carcinoma

doi: 10.1038/s41418-022-01051-7

Figure Lengend Snippet: A Dose responses of survival fractions of SK-Hep-1, SK-Hep-1R, HepG2 and HepG2R after IR. B Symmetric scatter diagram of differentially expressed genes (DEGs) between SK-Hep-1R and SK-Hep-1 cells. C Symmetric scatter diagram of DEGs between the HCC tumor and paracancerous tissues from TCGA and GEPIA dataset (download data: 2021-09-01). D Venn diagram of the co-expressed DEGs among above two groups (SK-Hep-1R/SK-Hep-1, tumor/normal tissues) and the survival genes for prognosis prediction of HCC from TCGA and GEPIA dataset. E Fold changes of SOCS2 and SMOX gene expressions in SK-Hep-1R cells and tumor tissues in comparison with SK-Hep-1 cells and normal tissues, respectively. F Kaplan-Meier curves of HCC survivals based on the expression status of SOCS2 gene according to TCGA and GEPIA dataset. G Box scatter diagrams of the relative expression level of SOCS2 in tumor and adjacent normal tissues according to 12 cohorts in HCCDB. The central mark is the median; the edges of the box are the 25th and 75th percentiles. H refers to HCCDB. H Boxplot of the relative expression level of SOCS2 in normal and tumor tissues of HCC patients with four pathological grade 1, 2, 3 or 4 from UALCAN database. The central mark is the median; the edges of the box are the 25th and 75th percentiles. I Representative immunofluorescence images of SOCS2 protein in tumor tissues of radioresistant HCC patients (HCC-R) ( n = 12) and radiosensitive HCC patients (HCC-S) ( n = 12). Nuclei were stained with DAPI (x4). Scale bars, 100 μm. * P < 0.05, ** P < 0.01 and *** P < 0.001 between indicated groups.

Article Snippet: The mutant SOCS2 plasmid was also designed by Genechem Technology.

Techniques: Expressing, Immunofluorescence, Staining

Journal: Cell Death and Differentiation

Article Title: SOCS2-enhanced ubiquitination of SLC7A11 promotes ferroptosis and radiosensitization in hepatocellular carcinoma

doi: 10.1038/s41418-022-01051-7

Figure Lengend Snippet: A Western blot assay of SOCS2 and tubulin proteins in SK-Hep-1, SK-Hep-1R, HepG2 and HepG2R cells. B , C Western blot assay of SOCS2 and tubulin proteins in SK-Hep-1 and HepG2 cells transfected with lvSOCS2, siSOCS2 or their negative control (lvNC, siNC). D Dose responses of survival factions of SK-Hep-1 and HepG2 cells with or without lvSOCS2 transfection. SK refers to SK-Hep-1, HG refers to HepG2. E Dose responses of survival factions of SK-Hep-1 and HepG2 cells with or without siSOCS2 transfection. F , G General view of tumor mass of each indicated group at 24 days after cell injection. H , I Tumor volume of above groups was examined every 3 days until 24 days after subcutaneously cell injection. J Representative immunohistochemistry images of the expressions of SOCS2, 4-HNE, GPX4 and SLC7A11 protein in the aforementioned xenograft tumors (x40). The red arrow indicated the spot or region where positive protein expression was present. Scale bars, 20 μm. * P < 0.05, ** P < 0.01 and *** P < 0.001 between indicated groups.

Article Snippet: The mutant SOCS2 plasmid was also designed by Genechem Technology.

Techniques: Western Blot, Transfection, Negative Control, Injection, Immunohistochemistry, Expressing

Journal: Cell Death and Differentiation

Article Title: SOCS2-enhanced ubiquitination of SLC7A11 promotes ferroptosis and radiosensitization in hepatocellular carcinoma

doi: 10.1038/s41418-022-01051-7

Figure Lengend Snippet: A Western blot assay of GPX4 and SOCS2 proteins and their relative levels in SK-Hep-1, SK-Hep-1R, HepG2 and HepG2R cells at 2, 4, 8, 24 h after 4 Gy IR or non-IR. B Western blot assay of SLC7A11, GPX4 and SOCS2 proteins in SK-Hep-1, SK-Hep-1R, HepG2 and HepG2R cells at 4 h after 4 Gy IR or non-IR. C , D Western blot assay of SLC7A11, GPX4 and SOCS2 proteins in SK-Hep-1, SK-Hep-1R, HepG2 and HepG2R cells with or without lvSOCS2 transfection at 4 h after 4 Gy IR or non-IR. Representative images ( F ) and quantification ( E ) of the relative fluorescence intensity of liperfluo in SK-Hep-1 and HepG2 cells transfected with lvSOCS2 at 4 h after 4 Gy IR or non-IR. Nuclei were stained with Hoechst (x40). Scale bars, 10 μm. * P < 0.05, ** P < 0.01 and *** P < 0.001 between indicated groups.

