Journal: Clinical and Experimental Immunology

Article Title: Low frequency, weak MCP‐1 secretion and exhausted immune status of peripheral monocytes were associated with progression of severe enterovirus A71‐infected hand, foot and mouth disease

doi: 10.1111/cei.13267

Figure Lengend Snippet: The expression levels of monocyte chemoattractant protein‐1 (MCP‐1), tumor necrosis factor (TNF)‐α, interleukin (IL)‐6, IL‐8, macrophage inflammatory protein (MIP)‐1α, MIP‐1β and IL‐1β in supernatant of stimulated monocytes were detected by enzyme‐linked immunosorbent assay (ELISA); 1 × 105 untouched monocytes of mild (n = 20) and severe (n = 20) cases were seeded in each well of 96‐well plates and stimulated with 20 ng/ml granulocyte–macrophage colony‐stimulating factor (GM‐CSF), GM‐CSF + lipopolysaccharide (LPS) (1 μg/ml), GM‐CSF + R848 (10 μg/ml) and GM‐CSF + enterovirus A71 (EV‐A71) [0·5 multiplicity of infection (MOI)/200 μl] for 48 h. The mean values and the standard deviation are indicated. Differences between groups were assessed using the non‐parametric Wilcoxon matched‐pairs test.

Article Snippet: Cells were stimulated with 20 ng/ml granulocyte–macrophage colony‐stimulating factor (GM‐CSF) (Invivogen, San Diego, CA, USA), GM‐CSF + lipopolysaccharides (LPS, TLR‐4 agonist) (1 μg/ml; Invivogen), GM‐CSF + R848 (Resiquimod; TLR‐7/8 agonist) (10 μg/ml; Invivogen), GM‐CSF + EV‐A71 (0·5 MOI/200 μl) for 48 h. Culture supernatants were collected for cytokine detection.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Infection, Standard Deviation

Journal: Clinical and Experimental Immunology

Article Title: Low frequency, weak MCP‐1 secretion and exhausted immune status of peripheral monocytes were associated with progression of severe enterovirus A71‐infected hand, foot and mouth disease

doi: 10.1111/cei.13267

Figure Lengend Snippet: Programmed death 1 ligand (PD‐L1) blockade stimulated the production of monocyte chemoattractant protein‐1 (MCP‐1) from activated peripheral blood mononuclear cells (PBMCs). (a,b) PBMCs co‐cultured with rhabdomyosarcoma (RD) cells [enterovirus A71 (EV‐A71+)] in the Transwell system. (a) Histograms showing representative results for enzyme‐linked immunosorbent assay (ELISA) antigen determination for MCP‐1, interleukin (IL)‐6, IFN‐γ and IL‐1β in supernatants of co‐cultured PBMCs and RD (EV‐A71 infected for 2 h) for 12 h. Samples of Transwell are shown as a schematic diagram in the right side of (b). (b) Histograms showing representative results for EV‐A71 replication influenced by anti‐PD‐L1 blockade, isotype antibody (negative control) and medium (blank). (c) PD‐L1 blockade was shown to increase intracellular MCP‐1 production of CD14+ monocytes in PBMCs activated by R848 or EV‐A71 virus; 2·5 × 106 PBMCs were settled in 96‐well plates with R10 medium and incubated with granulocyte–macrophage colony‐stimulating factor (GM‐CSF) + R848 + anti‐PD‐L1 antibody [or mouse immunoglobulin (Ig)G1κ isotype antibody] or GM‐CSF + EV‐A71 + anti‐PD‐L1 antibody (or isotype antibody) for 6 h. Cells performed intracellular cytokine staining with the following monoclonal antibodies: CD3‐AF700, CD14‐ef450 and MCP‐1‐PE. U.D. = undetectable. The experiments were repeated for four times. Differences between groups were assessed using the non‐parametric Wilcoxon matched‐pairs test.

