Journal: Clinical and Experimental Immunology

Article Title: Increased chemotaxis and activity of circulatory myeloid progenitor cells may contribute to enhanced osteoclastogenesis and bone loss in the C57BL/6 mouse model of collagen‐induced arthritis

doi: 10.1111/cei.12862

Figure Lengend Snippet: Development and assessment of collagen‐induced arthritis (CIA) in C57BL/6 mice. (a) Model of CIA; arthritis was induced by chicken collagen type II (CII) emulsified with complete Freund's adjuvant (CFA), with a booster dose of CII emulsified with incomplete FA (IFA). Mice were assessed at two time‐points defined as early arthritis (day 40/CIA40) and late arthritis (day 70/CIA70) for characterization of osteoclast progenitor subpopulations and analysis of bone metabolism. PBL = peripheral blood; SPL = spleen; BM = bone marrow; TMT = tarsometatarsal region of hind paws; OCP = osteoclast progenitor; CTX I = cross‐linked C‐telopeptide of type I collagen; μCT = micro‐computed tomography (micro‐CT). (b) Clinical scoring of CIA by a scale of 0–16 points (0–4 points for each paw) up to 70 days following immunization. The results shown are pooled data from six independent experiments, presented as mean ± standard deviation (n = 8–10 mice per group). (c) Serum levels of anti‐CII immunoglobulin (Ig)G1 and IgG2a antibodies in CIA analysed by enzyme‐linked immunosorbent assay (ELISA). Experiments were repeated three times, and representative results from one set of experiments are shown. Each symbol corresponds to the serum sample from individual mouse. AU = arbitrary unit. (d) Serum level of CTX I in CIA analysed by ELISA. The results shown are pooled data from three independent experiments (CIA, day 40). Each symbol corresponds to the serum sample from individual mouse. (e) Histological assessment of the first tarsometatarsal joint in mice developing arthritis (CIA, day 40) reveals osteitis and loss of subchondral bone, with prominent osteoclasts adjacent to the subchondral regions [haematoxylin and eosin (left), Goldner–Masson trichrome (middle) and tartrate‐resistant acid phosphatase (TRAP) stain (right)]. Magnification ×200. OST = osteitis; SB = subchondral bone; OC = osteoclasts. (f) Histomorphometric analysis of subchondral bone volume and number of active osteoclasts in control (ctrl) and arthritic mice (CIA, day 40). The experiments were repeated three times and data from a representative experiment are shown as mean ± standard deviation (n = 8 mice per group). *Significant difference from control at P < 0·05, Student's t‐test. [Colour figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Assessment of mouse cross‐linked C‐telopeptide of type I collagen Concentrations of mouse cross‐linked C‐telopeptide of type I collagen (CTX I) were determined in the mouse sera using a commercially available ELISA kit (Cusabio Biotech Co., Wuhan, China), according to the manufacturer's directions.

Techniques: Micro-CT, Standard Deviation, Enzyme-linked Immunosorbent Assay, Staining

Journal: Molecular Medicine Reports

Article Title: Continuous expression of CD83 on activated human CD4 + T cells is correlated with their differentiation into induced regulatory T cells

doi: 10.3892/mmr.2015.3796

Figure Lengend Snippet: Time-dependent expression of CD83, proliferation and the production of IL-2 and IFN-γ in CD4 + T cells. Following purity identification, the purified CD4 + T cells were either (A) directly stained with PE-labeled CD83 or (B–D) stimulated with anti-CD3/CD28 for 1–3 days, followed by staining with PE-labeled CD83. All samples were analyzed by flow cytometry and the typical flow cytometric histograms indicating the percentages of CD83-positive cells are shown. (E) Additionally, cell proliferation at days 1, 2 and 3 were determined using Cell Counting Kit 8. (F) The levels of IL-2 and IFN-γ in the supernatants from 1-, 2- and 3-day cultures of activated CD4 + T cells were analyzed by ELISA. Values are expressed as the mean ± standard deviation of three independent experiments ( ** P<0.01; * P<0.05). PE, phycoerythrin; CD, cluster of differentiation; IFN, interferon; IL, interleukin; neg.con, negative control.

Article Snippet: The levels of IL-2 and IFN-γ in CD4 + T-cell culture supernatants were measured in duplicate for each of the serial aliquots using a Human IL-2 Quantikine ELISA kit (cat. no. D2050) and Human IFN-γ Quantikine ELISA kit (cat. no. DIF50), purchased from R&D Systems (Minneapolis, MN, USA), according to the manufacturer's instructions.

Techniques: Expressing, Purification, Staining, Labeling, Flow Cytometry, Cell Counting, Enzyme-linked Immunosorbent Assay, Standard Deviation, Negative Control

Journal: Translational Neuroscience

Article Title: The role of melatonin in affecting cognitive dysfunction in acute sleep deprivation mice through the nuclear factor kappaB pathway and oxidative stress

doi: 10.1515/tnsci-2025-0379

Figure Lengend Snippet: Melatonin improved CD in male C57BL/6J ASD mice. MWM test was conducted to detect (a) escape latency, (b) time needed for first crossing, (c) number of crossings through the target platform zone, and (d) time spent in the target platform quadrant within 180 s of mice ( n = 12); (e) open field experiment was performed to assess mouse spontaneous activity ( n = 12); (f) elevated plus maze test was conducted to assess time spent in open and enclosed arms ( n = 12); (g) ELISA was used to measure AChE activity and ACh levels in the hippocampal CA1 region of SD mice ( n = 6). Data in panel (c) failed the Shapiro–Wilk test for normality and the Mann–Whitney U -test was applied for inter-group comparisons. All other data were normally distributed, with the results presented as mean ± standard deviation. (a) Escape latency in the MWM test was analyzed using two-way ANOVA, with the Šídák’s multiple comparison test used for post hoc analyses; (b)–(g) one-way ANOVA was applied for comparisons among multiple groups, and Tukey’s multiple comparison test for post hoc analysis. * P < 0.05, ** P < 0.01.