Article Snippet: The mutant SOCS2 plasmid was also designed by Genechem Technology.

Techniques: Western Blot, Transfection, Fluorescence, Staining

Journal: Cell Death and Differentiation

Article Title: SOCS2-enhanced ubiquitination of SLC7A11 promotes ferroptosis and radiosensitization in hepatocellular carcinoma

doi: 10.1038/s41418-022-01051-7

Figure Lengend Snippet: A Representative immunofluorescence images of SOCS2 and SLC7A11 proteins in the nonirradiated xenograft tumors (SK-Hep-1 and SK-Hep-1R) and HCC clinical tissues. Nuclei are stained with DAPI (×10). Scale bars, 100 μm. B , C Co-immunoprecipitation and Western blot assay of SOCS2 and SLC7A11 proteins in the whole cell lysates of SK-Hep-1 and HepG2 cells at 4 h after 4 Gy IR. D , E Point-fold line chart of SLC7A11 protein degradation according to Western blot assay (Fig. ). F , G Western blot analysis of SLC7A11, SOCS2 and tubulin proteins in SK-Hep-1 and HepG2 cells at 4 h after 4 Gy IR. MG-132 (10 μM) or leupeptin (50 μM) were added before IR. H Anti-Ub immunoblotting assay of SLC7A11 polyubiquitination in SK-Hep-1 and Hep2 cells at 4 h after 4 Gy IR. I Anti-Ub immunoblotting assay of SLC7A11 polyubiquitination in SK-Hep-1 and HepG2 cells transfected with lvSOCS2 at 4 h after 4 Gy IR. * P < 0.05, ** P < 0.01 and *** P < 0.001 between indicated groups.

Article Snippet: The mutant SOCS2 plasmid was also designed by Genechem Technology.

Techniques: Immunofluorescence, Staining, Immunoprecipitation, Western Blot, Transfection

Journal: Cell Death and Differentiation

Article Title: SOCS2-enhanced ubiquitination of SLC7A11 promotes ferroptosis and radiosensitization in hepatocellular carcinoma

doi: 10.1038/s41418-022-01051-7

Figure Lengend Snippet: A Schematic representation of three flag-fused SOCS2 constructs containing amino acids 1–40, 40–156 and 156–198. B Co-immunoprecipitation assay of SLC7A11 and different SOCS2 domains in the whole cell lysates of irradiated SK-Hep-1 and HepG2 cells. C Ubiquitination of SLC7A11 in irradiated SK-Hep-1 and HepG2 cells with or without ΔSH2 manipulation (ΔSH2, SH2 domain truncation mutant of SOCS2). D Schematic representation of three flag-fused SLC7A11 constructs containing amino acids 1–43, 43–470 and 470–501. E Co-immunoprecipitation assay of SOCS2 and different SLC7A11 domains in the whole cell lysates of irradiated SK-Hep-1 and HepG2 cells. F SLC7A11 polyubiquitination was detected by anti-Ub immunoblotting in 4 Gy irradiated SK-Hep-1 and HepG2 cells with or without ΔNTD manipulation (ΔNTD, NTD domain truncation mutant of SOCS2). G , H Dose responses of survival fractions of SK-Hep-1 and HepG2 cells transfected with SOCS2-ΔSH2 or SOCS2-WT plasmid. * P < 0.05, ** P < 0.01 and *** P < 0.001 between indicated groups.

Article Snippet: The mutant SOCS2 plasmid was also designed by Genechem Technology.

