Journal: Experimental and therapeutic medicine

Article Title: Blood purification treatment initiated at the time of sepsis diagnosis effectively attenuates serum HMGB1 upregulation and improves patient prognosis.

doi: 10.3892/etm.2017.4854

Figure Lengend Snippet: Figure 1. Serum and urine HMGB1 levels upon diagnosis of sepsis. (A) Serum and (B) urine levels of HMGB1 were measured by ELISA in the different patient groups prior to the initiation of treatment. Data are presented as the mean ± standard deviation. #P<0.05 vs. Control. HMGB1, high mobility group box protein 1; CRRT, continuous renal replacement treatment; HP, hemoperfusion.

Article Snippet: Serum and urine HMGB1 levels were measured using a Human HMGB1 ELISA kit (E-EL-H1554c, Elabscience Biotechnology Co., Ltd., Wuhan, China) according to the manufacturer's protocol.

Techniques: Biomarker Discovery, Enzyme-linked Immunosorbent Assay, Standard Deviation, Control

Journal: Experimental and therapeutic medicine

Article Title: Blood purification treatment initiated at the time of sepsis diagnosis effectively attenuates serum HMGB1 upregulation and improves patient prognosis.

doi: 10.3892/etm.2017.4854

Figure Lengend Snippet: Figure 2. Changes in serum and urine HMGB1 levels over 24 h. (A) Serum and (B) urine levels of HMGB1 in the sepsis (no apheresis intervention) group at different time points. Data are presented as the mean ± standard deviation. #P<0.05 vs. 0‑h value. HMGB1, high mobility group box protein 1.

Article Snippet: Serum and urine HMGB1 levels were measured using a Human HMGB1 ELISA kit (E-EL-H1554c, Elabscience Biotechnology Co., Ltd., Wuhan, China) according to the manufacturer's protocol.

Techniques: Standard Deviation

Journal: Experimental and therapeutic medicine

Article Title: Blood purification treatment initiated at the time of sepsis diagnosis effectively attenuates serum HMGB1 upregulation and improves patient prognosis.

doi: 10.3892/etm.2017.4854

Figure Lengend Snippet: Figure 3. Association of serum HMGB1 level with urine HMGB1 level and APACHE II score. Correlations were identified between (A) urine and serum HMGB1 levels, and (B) APACHE II score and serum HMGB1 level in the sepsis (no apheresis intervention) group. HMGB1, high mobility group box protein 1; APACHE II, Acute Physiology and Chronic Health Evaluation II.

Article Snippet: Serum and urine HMGB1 levels were measured using a Human HMGB1 ELISA kit (E-EL-H1554c, Elabscience Biotechnology Co., Ltd., Wuhan, China) according to the manufacturer's protocol.

Techniques:

Journal: Experimental and therapeutic medicine

Article Title: Blood purification treatment initiated at the time of sepsis diagnosis effectively attenuates serum HMGB1 upregulation and improves patient prognosis.

doi: 10.3892/etm.2017.4854

Figure Lengend Snippet: Figure 4. Changes in serum and urine HMGB1 levels in the different patient groups over 24 h. (A) Serum and (B) urine levels of HMGB1 were measured by ELISA at different time points after the initiation of treatments. Data are presented as the mean ± standard deviation. #P<0.05 vs. 0‑h value; *P<0.05 vs. sepsis group; ^P<0.05 vs. 12‑h value. HMGB1, high mobility group box protein 1; CRRT, continuous renal replacement treatment; HP, hemoperfusion.

Article Snippet: Serum and urine HMGB1 levels were measured using a Human HMGB1 ELISA kit (E-EL-H1554c, Elabscience Biotechnology Co., Ltd., Wuhan, China) according to the manufacturer's protocol.

