Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Suppression of ongoing experimental autoimmune encephalomyelitis by neutralizing the function of the p28 subunit of IL-27.

doi: 10.4049/jimmunol.173.10.6465

Figure Lengend Snippet: FIGURE 1. DNA vaccination-based anti-IL-27 Abs are highly specific. The Western blot shows that our DNA vaccination-based anti-IL-27 Ab binds mouse IL-27 p28 (lane 1; 27 kDa), but not recombinant mouse IL-18, IL-12, or TNF- (lanes 2, 3, and 4, respectively). A, Coomassie Blue staining verifies the appearance of each cytokine on the loaded gel. B, Western blot showing that of these cytokines, our DNA vaccination-based anti-IL-27 Ab binds only mouse IL-27 p28. These Ab also bound natural mouse IL-27 (verified by sequencing) from supernatant of activated MOGp35–55-specific cultured primary draining lymph node cells (not shown).

Article Snippet: Our recombinant mouse IL-27 p28, produced as described above, and commercially available recombinant mouse IL-18, IL-12, and TNF- (R&D Systems) were each subjected to Western blot analysis according to the protocol described in detail previously (29, 32), with the minor modification of using a 12% (rather than 8%) running gel.

Techniques: Western Blot, Recombinant, Staining, Sequencing, Cell Culture

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Suppression of ongoing experimental autoimmune encephalomyelitis by neutralizing the function of the p28 subunit of IL-27.

doi: 10.4049/jimmunol.173.10.6465

Figure Lengend Snippet: FIGURE 2. Anti-p28 Abs suppresses ongoing severe EAE. A, Four groups of 10 mice each were subjected to MOGp35–55-induced EAE. Beginning at the onset of disease (day 17), these mice were repeatedly (every other day) administered 100 g/mouse of anti-p28 Ab (f), IgG obtained from naive Lewis rats (Œ), or PBS (E). An observer blind to the experimental procedure scored EAE daily. The experiment summarized in Fig. 2 shows the results of one of three experiments performed under similar experimental conditions, with similar results. The mean maximal score SE represents six mice per group. The other four mice were killed on day 30 and subjected to histological evaluation (see Fig. 3). B, Five groups of six mice each were subjected to MOGp35–55-induced EAE. Beginning at the onset of disease (day 17), these mice were repeatedly (every other day) administered 100 g of anti-p28 Ab/mouse (f), anti-IL-18 Ab (F), anti-IL-1 Ab (), IgG obtained from Lewis rats previously subjected to an empty plasmid administration (Œ), or PBS (E). An observer blind to the experimental procedure scored EAE daily. Results are shown as the mean maximal score SE of six mice per group. C, Three groups of six mice each were subjected to induction of transferred EAE. Beginning at the onset of disease (day 5), these mice were repeatedly (every other day) administered 100 g of anti-p28 Ab/mouse (f), IgG obtained from naive Lewis rats (Œ), or PBS (E). An observer blind to the experimental procedure scored EAE daily. Results are shown as mean maximal score SE of six mice per group.

Article Snippet: Our recombinant mouse IL-27 p28, produced as described above, and commercially available recombinant mouse IL-18, IL-12, and TNF- (R&D Systems) were each subjected to Western blot analysis according to the protocol described in detail previously (29, 32), with the minor modification of using a 12% (rather than 8%) running gel.

Techniques: Plasmid Preparation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Suppression of ongoing experimental autoimmune encephalomyelitis by neutralizing the function of the p28 subunit of IL-27.

doi: 10.4049/jimmunol.173.10.6465

Figure Lengend Snippet: FIGURE 4. The beneficial effect of anti-IL-27 is dependent on the con- tinuing administration of protective Abs. Three groups of six mice each were subjected to active induction of EAE. Beginning 1 day after the onset of disease (day 17), these mice were treated with either a single dose of anti-p28 Ab (100 g/mouse; E) or with repeated administration (every other day) of this Ab (Œ) or PBS (f). An observer blind to the experi- mental procedure scored EAE daily. Results are shown as the mean max- imal score SE of six mice per group.

Article Snippet: Our recombinant mouse IL-27 p28, produced as described above, and commercially available recombinant mouse IL-18, IL-12, and TNF- (R&D Systems) were each subjected to Western blot analysis according to the protocol described in detail previously (29, 32), with the minor modification of using a 12% (rather than 8%) running gel.

Techniques:

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Suppression of ongoing experimental autoimmune encephalomyelitis by neutralizing the function of the p28 subunit of IL-27.

doi: 10.4049/jimmunol.173.10.6465

Figure Lengend Snippet: FIGURE 3. Anti-IL-27 therapy reduces the histological score of EAE. Histological evaluation was conducted 30 days after disease induction. Lumbar spinal cord samples from naive mice or from EAE mice treated with PBS, IgG from naive mice, or anti-IL-27 p28 Abs were subjected to histological analysis (nine sections each group). The arrowheads point to the parenchymal mononuclear cell infiltration. The scale for mononuclear cell infiltration used was: 0, no mononuclear cell infiltration; 1, one to five perivascular lesions per section with minimal parenchymal infiltration; 2, five to 10 perivascular lesions per section with parenchymal infiltration; and 3, 10 perivascular lesions per section with extensive parenchymal infiltration. The mean histological score SE was calculated for each group.

Article Snippet: Our recombinant mouse IL-27 p28, produced as described above, and commercially available recombinant mouse IL-18, IL-12, and TNF- (R&D Systems) were each subjected to Western blot analysis according to the protocol described in detail previously (29, 32), with the minor modification of using a 12% (rather than 8%) running gel.

Techniques:

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Suppression of ongoing experimental autoimmune encephalomyelitis by neutralizing the function of the p28 subunit of IL-27.

doi: 10.4049/jimmunol.173.10.6465

Figure Lengend Snippet: FIGURE 5. Protective administration of anti-IL-27 Abs decreases in vivo polarization of CD4 T cells into Th1 and suppresses IFN- production by Ag-specific T cells. C57BL/6 mice (three per group) were subjected to active induction of EAE and then to repeated administration (days 12, 14, and 16) of anti-IL-27 p28 Abs (100 g), PBS, or normal rat IgG. On day 17 cervical lymph node cells (that drain the autoimmune site) were subjected to intracellular staining of IL-4 and IFN-. A, FACS analysis of CD4 T cells in this experiment. This experiment represents results obtained in three different independent experiments with very similar data. Subsequently, cervical lymph node T cells from these mice were cultured in the presence of 100 M MOGp35–55. After 72 h of stimulation, supernatants were assayed for the protein level of IFN- (B) and IL-4 (not shown). This experiment represents results obtained in three different independent experiments with very similar data.

Article Snippet: Our recombinant mouse IL-27 p28, produced as described above, and commercially available recombinant mouse IL-18, IL-12, and TNF- (R&D Systems) were each subjected to Western blot analysis according to the protocol described in detail previously (29, 32), with the minor modification of using a 12% (rather than 8%) running gel.