Techniques: Construct, Co-Immunoprecipitation Assay, Irradiation, Mutagenesis, Western Blot, Transfection, Plasmid Preparation

Journal: Cell Death and Differentiation

Article Title: SOCS2-enhanced ubiquitination of SLC7A11 promotes ferroptosis and radiosensitization in hepatocellular carcinoma

doi: 10.1038/s41418-022-01051-7

Figure Lengend Snippet: A Immunoprecipitation and immunoblotting assay were selected to detect the type of polyubiquitination of Flag-SLC7A11 in SK-Hep-1 and HepG2 cells transfected with wild-type or K48/K63 mutant Ub after IR. B Western blot assay of SLC7A11, elongin B and elongin C proteins in 4 Gy irradiated SK-Hep-1 and HepG2 cells transfected with siRNA targeting elongin B or elongin C ( siEB/C ). C Amino acid sequences of SOCS2-BOX regions of SOCS1 (1), SOCS2 (2), SOCS6 (6) and SAB15 (15) proteins from different species. H refers to Human; C refers to Chicken; A refers to African clawed frog; R refers to Red flour beetle. D Schematic representation of two flag-fused SOCS2 plasmids with wild-type (S WT ) or L162C166/P162P166 (S LC→PP ) mutant. E Co-immunoprecipitation assay of S WT , S LC→PP , elongin B and elongin C in the whole cell lysates of irradiated SK-Hep-1 and HepG2 cells. F SLC7A11 polyubiquitination was detected by anti-Ub immunoblotting assay in 4 Gy irradiated SK-Hep-1 and HepG2 cells transfected with S WT or S LC→PP plasmid.

Article Snippet: The mutant SOCS2 plasmid was also designed by Genechem Technology.

Techniques: Immunoprecipitation, Western Blot, Transfection, Mutagenesis, Irradiation, Co-Immunoprecipitation Assay, Plasmid Preparation

Journal: Cell Death and Differentiation

Article Title: SOCS2-enhanced ubiquitination of SLC7A11 promotes ferroptosis and radiosensitization in hepatocellular carcinoma

doi: 10.1038/s41418-022-01051-7

Figure Lengend Snippet: A The pathway of SOCS2 sensitization. After IR, a rapid increase of SOCS2 led to a decrease of SLC7A11 and advanced ferroptosis and radiosensitization. B Diagram of the specific mechanism of SOCS2-induced ferroptosis. SOCS2-SH2 recognized SLC7A11-NTD, bound ubiquitin molecules which was linked to E2 ubiquitin-binding enzymes with the joint involvement of elongin B (EB) and elongin C (EC), and facilitated the transfer of ubiquitin molecules to the substrate protein SLC7A11 to promote SLC7A11 degradation. This degradation subsequently caused a reduction in cystine intake as well as a reduction in GSH and GPX4 and ultimately advanced ferroptosis. C The processes of SLC7A11 protein ubiquitination. In an ATP-dependent reaction, Ub was attached to the ubiquitin-activating enzyme E1 and activated, then delivered to the ubiquitin-binding enzyme E2. Next, the E3 ligase SOCS2 acted as a bridge, identifying E2-Ub and the substrate SLC7A11 to facilitate the association of Ub with SLC7A11. After several repetitions of the above process, a K48-ubiquitin chain was formed and attached to SLC7A11. Finally, the K48-ubiquitin chain was identified by the 26 S proteasome, leading to the degradation of SLC7A11 to fragments. Yellow arrows indicate the experimental results argued in this paper while black arrows represent results reported in other literature.

Article Snippet: The mutant SOCS2 plasmid was also designed by Genechem Technology.

Techniques: Binding Assay

Journal: Frontiers in Neuroscience

Article Title: Suppressor of Cytokine Signaling 2 Regulates Retinal Pigment Epithelium Metabolism by Enhancing Autophagy

doi: 10.3389/fnins.2021.738022

Figure Lengend Snippet: Abnormal RPE-Bruch’s membrane (BrM)-choriocapillaris complex of SOCS2 –/– mice. (A) PCR screening of SOCS2 +/– mice F1 founder to detect SOCS2 sequence from genomic DNA. (B) Primary culture of RPE cells isolated from WT mice. Scale bar, 200 μm. (C) Real-time quantitative PCR detected the mRNA expression of SOCS2 in the primary RPE of WT, heterozygote, and homozygote. Statistical significance was determined by using the one-way ANOVA. (D) Western blot detected the protein expression of SOCS2 in the RPE of WT and SOCS2 –/– mice. (E) SOCS2 expression and location in the retina of WT and SOCS2 –/– mice. Scale bar, 50 μm. (F) The body weights of the SOCS2 –/– ( n = 6, 8 weeks old, including males and females) and WT littermates ( n = 9, 10 weeks old, including males and females). (G) The structures of the retinal-RPE-choroid were detected by histology in WT and SOCS2 –/– mice ( n = 6, 1-year old, including males and females). Scale bar, 50 μm. (H) CML was detected by immunofluorescence on the sections of retinal-RPE-choroid from WT and SOCS2 –/– mice ( n = 6, 1-year old, including males and females). Scale bar, 50 μm. (I) BrM and deposits were detected by transmission electron micrographs on the sections of retinal-RPE-choroid from WT and SOCS2 –/– mice ( n = 6, 1-year old, including males and females). Scale bar, 1 μm. Abbreviations: SOCS2, suppressor of cytokine signaling 2; mRNA, messenger RNA; INL, inner nuclear layer; ONL, outer nuclear layer; ELM, external limiting membrane; RPE, retinal pigment epithelium; WT, wild type.