Article Snippet: ELISA kits were used to determine levels of IL-4 (Mouse IL-4 ELISA Kit, ml063156, Enzyme-linked Biotechnology, Shanghai, China), IL-10 (Mouse IL-10 ELISA Kit, ml037873, Enzyme-linked Biotechnology), TNF-α (Mouse TNF-α ELISA Kit, ml002095, Enzyme-linked Biotechnology), IL-1β (Mouse IL-1β ELISA Kit, ml301814, Enzyme-linked Biotechnology), acetylcholine (ACh) (mouse ACh ELISA kit, E-EL-0081, Elabscience, Wuhan, Hubei.

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Standard Deviation, Comparison

Journal: Translational Neuroscience

Article Title: The role of melatonin in affecting cognitive dysfunction in acute sleep deprivation mice through the nuclear factor kappaB pathway and oxidative stress

doi: 10.1515/tnsci-2025-0379

Figure Lengend Snippet: Melatonin mitigated neuroinflammation and oxidative stress in the hippocampal CA1 region of male C57BL/6J ASD mice. (a) HE staining to observe the structure of the hippocampal CA1 region of mice; (b) Nissl staining to observe the neurons of the hippocampal CA1 region of mice; ELISA to determine TNF-α, IL-1β, IL-4, and IL-10, (c) MDA and ROS levels, and SOD activity in the hippocampal CA1 region of mice (d); (e) western blot to measure PSD95 protein expression in the hippocampal CA1 region of mice; n = 6. All data were normally distributed as determined by the Shapiro–Wilk test and were presented as mean ± standard deviation. One-way ANOVA was applied for comparisons among multiple groups, and Tukey’s multiple comparison for post hoc analysis. Multiple comparisons were subjected to FDR correction using the Benjamini–Hochberg method ( q = 0.05). Corrected P -values were denoted as P adj , while uncorrected raw P -values were labeled as P . FDR correction ( q = 0.05) was applied to all multiple comparisons within each analytical category (inflammatory cytokines, oxidative stress markers). * P < 0.05, ** P < 0.01.

Article Snippet: ELISA kits were used to determine levels of IL-4 (Mouse IL-4 ELISA Kit, ml063156, Enzyme-linked Biotechnology, Shanghai, China), IL-10 (Mouse IL-10 ELISA Kit, ml037873, Enzyme-linked Biotechnology), TNF-α (Mouse TNF-α ELISA Kit, ml002095, Enzyme-linked Biotechnology), IL-1β (Mouse IL-1β ELISA Kit, ml301814, Enzyme-linked Biotechnology), acetylcholine (ACh) (mouse ACh ELISA kit, E-EL-0081, Elabscience, Wuhan, Hubei.

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Activity Assay, Western Blot, Expressing, Standard Deviation, Comparison, Labeling

Journal: Translational Neuroscience

Article Title: The role of melatonin in affecting cognitive dysfunction in acute sleep deprivation mice through the nuclear factor kappaB pathway and oxidative stress

doi: 10.1515/tnsci-2025-0379

Figure Lengend Snippet: Suppression of the NF-κB pathway improved neuroinflammation and oxidative stress in the hippocampal CA1 region of male C57BL/6J ASD mice. (a/f) Western blot to determine p-p65/p65 ratio and PSD95 protein expression in the hippocampal CA1 region of mice; (b) HE staining to observe the structure of the hippocampal CA1 region of mice; (c) Nissl staining to observe the neuronal status of the hippocampal CA1 region of mice; ELISA to determine (d) TNF-α, IL-1β, IL-4, and IL-10 levels and (e) MDA levels, ROS, and SOD activity in the hippocampal CA1 region; n = 6. Data were expressed as mean ± standard deviation. The t -test was adopted for inter-group comparisons. Multiple comparisons were subjected to FDR correction using the Benjamini–Hochberg method ( q = 0.05). Corrected P -values were denoted as P adj , while uncorrected raw P -values were labeled as P . FDR correction ( q = 0.05) was applied to all multiple comparisons within each analytical category (inflammatory cytokines, oxidative stress markers). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: ELISA kits were used to determine levels of IL-4 (Mouse IL-4 ELISA Kit, ml063156, Enzyme-linked Biotechnology, Shanghai, China), IL-10 (Mouse IL-10 ELISA Kit, ml037873, Enzyme-linked Biotechnology), TNF-α (Mouse TNF-α ELISA Kit, ml002095, Enzyme-linked Biotechnology), IL-1β (Mouse IL-1β ELISA Kit, ml301814, Enzyme-linked Biotechnology), acetylcholine (ACh) (mouse ACh ELISA kit, E-EL-0081, Elabscience, Wuhan, Hubei.

Techniques: Western Blot, Expressing, Staining, Enzyme-linked Immunosorbent Assay, Activity Assay, Standard Deviation, Labeling

Journal: Translational Neuroscience

Article Title: The role of melatonin in affecting cognitive dysfunction in acute sleep deprivation mice through the nuclear factor kappaB pathway and oxidative stress

doi: 10.1515/tnsci-2025-0379

Figure Lengend Snippet: Inhibition of the NF-κB pathway notably attenuated CD in male C57BL/6J ASD mice. MWM test to determine (a) escape latency, (b) first crossing time, (c) number of crossings through the target platform zone, and (d) time spent in the target platform quadrant within 180 s of mice ( n = 12); (e) open field experiment to assess spontaneous activity of mice ( n = 12); (f) elevated plus maze test was conducted to assess time spent in open and enclosed arms ( n = 12); (g) ELISA was used to measure AChE activity and ACh levels in the hippocampal CA1 region of SD mice ( n = 6). Data were expressed as mean ± standard deviation. (a) Escape latency in the MWM test was analyzed using two-way ANOVA, with the Šídák’s multiple comparison test used for post hoc analyses; (b)–(g) the t test was utilized for inter-group comparisons. ** P < 0.01.