Article Snippet: Cells were stimulated with 20 ng/ml granulocyte–macrophage colony‐stimulating factor (GM‐CSF) (Invivogen, San Diego, CA, USA), GM‐CSF + lipopolysaccharides (LPS, TLR‐4 agonist) (1 μg/ml; Invivogen), GM‐CSF + R848 (Resiquimod; TLR‐7/8 agonist) (10 μg/ml; Invivogen), GM‐CSF + EV‐A71 (0·5 MOI/200 μl) for 48 h. Culture supernatants were collected for cytokine detection.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Infection, Negative Control, Virus, Incubation, Staining

Journal: Clinical and Experimental Immunology

Article Title: Low frequency, weak MCP‐1 secretion and exhausted immune status of peripheral monocytes were associated with progression of severe enterovirus A71‐infected hand, foot and mouth disease

doi: 10.1111/cei.13267

Figure Lengend Snippet: Untouched monocytes were negatively isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors and treated by anti‐programmed death ligand 1 (PD‐L1) monoclonal antibody (MIH1) or mouse immunoglobulin (Ig)G1κ isotype control antibodies for 6 h. The monocytes were co‐cultured with 20 ng/ml granulocyte–macrophage colony‐stimulating factor (GM‐CSF) and 0·5 multiplicity of infection (MOI) enterovirus A71 (EV‐A71) or 10 µg/ml R848 into 96‐well tissue culture plates for 3 days and the culture supernatants were collected for monocyte chemoattractant protein‐1 (MCP‐1) detection. Differences between groups were assessed using the non‐parametric Wilcoxon matched‐pairs test.

Article Snippet: Cells were stimulated with 20 ng/ml granulocyte–macrophage colony‐stimulating factor (GM‐CSF) (Invivogen, San Diego, CA, USA), GM‐CSF + lipopolysaccharides (LPS, TLR‐4 agonist) (1 μg/ml; Invivogen), GM‐CSF + R848 (Resiquimod; TLR‐7/8 agonist) (10 μg/ml; Invivogen), GM‐CSF + EV‐A71 (0·5 MOI/200 μl) for 48 h. Culture supernatants were collected for cytokine detection.

Techniques: Isolation, Cell Culture, Infection

Journal: Oncology Letters

Article Title: SPAG6 silencing induces autophagic cell death in SKM-1 cells via the AMPK/mTOR/ULK1 signaling pathway

doi: 10.3892/ol.2020.11607

Figure Lengend Snippet: Inhibiting autophagy with autophagy inhibitors attenuates SPAG6 knockdown-induced apoptosis. Cells were treated with SPAG6-shRNA lentivirus or NC-shRNA lentivirus alone, or co-treated with 3-MA or CQ and SPAG6-shRNA. (A) Relative protein expression levels of SPAG6, LC3, Beclin1 and P62 in each group were detected via western blot analysis. (B) Transmission electron microscopy revealed the intracellular ultrastructures of each group (magnification, ×20,000). The images display a representative experiment from three independent experiments. Red arrows indicate autophagic vacuoles. Scale bar, 1 µm. (C) Number of autophagosomes per field was quantified. (D) Relative protein expression levels of SPAG6, Bcl-2, Bax and cleaved caspase-3 in each group were detected using western blot analysis. (E) Following annexin V APC/DAPI double-staining, the total apoptosis rate of SKM-1 cells treated with NC-shRNA or SPAG6-shRNA alone and SKM-1 cells co-treated with SPAG6-shRNA and 3-MA or CQ was detected using flow cytometry. The images display a representative experiment from three independent experiments. (F) Analysis of the apoptotic rate shown in (E) Data are presented as the mean ± standard deviation of three independent experiments. *P<0.05; **P<0.01 vs. NC-shRNA, # P<0.05; ## P<0.01 vs. SPAG6-shRNA. NC, negative control; shRNA, short hairpin RNA; SPAG6, sperm-associated antigen 6; LC3, microtubule-associated protein 1 light chain 3; Beclin1, autophagy-associated protein 6; p62, sequestosome 1; CQ, chloroquine; 3-MA, 3-methyladenine; APC, allophycocyanin; LL, lower left; UL, upper left; LR, lower right; UR, upper right; ns, not significant.

Article Snippet: Cells were grown in complete medium with 3-methyladenine (3-MA; 5 mM/l), chloroquine (CQ; 10 μM/l) or the AMPK inhibitor Compound C (5 μM/l), purchased from Selleck Chemicals and all cells were incubated at 37°C with 5% CO 2 .

