Journal: Redox Biology

Article Title: A role for peroxiredoxins in H 2 O 2 - and MEKK-dependent activation of the p38 signaling pathway

doi: 10.1016/j.redox.2019.101340

Figure Lengend Snippet: Mammalian MEKK4 is redox-sensitive and interacts with cytosolic peroxiredoxin Prx2 in response to H 2 O 2 . ( A, A ′) A tagged version of Prx2 (Prx2-SBP) was expressed in HEK293T cells. Cells were treated with H 2 O 2 (100 μM) and lysed at indicated time points. Prx2-SBP was affinity-purified using streptavidin beads. Precipitates ( A ) and whole cell lysates (WCL) ( A ′) were analyzed by SDS-PAGE under reducing (R) and non-reducing (NR) conditions followed by immunoblotting (IB). The immunoblots are representative of 3 independent experiments (n = 3). ( B, B′ ) Complementary affinity purification experiment: A tagged version of MEKK4 (MEKK4-SBP) was expressed in HEK293T cells. Cells were treated with H 2 O 2 (100 μM) and lysed at indicated time points. MEKK4-SBP was affinity-purified using streptavidin beads. Precipitates ( B ) and WCL ( B ′) were analyzed by SDS-PAGE under R and NR conditions followed by IB. e.v.: empty vector, PD: pull down, *: endogenous protein, X: unknown protein. The immunoblots are representative of 3 independent experiments (n = 3).

Article Snippet: Jafrac1 was detected with polyclonal goat anti-Prx2 antibody (R&D Systems #AF3489), Drosophila α -tubulin with a monoclonal mouse antibody (Hybridoma Bank AA4.3), Drosophila p-p38 (Thr180/Tyr182) with a monoclonal rabbit antibody (Cell Signaling Technology #4631), Drosophila p38 with a goat polyclonal antibody (Santa Cruz Biotechnology sc-15714) diluted 1:300, myc-tag with a rabbit monoclonal antibody (Cell Signaling Technology #2278), SBP-tag with a mouse monoclonal antibody (Santa Cruz Biotechnology sc-101595), human Prx2 with a rabbit monoclonal antibody (abcam #109367), MEKK4 with a mouse monoclonal antibody (sc-166196), human p-p38 with a mouse monoclonal antibody (BD #6122889) diluted 1:2000, human p38 with a rabbit polyclonal antibody (Cell Signaling Technology #9212) and human β-tubulin with a rabbit monoclonal antibody (Cell Signaling Technology #2128).

Techniques: Affinity Purification, SDS Page, Western Blot, Plasmid Preparation

Journal: Emerging Infectious Diseases

Article Title: Livestock Susceptibility to Infection with Middle East Respiratory Syndrome Coronavirus

doi: 10.3201/eid2302.161239

Figure Lengend Snippet: Presence of MERS-CoV receptor DPP4 (IHC) and of mucosubstances (PAS) in upper and lower respiratory tract tissues from sheep, pigs, llamas, and horses. A) In the nose, DPP4 (red cytoplasmic or membrane staining) was present on the lining epithelium of pigs, llamas, and horses but not sheep. PAS staining (magenta) demonstrated more mucous cells in the lining epithelium of sheep and horses and a layer of mucus on the lining epithelium of the horses. B) DPP4 (red cytoplasmic or membrane staining) was present on the lining epithelium of the trachea, bronchus/bronchioles, and alveoli in the pigs, llamas and horses but not in the sheep. Original magnification ×400 for all images. DPP4, dipeptidyl peptidase-4; IHC, immunohistochemistry; MERS-CoV, Middle East respiratory syndrome coronavirus; PAS, periodic acid–Schiff; term., terminal.

Article Snippet: In brief, we performed DPP4 IHC staining by using 5 μg/mL of polyclonal goat IgG anti-human DPP4 antibody (R&D Systems, Abingdon, UK) and peroxidase-labeled rabbit anti-goat IgG (1:200; DAKO; Agilent Technologies Company, Santa Clara, CA, USA) as a secondary antibody.