Techniques: In Vivo, Staining, Cell Culture

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Suppression of ongoing experimental autoimmune encephalomyelitis by neutralizing the function of the p28 subunit of IL-27.

doi: 10.4049/jimmunol.173.10.6465

Figure Lengend Snippet: FIGURE 6. Neutralizing the function of IL-27 reduces IFN- produc- tion by IFN--producing T cells. A, C57BL/6 mice (three per group) were subjected to active induction of EAE and then to repeated administration (days 3 and 6) of 100 g of anti-IL-27 p28 Abs (group 3), PBS (group 2), or normal rat IgG (group 1). On day 9, spleen cells were subjected to spot ELISA as previously described (38). A, Relative number of positive spots per 107 cultured cells. The average size of positive spots was analyzed. B, The MOGp33–55-specific CD4 T cell line was cultured with or without 100 M MOGp33–55. Cultured cells were supplemented with anti-IL-27 Abs at a final concentration of 10 g/ml (), normal rat IgG (f), or PBS (E). After 60 h of incubation, cells were plates in spot ELISA plates for an additional 24 h for the detection of IFN--positive spots (38). Number of positive spots (y-axis) and spot sizes (x-axis; logarithmic scale) determined as previously described (46).

Article Snippet: Our recombinant mouse IL-27 p28, produced as described above, and commercially available recombinant mouse IL-18, IL-12, and TNF- (R&D Systems) were each subjected to Western blot analysis according to the protocol described in detail previously (29, 32), with the minor modification of using a 12% (rather than 8%) running gel.

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Concentration Assay, Incubation

Journal: iScience

Article Title: Atypical B cells consist of subsets with distinct functional profiles

doi: 10.1016/j.isci.2023.108496

Figure Lengend Snippet:

Article Snippet: One day prior to sorting B cells, wells of a 96-well plate were each seeded with 30,000 adherent, CD40L-expressing 3T3 cells (kind gift from Dr. Mark Connors, NIH) in 100 μl IMDM/Glutamax/ 10% FBS containing 2× MycoZap Plus-PR (Lonza #VZA-2021), 100 ng/ml human IL-2 (GoldBio #1110-02-50), and 100 ng/ml human IL-21 (GoldBio #1110-21-10) to promote expansion and differentiation of B cells into antibody-secreting cells.

Techniques: Staining, Recombinant, Cell Isolation, Multiplex Assay, Sequencing, Plasmid Preparation, Software

Journal: The EMBO Journal

Article Title: The ion channel CALHM6 controls bacterial infection‐induced cellular cross‐talk at the immunological synapse

doi: 10.15252/embj.2022111450

Figure Lengend Snippet:

Article Snippet: Recombinant mouse il‐10 (carrier‐free) , Biolegend , 575804.

Techniques: Western Blot, Protease Inhibitor, Sample Prep, Flow Cytometry, Staining, Purification, Microscopy, Membrane, Recombinant, Activation Assay, Transfection, Retroviral, Transduction, Reverse Transcription, Enzyme-linked Immunosorbent Assay, Cytotoxicity Assay, Lactate Dehydrogenase Assay, Selection, Plasmid Preparation, ATP Bioluminescent Assay, SYBR Green Assay, Gel Extraction, Generated, Expressing, Sequencing, Gene Expression, Software

Journal: Cellular & molecular immunology

Article Title: The membrane-associated ubiquitin ligases MARCH2 and MARCH3 target IL-5 receptor alpha to negatively regulate eosinophilic airway inflammation.

doi: 10.1038/s41423-022-00907-9

Figure Lengend Snippet: Fig. 1 MARCH2/3 negatively regulates IL-5-triggered signaling by downregulating IL-5Rα. A Effects of MARCH protein overexpression on the IL- 5Rα protein level. HEK293 cells were transfected with the indicated plasmids for 24 h before immunoblot analysis with the indicated antibodies. B MARCH2 and MARCH3 promote the degradation of IL-5Rα but not IL-5Rβ in a dose-dependent manner. HEK293 cells were transfected with the indicated plasmids for 24 h before immunoblot analysis with the indicated antibodies. C Effects of MARCH2, MARCH3 and MARCH2/3 deficiency on the IL-5Rα protein level. TF-1-IL5 cells were starved overnight by removal of recombinant IL-5 from the medium and were then stimulated with IL-5 (100 ng/mL) for the indicated times before immunoblot analysis with the indicated antibodies. The lower graph shows the levels of IL-5Rα and IL-5Rβ relative to β-actin, as quantified by ImageJ. D Effects of MARCH2, MARCH3 and MARCH2/3 deficiency on IL-5-induced phosphorylation of STAT5Y694/699 in TF-1-IL5 cells. KO and control TF-1-IL5 cells were starved overnight by removal of recombinant IL-5 from the medium and were then left untreated or treated with IL-5 (100 ng/mL) for the indicated times before immunoblot analysis. E Effects of MARCH2, MARCH3 and MARCH2/3 deficiency on the transcription of downstream genes induced by IL-5 and GM-CSF in TF-1-IL5 cells. KO and control TF-1-IL5 cells were starved overnight and then left untreated or treated with IL-5 (20 ng/mL) or GM-CSF (20 ng/mL) for the indicated times before qPCR analysis. The data shown are the means ± SEMs; n = 3 technical replicates. **P < 0.01; n.s. not significant. IB immunoblot. All experiments were repeated at least two times with similar results

Article Snippet: The following items were purchased from the indicated companies: RNAiso Plus (Takara Bio, 9109); M-MLV reverse transcriptase (Invitrogen, 28025-013); RiboLock RNase Inhibitor (Thermo Scientific, EO0382); SYBR Green (Bio-Rad, 172-5274); MG132 (MCE, HY-13259); 3-MA (MCE, 5142-23-4); polybrene (Millipore, TR-1003-G); RPMI-1640 medium (Gibco, C11875500BT); Dulbecco’s modified Eagle’s medium (Gibco, C11995500BT); fetal bovine serum (FBS; CellMAX, SA211.02); penicillin/streptomycin (Gibco, 15140-122); ELISA kits for mouse Il-4 (BioLegend, 431101), Il-5 (BioLegend, 431204) and Il-33 (R&D Systems, DY3626-05); recombinant human IL-5, GM-CSF, and murine Il-5 (Peprotech, 200-05, 300-03 and 215-15, respectively); mouse monoclonal antibodies against IL-3/IL-5/GM-CSFRβ (Santa Cruz Biotechnology, sc-398246), FLAG® M2 (Sigma‒Aldrich, F3165), β-actin (Sigma‒Aldrich, A2228), hemagglutinin (HA; BioLegend, 901515), and Myc (Cell Signaling Technology, 2276); rabbit antibodies against p-Y694/699-STAT5 (Cell Signaling Technology, 4322), STAT5 (Cell Signaling Technology, 94205), human IL-5Rα (Abbexa, abx103030), murine March2 (Abcam, ab220292), and ubiquitin linkage-specific K27 (Abcam, ab181537); a goat antibody against human IL-5Rα (Thermo Fisher, PA5-47340); HRP-conjugated mouse anti-HA (Sigma Aldrich, H6533); monoclonal anti-FLAG® M2-HRP (Sigma‒Aldrich, A8592); mouse anti-goat IgG-HRP (Santa Cruz Biotechnology, sc-2354); goat anti-rabbit IgG HRP (Abmart, M21007); anti-mouse Ig HRP (Rockland Immunochemicals, 18-8817-33); and anti-rabbit IgG HRP (Rockland Immunochemicals, 18-8816-33).