Article Snippet: ELISA kits were used to determine levels of IL-4 (Mouse IL-4 ELISA Kit, ml063156, Enzyme-linked Biotechnology, Shanghai, China), IL-10 (Mouse IL-10 ELISA Kit, ml037873, Enzyme-linked Biotechnology), TNF-α (Mouse TNF-α ELISA Kit, ml002095, Enzyme-linked Biotechnology), IL-1β (Mouse IL-1β ELISA Kit, ml301814, Enzyme-linked Biotechnology), acetylcholine (ACh) (mouse ACh ELISA kit, E-EL-0081, Elabscience, Wuhan, Hubei.

Techniques: Inhibition, Activity Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation, Comparison

Journal: Translational Neuroscience

Article Title: The role of melatonin in affecting cognitive dysfunction in acute sleep deprivation mice through the nuclear factor kappaB pathway and oxidative stress

doi: 10.1515/tnsci-2025-0379

Figure Lengend Snippet: Activation of the NF-κB signaling promoted oxidative stress and CD in the hippocampal CA1 region of male C57BL/6J ASD mice. (a/f) Western blot for determination of p-p65, p65, and PSD95 protein expression levels in the hippocampal CA1 region ( n = 6); (b) HE staining for observation of the structure of the mouse hippocampal CA1 region ( n = 6); (c) Nissl staining for assessment of neuronal status in the mouse hippocampal CA1 region ( n = 6); (d) ELISA for measurement of levels of TNF-α, IL-1β, IL-4, and IL-10 levels in the hippocampal CA1 region of mice ( n = 6); (e) levels of MDA, ROS, and SOD activity in the mouse hippocampal CA1 region ( n = 6); (f) western blot for determination of PSD95 protein expression; MWM test to detect (g) escape latency, (h) time needed for first crossing, (i) number of crossings through the target platform zone within 180 s, and (j) time spent in the target platform quadrant within 180 s of mice ( n = 12); (k) open field experiment to assess mouse spontaneous activity ( n = 12); (l) elevated plus maze test to quantify time spent in open and enclosed arms ( n = 12); (m) ELISA was used to measure AChE activity and ACh levels in the hippocampal CA1 region of SD mice ( n = 6). Data were presented as mean ± standard deviation. (g) Escape latency was analyzed using two-way ANOVA, with the Šídák’s multiple comparison test used for post hoc analyses; other panels: inter-group comparisons were analyzed by the t -test. Multiple comparisons underwent FDR correction via the Benjamini–Hochberg method ( q = 0.05). Corrected P -values were denoted as P adj and uncorrected raw P -values as P . FDR correction ( q = 0.05) was applied to all multiple comparisons within each analytical category (inflammatory cytokines, oxidative stress markers). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: ELISA kits were used to determine levels of IL-4 (Mouse IL-4 ELISA Kit, ml063156, Enzyme-linked Biotechnology, Shanghai, China), IL-10 (Mouse IL-10 ELISA Kit, ml037873, Enzyme-linked Biotechnology), TNF-α (Mouse TNF-α ELISA Kit, ml002095, Enzyme-linked Biotechnology), IL-1β (Mouse IL-1β ELISA Kit, ml301814, Enzyme-linked Biotechnology), acetylcholine (ACh) (mouse ACh ELISA kit, E-EL-0081, Elabscience, Wuhan, Hubei.

Techniques: Activation Assay, Western Blot, Expressing, Staining, Enzyme-linked Immunosorbent Assay, Activity Assay, Standard Deviation, Comparison

Journal: Endocrinology and Metabolism

Article Title: Fancd2os Reduces Testosterone Production by Inhibiting Steroidogenic Enzymes and Promoting Cellular Apoptosis in Murine Testicular Leydig Cells

doi: 10.3803/enm.2022.1431

Figure Lengend Snippet: Fig. 4. Fancd2 opposite-strand (Fancd2os) reduces testosterone production and steroidogenic enzyme expression in TM3 cells. The testoster one levels in Fancd2os-overexpressing (A) or knockdown TM3 cells (B) were detected by enzyme-linked immunosorbent assays (n=3 per group). (C, D, E) Relative quantities of mRNA expression of steroidogenic acute regulatory protein (StAR), P450 cholesterol side-chain cleavage (P450scc), and 3β-hydroxysteroid dehydrogenase (3β-HSD) in Fancd2os-overexpressing TM3 cells or Fancd2os knockdown TM3 cells (F, G, H) were determined real-time polymerase chain reaction using β-actin as a housekeeping gene. Each bar represents the mean± standard deviation from three separate experiments. Significant difference compared to TM3, vector/TM3 or NC/TM3. aP<0.05; bP<0.01.

Article Snippet: The testosterone concentration of both serum and cell supernatants was measured using a testosterone ELISA Kit (Elabscience, Houston, TX, USA) according to the manufacturer’s protocol.

Techniques: Expressing, Knockdown, Real-time Polymerase Chain Reaction, Standard Deviation, Plasmid Preparation

Journal: Endocrinology and Metabolism

Article Title: Fancd2os Reduces Testosterone Production by Inhibiting Steroidogenic Enzymes and Promoting Cellular Apoptosis in Murine Testicular Leydig Cells

doi: 10.3803/enm.2022.1431

Figure Lengend Snippet: Fig. 6. Higher Fancd2 opposite-strand (Fancd2os) levels in older mouse Leydig cells result in cellular apoptosis and lower serum testosterone production. (A) The serum testosterone levels from mice of different ages were measured using enzyme-linked immunosorbent assays. (B, C) The testis tissues from different aged mice were sliced and then Fancd2os protein expression and apoptosis were analyzed using immuno chemistry and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, respectively. ST represents seminiferous tubule. Black arrows and red arrows indicate Fancd2os-positive cells and TUNEL-positive cells, respectively. Scale bar repre sents 25 μm. The results are given as the mean±standard deviation (n=3). (D) The correlations between Fancd2os expression (B) and the TUNEL-positive staining rate (C) were analyzed using Pearson correlation coefficients. An r≥0.5 was considered to indicate a strong correla tion, and P<0.05 was considered statistically significant. aP<0.05 compared with juvenile mice; bP<0.05 compared with young mice; cP<0.05 compared with middle-aged mice.