Techniques: Membrane, Staining, Immunohistochemistry

Journal: Human reproduction (Oxford, England)

Article Title: Interleukin 1 regulates its own receptors in human endometrial cells via distinct mechanisms.

doi: 10.1093/humrep/dep192

Figure Lengend Snippet: Figure 1 Effect of IL1B on mbIL1R, mbIL1R2 and IL1RA expression in KLE cells. Confluent KLE cell cultures were treated with 0.1 ng/ml IL1B for varying periods of time (0–24 h) (A) or with different concentrations of IL1B (0, 0.1, 1 and 10 ng/ml) for 12 h (B). Cells were recovered to evaluate IL1R1, IL1R2 and IL1RA protein expression in total cell protein extracts by western blot. a-Tubulin was also probed on the same membranes to ensure equal protein loading. The intensity of mbIL1Rs, IL1RA and corresponding a-tubulin bands was evaluated by densitometric analysis (C and D). Values were normalized to a-tubulin band intensity and expressed as % of control (ratio of normalized mbIL1R1, mbIL1R2 or IL1RA band intensity detected following treatment with IL1B to that detected following incubation with the control culture medium alone for an equivalent period of time). *P , 0.05 and **P , 0.01 as compared with control. Data are from three different experiments. MM, minimal medium; mb, membrane-bound; s, soluble.

Article Snippet: IL1R2 ELISA is based on the use of a mouse monoclonal anti-human IL1R2 antibody for capture and a goat polyclonal anti-human IL1R2 antibody for detection (R&D systems).

Techniques: Expressing, Western Blot, Control, Incubation, Membrane

Journal: Human reproduction (Oxford, England)

Article Title: Interleukin 1 regulates its own receptors in human endometrial cells via distinct mechanisms.

doi: 10.1093/humrep/dep192

Figure Lengend Snippet: Figure 3 Immunocytofluorescence of IL1R1, IL1R2 and IL1RA in KLE cells. Cells cultured in chamber slides were incubated for 24 h with the culture medium alone or containing 0.1 ng/ml IL1B. Detection of IL1R1 (A and C), IL1R2 (E and G) and IL1RA (I and K) was performed by immunocytochemical staining using specific monoclonal mouse antibodies. Note the increase in IL1R1 (C), IL1R2 (G) and IL1RA (K) immunofluorescent signal in KLE cells exposed to IL1B by comparison with non-stimulated cells (A, E and I, respectively). No immunofluorescence was observed in the absence of primary antibodies (B, F and J) or the presence of mouse IgGs (D, H and L) (controls). Data are representative of four different experiments.

Article Snippet: IL1R2 ELISA is based on the use of a mouse monoclonal anti-human IL1R2 antibody for capture and a goat polyclonal anti-human IL1R2 antibody for detection (R&D systems).

Techniques: Cell Culture, Incubation, Staining, Comparison

Journal: Human reproduction (Oxford, England)

Article Title: Interleukin 1 regulates its own receptors in human endometrial cells via distinct mechanisms.

doi: 10.1093/humrep/dep192

Figure Lengend Snippet: Figure 4 Effect of IL1B on IL1R1, IL1R2 and IL1RA mRNA expression in KLE cells. Confluent KLE cell cultures were treated with 0.1 ng/ml IL1B for varying periods of time (0–24 h) (A, B and C, respectively) or with different con- centrations of IL1B (0, 0.1, 1 and 10 ng/ml) for 12 h (D, E and F, respectively). Total RNA was extracted and reverse transcribed, IL1R1, IL1R2, IL1RA and GAPDH cDNAs were amplified by Real-Time PCR as described in Materials and Methods and IL1R1, IL1R2 and IL1RA mRNA levels were normalized to GAPDH mRNA levels. Data were expressed as % of control (ratio of IL1R1, IL1R2 or IL1RA mRNA levels found in cells incubated with IL1 to those found in cells incubated with the control basal culture medium for an equivalent period of time). *P , 0.05, **P , 0.01 and ***P , 0.001 as compared with control. Data are from three different experiments.