Techniques: Over Expression, Transfection, Western Blot, Recombinant, Phospho-proteomics, Control

Journal: Cellular & molecular immunology

Article Title: The membrane-associated ubiquitin ligases MARCH2 and MARCH3 target IL-5 receptor alpha to negatively regulate eosinophilic airway inflammation.

doi: 10.1038/s41423-022-00907-9

Figure Lengend Snippet: Fig. 2 MARCH2/3 mediates K27-linked polyubiquitination and lysosomal degradation of IL-5Rα. A MARCH2 and MARCH3 interact strongly with IL-5Rα and weakly with IL-5Rβ. HEK293 cells were transfected with the indicated plasmids for 20 h before coimmunoprecipitation (co-IP) and immunoblot analysis with the indicated antibodies. B Endogenous MARCH2 and MARCH3 associate with IL-5Rα. TF-1-IL-5 cells were starved overnight by removal of recombinant IL-5 from the medium and were then left untreated or treated with IL-5 (100 ng/mL) for the indicated times before coimmunoprecipitation and immunoblot analysis with the indicated antibodies. C MARCH2 and MARCH3 but not their E3 ligase-inactive mutants mediate the polyubiquitination of IL-5Rα. HEK293 cells were transfected with the indicated plasmids for 24 h before coimmunoprecipitation and immunoblot analysis with the indicated antibodies. D MARCH2 and MARCH3 mediate K27-linked polyubiquitination of IL-5Rα. HEK293 cells were transfected with the indicated plasmids for 18-20 h before coimmunoprecipitation and immunoblot analysis with the indicated antibodies. E Effects of MARCH2/3 deficiency on IL-5-induced K27-linked polyubiquitination of IL-5Rα. Control and MARCH2/3-deficient TF-1-IL5 cells were starved overnight by removal of recombinant IL-5 from the medium and were then left untreated or treated with IL-5 (100 ng/mL) for the indicated times before coimmunoprecipitation and immunoblot analysis. KO, K only; KR, K mutated to R. F Effects of inhibitors on MARCH2- and MARCH3-mediated downregulation of IL-5Rα. HEK293 cells were transfected with the indicated plasmids for 12 h and were then treated with NH4Cl (25 mM), MG132 (100 μM) or 3-MA (3.35 mM) for 6 h before immunoblot analysis with the indicated antibodies. G NH4Cl inhibits IL-5Rα degradation following CHX treatment. HEK293 cells were treated with CHX (0.1 mM) for 3 h and were then treated with NH4Cl (25 mM), MG132 (100 μM) or 3-MA (3.35 mM) for an additional 3 h before immunoblot analysis with the indicated antibodies. IB immunoblot, IP immunoprecipitation. All experiments were repeated at least two times with similar results

Article Snippet: The following items were purchased from the indicated companies: RNAiso Plus (Takara Bio, 9109); M-MLV reverse transcriptase (Invitrogen, 28025-013); RiboLock RNase Inhibitor (Thermo Scientific, EO0382); SYBR Green (Bio-Rad, 172-5274); MG132 (MCE, HY-13259); 3-MA (MCE, 5142-23-4); polybrene (Millipore, TR-1003-G); RPMI-1640 medium (Gibco, C11875500BT); Dulbecco’s modified Eagle’s medium (Gibco, C11995500BT); fetal bovine serum (FBS; CellMAX, SA211.02); penicillin/streptomycin (Gibco, 15140-122); ELISA kits for mouse Il-4 (BioLegend, 431101), Il-5 (BioLegend, 431204) and Il-33 (R&D Systems, DY3626-05); recombinant human IL-5, GM-CSF, and murine Il-5 (Peprotech, 200-05, 300-03 and 215-15, respectively); mouse monoclonal antibodies against IL-3/IL-5/GM-CSFRβ (Santa Cruz Biotechnology, sc-398246), FLAG® M2 (Sigma‒Aldrich, F3165), β-actin (Sigma‒Aldrich, A2228), hemagglutinin (HA; BioLegend, 901515), and Myc (Cell Signaling Technology, 2276); rabbit antibodies against p-Y694/699-STAT5 (Cell Signaling Technology, 4322), STAT5 (Cell Signaling Technology, 94205), human IL-5Rα (Abbexa, abx103030), murine March2 (Abcam, ab220292), and ubiquitin linkage-specific K27 (Abcam, ab181537); a goat antibody against human IL-5Rα (Thermo Fisher, PA5-47340); HRP-conjugated mouse anti-HA (Sigma Aldrich, H6533); monoclonal anti-FLAG® M2-HRP (Sigma‒Aldrich, A8592); mouse anti-goat IgG-HRP (Santa Cruz Biotechnology, sc-2354); goat anti-rabbit IgG HRP (Abmart, M21007); anti-mouse Ig HRP (Rockland Immunochemicals, 18-8817-33); and anti-rabbit IgG HRP (Rockland Immunochemicals, 18-8816-33).

Techniques: Transfection, Co-Immunoprecipitation Assay, Western Blot, Recombinant, Control, Immunoprecipitation

Journal: Cellular & molecular immunology

Article Title: The membrane-associated ubiquitin ligases MARCH2 and MARCH3 target IL-5 receptor alpha to negatively regulate eosinophilic airway inflammation.

doi: 10.1038/s41423-022-00907-9

Figure Lengend Snippet: Fig. 3 MARCH2 and MARCH3 mediate the polyubiquitination of IL-5Rα at K379 and K383, respectively. A Sequence alignment of the cytoplasmic domains of IL-5Rα in the indicated species. The sequences correspond to aa 363-420 in human IL-5Rα. B MARCH2 and MARCH3 mediate K27-linked polyubiquitination of IL-5Rα at K379 and K383, respectively. HEK293 cells were transfected with the indicated plasmids for 18 h before coimmunoprecipitation and immunoblot analysis with the indicated antibodies. C Effects of MARCH2 and MARCH3 on the stability of IL-5Rα and its mutants. HEK293 cells were transfected with the indicated plasmids for 24 h before immunoblot analysis with the indicated antibodies. D IL-5Rα colocalizes with MARCH2 and MARCH3. HEK293 cells were transfected with FLAG-tagged IL-5Rα, HA-tagged MARCH2 and HA-tagged MARCH3 for 20 h. The cells were stained with LysoTracker Red (200 nM) for 1 h and were then fixed with 4% paraformaldehyde and stained with mouse anti-FLAG and rabbit anti-HA antibodies. IL-5Rα was labeled with an Alexa Fluor 488-conjugated secondary antibody, and MARCH2 and MARCH3 were labeled with an Alexa Fluor 647-conjugated secondary antibody. Images were acquired under a confocal microscope with a ×63 oil objective. E Reconstitution of IL-5Rα-deficient TF-1-IL5 cells with wild-type IL-5Rα and its mutants. IL-5Rα-deficient TF-1-IL5 cells were reconstituted with wild-type IL-5Rα, IL-5RαK379R, IL-5RαK383R, IL-5RαK379/383R or empty vector by lentiviral- mediated transduction. The reconstituted cells were starved overnight by removal of recombinant IL-5 from the medium and were then stimulated with IL-5 (20 ng/ml) for the indicated times before qPCR analysis of the mRNA levels of the indicated genes. The data shown are the means ± SEMs; n = 3 technical replicates. *P < 0.05; **P < 0.01; n.s. not significant. IB immunoblot, IP immunoprecipitation. All experiments were repeated at least twice with similar results