Techniques: Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: British journal of pharmacology

Article Title: Veklury® (remdesivir) formulations inhibit initial membrane-coupled events of SARS-CoV-2 infection due to their sulfobutylether-β-cyclodextrin content.

doi: 10.1111/bph.16063

Figure Lengend Snippet: FIGURE 2 Methyl-β cyclodextrin (MβCD)-induced reductions in ACE2 binding of Wuhan-Hu-1 strain of SARS-CoV-2 (WT) and Delta SARS- CoV-2 spike receptor-binding domains (RBDs) negatively correlate with the applied RBD concentrations. HEK/ACE2 + TMPRSS2 cells were treated with different concentrations of MβCD ranging between 0.25 and 10 mM. Fluorescence intensities of cells were measured using flow cytometry for 5 min continuously starting immediately after the addition of GFP-conjugated WT (a) or Delta (b) SARS-CoV-2 spike RBDs applied at various concentrations between 0.1 and 5 μgml1, or 0.05 and 2.5 μgml1, respectively. Moving averages of time-correlated fluorescence intensities of at least 50,000 cells of normal morphology were determined with a window size of 10 s and subsequently normalized to the average of intensities obtained in the last time window of untreated control samples. Mean ± SD values were calculated from data of six independent samples per treatment condition and plotted as a function of time (starting 20 s after RBD addition). The extents of inhibition of WT (a) and Delta (b) RBD binding at the end of the measurement time in response to various MβCD concentrations were calculated and plotted as mean ± SD as a function of the applied RBD concentration in the right panels. Asterisks indicate significant differences of samples treated with the lowest versus highest RBD concentrations at each applied CD concentration (*P < 0.05; two-way ANOVA followed by Tukey's HSD test). See details of statistical analysis in Table S2.

Article Snippet: When using Omicron spike trimer conjugated with Alexa Fluor 488 (R&D Systems; AFG11060), experiments were repeated after SBECD, Veklury® P or Veklury® S treatments in HEK/ACE2 + TMPRSS2 and Calu-3 cells using the trimer at 0.5 and 2.5 μg ml 1, respectively.

Techniques: Binding Assay, Fluorescence, Flow Cytometry, Control, Inhibition, Concentration Assay

Journal: British journal of pharmacology

Article Title: Veklury® (remdesivir) formulations inhibit initial membrane-coupled events of SARS-CoV-2 infection due to their sulfobutylether-β-cyclodextrin content.

doi: 10.1111/bph.16063

Figure Lengend Snippet: FIGURE 6 Veklury® (remdesivir) formulations effectively decrease ACE2 binding of Omicron SARS-CoV-2 spike receptor-binding domain (RBD) and the cellular uptake of Omicron SARS-CoV-2 spike trimer due to their SBECD content. (a) HEK/ACE2 + TMPRSS2 (left panel) or Calu-3 cells (right panel) were treated with SBECD, Veklury® P (powder) or Veklury® S (solution) at CD concentrations of 1, 5 and 10 mM. Then, HEK/ ACE2 + TMPRSS2 cells were incubated in the presence of 0.2-μgml1 Omicron SARS-CoV-2 spike RBD–GFP, whereas Calu-3 cells were labelled with 1-μgml1 Omicron RBD–GFP for 4 min. The emitted fluorescence intensities of individual cells were subsequently measured using flow cytometry, and the mean intensity was calculated from data of at least 10,000 cells of normal morphology per sample. The average values of six independent measurements (±SD) were calculated, normalized to the mean value determined in untreated control samples and plotted as a function of the applied concentrations of CD. Asterisks indicate significant differences of samples treated with the highest applied concentrations compared to the control samples (*P < 0.05; two-way ANOVA followed by Tukey's HSD test). See details of statistical analysis in Table S6. (b) HEK/ACE2 + TMPRSS2 (left panel) or Calu-3 cells (right panel) treated with SBECD, Veklury® P or Veklury® S at CD concentrations of 1 and 5 mM for 1 h were subsequently incubated for 4 h in the presence of Omicron SARS-CoV-2 spike trimers conjugated with Alexa Fluor 488. The average fluorescence intensity values emitted by Alexa Fluor 488-labelled trimers were calculated using data of intracellular pixels identified using the membrane marker N-[3-(40-dihexylamino-3-hydroxy-flavonyl-6-oxy)-propyl]N,N-dimethyl-N-(3-sulfopropyl)-ammonium inner salt (F66) as described in Figure 5 for individual cells, which were subsequently normalized to the median value determined in untreated control samples. The number of cells obtained from five independent experiments and involved in the analysis was from left to right: 382, 348, 373, 396, 388, 402 and 393 for Omicron trimer uptake in HEK/ACE2 + TMPRSS2 and 308, 250, 256, 223, 222, 232 and 296 for Omicron trimer uptake in Calu-3. Data points indicated in the figure were obtained from individual cells and plotted along with median values with quartiles. Grey shaded areas show ranges between 0.5 and 1. Asterisks indicate significant differences compared to the control samples (*P < 0.05; one-way ANOVA followed by Tukey's HSD test). See details of statistical analysis in Table S6.