Article Snippet: IL1R2 ELISA is based on the use of a mouse monoclonal anti-human IL1R2 antibody for capture and a goat polyclonal anti-human IL1R2 antibody for detection (R&D systems).

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Control, Incubation

Journal: Human reproduction (Oxford, England)

Article Title: Interleukin 1 regulates its own receptors in human endometrial cells via distinct mechanisms.

doi: 10.1093/humrep/dep192

Figure Lengend Snippet: Figure 5 Effect of IL1B on IL1R1, IL1R2 and IL1RA mRNA stability and gene expression in KLE cells. Confluent KLE cell cultures were stimulated with IL1B (1 ng/mll) for 12 h. Actinomycin D (10 mg/ml) was added to stop the de novo RNA synthesis, and cells were harvested after 0, 2, 4 and 8 h of incubation with actinomycin D. Total RNA was extracted and reverse transcribed. cDNA was analyzed by real-time PCR with specific primers for IL1R1, IL1R2, IL1RA and GAPDH. Levels of IL1R1, IL1R2 and IL1RA mRNA were normalized to those of GAPDH to assess the kinetics of IL1R1 (A), IL1R2 (B) and IL1RA (C) mRNA degradation. Data were expressed as % of control (ratio of IL1R1, IL1R2 or IL1RA mRNA levels found at different periods of time following the arrest of de novo mRNA transcription to that found at the time of arrest); data are from three different experiments. To evaluate IL1R1, IL1R2 and IL1RA transcriptional activation (D), confluent KLE cell cultures were stimulated with IL1B (1 ng/ml) for 6 h, cell nuclei were isolated and nuclear mRNA transcription was analyzed by nuclear run-on as described in Materials and Methods. DNA samples immobilized onto nylon membranes were as follows: lane 1, IL1R1 cDNA; lane 2, IL1R2 cDNA; lane 3, IL1RA; lane 4, 28S cDNA; and lane 5, pBluescript plasmid DNA. Radioactive transcripts were from KLE cells stimulated in MM or with 1 ng/ml IL1B. Data are repre- sentative of four different experiments.

Article Snippet: IL1R2 ELISA is based on the use of a mouse monoclonal anti-human IL1R2 antibody for capture and a goat polyclonal anti-human IL1R2 antibody for detection (R&D systems).

Techniques: Gene Expression, Incubation, Reverse Transcription, Real-time Polymerase Chain Reaction, Control, Activation Assay, Isolation, Plasmid Preparation

Journal: Human reproduction (Oxford, England)

Article Title: Interleukin 1 regulates its own receptors in human endometrial cells via distinct mechanisms.

doi: 10.1093/humrep/dep192

Figure Lengend Snippet: Figure 6 Effect of IL1B on mbIL1R1, mbIL1R2 and IL1RA expression in endometrial epithelial cells. Confluent endometrial epithelial cell cultures were treated with different concentrations of IL1B (0, 0.1, 1 and 10 ng/ml) for 12 h. Cells were recov- ered to evaluate IL1R1, IL1R2 and IL1RA protein expression in total cell protein extracts by Western blot (A). a-Tubulin was probed on the same membranes to ensure equal protein loading. Culture supernatants were recovered to evaluate sIL1R2 release (C) and IL1RA secretion (D) by ELISA; data were from four different endometrial samples and expressed as % of control (ratio of sIL1R2 or IL1RA concentrations detected in the presence of IL1 to those detected in the control basal culture medium for an equivalent period of 12 h). *P , 0.05, as compared with control. The intensity of mbIL1Rs, IL1RA and corresponding a-tubulin bands was evaluated by densitometric analysis (B). Values were normalized to a-tubulin band intensity and expressed as % of control (ratio of normalized mbIL1R1, mbIL1R2 or IL1RA band intensity detected following treatment with IL1B to that detected following incubation with the control culture medium alone for an equivalent period of time). *P , 0.05 and **P , 0.01 as compared with control. Data were from three different endometrial samples. sIL1R1 and sIL1R2 release and IL1RA secretion from endometrial epithelial cell cultures was also analyzed by metabolic labeling with 35S-cysteine and immunoprecipitation using a goat anti-IL1R1, goat anti-IL1R2 or rabbit anti- IL1RA antibody (E). Immunoprecipitated proteins were then analyzed by SDS-PAGE as described in Materials and Methods (representative data from three different endometrial samples); mb, membrane-bound; s, soluble.