Article Snippet: The following items were purchased from the indicated companies: RNAiso Plus (Takara Bio, 9109); M-MLV reverse transcriptase (Invitrogen, 28025-013); RiboLock RNase Inhibitor (Thermo Scientific, EO0382); SYBR Green (Bio-Rad, 172-5274); MG132 (MCE, HY-13259); 3-MA (MCE, 5142-23-4); polybrene (Millipore, TR-1003-G); RPMI-1640 medium (Gibco, C11875500BT); Dulbecco’s modified Eagle’s medium (Gibco, C11995500BT); fetal bovine serum (FBS; CellMAX, SA211.02); penicillin/streptomycin (Gibco, 15140-122); ELISA kits for mouse Il-4 (BioLegend, 431101), Il-5 (BioLegend, 431204) and Il-33 (R&D Systems, DY3626-05); recombinant human IL-5, GM-CSF, and murine Il-5 (Peprotech, 200-05, 300-03 and 215-15, respectively); mouse monoclonal antibodies against IL-3/IL-5/GM-CSFRβ (Santa Cruz Biotechnology, sc-398246), FLAG® M2 (Sigma‒Aldrich, F3165), β-actin (Sigma‒Aldrich, A2228), hemagglutinin (HA; BioLegend, 901515), and Myc (Cell Signaling Technology, 2276); rabbit antibodies against p-Y694/699-STAT5 (Cell Signaling Technology, 4322), STAT5 (Cell Signaling Technology, 94205), human IL-5Rα (Abbexa, abx103030), murine March2 (Abcam, ab220292), and ubiquitin linkage-specific K27 (Abcam, ab181537); a goat antibody against human IL-5Rα (Thermo Fisher, PA5-47340); HRP-conjugated mouse anti-HA (Sigma Aldrich, H6533); monoclonal anti-FLAG® M2-HRP (Sigma‒Aldrich, A8592); mouse anti-goat IgG-HRP (Santa Cruz Biotechnology, sc-2354); goat anti-rabbit IgG HRP (Abmart, M21007); anti-mouse Ig HRP (Rockland Immunochemicals, 18-8817-33); and anti-rabbit IgG HRP (Rockland Immunochemicals, 18-8816-33).

Techniques: Sequencing, Transfection, Western Blot, Staining, Labeling, Microscopy, Plasmid Preparation, Transduction, Recombinant, Immunoprecipitation

Journal: Cellular & molecular immunology

Article Title: The membrane-associated ubiquitin ligases MARCH2 and MARCH3 target IL-5 receptor alpha to negatively regulate eosinophilic airway inflammation.

doi: 10.1038/s41423-022-00907-9

Figure Lengend Snippet: Fig. 5 March2/3 deficiency promotes eosinophilic airway inflammation. A A schematic timeline of the mouse model of allergic asthma. B Effects of March deficiency on the percentages and numbers of CCR3+Siglec-F+ eosinophils in the bone morrow. Bone marrow cells from OVA-challenged, age-matched wild-type, March2–/–, March3–/– and March2–/–/3–/– mice were collected 24 h after the final OVA challenge and analyzed by flow cytometry. The data shown are the means ± SDs; n = 6 mice per group. C Effects of March deficiency on the percentage of CCR3+Siglec-F+ eosinophils in the peripheral blood. Peripheral blood leukocytes from OVA-challenged, age-matched wild-type, March2–/–, March3–/– and March2–/–/3–/– mice were collected 24 h after the final OVA challenge and analyzed by flow cytometry. The data shown are the means ± SDs; n = 6 mice per group. D Effects of March deficiency on the percentages and numbers of Lin-CD34+Sca1-c-KitintIl-5rα+ eosinophil progenitors in the bone marrow. Bone marrow cells from OVA-challenged, age-matched wild-type, March2–/–, March3–/–and March2–/–/3–/– mice were collected 24 h after the final OVA challenge and analyzed by flow cytometry. The data shown are the means ± SDs; n = 6 mice per group. E H&E and PAS staining of lung tissues. OVA-challenged, age-matched wild-type, March2–/–, March3–/– and March2–/–/3–/– mice were sacrificed 24 h after the final OVA challenge. Lung tissues were collected, fixed with 4% paraformaldehyde and embedded in paraffin. The sections were stained with hematoxylin and eosin (H&E) or periodic acid-Schiff (PAS). Scale bar, 200 μm. F Histological inflammation scores. Immune cell infiltration and peribronchial mucus production were scored based on the above H&E- and PAS-stained sections. The data shown are the means ± SDs; n = 6 mice per group, 4–5 images per mouse. G Effects of March deficiency on the levels of Il-4, Il-5 and Il-33 in the peripheral blood. Sera from OVA-challenged, age-matched wild-type, March2–/–, March3–/–and March2–/–/3–/– mice were collected 24 h after the final OVA challenge for ELISA. The data shown are the means ± SDs; n = 6 mice per group. WT wild-type. *P < 0.05; **P < 0.01; n.s. not significant. The data in (B)–(G) were combined from two independent experiments

Article Snippet: The following items were purchased from the indicated companies: RNAiso Plus (Takara Bio, 9109); M-MLV reverse transcriptase (Invitrogen, 28025-013); RiboLock RNase Inhibitor (Thermo Scientific, EO0382); SYBR Green (Bio-Rad, 172-5274); MG132 (MCE, HY-13259); 3-MA (MCE, 5142-23-4); polybrene (Millipore, TR-1003-G); RPMI-1640 medium (Gibco, C11875500BT); Dulbecco’s modified Eagle’s medium (Gibco, C11995500BT); fetal bovine serum (FBS; CellMAX, SA211.02); penicillin/streptomycin (Gibco, 15140-122); ELISA kits for mouse Il-4 (BioLegend, 431101), Il-5 (BioLegend, 431204) and Il-33 (R&D Systems, DY3626-05); recombinant human IL-5, GM-CSF, and murine Il-5 (Peprotech, 200-05, 300-03 and 215-15, respectively); mouse monoclonal antibodies against IL-3/IL-5/GM-CSFRβ (Santa Cruz Biotechnology, sc-398246), FLAG® M2 (Sigma‒Aldrich, F3165), β-actin (Sigma‒Aldrich, A2228), hemagglutinin (HA; BioLegend, 901515), and Myc (Cell Signaling Technology, 2276); rabbit antibodies against p-Y694/699-STAT5 (Cell Signaling Technology, 4322), STAT5 (Cell Signaling Technology, 94205), human IL-5Rα (Abbexa, abx103030), murine March2 (Abcam, ab220292), and ubiquitin linkage-specific K27 (Abcam, ab181537); a goat antibody against human IL-5Rα (Thermo Fisher, PA5-47340); HRP-conjugated mouse anti-HA (Sigma Aldrich, H6533); monoclonal anti-FLAG® M2-HRP (Sigma‒Aldrich, A8592); mouse anti-goat IgG-HRP (Santa Cruz Biotechnology, sc-2354); goat anti-rabbit IgG HRP (Abmart, M21007); anti-mouse Ig HRP (Rockland Immunochemicals, 18-8817-33); and anti-rabbit IgG HRP (Rockland Immunochemicals, 18-8816-33).

Techniques: Cytometry, Staining, Enzyme-linked Immunosorbent Assay

Journal: eLife

Article Title: A systematic CRISPR screen reveals an IL-20/IL20RA-mediated immune crosstalk to prevent the ovarian cancer metastasis

doi: 10.7554/eLife.66222

Figure Lengend Snippet: ( A ) Growth curves of IL20RA-reconstituted or control (Vec) ID8 cells under the stimulation of IL-20. ( B ) Growth curves of shIL20RA- or shCtrl-transfected SK-OV-3 cells under the stimulation of IL-20. ( C, D ) Transwell cell migration assay of shIL20RA- or shCtrl-transfected SK-OV-3 cells in the presence of IL-20, which were quantified in ( D ) (shown as means ± SEM from three independent experiments, ns, not significant, by unpaired two-sided Student’s t-test). Scale bar: 50 μm.