Techniques: Knockdown, shRNA, Expressing, Western Blot, Transmission Assay, Electron Microscopy, Double Staining, Flow Cytometry, Standard Deviation, Negative Control

Journal: Development & Reproduction

Article Title: Regulation of Transcriptional Coactivator with PDZ-Binding Motif (TAZ) Expression by Estrogen in the Mouse Uterine Endometrium

doi: 10.12717/DR.2025.29.2.31

Figure Lengend Snippet: (A, B) OVX mice were treated with an estrogen receptor selective antagonist ICI 182,780 (ICI, 500 μg/mouse) 30 min before estrogen (E 2 , 200 ng/mouse) injection and sacrificed at 6 h after E 2 administration. TAZ expression was analyzed by western blot analysis. The relative protein intensity of TAZ in western blot was analyzed by the ImageJ program. Data were shown with mean±SEM. The one-way ANOVA analysis and Tukey’s test were used to calculate the p-value, ** p <0.01, *** p <0.001. (C) Immunofluorescence images showed the localization and expression of TAZ (red) in uteri of OVX mice treated with oil, E 2 , or a combination of ICI and E 2 . Normal rabbit IgG (IgG control) was used as a negative control. DAPI (blue) was used for nuclei staining. The scale bars indicate 20 μm. Taz , transcriptional coactivator with PDZ-binding motif; LE, luminal epithelium; GE, glandular epithelium; OVX, ovariectomized.

Article Snippet: To determine whether the expression of TAZ in the mouse uterus is dependent on estrogen receptors, an estrogen receptor antagonist ICI 182,780 (500 μg/mouse, Medchemexpress, Princeton, NJ, USA) was pretreated 30 min before estrogen treatment ( ).

Techniques: Injection, Expressing, Western Blot, Immunofluorescence, Control, Negative Control, Staining, Binding Assay

Journal: PLoS ONE

Article Title: Characterization of a Novel Small Molecule Subtype Specific Estrogen-Related Receptor α Antagonist in MCF-7 Breast Cancer Cells

doi: 10.1371/journal.pone.0005624

Figure Lengend Snippet: Short hairpin plasmids expressing short hairpin RNA (shRNA) specific for knocking-down ERRα were transfected separately into MCF-7 cells. In addition, a shRNA expressing a scrambled artificial non-specific sequence was transfected. (-) control. (A) MCF-7/shRNA ERRα3 cells underwent four rounds (I–IV) of fluorescent activated cell sorting (FACS) to enrich for GFP expressing cells. FACS is described in Materials and Methods . (B) ERRα ( ESRRA ), ACADM , and PGC-1α ( PPARGC1A ) mRNA expression was measured by real-time RT-PCR. After 4 rounds of FACS, MCF-7/shRNA ERRα3 cells ERRα ( ESRRA ) mRNA levels were significantly reduced by 79%, ACADM levels by 75%, and PGC-1α ( PPARGC1A ) by 71% (*, P<0.05). Differences in relative mRNA expression between MCF-7/shRNA (-) and MCF-7/shRNA ERRα3 were measured by ANOVA followed by a student t-test with a 0.05 significance level. (C) MCF-7, MCF-7/shRNA (-), MCF-7/shRNA ERRα2, and MCF-7/shRNA ERRα3 cells ERRα protein expression was measured by Western blot, equal loading of protein was assessed by Coomassie blue staining of gels, and densitometric quantification are described in Materials and Methods . Results shown are representative of three independent experiments. MCF-7/shRNA ERRα2 exhibited 59% less protein verses the negative control, while MCF-7/shRNA ERRα3 cells ERRα protein levels were reduced by 69%. (D) ERα ( ESR1 ), aromatase ( CYP19A1 ), osteopontin ( SPP1 ), and pS2 ( TFF1 ) mRNA expression was measured in MCF-7/shRNA (-), MCF-7/shRNA ERRα2, and MCF-7/shRNA ERRα3 cells by real-time RT-PCR after 4 rounds of FACS. Knocking-down ERRα by shRNA (RNAi) led to significant decrease (*, P<0.05) in expression of ERRα target genes aromatase ( CYP19A1 ), osteopontin ( SPP1 ), and pS2 ( TFF1 ) while ERα ( ESR1 ) levels were not affected. All real-time RT-PCR results are representative of three independent experiments performed in triplicate.

Article Snippet: Primer/probe sets for target genes: human ERRα ( ESRRA ) (Hs00607062_gH), human ERα ( ESR1 ) (Hs00174860_m1), human PGC-1α ( PPARGC1A ) (Hs00173304_m1), human PDK4 ( PDK4 ) (Hs00176875_m1), human osteopontin ( SPP1 ) (Hs00959010_m1), human pS2 ( TFF1 ) (Hs00170216_m1), human ACADM ( ACADM ) (Hs00163494_m1) and 18S rRNA endogenous control (4308329) were purchased from Applied Biosystems.