Article Snippet: IL1R2 ELISA is based on the use of a mouse monoclonal anti-human IL1R2 antibody for capture and a goat polyclonal anti-human IL1R2 antibody for detection (R&D systems).

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Control, Incubation, Labeling, Immunoprecipitation, SDS Page, Membrane

Journal: Human reproduction (Oxford, England)

Article Title: Interleukin 1 regulates its own receptors in human endometrial cells via distinct mechanisms.

doi: 10.1093/humrep/dep192

Figure Lengend Snippet: Figure 7 Effect of IL1B on IL1R1, IL1R2 and IL1RA mRNA expression in endometrial epithelial cells. Confluent cultures were treated with 0.1 ng/ml IL1B with different concentrations of IL1B (0, 0.1, 1 and 10 ng/ml) for 12 h. Total RNA was extracted and reverse transcribed, IL1R1, IL1R2, IL1RA and GAPDH cDNAs were amplified by Real-Time PCR as described in Materials and Methods and IL1R1 (A), IL1R2 (B) and IL1RA (C) mRNA levels were normalized to GAPDH mRNA levels. Data were from three different endometrial samples and expressed as % of control (ratio of IL1R1, IL1R2 or IL1RA mRNA levels found in cells incubated with IL1 to those found in cells incubated with the control basal culture medium for an equivalent period of 12 h). *P , 0.05 and **P , 0.01 as compared with control.

Article Snippet: IL1R2 ELISA is based on the use of a mouse monoclonal anti-human IL1R2 antibody for capture and a goat polyclonal anti-human IL1R2 antibody for detection (R&D systems).

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Control, Incubation

Journal: Journal of Inflammation (London, England)

Article Title: The PPAR-γ antagonist GW9662 elicits differentiation of M2c-like cells and upregulation of the MerTK/Gas6 axis: a key role for PPAR-γ in human macrophage polarization

doi: 10.1186/s12950-015-0081-4

Figure Lengend Snippet: GW9662 induces M2c-like cells that upregulate MerTK and its ligand Gas6. (A-C) Healthy monocytes were cultured in serum-free medium in the absence of cytokines or growth factors (M0 differentiation), with or without the PPAR-γ antagonist GW9662 (2.5-10 μM), for 4 days; when specified, the PPAR-γ agonist rosiglitazone (1 μM) was added. Expression of MerTK, CD163 and CD16 was measured by flow cytometry. (D-E) Gas6 production levels were quantified by ELISA in culture medium, upon incubation with or without GW9662 (2.5-10 μM) of otherwise untreated cells (M0 conditions), LPS (50 ng/ml; M1 conditions) or IL-4 (20 ng/ml; M2a conditions) exposed cells. (A-E) Pooled data are represented as mean values ± SEM. Analysis was performed using one-way repeated measures ANOVA with Newman-Keuls multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. When not specified by additional graphic signs, statistical annotations (asterisks) refer to comparisons with respect to the relative GW9662 untreated control group. Each set of data is representative of three independent experiments.

Article Snippet: Biotinylated goat polyclonal anti-human Gas6 antibody (R&D Systems), followed by HRP-conjugated streptavidin (Biolegend), was used for detection.