Article Snippet: Plasmid cytomegalovirus 3 (pCMV3)-Human-SOCS2-orange fluorescent protein (OFP) expression plasmid (HG11285-ACR), control vector OFP expression plasmids (CV025), pCMV3-Human-SOCS2-green fluorescent protein (GFP) (HG11285-ACG), and control vectors GFP expression plasmids (CV026) were purchased from the Sino Biological Incorporation (Wayne, PA, United States).

Techniques: Sequencing, Isolation, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Immunofluorescence, Transmission Assay

Journal: Frontiers in Neuroscience

Article Title: Suppressor of Cytokine Signaling 2 Regulates Retinal Pigment Epithelium Metabolism by Enhancing Autophagy

doi: 10.3389/fnins.2021.738022

Figure Lengend Snippet: RPE lacking the SOCS2 gene exhibited impaired autophagy. (A) In vivo , autophagic flux was detected by injecting 2 μl of 1 × 10 12 vg/ml adeno-associated virus 2 (AAV2)-mRFP-GFP-LC3 in the eyeball of adult WT and SOCS2 –/– mice subretinally. About 2-weeks later, mice were starved for 24 h before euthanasia, and RPE/choroid flat mounts were isolated from mice ( n = 8, 10-week old, including males and females). (B) The numbers of autophagosomes and autolysosomes were evaluated by counting yellow and RFP dots. Puncta dots per horizon are shown as the mean ± SEM. (C) LC3B puncta dots (green) and p62 puncta dots (green) were stained by immunofluorescence in mouse primary RPE cells that were starved and treated with or without 30 μM CQ for 24 h. One group of SOCS2 –/– RPE re-expressed SOCS2 by transient transfection. (D,E) Fluorescence intensity of LC3B and p62 were quantified by the ImageJ software. (F) Immunoblotting for LC3B and p62 was performed in mouse primary RPE with different treatments. (G) The ratio of LC3B-II/LC3B-I was quantified by ImageJ software and normalized to β-actin. (H) p62 was quantified by ImageJ software and normalized to β-actin. Values for the control were set to 1. Statistical significance was determined by using the two-tailed unpaired t -test.

Article Snippet: Plasmid cytomegalovirus 3 (pCMV3)-Human-SOCS2-orange fluorescent protein (OFP) expression plasmid (HG11285-ACR), control vector OFP expression plasmids (CV025), pCMV3-Human-SOCS2-green fluorescent protein (GFP) (HG11285-ACG), and control vectors GFP expression plasmids (CV026) were purchased from the Sino Biological Incorporation (Wayne, PA, United States).

Techniques: In Vivo, Isolation, Staining, Immunofluorescence, Transfection, Fluorescence, Software, Western Blot, Two Tailed Test

Journal: Frontiers in Neuroscience

Article Title: Suppressor of Cytokine Signaling 2 Regulates Retinal Pigment Epithelium Metabolism by Enhancing Autophagy

doi: 10.3389/fnins.2021.738022

Figure Lengend Snippet: SOCS2 promoted autophagy in ARPE-19 cells. (A) The mRNA expression of SOCS2 was detected by real-time quantitative PCR in ARPE-19 after transient transfection with pCMV3-Human-SOCS2-OFP expression plasmids or control vectors. The two-tailed unpaired t -test was used. (B,C) Western blot images of SOCS2-OFP and SOCS2-GFP were detected by the SOCS2 antibody to check the overexpression of SOCS2. (D) LC3B puncta dots (green) and p62 puncta dots (green) were stained by immunofluorescence in ARPE-19 cells transfected with pCMV3-Human-SOCS2-OFP expression plasmids or control vectors for 24 h, then cells were starved and treated with or without 30 μM CQ for 24 h. (E,F) Fluorescence intensity of LC3B and p62 was quantified by ImageJ software. (G) Western blot detected the expression of LC3B and p62 in SOCS2 overexpressing ARPE-19 cells that were treated with or without 30 μM CQ for 24 h. (H) The ratio of LC3B-II/LC3B-I was quantified by ImageJ software and normalized to β-actin. Values for the control were set to 1. (I) p62 was quantified by ImageJ software and normalized to β-actin. Values for the control were set to 1. Statistical significance was determined by using the two-tailed unpaired t -test.