Article Snippet: To detect the activation of signaling pathway, ID8 and SK-OV-3 cells were stimulated with 2.5 ng/mL murine and human recombinant IL-20 protein (Sino Biological) for 24 hr, respectively.

Techniques: Transfection, Cell Migration Assay

Journal: eLife

Article Title: A systematic CRISPR screen reveals an IL-20/IL20RA-mediated immune crosstalk to prevent the ovarian cancer metastasis

doi: 10.7554/eLife.66222

Figure Lengend Snippet: ( A ) Schematic of the in vitro crosstalk experiment using conditioned medium (CM) from OC cells to educate macrophages. ( B ) qRT-PCR analysis of macrophage marker genes in RAW 264.7 cells stimulated with CM of IL-20-stimulated/unstimulated IL20RA-reconsitituted (CM IL20RA ) or control (CM Vec ) ID8 cells for 72 hr. ( C, D ) Representative image of bone marrow-derived macrophage (BMDM) differentiated from bone marrow cell with the treatment of macrophage colony stimulating factor for 7 days ( C ), which were further characterized by qRT-PCR analysis of its marker gene Cd68 ( D ). Scale bar: 20 μm. ( E ) qRT-PCR analysis of macrophage marker genes in BMDM treated with CM of IL-20-stimulated/unstimulated IL20RA-reconsitituted (CM IL20RA ) or control (CM Vec ) ID8 cells for 72 hr. ( F, G ) Image of macrophages differentiated from THP-1 cells with the treatment of phorbol-12-myristate-13-acetate for 48 hr ( F ) and further characterization by qRT-PCR analysis of CD68 ( G ). Scale bar: 20 μm. ( H ) qRT-PCR analysis of macrophage marker genes in THP-1-derived macrophages treated with CM from IL-20-stimulated/unstimulated shIL20RA or control shRNA (shCtrl)-transfected SK-OV-3 cells for 72 hr. All the qRT-PCR data are shown as means ± SEM from three independent experiments, *p<0.05, **p<0.01, ***p<0.001, by unpaired two-sided Student’s t-test. Figure 4—source data 1. An Excel sheet with numerical quantification data.

Article Snippet: To detect the activation of signaling pathway, ID8 and SK-OV-3 cells were stimulated with 2.5 ng/mL murine and human recombinant IL-20 protein (Sino Biological) for 24 hr, respectively.

Techniques: In Vitro, Quantitative RT-PCR, Marker, Derivative Assay, shRNA, Transfection

Journal: eLife

Article Title: A systematic CRISPR screen reveals an IL-20/IL20RA-mediated immune crosstalk to prevent the ovarian cancer metastasis

doi: 10.7554/eLife.66222

Figure Lengend Snippet: ( A ) Schematic of the in vitro co-culture experiments. ( B, C ) Transwell migration assay to show the migrated RAW 264.7 cells co-cultured with IL20RA-reconstituted or control (Vec) ID8 cells ( B ), which were quantified in ( C ) (shown as means ± SEM from three independent experiments, ns, not significant, by unpaired two-sided Student’s t-test). Scale bar: 50 μm. ( D ) qRT-PCR analysis of macrophage marker genes in bone marrow-derived macrophage co-cultured with IL20RA-reconstituted or control (Vec) ID8 cells for 72 hr. ( E ) qRT-PCR analysis of macrophage marker genes in THP-1-derived macrophages co-cultured with shIL20RA- or shCtrl-transfected SK-OV-3 cells for 72 hr. ( F ) qRT-PCR analysis of macrophage marker genes in RAW 264.7 cells stimulated with IL-20 protein for 72 hr. ( G ) qRT-PCR analysis of macrophage marker genes in RAW 264.7 cells stimulated with IL-24 protein for 72 hr. All the qRT-PCR data are shown as means ± SEM from three independent experiments, *p<0.05, **p<0.01, ***p<0.001, by unpaired two-sided Student’s t-test.

Article Snippet: To detect the activation of signaling pathway, ID8 and SK-OV-3 cells were stimulated with 2.5 ng/mL murine and human recombinant IL-20 protein (Sino Biological) for 24 hr, respectively.

Techniques: In Vitro, Co-Culture Assay, Transwell Migration Assay, Cell Culture, Quantitative RT-PCR, Marker, Derivative Assay, Transfection

Journal: eLife

Article Title: A systematic CRISPR screen reveals an IL-20/IL20RA-mediated immune crosstalk to prevent the ovarian cancer metastasis

doi: 10.7554/eLife.66222

Figure Lengend Snippet: ( A, B ) qRT-PCR analysis of IL20RA ligands ( Il19 , Il20, and Il24 ) in peritoneal organs (intestinal, abdominal wall, ovary) ( A ) or peritoneal macrophages (CD11b + F4/80 + ) ( B ) taken from C57BL/6 mice with intraperitoneal injection of ID8 cells (OC) or phosphate-buffered saline (PBS) control (Ctrl) 9 days before. Data are shown as means ± SEM, n = 18 for Ctrl group and n = 6 for OC group, ***p<0.001, ns, not significant, by unpaired two-sided Student’s t-test. ( C ) qRT-PCR analysis of IL20RA ligands in IL20RA-reconstituted or control (Vec) ID8 cells (means ± SEM, ns, not significant). ( D ) ELISA measurement of IL-20 and IL-24 in peritoneal flushing fluid from mice with intraperitoneal injection of ID8 cells (OC) or PBS control (Ctrl) 9 days before. n = 6 for each group. Data are shown as means ± SEM, ***p<0.001, by unpaired two-sided Student’s t-test. ( E ) qRT-PCR analysis of the abdominal walls dissected from C57BL/6 mice and co-cultured with medium (Ctrl) or ID8 cells for 48 hr (means ± SEM from three independent experiments, ***p<0.001, ns, not significant, by unpaired two-sided Student’s t-test). ( F ) Hematoxylin and eosin staining of the abdominal wall of C57BL/6 mice. Scale bar: 50 μm (upper panel); 20 μm (lower panel). ( G ) Immunohistochemical staining of IL-20 and IL-24 in abdominal walls dissected from mice with intraperitoneal injection of ID8 cells (OC) or PBS control (Ctrl) 9 days before. Scale bar: 20 μm. Figure 6—source data 1. An Excel sheet with numerical quantification data.

Article Snippet: To detect the activation of signaling pathway, ID8 and SK-OV-3 cells were stimulated with 2.5 ng/mL murine and human recombinant IL-20 protein (Sino Biological) for 24 hr, respectively.

Techniques: Quantitative RT-PCR, Injection, Enzyme-linked Immunosorbent Assay, Cell Culture, Staining, Immunohistochemical staining

Journal: eLife

Article Title: A systematic CRISPR screen reveals an IL-20/IL20RA-mediated immune crosstalk to prevent the ovarian cancer metastasis

doi: 10.7554/eLife.66222

Figure Lengend Snippet: ( A, B ) Immunofluorescent staining of IL-20/IL-24 (red), cytokeratin CK18 (green), and DAPI (blue) in peritoneum dissected from mice with intraperitoneal injection of ID8 cells (OC) or phosphate-buffered saline control (Ctrl) 9 days before. Scale bar: 30 μm.

Article Snippet: To detect the activation of signaling pathway, ID8 and SK-OV-3 cells were stimulated with 2.5 ng/mL murine and human recombinant IL-20 protein (Sino Biological) for 24 hr, respectively.