Techniques: Cell Culture, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Incubation, Control

Journal: Cell Death & Disease

Article Title: Downregulation of SREBP inhibits tumor growth and initiation by altering cellular metabolism in colon cancer

doi: 10.1038/s41419-018-0330-6

Figure Lengend Snippet: a , b The real-time PCR analysis of SREBP1 and SREBP2 expression in control and knockdown DLD1 ( a ) and Pt130 ( b ) cells. Two different shRNA targeting sequences were used for knocking down SREBP1 or SREBP2. c , d The expression of SREBP1 and SREBP2 proteins were downregulated in stable DLD1 ( c ) and Pt130 ( d ) knockdown cells. Protein lysates from control and knockdown cells were analyzed by western blot. Both the precursor and mature forms of SREBP1 and SREBP2 proteins were detected. e , f Knockdown of SREBP1 and SREBP2 decreased the expression of downstream genes related to fatty acid synthesis and metabolism in DLD1 ( e ) and Pt130 ( f ) cells. Data represent the mean ± SD (* p < 0.001 and # p < 0.05 compared to sh-Control). g Knockdown of SREBP1 or SREBP2 decreased lipogenesis in colon cancer cells. Total cellular lipids were extracted from control and SREBP knockdown DLD1 and Pt130 cells and the levels of free fatty acids, cholesterol, and triglyceride were measured and normalized to the amount of total proteins. Data represent the mean ± SD (* p < 0.001, § p < 0.01, and # p < 0.05 compared to sh-Control)

Article Snippet: The SREBP1 antibody was from BD Biosciences (CA, USA), whereas the SREBP2 antibody was from R&D Systems (MN, USA).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Control, Knockdown, shRNA, Western Blot

Journal: Cell Death & Disease

Article Title: Downregulation of SREBP inhibits tumor growth and initiation by altering cellular metabolism in colon cancer

doi: 10.1038/s41419-018-0330-6

Figure Lengend Snippet: a , b Knockdown of SREBP1 or SREBP2 decreased the rate of proliferation in DLD1 ( a ) and Pt130 ( b ) cells. Equal number of control and SREBP knockdown cells were allowed to grow for 4 days and the number of cells were counted each day. Data represent the mean ± SD ( § p < 0.01 and # p < 0.05 compared to sh-Control). c , d Knockdown of SREBP1 or SRBEP-2 decreased the formation of tumor spheroids in the stem cell suspension medium. Control and SREBP knockdown DLD1 ( c ) and Pt130 ( d ) cells were seeded as single cells in the stem cell suspension medium and the number of colonies formed was determined after 6 days. Data represent the mean ± SD ( ¶ p < 0.0001 and * p < 0.001 compared to sh-Control). e Knockdown of SREBP1 or SRBEP2 reduced the expression of genes associated with colon cancer stem cells. The relative expression of CD44 , CD133 , LGR5 , and AXIN2 mRNA was determined using real-time PCR in control and SREBP knockdown DLD1 and Pt130 cells. Data represent the mean ± SD ( § p < 0.01 and # p < 0.05 compared to sh-Control)

Article Snippet: The SREBP1 antibody was from BD Biosciences (CA, USA), whereas the SREBP2 antibody was from R&D Systems (MN, USA).

Techniques: Knockdown, Control, Suspension, Expressing, Real-time Polymerase Chain Reaction

Journal: Cell Death & Disease

Article Title: Downregulation of SREBP inhibits tumor growth and initiation by altering cellular metabolism in colon cancer

doi: 10.1038/s41419-018-0330-6

Figure Lengend Snippet: a Representative OCR measurements obtained from the Mito tress test performed in control (sh-Control) and SREBP knockdown (sh-SREBP1 and sh-SREBP2) Pt130 cells using the Seahorse XF96 Extracellular Flux analyzer. Oligomycin, FCCP, and antimycin A (Anti-A) were added at the indicated points. The shaded areas indicate the OCR levels associated with basal and maximal respiration, ATP turnover and reserved capacity, respectively. b Experiments as shown in a were quantified and the relative levels of OCR associated with basal and maximal respiration, ATP turnover and reserved capacity were calculated. Data represent the mean ± SEM ( ¶ p < 0.0001 and # p < 0.05 compared to Sh-Control). c Representative ECAR measurements obtained from the glycolysis stress test performed in control and SREBP knockdown Pt130 cells using the Seahorse XF96 Extracellular Flux analyzer. Glucose, oligomycin, and 2-deoxyglucose (2-DG) were added at the indicated points. The shaded areas indicate the ECAR levels associated with glycolysis, glycolytic capacity, and glycolytic reserve, respectively. d Experiments as shown in c were quantified and ECAR associated with glycolysis, glycolytic capacity, and glycolytic reserve was calculated. Data represent the mean ± SEM ( ¶ p < 0.0001 compared to Sh-Control). e Knockdown of SREBP resulted in metabolic shifts in Pt130 cells. The energetic map of control and SREBP knockdown Pt130 cells was based on the measurements obtained in a and c . The OCR and ECAR data shown in the map represent the OCR of basal respiration from the Mito stress test and the glycolysis-related ECAR from the glycolysis stress test, respectively (data represent the mean ± SEM)