Techniques: Expressing, shRNA, Transfection, Sequencing, Control, FACS, Quantitative RT-PCR, Western Blot, Staining, Negative Control

Journal:

Article Title: Potent antiarthritic properties of a glucocorticoid derivative, NCX-1015, in an experimental model of arthritis

doi: 10.1073/pnas.022641099

Figure Lengend Snippet: Modulation of CIA in the rat by prednisolone and the GC derivatives. (A) Chemical structure of prednisolone and the chains added to position 21 to yield either NCX-1015 or NCX-1016. (B–D) Rats were treated daily i.p. with NCX-1015 (▴, 4 μmol/kg; ▵, 0.4 μmol/kg), prednisolone (●, 4 μmol/kg; ○, 0.4 μmol/kg), NCX-1016 (♦, 4 μmol/kg) or vehicle (■, 100 μl peanut oil) from day 12 to day 18 after collagen II injection. A group of naïve rats (□) also was used. During the development of the arthritis, plethysmometry was used to assess paw volume (B), and rats' paws and ankles were individually assessed and scored (C), as described in Materials and Methods. After killing the animals (day 18), anklebone thickness was measured with calipers (D). Data are expressed as means ± SEM (n = 10 per group). *, P ≤ 0.05 vs. vehicle-treated group and #, P ≤ 0.05 vs. naïve animals.

Article Snippet: The negative control compound, deprived of the nitrooxy group, NCX-1016 (Mw 494.6) was synthesized at NicOx laboratories in Milan, Italy, as follows: prednisolone (33.3 mmol in tetrahydrofurane) was added to 49.9 mmol of 4-(hydroxymethyl)benzoyl chloride and triethylamine and stirred for 24 h at room temperature.

Techniques: Injection

Journal:

Article Title: Potent antiarthritic properties of a glucocorticoid derivative, NCX-1015, in an experimental model of arthritis

doi: 10.1073/pnas.022641099

Figure Lengend Snippet: Effect of CIA and GC treatment on serum levels of cytokines and pyridinoline. (A–C) Prednisolone (Pred), NCX-1015, NCX-1016, or vehicle (V) treatment was the same as in Fig. ​Fig.11 (i.p. from day 12 at the indicated doses in μmol/kg), whereas in D, drugs or vehicle (V) were given orally. A group of naïve rats also was used. (A) IL-1β, (B) IL-6 and (C and D) pyridinoline concentration as measured by using specific ELISA. Data are expressed as means ± SEM (n = 10 per group). *, P ≤ 0.05 vs. vehicle-treated group and #, P ≤ 0.05 vs. naïve animals.

Article Snippet: The negative control compound, deprived of the nitrooxy group, NCX-1016 (Mw 494.6) was synthesized at NicOx laboratories in Milan, Italy, as follows: prednisolone (33.3 mmol in tetrahydrofurane) was added to 49.9 mmol of 4-(hydroxymethyl)benzoyl chloride and triethylamine and stirred for 24 h at room temperature.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Potent antiarthritic properties of a glucocorticoid derivative, NCX-1015, in an experimental model of arthritis

doi: 10.1073/pnas.022641099

Figure Lengend Snippet: Bone resorption measured in rat primary osteoclasts. A mixed population of rat bone cells was incubated overnight with prednisolone (Pred), NCX-1015, NCX-1016, or vehicle (V). Osteoclast activity was measured as described in Materials and Methods. (A) Osteoclast bone resorptive activity after 18-h incubation with increasing concentrations of prednisolone. *, P < 0.05 vs. vehicle (dose 0). (B) NCX-1016 mimicked prednisolone effect, whereas NCX-1015 was inactive. *, P < 0.05 vs. vehicle-treated cells. (C) Addition of the NO-donor NOC-18 (1 μM) to 1 nM prednisolone or NCX-1016 abolished the increase in bone resorption. (D) In the presence of the guanylate cyclase inhibitor (ODQ, 5 μM), 1 nM NCX-1015 stimulated osteoclast activity. All results are the means ± SEM of at least three to four experiments performed in triplicate.

Article Snippet: The negative control compound, deprived of the nitrooxy group, NCX-1016 (Mw 494.6) was synthesized at NicOx laboratories in Milan, Italy, as follows: prednisolone (33.3 mmol in tetrahydrofurane) was added to 49.9 mmol of 4-(hydroxymethyl)benzoyl chloride and triethylamine and stirred for 24 h at room temperature.

Techniques: Incubation, Activity Assay