Article Snippet: The testosterone concentration of both serum and cell supernatants was measured using a testosterone ELISA Kit (Elabscience, Houston, TX, USA) according to the manufacturer’s protocol.

Techniques: Expressing, End Labeling, TUNEL Assay, Standard Deviation, Staining

Journal: Cancer Immunology Research

Article Title: Granulocyte–Macrophage Colony-Stimulating Factor Influence on Soluble and Membrane-Bound ICOS in Combination with Immune Checkpoint Blockade

doi: 10.1158/2326-6066.CIR-22-0702

Figure Lengend Snippet: Comparison between human ICOS full length and splice variant. A, Schematic representation of full length and spliced variant of human ICOS. The full length of ICOS consists of 5 exons. The gray boxes denote untranslated regions, and the black boxes denote coding sequences. The transmembrane domain (TM) is located in exon 3 and labeled with a green bar. The splicing region is shown in red. B, Comparison of cDNA nucleotide sequences, amino acid sequences, and 5′UTR sequences between ICOS-FL and the splice variant. Nucleotide sequences of cDNA for ICOS-FL (b1) and the splice variant of ICOS (b4) are represented; spliced region of ICOS is shown in red and delineated by red brackets; the nucleotides and amino acids of TM are shown in green. Asterisk indicates the stop codon. Amino acid sequences of full length of ICOS (b2) and the splice variant of ICOS (b5) are shown. The underline depicts additional differences in amino acid sequence compared with ICOS-FL. “Stop” represents a stop codon. 5′UTR sequences of ICOS-FL (b3) and the splice variant of ICOS (b6) are shown. The 5′UTR of the ICOS-SV lacks 32 nucleotides compared with the 5′UTR of ICOS-FL shown in red and delineated by red brackets. C, Schematic diagram of ICOS-FL and the ICOS-SV protein. Thirty-two out of 38 amino acids in the intracellular domain are spliced, as shown in red.

Article Snippet: To evaluate soluble ICOS-SVs in the sera of melanoma patients or cell culture supernatants, the Human ICOS DuoSet ELISA kit (DY169, R&D Systems) was used according to the manufacturer's protocol.

Techniques: Comparison, Variant Assay, Labeling, Sequencing

Journal: Cancer Immunology Research

Article Title: Granulocyte–Macrophage Colony-Stimulating Factor Influence on Soluble and Membrane-Bound ICOS in Combination with Immune Checkpoint Blockade

doi: 10.1158/2326-6066.CIR-22-0702

Figure Lengend Snippet: Secretion of the ICOS-SV from ICOS-SV–overexpressing cells. A, RT-PCR to detect the ICOS-SV in human T cells. Total RNA was isolated from purified T cells in PBMCs of a healthy donor. Expression of the ICOS variant was examined by RT-PCR with specific primers. RT-PCR of ICOS-FL is shown as a control. B, DNA sequencing of ICOS-SV cDNA from human T cells. The cDNA of the PCR product was cloned into a TOPO TA vector. The ICOS-SV was confirmed by DNA sequencing using an M13 forward primer. Splicing point is shown by the red arrow. C, DNA sequencing of ICOS-FL cDNA is shown as a control. No splicing occurs in the region corresponding to the position in ( B ), as shown in blue. D, Expression of ICOS-SV and ICOS-FL in 293T cells determined by immunoblot. 293T cells were transduced with recombinant GFP retroviral vector encoding either ICOS-SV or ICOS-FL as a positive control. The parental cells and the GFP-empty vector–transduced cells were used as negative controls. Sample loading was normalized to actin. E, Cell culture supernatants were collected from the cells described in ( A ). Secreted ICOS-SV was examined by ELISA. Two-tailed unpaired t test was used to compare ICOS secretion between the two groups. ***, P < 0.001. Standard deviation of the mean (SD) is shown. F, To validate the secreted ICOS variant protein in the cell supernatant, immunoprecipitation, SDS-PAGE, and immunoblotting assays were performed. Sample loading was normalized to cell numbers.

Article Snippet: To evaluate soluble ICOS-SVs in the sera of melanoma patients or cell culture supernatants, the Human ICOS DuoSet ELISA kit (DY169, R&D Systems) was used according to the manufacturer's protocol.

Techniques: Reverse Transcription Polymerase Chain Reaction, Isolation, Purification, Expressing, Variant Assay, Control, DNA Sequencing, Clone Assay, Plasmid Preparation, Western Blot, Transduction, Recombinant, Retroviral, Positive Control, Cell Culture, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Standard Deviation, Immunoprecipitation, SDS Page