Techniques: Staining, Injection

Journal: eLife

Article Title: A systematic CRISPR screen reveals an IL-20/IL20RA-mediated immune crosstalk to prevent the ovarian cancer metastasis

doi: 10.7554/eLife.66222

Figure Lengend Snippet: ( A ) qRT-PCR analysis of Oas1a and Il18 upon IL20RA reconstitution in ID8 cells. ( B ) The secreted IL-18 from IL20RA-reconstituted and control ID8 cells was measured by ELISA. ( C ) Western blot analysis of indicated proteins in IL20RA-reconstituted and control ID8 cells under the stimulation of IL-20. ( D ) Western blot analysis of indicated proteins in SK-OV-3 cells stimulated with IL-20 or phosphate-buffered saline (PBS) (Ctrl) for 24 hr. ( E ) ELISA measurement of IL-18 secreted from SK-OV-3 cells stimulated by IL-20 for 24 hr. ( F ) Schematic of the Oas1a promoter and Il18 promoter with predicted STAT3-binding sites and the primer sets. ( G ) IL20RA-reconstituted ID8 cells were stimulated with IL-20 or PBS (Ctrl) for 24 hr before the STAT3 binding on Oas1a promoter and Il18 promoter was analyzed by ChIP-qPCR. ( H, I ) Immunohistochemical analysis of IL20RA, p-STAT3, OAS1, and IL-18 in human primary ovarian cancer tissues (Pri) and paired peritoneal metastatic nodules (Met) ( H ) and quantification ( I ). **p<0.01; ***p<0.001, by paired two-sided Student’s t-test. Scale bar: 20 μm. ( J ) qRT-PCR analysis of macrophage marker genes in RAW 264.7 cells stimulated with IL-20 protein for 72 hr. ( K ) qRT-PCR analysis of macrophage marker genes in RAW 264.7 cells treated by CM IL20RA IL-20 (+) or CM Vec IL-20 (+) together with IL-18 neutralizing antibody (nAb) or nonspecific lgG (IgG) for 72 hr. All the qRT-PCR and ELISA data are shown as means ± SEM from three independent experiments, *p<0.05, **p<0.01, ***p<0.001, by unpaired two-sided Student’s t-test. Figure 7—source data 1. An Excel sheet with numerical quantification data.

Article Snippet: To detect the activation of signaling pathway, ID8 and SK-OV-3 cells were stimulated with 2.5 ng/mL murine and human recombinant IL-20 protein (Sino Biological) for 24 hr, respectively.

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot, Binding Assay, Immunohistochemical staining, Marker

Journal: eLife

Article Title: A systematic CRISPR screen reveals an IL-20/IL20RA-mediated immune crosstalk to prevent the ovarian cancer metastasis

doi: 10.7554/eLife.66222

Figure Lengend Snippet: ( A ) qRT-PCR analysis of Il20rb in ID8 Vec and ID8 IL20RA cells transfected with shCtrl or shIL20RB (means ± SEM, ***p<0.001, by unpaired two-sided Student’s t-test). ( B ) Western blot analysis of indicated proteins in IL20RB-silenced ID8 Vec and ID8 IL20RA cells and their control cells under the stimulation of IL-20. ( C ) qRT-PCR analysis of macrophage marker genes in RAW 264.7 cells stimulated with conditioned medium of IL-20-stimulated IL20RB-silenced ID8 Vec and ID8 IL20RA cells and their control cells for 72 hr. Data are shown as means ± SEM from three independent experiments, *p<0.05, **p<0.01, ***p<0.001, ns, not significant, by unpaired two-sided Student’s t-test. ( D–G ) Representative images of ascites formation ( D ) and the metastatic nodules in peritoneal cavity ( E ) at day 45 after intraperitoneal injected with ID8 Vec , ID8 IL20RA , and ID8 IL20RA/shIL20RB cells. The quantification of ascites and metastatic nodules is shown in ( F ) and ( G ), respectively. Data are shown as means ± SEM, n = 6, ***p<0.001, ns, not significant, by unpaired two-sided Student’s t-test.

Article Snippet: To detect the activation of signaling pathway, ID8 and SK-OV-3 cells were stimulated with 2.5 ng/mL murine and human recombinant IL-20 protein (Sino Biological) for 24 hr, respectively.

Techniques: Quantitative RT-PCR, Transfection, Western Blot, Marker, Injection

Journal: eLife

Article Title: A systematic CRISPR screen reveals an IL-20/IL20RA-mediated immune crosstalk to prevent the ovarian cancer metastasis

doi: 10.7554/eLife.66222

Figure Lengend Snippet: ( A ) qRT-PCR analysis of potential IL20RA ligands from the abdominal wall taken from C57BL/6 mice with intraperitoneal injection of SK-OV-3 cells or phosphate-buffered saline control (Ctrl) 9 days before. ( B ) qRT-PCR analyses of macrophage marker genes in RAW 264.7 cells stimulated with human IL-18 protein for 72 hr. ( C ) qRT-PCR analysis of macrophage marker genes in RAW 264.7 cells treated with conditioned medium from IL-20-stimulated shIL20RA or control shRNA (shCtrl)-transfected SK-OV-3 cells for 72 hr. All the qRT-PCR data are shown as means ± SEM from three independent experiments, *p<0.05, **p<0.01, ***p<0.001, by unpaired two-sided Student’s t-test.

Article Snippet: To detect the activation of signaling pathway, ID8 and SK-OV-3 cells were stimulated with 2.5 ng/mL murine and human recombinant IL-20 protein (Sino Biological) for 24 hr, respectively.

Techniques: Quantitative RT-PCR, Injection, Marker, shRNA, Transfection

Journal: eLife

Article Title: A systematic CRISPR screen reveals an IL-20/IL20RA-mediated immune crosstalk to prevent the ovarian cancer metastasis

doi: 10.7554/eLife.66222

Figure Lengend Snippet:

Article Snippet: To detect the activation of signaling pathway, ID8 and SK-OV-3 cells were stimulated with 2.5 ng/mL murine and human recombinant IL-20 protein (Sino Biological) for 24 hr, respectively.

Techniques: Transfection, Construct, shRNA, Recombinant, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

Journal: Cell reports

Article Title: Impact of eIF2α phosphorylation on the translational landscape of mouse embryonic stem cells