Article Snippet: The SREBP1 antibody was from BD Biosciences (CA, USA), whereas the SREBP2 antibody was from R&D Systems (MN, USA).

Techniques: Control, Knockdown

Journal: Cell Death & Disease

Article Title: Downregulation of SREBP inhibits tumor growth and initiation by altering cellular metabolism in colon cancer

doi: 10.1038/s41419-018-0330-6

Figure Lengend Snippet: a , b Knockdown of SREBP1 and SREBP2 reduced fatty acid oxidation (FAO) in DLD1 ( a ) and Pt130 ( b ) cells. The rate of cellular FAO was determined by subjecting control and SREBP knockdown cells to the FAO assays using the Seahorse XF96 Extracellular Flux analyzer. Data represent the mean ± SEM ( § p < 0.01 compared to Sh-Control). c Knockdown of SCAP reduced FAO in DLD1 and Pt130 cells. The rate of cellular FAO was determined using Seahorse FAO assays. Data represent the mean ± SEM ( # p < 0.05, compared to Sh-Control)

Article Snippet: The SREBP1 antibody was from BD Biosciences (CA, USA), whereas the SREBP2 antibody was from R&D Systems (MN, USA).

Techniques: Knockdown, Control

Journal: Cell Death & Disease

Article Title: Downregulation of SREBP inhibits tumor growth and initiation by altering cellular metabolism in colon cancer

doi: 10.1038/s41419-018-0330-6

Figure Lengend Snippet: a Control, SREBP1, and SREBP2 knockdown Pt130 cells were injected subcutaneously into NSG mice. The size of the tumors was measured every 3 days starting at day 14. Data represent the mean ± SEM ( ¶ p < 0.0001 compared to Sh-Control group). b On day 29, tumors were excised and weighted. Data represent the mean ± SEM ( ¶ p < 0.0001 and § p < 0.01 compared to Sh-Control group). c Detection of proliferating cells in tumors with IHC staining using the anti-Ki67 antibody. Scale bar, 25 μm. d Numbers of Ki67-positive cells were quantified in 500 randomly chosen tumor cells. Data in the graph represent the mean ± SD ( ¶ p < 0.0001 compared to Sh-Control group). e Detection of apoptotic cells in tumors with IHC staining using the anti-cleaved caspase-3 antibody. Scale bar, 25 μm. f Numbers of cleaved caspase-3-positive cells were quantified in 500 randomly chosen tumor cells. Data in the graph represent the mean ± SD ( ¶ p < 0.0001 compared to Sh-Control group). g Knockdown of SREBP reduced the expression of genes associated with colon cancer stem cells. The relative expression of CD44 , CD133 , LGR5 , and AXIN2 mRNA was determined using real-time PCR in tumors derived from control, SREBP1 and SREBP2 knockdown Pt130 cells. Data represent the mean ± SD (* p < 0.001, § p < 0.01, and # p < 0.05 compared to Sh-Control group). h Tumor initiation experiments were performed using control, SREBP1, and SREBP2 knockdown Pt130 cells. The cells were mixed with Matrigel and subcutaneously inoculated into NSG mice at 1000 cells per site and total of eight injections were used for each cell line. The number of tumors formed was determined 3 months post inoculation.

Article Snippet: The SREBP1 antibody was from BD Biosciences (CA, USA), whereas the SREBP2 antibody was from R&D Systems (MN, USA).

Techniques: Control, Knockdown, Injection, Immunohistochemistry, Expressing, Real-time Polymerase Chain Reaction, Derivative Assay