Journal: Cancer Immunology Research

Article Title: Granulocyte–Macrophage Colony-Stimulating Factor Influence on Soluble and Membrane-Bound ICOS in Combination with Immune Checkpoint Blockade

doi: 10.1158/2326-6066.CIR-22-0702

Figure Lengend Snippet: Suppressive effects of sICOS-SV on the costimulation of T cells. A, CHO-K1 cells were transduced with either ICOSL-expressing lentiviral vector or GFP-empty vector (CHO-ICOSL − ) as a negative control. Expression of ICOSL on the cell surface of CHO-K1 was examined by flow cytometry using anti-ICOSL (open histogram with solid line) or lentivirus GFP-empty vector (open histogram with dotted line). Isotype antibody was used as a negative control (filled histogram). B, ICOSL-expressing CHO-K1 cells were stained with ICOS-SV Ig (red line), control IgG Ig (blue line), or PD-1 Ig (orange line) and analyzed by flow cytometry. Both soluble control Ig and PD-1 Ig were used as negative controls. GFP-empty vector-expression CHO-K1 cells stained with ICOS-SV Ig (green line) were used as another negative control for the binding assay. Isotype antibody control was used as a negative control (gray filled histogram). C, CD154 expression was determined by FACS analysis. Isotype control antibody is shown in gray. Activated T cells treated with a suboptimal dose of anti-CD3 were incubated with control Ig + CHO-ICOSL − cells (CHO cells transduced with GFP-empty vector, blue), control Ig + CHO-ICOSL + cells (green), PD-1 Ig + CHO-ICOSL + cells (red), or ICOS-SV Ig + CHO-ICOSL + cells (yellow). Both control Ig and PD-1 Ig were used as negative controls. D, Two-tailed unpaired t test was used to compare expression of CD154 between two groups. **, P < 0.01; ***, P < 0.001. Standard deviation of the mean (SD) is shown. E, CD69 expression was determined by FACS analysis. Isotype control antibody is shown in gray. Activated T cells treated with a suboptimal dose of anti-CD3 were incubated with control Ig + CHO-ICOSL − cells (CHO cells transduced with GFP-empty vector, blue), control Ig + CHO-ICOSL + cells (green), PD-1 Ig + CHO-ICOSL + cells (red), or ICOS-SV Ig + CHO-ICOSL + cells (yellow). Both control Ig and PD-1 Ig were used as negative controls. F, Two-tailed unpaired t test was used to compare expression of CD69 between the two groups. **, P < 0.01; ***, P < 0.001. Standard deviation of the mean (SD) is shown. G, CHO cells transduced with GFP-empty vector (CHO-ICOSL − ) were used as negative controls. Sample loading was normalized to total pan-Akt. H, T-cell proliferation was determined by a [ 3 H]-TdR thymidine incorporation assay. CHO cells transduced with empty vector (CHO-ICOSL − ) were used as negative controls. Two-tailed unpaired t test was used to compare 3 H uptake between the two groups, respectively. *, P < 0.1. Standard deviation of the mean (SD) is shown. I, Schematic diagram of ICOS/ICOSL costimulatory T-cell proliferation, as well as the blocking function of the sICOS-SV. Depicted are the cytoplasmic tail sequences of ICOS-FL and sICOS-SV isoforms. The YMFM Src Homology 2 (SH2) binding motif in the cytoplasmic tail of the ICOS-FL is highlighted in pink. Upon ICOS engagement by the ICOSL on CHO cells, the unique YMFM motif recruits a p85a and a p50a subunits of PI3K, resulting in the elevated phosphorylation of Akt, thereby inducing PI3K activity. In contrast, the ICOS-SV, a truncated isoform lacking the YMFM motif in its cytoplasmic tail, cannot elicit phosphorylation of Akt. Consequently, it fails to promote T-cell proliferation. The secreted ICOS-SV (red) competes with membrane-bound ICOS for binding to ICOSL, thereby blocking the interaction between ICOSL and membrane ICOS. As a result, the sICOS-SV suppresses phosphorylation of Akt and T-cell proliferation, leading to the inhibition of T-cell immunity. The diagram was created with BioRender.com. All data shown are representative of at least 2 independent experiments.

Article Snippet: To evaluate soluble ICOS-SVs in the sera of melanoma patients or cell culture supernatants, the Human ICOS DuoSet ELISA kit (DY169, R&D Systems) was used according to the manufacturer's protocol.

Techniques: Transduction, Expressing, Plasmid Preparation, Negative Control, Flow Cytometry, Staining, Control, Binding Assay, Incubation, Two Tailed Test, Standard Deviation, Thymidine Incorporation Assay, Blocking Assay, Phospho-proteomics, Activity Assay, Membrane, Inhibition

Journal: Cancer Immunology Research

Article Title: Granulocyte–Macrophage Colony-Stimulating Factor Influence on Soluble and Membrane-Bound ICOS in Combination with Immune Checkpoint Blockade

doi: 10.1158/2326-6066.CIR-22-0702

Figure Lengend Snippet: sICOS from melanoma patients inhibits T-cell activation and proliferation induced by GM-CSF–driven DCs in MLRs. A, CD4 + T cells (responders) were stimulated by allogeneic GM-CSF–driven MoDCs (stimulators: negative control DCs, DCs generated by GM-CSF/IL4 + anti-IgG, or DCs generated by GM-CSF/IL4 + anti-CD116) in MLRs. T cells were assessed for activation via flow cytometry using anti-ICOS (blue), anti-GITR (green), or anti-CD25 (gray). Isotype control antibodies were used as negative controls. B, Statistical analysis of the percentage of CD4 + ICOS + , CD4 + GITR + , and CD4 + CD25 + T-cell populations from different groups as described above. C, Soluble ICOS levels were determined by ELISA using supernatants from the MLRs described above. D – E, Serum from sICOS-high and sICOS-negative patients were added to the MLRs, and T-cell proliferation ( D ) and CD69 expression ( E ) were evaluated. Additionally, sICOS was depleted from sICOS-high serum to assess its effects on T cells. F, Statistical analysis of the percentage of CD4 + CD69 + T-cell populations from different groups as described above. All data shown are representative of at least 2 independent experiments. Two-tailed unpaired t test was used between the two groups. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant ( P > 0.05). Standard deviation of the mean (SD) is shown.