doi: 10.1016/j.celrep.2023.113615

Figure Lengend Snippet: (A) Ribosome (Ribo-seq) and RNA (RNA-seq) coverage plots for Atf4 mRNA in eIF2α +/+ and eIF2α A/A mESCs under LIF conditions. (B) The percentage of Atf4 mRNAs in each fraction of polysome profiles (left) and total input mRNA level (right) of eIF2α +/+ and eIF2α A/A mESCs cultured in LIF medium were quantified by RT-qPCR. *p < 0.05, (n = 3), two-way ANOVA test with correction for multiple comparisons using the Benjamini-Hochberg method. Data presented as mean ± SEM. (C) Ribo-seq and RNA-seq coverage plot of Epop in eIF2α +/+ and eIF2α A/A mESCs under LIF conditions. (D) The percentage of Epop mRNAs in each fraction of polysome profiles (left) and total input mRNA level (right) of eIF2α +/+ and eIF2α A/A mESCs cultured in LIF medium were quantified by RT-qPCR. *p < 0.05, **p < 0.01, (n = 3), two-way ANOVA test with correction for multiple comparisons using the Benjamini-Hochberg method. Data presented as mean ± SEM. (E) Position and amino acid conservation of Epop uORF-encoded protein in several mammals. The number of nucleotides (nt) between the cap and the start of uORF is indicated in blue for each species. The distance of the uORF start site to the CDS start site, and the length of the uORF-encoded protein are indicated in red and green, respectively, for each species. (F) Amino acid conservation of N-terminal region of EPOP uORF-encoded peptide in mammalian species. (G) Conservation of nucleotides flanking the start site of Epop uORF in mammalian species. (H) Left: UCSC Genome browser views for ATF4 binding events in GCN2 +/+ wild-type (WT) and GCN2 −/− knockout (KO) cells that were exposed to control (+Leu) or leucine-deficient (−Leu) medium. ATF4 binding profiles in ATF4 +/+ (WT) and ATF4 −/− (KO) cells treated with tunicamycin (Tm) (GSE35681) are shown below the panel. Right: the heatmap shows fold enrichment for ATF4 ChIP-seq target eIF1. (I and J) RT-qPCR analysis of N2A cells transduced with the indicated siRNA or shRNAs. Values were normalized to β-actin level. *p < 0.05, **p < 0.01. Data are presented as mean ± SEM (n = 3) t test. (K and L) Western blot analysis and quantification of ATF4 and eIF1 in N2A cells transduced with shCtrl or shRNA against ATF4. Values were normalized to β-actin level. Results are presented as mean ± SEM (n = 3). *p < 0.05, t test.

Article Snippet: Murine N2A (ATCC CCL-131) cells with neuronal and ameboid stem cell-like morphology and HEK293T (ATCC CRL-3216) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin-streptomycin and glutamine at 37°C and 5% CO2.

Techniques: RNA Sequencing, Cell Culture, Quantitative RT-PCR, Binding Assay, Knock-Out, Control, ChIP-sequencing, Transduction, Western Blot, shRNA

Journal: Cell reports

Article Title: Impact of eIF2α phosphorylation on the translational landscape of mouse embryonic stem cells

doi: 10.1016/j.celrep.2023.113615

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Murine N2A (ATCC CCL-131) cells with neuronal and ameboid stem cell-like morphology and HEK293T (ATCC CRL-3216) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin-streptomycin and glutamine at 37°C and 5% CO2.

Techniques: Recombinant, Mass Spectrometry, SYBR Green Assay, Reverse Transcription, Phospho-proteomics, Derivative Assay, Plasmid Preparation, Control, Software

Journal:

Article Title: Nuclear Factor YY1 Inhibits Transforming Growth Factor ?- and Bone Morphogenetic Protein-Induced Cell Differentiation

doi: 10.1128/MCB.23.13.4494-4510.2003

Figure Lengend Snippet: YY1 represses TGF-β and BMP immediate-early gene responses. (A) YY1 represses PAI-1 induction by TGF-β1. Northern analysis of PAI-1 mRNA from Mv1Lu cells mock treated (−) or treated (+) for 24 h with 10 ng of TGF-β1 per ml in the absence of YY1 (−) or in the presence of increasing doses of YY1 (Ad-YY1) (triangle; MOI of 20 or 100) or control GFP (Ad-GFP) (MOI of 20 or 100) adenovirus is shown. Sample loading and blotting efficiency was monitored with β-actin mRNA on the same blot. (B) YY1 represses Id-1 induction by TGF-β1. Immunoblot analysis of endogenous Id-1 expression in MDA-MB-468 cells infected with the indicated MOI of recombinant adenoviruses and then treated with vehicle (−) or 2.5 ng of TGF-β1 per ml (+) for 1 h is shown. Arrows mark the positions of endogenous Id-1, adenoviral YY1, adenoviral Flag-Smad4 (F-Smad4), endogenous phosphorylated Smad2 (Smad2-P), and endogenous β-catenin, which serves as a loading and blotting efficiency control. (C) YY1 represses Id-1 induction by BMP-7. Immunoblot analysis of endogenous Id-1 expression in MDA-MB-468 cells is as for panel B but in response to 300 ng of BMP-7 per ml. Infection conditions were as for panel B, and arrows mark the same proteins as in panel B except for endogenous phosphorylated Smad1 (Smad1-P) instead of Smad2. Asterisks mark nonspecific protein bands. (D) YY1 represses PAI-1 promoter induction by TGF-β1. PAI-1 (800)-luciferase assays were performed in HepG2 cells with increasing amounts of YY1 plasmid (triangle; 0.1 to 1.0 μg) in the absence (−) or presence (+) of overnight stimulation with 2.5 ng of TGF-β1 per ml. The averaged data for normalized reporter activity are shown with standard errors derived from triplicate transfections repeated at least twice independently. (E) YY1 represses the Smad-specific 12xCAGA promoter. The assays were similar to those for panel D, with the 12xCAGA-luciferase reporter in HepG2 cells after overnight stimulation with 2.5 ng of TGF-β1 per ml. Data are reported as in panel D. (F) YY1 represses the Smad-specific Id1-BRE2 promoter. The assays were similar to those for panel D, with the Id1-BRE2-luciferase reporter in C2C12 cells after overnight stimulation with 100 ng of BMP-6 per ml. Data are reported as in panel D.

Article Snippet: Murine C2C12 myoblasts, murine mammary epithelial NMuMG cells, mink lung epithelial Mv1Lu cells, monkey kidney COS-7 cells, human embryonic kidney 293T cells, human mammary MDA-MB-468 cells, and human hepatoma HepG2 cells were obtained and cultured according to protocols from the American Type Culture Collection.

Techniques: Northern Blot, Control, Western Blot, Expressing, Infection, Recombinant, Luciferase, Plasmid Preparation, Activity Assay, Derivative Assay, Transfection

Journal:

Article Title: Nuclear Factor YY1 Inhibits Transforming Growth Factor ?- and Bone Morphogenetic Protein-Induced Cell Differentiation

doi: 10.1128/MCB.23.13.4494-4510.2003

Figure Lengend Snippet: YY1 interacts physically with Smads in vitro. (A) Schematic representation of Smad proteins with their conserved MH1, the DNA-binding β-hairpin loop (black boxes), MH2, and the less conserved linker sequences. The strength of Smad protein interaction with YY1 is quantified based on the data in panels B and C. (B) Endogenous YY1 from C2C12 cells interacts with the indicated GST-Smad fusions with different relative affinities (lanes 2 to 5). A total C2C12 lysate immunoblot is shown for comparison (lane 6). Ponceau S staining of the immunoblot shows the immobilized GST-Smads and GST protein. (C) YY1 interacts with the MH1 domain of Smad4. Experiment similar to those for panel B were performed with endogenous YY1 from HaCaT cells. Immobilized GST fusion proteins are shown in a Coomassie brilliant blue (CBB) stain of the gel. In panels B and C, arrows mark the specific protein bands.

Article Snippet: Murine C2C12 myoblasts, murine mammary epithelial NMuMG cells, mink lung epithelial Mv1Lu cells, monkey kidney COS-7 cells, human embryonic kidney 293T cells, human mammary MDA-MB-468 cells, and human hepatoma HepG2 cells were obtained and cultured according to protocols from the American Type Culture Collection.