Article Snippet: When using Omicron spike trimer conjugated with Alexa Fluor 488 (R&D Systems; AFG11060), experiments were repeated after SBECD, Veklury® P or Veklury® S treatments in HEK/ACE2 + TMPRSS2 and Calu-3 cells using the trimer at 0.5 and 2.5 μg ml 1, respectively.

Techniques: Binding Assay, Incubation, Fluorescence, Flow Cytometry, Control, Membrane, Marker

Journal: OncoImmunology

Article Title: Carbon ion radiotherapy triggers immunogenic cell death and sensitizes melanoma to anti-PD-1 therapy in mice

doi: 10.1080/2162402x.2022.2057892

Figure Lengend Snippet: Figure 1. CIRT-induced immunogenic cell death in vitro. Human osteosarcoma U2OS cells were irradiated by X-ray and carbon ion radiotherapy (CIRT), and biomarkers of immunogenic cell death were evaluated at 6 h and 24 h after irradiation. Mitoxantrone (MTX, 1 µM) was used as a prototype immunogenic cell death inducer Flow cytometry, immunofluorescence, and immunoblot assays showed that CIRT induced CALR exposure and elevated the phosphorylation level of eIF2α, enhanced ATP release by quinacrine staining and ELISA, augmented HMGB1 exodus from the nucleus, and significantly up-regulated the expression of type I interferon at the mRNA level. Representative images and quantification are shown (mean ± SD of triplicate assessments, Student’s t test, **p < .01, ***p < .001).

Article Snippet: The HMGB1 (#E-EL-M0676c) and IFN-γ (#E-EL-M0048c) ELISA kits were purchased from Elabscience (Wuhan, China); the ENLITEN ATP assay kits (#ff2000) was purchased from Promega (Madison, WI, USA).

Techniques: In Vitro, Irradiation, Flow Cytometry, Immunofluorescence, Western Blot, Phospho-proteomics, Staining, Enzyme-linked Immunosorbent Assay, Expressing

Journal: OncoImmunology

Article Title: Carbon ion radiotherapy triggers immunogenic cell death and sensitizes melanoma to anti-PD-1 therapy in mice

doi: 10.1080/2162402x.2022.2057892

Figure Lengend Snippet: Figure 2. CIRT-induced immunogenic cell death in vivo. 5 × 105 B16 and S91 cells were irradiated with 5 GyE of carbon ion beams or treated with 2 μM MTX for 24 h respectively, then subcutaneously inoculated in C57BL/6 mice. 2 weeks after, rechallenged with 5 × 104 B16 and S91 cells and the tumor growth was documented regularly. C57BL/6 mice bearing subcutaneous B16 or S91 melanoma were locally irradiated with X-rays (XR, 5 Gy) or carbon ions (CIRT, 5 GyE) at the tumor site. Mitoxantrone (MTX, 2.0 mg/Kg)-treated animals were used as positive control. Eight days after irradiation, CALR exposure, eIF2α phosphorylation, and HMGB1 exodus were examined by immunofluorescence. The secretion of HMGB1, ATP, and IFN-γ in the serum was determined by ELISA; and infiltration of IFN-γ expressing cell in the tumor bed was assessed by immunohistocheistry. Representative images and quantification are shown (mean ± SD of triplicate assessments, Student’s t test, ***p < .001).