Article Snippet: To evaluate soluble ICOS-SVs in the sera of melanoma patients or cell culture supernatants, the Human ICOS DuoSet ELISA kit (DY169, R&D Systems) was used according to the manufacturer's protocol.

Techniques: Activation Assay, Negative Control, Generated, Flow Cytometry, Control, Enzyme-linked Immunosorbent Assay, Expressing, Two Tailed Test, Standard Deviation

Journal: Molecular medicine reports

Article Title: Intraperitoneal injection of thalidomide alleviates early osteoarthritis development by suppressing vascular endothelial growth factor expression in mice.

doi: 10.3892/mmr.2018.8980

Figure Lengend Snippet: Figure 2. mRNA expression levels of VEGF and MMP‑13 in the knee articular cartilage of mice among the Sham, Dmm and Dmm+Th groups (n=4 in each group). (A) Relative mRNA expression levels of VEGF in the medial articular cartilage. (B) Relative mRNA expression levels of MMP‑13 in the medial articular cartilage. The values are presented as the mean ± standard deviation. *P<0.05 compared with the Sham group; #P<0.05 compared with the Dmm group. Dmm, destabilization of the medial meniscus; MMP‑13, matrix metalloproteinase‑13; Th, thalidomide; VEGF, vascular endothelial growth factor.

Article Snippet: An ELISA kit of VEGF (E-EL-M1292c) was purchased from Elabscience Biotechnology Co., Ltd., Wuhan, China.

Techniques: Expressing, Standard Deviation

Journal: Molecular medicine reports

Article Title: Intraperitoneal injection of thalidomide alleviates early osteoarthritis development by suppressing vascular endothelial growth factor expression in mice.

doi: 10.3892/mmr.2018.8980

Figure Lengend Snippet: Figure 3. Immunohistochemical analysis of VEGF expression in the knee articular cartilage of mice among the Sham, Dmm and Dmm+Th groups (n=4 in each group). (A) Immunohistochemistry staining of VEGF in the articular cartilage of the medial tibial plateau (magnification, x400, scale bar=100 µm). (B) Quantification of VEGF positive cells, based on the results of immunohistochemistry staining. The values are presented as the mean ± standard deviation. *P<0.05 compared with the Sham group; #P<0.05 compared with the Dmm group. Dmm, destabilization of the medial meniscus; Th, thalidomide; VEGF, vascular endothelial growth factor.

Article Snippet: An ELISA kit of VEGF (E-EL-M1292c) was purchased from Elabscience Biotechnology Co., Ltd., Wuhan, China.

Techniques: Immunohistochemical staining, Expressing, Immunohistochemistry, Staining, Standard Deviation

Journal: Molecular medicine reports

Article Title: Intraperitoneal injection of thalidomide alleviates early osteoarthritis development by suppressing vascular endothelial growth factor expression in mice.

doi: 10.3892/mmr.2018.8980

Figure Lengend Snippet: Figure 5. ELISA analysis of serum VEGF concentration of mice among the Sham, Dmm and Dmm+Th groups (n=8 in each group). The values are presented as the mean ± standard deviation. *P<0.05 compared with the Sham group; #P<0.05 compared with the Dmm group. Dmm, destabilization of the medial meniscus; Th, thalidomide; VEGF, vascular endothelial growth factor.

Article Snippet: An ELISA kit of VEGF (E-EL-M1292c) was purchased from Elabscience Biotechnology Co., Ltd., Wuhan, China.

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay, Standard Deviation

Journal: PLoS Pathogens

Article Title: CCR5 Is Essential for NK Cell Trafficking and Host Survival following Toxoplasma gondii Infection

doi: 10.1371/journal.ppat.0020049

Figure Lengend Snippet: (A and B) Proliferation. Magnetically purified positively selected CD4 + (A) and CD8 + (B) T cells (>95% pure) isolated from the spleens of C57BL/6x129 on day 7 PI. Cells were stimulated either with ConA (2.5 μg/ml) or Toxoplasma lysate antigen (15 μg/ml). After 72 h incubation, proliferation was measured by 3 H thymidine incorporation. Data are represented as mean cpm ± standard deviation and are representative of two experiments. (C and D) IFNγ secretion. 10 6 purified CD4 + (C) and CD8 + (D) T cells from 7 d infected C57BL/6x129 mice were cultured in presence of 15 μg/ml of Toxoplasma lysate antigen and irradiated feeder cells (5 × 10 5 cells/well) in 24-well plates. After 72 h of incubation the supernatants were collected, centrifuged, and assayed for IFNγ production by ELISA. (E) Intracellular IFNγ production. Female CCR5 −/− (5–8 wk old) and wild-type mice were infected perorally with T. gondii cysts and splenocytes were harvested at day 7 PI, pooled (three mice per group), and cultured in vitro with phorbol 12-myristate 13-acetate, ionomycin, and monensin for 4 h. The cultured cells were stained for CD4 or CD8 before intracellular staining for IFNγ. Data are presented as percentage (mean ± standard deviation) of CD4 + or CD8 + T cells positive for IFNγ and are pooled from two experiments.

Article Snippet: Supernatants were analyzed for IFNγ by ELISA using mouse IFNγ immunoassay kit (R&D Systems, Minneapolis, Minnesota, United States).

Techniques: Purification, Isolation, Incubation, Standard Deviation, Infection, Cell Culture, Irradiation, Enzyme-linked Immunosorbent Assay, In Vitro, Staining

Journal: Cancer science

Article Title: RNA interference-mediated signal transducers and activators of transcription 3 gene silencing inhibits invasion and metastasis of human pancreatic cancer cells.

doi: 10.1111/j.1349-7006.2007.00485.x

Figure Lengend Snippet: Fig. 1. Expression of signal transducers and activators of transcription 3 (STAT3) in SW1990 cells and HeLa cells. (a) STAT3 expression was determined by Western blot analysis. Detection of β-actin was used as a loading control. HeLa cell line served as a positive control of STAT3 expression. (b) STAT3 distribution in SW1990 cells was examined by immunocytochemistry (400 ×), the buffy particle corresponded for STAT3 (shown by arrow).