Techniques: In Vitro, Binding Assay, Western Blot, Comparison, Staining

Journal:

Article Title: Nuclear Factor YY1 Inhibits Transforming Growth Factor ?- and Bone Morphogenetic Protein-Induced Cell Differentiation

doi: 10.1128/MCB.23.13.4494-4510.2003

Figure Lengend Snippet: Endogenous YY1 restricts TGF-β and BMP signaling levels. (A and B) Antisense YY1 reverses the inhibitory effect of YY1 and increases the TGF-β1 responsiveness of a Smad-dependent gene promoter in Mv1Lu cells. (A) Induction of the 12xCAGA reporter by overnight stimulation with 0.5 ng of TGF-β1 per ml in the presence of a constant amount (0.5 μg) of sense YY1 (S-YY1) and increasing amounts (0.03, 0.1, 0.3, and 1.0 μg) of antisense YY1 (AS-YY1) plasmids. (B) Increased responsiveness of the 12xCAGA reporter to TGF-β1 (0.5 ng/ml) in the absence (empty bars) or presence (full bars) of antisense YY1 (0.3 μg DNA). (C) RNAi against endogenous YY1 dramatically enhances the inducibility of the 12xCAGA-luciferase reporter by overnight stimulation with 0.5 ng of TGF-β1 per ml of HaCaT cells transfected with mock or YY1 siRNA oligonucleotides. (D) RNAi against YY1 efficiently decreases endogenous YY1 protein levels in C2C12 cells. Immunoblotting with the indicated antibodies of cell extracts from C2C12 cells transfected with no (−), an unrelated (control), or different doses of (triangle) specific YY1 siRNA oligonucleotides. (E) RNAi against YY1 dramatically enhances the inducibility of the Id1-BRE2-luciferase reporter by overnight stimulation with 300 ng of BMP-7 per ml in C2C12 cells transfected with mock, control siRNA or YY1 siRNA oligonucleotides. (F) RNAi against endogenous YY1 enhances the TGF-β1-inducible levels of endogenous PAI-1 protein. Immunoblotting of HaCaT cell extracts transfected with no (mock), control siRNA, or specific anti-YY1 siRNA oligonucleotides and treated (+) or not (−) with 2.5 ng of TGF-β1 per ml for 4 h with the indicated antibodies is shown. The asterisk marks nonspecific protein bands. The numbers below the top two panels indicate densitometric values of the specific bands normalized over the corresponding α-tubulin bands and expressed relative to the control band of lane 1, which is set to 1. (G) RNAi against endogenous YY1 enhances the inducibility of the endogenous PAI-1 gene by TGF-β1. Real-time PCR analysis of human PAI-1 mRNA from HaCaT cells transfected with no (mock), control siRNA, or specific anti-YY1 siRNA oligonucleotides and treated (+) or not (−) with 5 ng of TGF-β1 per ml for 16 h is shown. The bar graph plots average expression values and standard deviations of PAI-1 mRNA levels normalized to control GAPDH mRNA levels. The data in panels A, B, C, and E are presented as in Fig. ​Fig.1D1D.

Article Snippet: Murine C2C12 myoblasts, murine mammary epithelial NMuMG cells, mink lung epithelial Mv1Lu cells, monkey kidney COS-7 cells, human embryonic kidney 293T cells, human mammary MDA-MB-468 cells, and human hepatoma HepG2 cells were obtained and cultured according to protocols from the American Type Culture Collection.

Techniques: Luciferase, Transfection, Western Blot, Control, Real-time Polymerase Chain Reaction, Expressing

Journal:

Article Title: Nuclear Factor YY1 Inhibits Transforming Growth Factor ?- and Bone Morphogenetic Protein-Induced Cell Differentiation

doi: 10.1128/MCB.23.13.4494-4510.2003

Figure Lengend Snippet: Ectopic expression of YY1 inhibits TGF-β- and BMP-induced transdifferentiation. (A) Top panels, YY1 inhibits mammary NMuMG cell EMT induced by TGF-β1. NMuMG cells were infected with Ad-GFP (MOI, 25) or Ad-YY1 (MOI, 10) adenovirus, treated for 24 h with vehicle (control) or with 2.5 ng of TGF-β1 per ml, and subjected to actin direct fluorescence microscopy. Bottom panels, YY1 inhibits NMuMG EMT induced by the TGF-β type I receptor. EMT induced by caALK-5 receptor (MOI, 60) is blocked by coinfection with the YY1 virus at an MOI of 20 (Ad-YY1) or with the Smad7 virus at an MOI of 20 (Ad-Smad7). Bar, 20 μm. (B) YY1 blocks BMP-7-induced preosteoblast differentiation of C2C12 myoblasts. In situ ALP staining of C2C12 cells treated with vehicle (control) or 300 ng of BMP-7 per ml for 3 days after infection with Ad-GFP (MOI, 20) or Ad-YY1 (MOI, 4) virus is shown. Bar, 80 μm.

Article Snippet: Murine C2C12 myoblasts, murine mammary epithelial NMuMG cells, mink lung epithelial Mv1Lu cells, monkey kidney COS-7 cells, human embryonic kidney 293T cells, human mammary MDA-MB-468 cells, and human hepatoma HepG2 cells were obtained and cultured according to protocols from the American Type Culture Collection.

Techniques: Expressing, Infection, Control, Fluorescence, Microscopy, Virus, In Situ, Staining

Journal:

Article Title: Nuclear Factor YY1 Inhibits Transforming Growth Factor ?- and Bone Morphogenetic Protein-Induced Cell Differentiation

doi: 10.1128/MCB.23.13.4494-4510.2003

Figure Lengend Snippet: Smad-dependent myoblast-to-preosteoblast differentiation is regulated by YY1. (A) ALP staining of C2C12 cells transiently transfected with the indicated plasmids for caALK-6, Smad1, Smad4, and sense and antisense (AS) YY1. Representative photomicrographs are shown. Bar, 80 μm. (B) Bar graph of the C2C12 differentiation data from panel A, expressing the relative number of ALP-positive cells compared to the total number of cells counted in the whole transfected culture. Values are averages from duplicate cell cultures with standard errors. The percentage of ALP-positive cells detected after cotransfection with caALK-6, Smad1, and Smad4 was set at 100% (dashed line), relative to which all other conditions are plotted. Triangles represent 0.01, 0.1, and 0.3 μg of transfected YY1 and 0.1 and 0.3 μg of AS-YY1 plasmid DNA. (C) RNAi against endogenous YY1 enhances the preosteoblastic differentiation of C2C12 cells in response to BMP Smads. ALP enzymatic activity measured in cell extracts of transfected C2C12 cells with the indicated expression vectors in the absence (−) or presence (+) of YY1 siRNA oligonucleotides is shown. Average values of triplicate transfected-extract measurements with standard errors are expressed as nanomoles of released p-nitrophenol (p-NP) per minute of reaction per milligram of total protein in the extract. (D) C2C12 preosteoblastic differentiation is not altered by YY1 mutants that fail to interact with Smads. ALP enzymatic activity measured in transfected C2C12 cells with the indicated expression vectors is shown. The data are expressed as in panel C. The triangle represents 0.03, 0.1, 0.3, and 0.5 μg of transfected YY1 plasmid DNA, and the YY1 deletion mutants (0.1 μg each) are listed as in Fig. ​Fig.4.4. The dashed line corresponds to the specific activity of the receptor-plus-Smad condition.

Article Snippet: Murine C2C12 myoblasts, murine mammary epithelial NMuMG cells, mink lung epithelial Mv1Lu cells, monkey kidney COS-7 cells, human embryonic kidney 293T cells, human mammary MDA-MB-468 cells, and human hepatoma HepG2 cells were obtained and cultured according to protocols from the American Type Culture Collection.

Techniques: Staining, Transfection, Expressing, Cotransfection, Plasmid Preparation, Activity Assay