Article Snippet: Plasmid cytomegalovirus 3 (pCMV3)-Human-SOCS2-orange fluorescent protein (OFP) expression plasmid (HG11285-ACR), control vector OFP expression plasmids (CV025), pCMV3-Human-SOCS2-green fluorescent protein (GFP) (HG11285-ACG), and control vectors GFP expression plasmids (CV026) were purchased from the Sino Biological Incorporation (Wayne, PA, United States).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Transfection, Two Tailed Test, Western Blot, Over Expression, Staining, Immunofluorescence, Fluorescence, Software

Journal: Frontiers in Neuroscience

Article Title: Suppressor of Cytokine Signaling 2 Regulates Retinal Pigment Epithelium Metabolism by Enhancing Autophagy

doi: 10.3389/fnins.2021.738022

Figure Lengend Snippet: Ubiquitylated proteins recognized by SOCS2 were transferred into autophagosomes and autolysosomes. (A) The endogenous detection of protein interaction between SOCS2 and ubiquitin was detected in starved ARPE-19 by Duolink in situ fluorescence. The red fluorescence indicated the interaction of SOCS2 and ubiquitin. (B) Immunofluorescence staining with ubiquitin (red) and DAPI (blue) was performed in starved ARPE-19 with transient transfection of GFP control vectors or SOCS2-GFP plasmids. The yellow dots indicated the colocalization of SOCS2 and ubiquitin in merged pictures. (C) Immunofluorescence staining with the antibody p62 (red) and DAPI (blue) in ARPE-19 with the same treatment of B. The yellow dots indicated the colocalization of SOCS2 and p62 in merged pictures. (D) The immunoprecipitation of p62 and SOCS2 was detected in ARPE-19 with the same treatment of B. (E) Immunofluorescence staining was performed with the antibodies against p62 (green) and ubiquitin (red) in starved ARPE-19. (F) Immunofluorescence staining with the antibody LC3B (red) in ARPE-19 with the same treatment of B. (G) The immunoprecipitation of LC3B and SOCS2 was detected in ARPE-19 with the same treatment of B. (H) The endogenous detection of protein interaction between SOCS2 and LAMP2 was detected in starved ARPE-19 by Duolink in situ fluorescence. The red fluorescence indicated the interaction of SOCS2 and LAMP2. (I) Immunofluorescence staining with the antibody LAMP2 in ARPE-19 after transient transfection of SOCS2-OFP plasmids or OFP control vectors. (J,K) The immunoprecipitation of p62 and phosphorylated mammalian target of rapamycin (p-mTOR), SOCS2, and p-mTOR were detected in ARPE-19 with the same treatment of B.

Article Snippet: Plasmid cytomegalovirus 3 (pCMV3)-Human-SOCS2-orange fluorescent protein (OFP) expression plasmid (HG11285-ACR), control vector OFP expression plasmids (CV025), pCMV3-Human-SOCS2-green fluorescent protein (GFP) (HG11285-ACG), and control vectors GFP expression plasmids (CV026) were purchased from the Sino Biological Incorporation (Wayne, PA, United States).

Techniques: In Situ, Fluorescence, Immunofluorescence, Staining, Transfection, Immunoprecipitation

Journal: Frontiers in Neuroscience

Article Title: Suppressor of Cytokine Signaling 2 Regulates Retinal Pigment Epithelium Metabolism by Enhancing Autophagy

doi: 10.3389/fnins.2021.738022

Figure Lengend Snippet: SOCS2 regulated the phosphorylation of glycogen synthase kinase 3β (GSK3β) and mTOR. (A) Western blot images of p-GSK3β, GSK3β, and p-mTOR were detected in the primary RPE from WT and SOCS2 –/– . (B,C,D) Data shown are the mean of GSK3β, p-GSK3β, and p-mTOR bands quantified by ImageJ software and normalized to β-actin. Values for the control were set to 1. (E) Phosphorylation of GSK3β and mTOR was detected by immunoblot in ARPE-19 after transient transfection with SOCS2-OFP expression plasmids or OFP control vectors. (F,G,H) Data shown are the mean of p-GSK3β, GSK3β, and p-mTOR bands quantified by ImageJ software and normalized to β-actin. The two-tailed unpaired t -test was used.