Article Snippet: The HMGB1 (#E-EL-M0676c) and IFN-γ (#E-EL-M0048c) ELISA kits were purchased from Elabscience (Wuhan, China); the ENLITEN ATP assay kits (#ff2000) was purchased from Promega (Madison, WI, USA).

Techniques: In Vivo, Irradiation, Positive Control, Phospho-proteomics, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Expressing

Journal: OncoImmunology

Article Title: Carbon ion radiotherapy triggers immunogenic cell death and sensitizes melanoma to anti-PD-1 therapy in mice

doi: 10.1080/2162402x.2022.2057892

Figure Lengend Snippet: Figure 3. Combination therapy with CIRT and anti-PD-1 stimulates immunogenic cell death. C57BL/6 mice bearing subcutaneous B16 and S91 melanoma (Model) were injected intraperitoneally with anti-PD-1 (α-PD-1) or locally irradiated with 5 GyE of carbon ions (CIRT) at tumor sites, or received treatment with 5 Gy CIRT plus anti-PD-1 (CIRT+α-PD-1). Eight days after irradiation, we examined CALR exposure, eIF2α phosphorylation, and HMGB1 exodus by immunofluorescence; secretion of IFN-γ in serum by ELISA; and IFN-γ infiltration in the tumor by immunohistochemical assay. Representative images and quantification are shown (mean ± SD of triplicate assessments, Student’s t test, ***p < .001).

Article Snippet: The HMGB1 (#E-EL-M0676c) and IFN-γ (#E-EL-M0048c) ELISA kits were purchased from Elabscience (Wuhan, China); the ENLITEN ATP assay kits (#ff2000) was purchased from Promega (Madison, WI, USA).

Techniques: Injection, Irradiation, Phospho-proteomics, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining

Journal:

Article Title: CTLA-4 regulates allergen response by modulating GATA-3 protein level per cell

doi: 10.1111/j.1365-2567.2007.02537.x

Figure Lengend Snippet: Effects of in vitro CTLA-4 blockade on GATA-3 protein level/cell and frequency of IL-4-producing cells. Naive CD4+ T cells purified from the spleens of normal mice were induced to differentiate with anti-CD3 mAb, a recombinant form of CD80-Fc, IL-4 and anti-IFN-γ mAb for 3 days. Anti-CTLA-4 mAb was added in soluble form at increasing concentrations (0·1–10 μg/ml). (a) Mean fluorescence intensity for GATA-3 staining, (b) percentage of IL-4+ cells.

Article Snippet: 18 CD4 cell culture For in vitro experiments, splenic naive CD4 cells were purified from non-immunized BALB/c mice by immuno-magnetic cell sorting as previously described 19 and cultured in 24-well plates precoated with anti-CD3ε mAb (clone 145-2C11; 10 μg/ml) and/or recombinant mouse B7-1(CD80)/Fc chimeric protein (R & D Systems, cat. 740-B1; 0·33 μg/ml).

Techniques: In Vitro, Purification, Recombinant, Fluorescence, Staining

Journal: Investigational New Drugs

Article Title: The kinesin Eg5 inhibitor K858 exerts antiproliferative and proapoptotic effects and attenuates the invasive potential of head and neck squamous carcinoma cells

doi: 10.1007/s10637-022-01238-2

Figure Lengend Snippet: A – Immunofluorescence of cyclin B1 in untreated SCC-15 cells ( a ) and SCC-15 cells treated with 1 μM K858 for 24 h shown with ( b ) and without ( c ) nuclear staining. Bar, 7 μm. B – Western blot of cyclin B1 and tubulin in untreated (CTR) HNSCC cells and HNSCC cells treated with 1 μM K858 for 24 h

Article Snippet: Alternatively, after fixation, the cells were permeabilized with 0.1% Triton for 10 min at RT, incubated with 3% bovine serum albumin (BSA) for 1 h at RT and then incubated at 4 °C overnight with a primary rabbit polyclonal antibody to cyclin B1 with 0.1% BSA (1:200; Elabscience, TX, USA).

Techniques: Immunofluorescence, Staining, Western Blot