Article Snippet: Membranes were incubated in blocking buffer (1 × TBS, 0.1% Tween 20, and 5% non-fat dry milk) for 1 h at room temperature, followed by hybridization with antip-STAT3[tyr-705] antibody (Cell Signal, 1:1000 dilution), anti-STAT3 antibody (Cell Signal, 1:1000 dilution), anti-MMP-2 antibody (Santa Cruz, 1:500 dilution), anti-VEGF antibody (Santa Cruz, 1:500 dilution) or antiβ-actin antibody (Labvision, Fremont, CA, USA, 1:100 dilution), at 4°C overnight.

Techniques: Expressing, Western Blot, Control, Positive Control, Immunocytochemistry

Journal: Cancer science

Article Title: RNA interference-mediated signal transducers and activators of transcription 3 gene silencing inhibits invasion and metastasis of human pancreatic cancer cells.

doi: 10.1111/j.1349-7006.2007.00485.x

Figure Lengend Snippet: Fig. 2. Effects of signal transducers and activators of transcription 3 (STAT3) specific shRNA expression vector on STAT3 expression. (a) Reverse transcription-polymerase chain reaction (RT-PCR) analysis. The RNA samples (2 µg in each) extracted from SW1990 cells, SW1990 cells transfected with pRNAT-Con, pRNAT-STAT3-siRNA-I, pRNAT-STAT3- siRNA-II and pRNAT-STAT3-siRNA-III were subjected to RT-PCR for STAT3 and β-actin mRNAs as described in Materials and Methods. RT- PCR for β-actin was carried out in parallel to show an equal amount of total RNA in the sample. (b) Western blot analysis. Cellular whole and nuclear protein extracts (100 µg in each) were prepared from SW1990 cells, SW1990 cells transfected with pRNAT-Con, pRNAT-STAT3-siRNA-I, pRNAT-STAT3-siRNA-II and pRNAT-STAT3-siRNA-III. The expression of STAT3 protein was determined using Western blot analysis with an anti-STAT3 antibody. The expression of p-STAT3 protein was deter- mined by hybridizing the same membrane filter with an antip-STAT3 antibody. The levels of β-actin expression were determined as a control for equivalent protein loading. Results shown are for one represent- ative experiment of three.

Article Snippet: Membranes were incubated in blocking buffer (1 × TBS, 0.1% Tween 20, and 5% non-fat dry milk) for 1 h at room temperature, followed by hybridization with antip-STAT3[tyr-705] antibody (Cell Signal, 1:1000 dilution), anti-STAT3 antibody (Cell Signal, 1:1000 dilution), anti-MMP-2 antibody (Santa Cruz, 1:500 dilution), anti-VEGF antibody (Santa Cruz, 1:500 dilution) or antiβ-actin antibody (Labvision, Fremont, CA, USA, 1:100 dilution), at 4°C overnight.

Techniques: shRNA, Expressing, Plasmid Preparation, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Transfection, Western Blot, Membrane, Control

Journal: Cancer science

Article Title: RNA interference-mediated signal transducers and activators of transcription 3 gene silencing inhibits invasion and metastasis of human pancreatic cancer cells.

doi: 10.1111/j.1349-7006.2007.00485.x

Figure Lengend Snippet: Fig. 3. Effects of stable transfection of pRNAT-STAT3-siRNA-II vector on signal transducers and activators of transcription 3 (STAT3) expres- sion. (a) Reverse transcription-polymerase chain reaction (RT-PCR) analysis. The RNA samples (2 µg in each) extracted from SW1990 cells, SW1990 cells transfected with a control vector (SW1990-Con), and SW1990 cells transfected with STAT3-RNAi (SW1990-RNAi-a and SW1990-RNAi-b) were subjected to RT-PCR for STAT3 and β-actin mRNAs as described in Materials and Methods. RT-PCR for β-actin was carried out in parallel to show an equal amount of total RNA in the sample. (b) Western blot analysis. Cellular whole and nuclear protein extracts (100 µg in each) were prepared from SW1990 cells, SW1990 cells transfected with a control vector (SW1990-Con), and SW1990 cells transfected with STAT3-RNAi (SW1990-RNAi-a and SW1990-RNAi-b). The expression of STAT3 protein was determined using Western blot analysis with an anti-STAT3 antibody. The expression of p-STAT3 protein was determined by hybridizing the same membrane filter with an antip-STAT3 antibody. The levels of β-actin expression were determined as a control for equivalent protein loading. (c) STAT3 DNA-binding activity. Cell nuclear protein extracts (10 µg in each) were prepared from SW1990 cells, SW1990 cells transfected with a control vector (SW1990-Con), and SW1990 cells transfected with STAT3-RNAi (SW1990-RNAi-a and SW1990-RNAi-b) and subjected to electrophoretic mobility shift analysis (EMSA) using biotin end-labeled oligonucleotide probes containing a consensus-binding motif for STAT3. Results shown are for one representative experiment of three.

Article Snippet: Membranes were incubated in blocking buffer (1 × TBS, 0.1% Tween 20, and 5% non-fat dry milk) for 1 h at room temperature, followed by hybridization with antip-STAT3[tyr-705] antibody (Cell Signal, 1:1000 dilution), anti-STAT3 antibody (Cell Signal, 1:1000 dilution), anti-MMP-2 antibody (Santa Cruz, 1:500 dilution), anti-VEGF antibody (Santa Cruz, 1:500 dilution) or antiβ-actin antibody (Labvision, Fremont, CA, USA, 1:100 dilution), at 4°C overnight.