Article Snippet: Plasmid cytomegalovirus 3 (pCMV3)-Human-SOCS2-orange fluorescent protein (OFP) expression plasmid (HG11285-ACR), control vector OFP expression plasmids (CV025), pCMV3-Human-SOCS2-green fluorescent protein (GFP) (HG11285-ACG), and control vectors GFP expression plasmids (CV026) were purchased from the Sino Biological Incorporation (Wayne, PA, United States).

Techniques: Western Blot, Software, Transfection, Expressing, Two Tailed Test

Journal: Oncology Letters

Article Title: Knockdown of m6A methyltransferase METTL3 in gastric cancer cells results in suppression of cell proliferation

doi: 10.3892/ol.2020.11794

Figure Lengend Snippet: Manipulation of SOCS2 expression alters the proliferation of AGS cells. Knockdown of SOCS2 in AGS-WT cells promoted cell proliferation in AGS cells. Cells were treated with SOCS2-siRNA to knock down SOCS2. Cells treated with a non-specific control siRNA were used as controls. AGS cell treatment with SOCS2-siRNA significantly knocked down SOCS2 expression in AGS-WT cells at (A) RNA and (B) protein levels. *P<0.05 vs. Ctrl siRNA. (C) A significantly higher cell proliferation rate was detected by using an MTS assay in cells treated with siRNA-SOCS2 when compared with cells treated with Ctrl-siRNA. *P<0.05 vs. Ctrl siRNA. Overexpression of SOCS2 inhibited cell AGS cell proliferation. Cells were transfected with pCMV6-SOCS2 (full-length-SOCS2) to overexpress SOCS2. Cells treated with the pCMV6 empty vector were used as controls. SOCS2 overexpression in AGS-WT cells following transfection of pCMV6-SOCS2 was confirmed via reverse transcription-quantitative PCR and western blotting, respectively, at (D) RNA and (E) protein levels. *P<0.05 vs. pCMV6-Ctrl. A significantly lower cell proliferation rate was detected using an MTS assay in cells transfected with pCMV6-SOCS2 compared with pCMV6 empty vector-treated cells (F) Representative blotting images are presented in B and E. Densitometric levels of positive band for SOCS2 were quantified and are expressed as the ratio to GAPDH. *P<0.05 vs. pCMV6-Ctrl. SOCS, suppressor of cytokine signaling; WT, wild type; siRNA, small interfering RNA; Ctrl, control; OD, optical density.

Article Snippet: The pCMV6 plasmid with full-length SOCS2 sequence (pCMV6-SOCS2) was purchased from Origene (RC203163).

Techniques: Expressing, Knockdown, Control, MTS Assay, Over Expression, Transfection, Plasmid Preparation, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Small Interfering RNA

Journal: Oncology Reports

Article Title: m 6 A methyltransferase METTL3 maintains colon cancer tumorigenicity by suppressing SOCS2 to promote cell proliferation

doi: 10.3892/or.2020.7665

Figure Lengend Snippet: Modulation of SOCS2 expression alters the proliferation ability of SW480 cells. (A-C) siRNA (si-SOCS2) was used to successfully knock down SOCS2 expression in SW480 cells. Knockdown of SOCS2 had minor impact on METTL3 expression but significantly increased LGR5 expression in SW480 cells (*P<0.05, compared with the si-Ctrl group). (D) Knockdown of SOCS2 enhanced SW480 cell proliferation, as determined by an MTS assay (*P<0.05, compared with the si-Ctrl group). (E-G) Transfection of the pCMV6-SOCS2 plasmid upregulated SOCS2 expression and decreased LGR5 expression in SW480 cells (*P<0.05, compared with the pCMV6-Entry group). SOCS2 overexpression had minor impact on METTL3 expression (P>0.05, compared with the pCMV6-Entry group). (H) SOCS2 overexpression decreased SW480 cell proliferation, as determined by an MTS assay (*P<0.05, compared with the pCMV6-Entry group). SOCS2, suppressor of cytokine signaling 2; METTL3, methyltransferase like 3; LGR5, leucine-rich repeat-containing G protein-coupled receptor 5.

Article Snippet: The pCMV6 plasmid containing the full-length SOCS2 sequence (FULL-SOCS2) was purchased from Origene (RC203163).

Techniques: Expressing, Knockdown, MTS Assay, Transfection, Plasmid Preparation, Over Expression