Techniques: Stable Transfection, Plasmid Preparation, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Transfection, Control, Western Blot, Expressing, Membrane, Binding Assay, Activity Assay, Electrophoretic Mobility Shift Assay, Labeling

Journal: Cancer science

Article Title: RNA interference-mediated signal transducers and activators of transcription 3 gene silencing inhibits invasion and metastasis of human pancreatic cancer cells.

doi: 10.1111/j.1349-7006.2007.00485.x

Figure Lengend Snippet: Fig. 4. Effects of silence of signal transducers and activators of transcription 3 (STAT3) gene on SW1990 cell invasion in vitro. SW1990-RNAi-a, SW1990-RNAi-b, SW1990-Con and the parental cell lines were seeded into the upper compart- ments of invasion chambers. Cells were allowed to invade for 48 h at 37°C. The tumor cells that invaded the ECMatrix and migrated through the polycarbonate membrane were stained by the staining solution and dissolved in 10% acetic acid. The dye/solution mixture was transferred onto a 96-well plate for colorimetric reading of optical density (OD) at 560 nm. The number of migrated cells that penetrated through the ECMatrix-coated filters was expressed as the OD at 560 nm. The standard deviation bars represent replicates within the assay. *P < 0.001 compared with that of parental SW1990 cells. Results shown are for one representative experiment of three.

Article Snippet: Membranes were incubated in blocking buffer (1 × TBS, 0.1% Tween 20, and 5% non-fat dry milk) for 1 h at room temperature, followed by hybridization with antip-STAT3[tyr-705] antibody (Cell Signal, 1:1000 dilution), anti-STAT3 antibody (Cell Signal, 1:1000 dilution), anti-MMP-2 antibody (Santa Cruz, 1:500 dilution), anti-VEGF antibody (Santa Cruz, 1:500 dilution) or antiβ-actin antibody (Labvision, Fremont, CA, USA, 1:100 dilution), at 4°C overnight.

Techniques: In Vitro, Membrane, Staining, Standard Deviation

Journal: Cancer science

Article Title: RNA interference-mediated signal transducers and activators of transcription 3 gene silencing inhibits invasion and metastasis of human pancreatic cancer cells.

doi: 10.1111/j.1349-7006.2007.00485.x

Figure Lengend Snippet: Fig. 5. Effects of silence of signal transducers and activators of transcription 3 (STAT3) gene on SW1990 cell metastasis in vivo. In total, 5 × 105 SW1990 cells, SW1990 cells transfected with a control vector (SW1990-Con), and SW1990 cells transfected with STAT3-RNAi (SW1990-RNAi-a and SW1990-RNAi-b) were injected i.v. into groups of nude mice (n = 6). The mice were killed on day 21 or when mice became moribund. The number of experimental lung metastases was determined with the aid of a dissecting microscope. Results were expressed as the median number and range of lung metastasis nodules. *P < 0.001 compared with that of parental SW1990 cells. Results shown here are for one representative experiment of three. (a) H&E staining of formalin-fixed, paraffin-embedded lung tissue of nude mice (100 ×). The metastasis nodus are indicated by arrows. (b) H&E staining of formalin-fixed, paraffin-embedded metastasis nodus of lung tissue of nude mice (400 ×). (c) In SW1990 cells injected nude mice, STAT3, matrix metalloproteinases-2 (MMP-2) and vascular endothelial growth factor (VEGF) distribution in metastasis nodus of lung tissue was examined by immunohistochemistry (400 ×), the buffy particle corresponded for STAT3, MMP-2 and VEGF. Immunohistochemistry showed that STAT3, MMP-2 and VEGF were distributed in cytoplasm.

Article Snippet: Membranes were incubated in blocking buffer (1 × TBS, 0.1% Tween 20, and 5% non-fat dry milk) for 1 h at room temperature, followed by hybridization with antip-STAT3[tyr-705] antibody (Cell Signal, 1:1000 dilution), anti-STAT3 antibody (Cell Signal, 1:1000 dilution), anti-MMP-2 antibody (Santa Cruz, 1:500 dilution), anti-VEGF antibody (Santa Cruz, 1:500 dilution) or antiβ-actin antibody (Labvision, Fremont, CA, USA, 1:100 dilution), at 4°C overnight.

Techniques: In Vivo, Transfection, Control, Plasmid Preparation, Injection, Microscopy, Staining, Formalin-fixed Paraffin-Embedded, Immunohistochemistry

Journal: Cancer science

Article Title: RNA interference-mediated signal transducers and activators of transcription 3 gene silencing inhibits invasion and metastasis of human pancreatic cancer cells.

doi: 10.1111/j.1349-7006.2007.00485.x

Figure Lengend Snippet: Fig. 6. Effects of silence of signal transducers and activators of transcription 3 (STAT3) gene on the expression of matrix metalloproteinases-2 (MMP-2) and vascular endothelial growth factor (VEGF). (a) Reverse transcription-polymerase chain reaction (RT-PCR) analysis. The RNA samples (2 µg in each) extracted from SW1990 cells, SW1990 cells transfected with a control vector (SW1990-Con), and SW1990 cells transfected with STAT3- RNAi (SW1990-RNAi-a and SW1990-RNAi-b) were subjected to RT-PCR for MMP-2, VEGF, and β-actin mRNAs as described in Materials and Methods. RT-PCR for β-actin was carried out in parallel to show an equal amount of total RNA in the sample. (b) Western blot analysis. Cellular whole protein extracts (100 µg in each) were prepared from SW1990 cells, SW1990 cells transfected with a control vector (SW1990-Con), and SW1990 cells transfected with STAT3-RNAi (SW1990-RNAi-a and SW1990-RNAi-b). The expression of MMP-2 protein was determined using Western blot analysis with an anti-MMP-2 antibody. The expression of VEGF protein was determined by hybridizing the same membrane filter with an anti-VEGF antibody. The levels of β-actin expression were determined as a control for the equivalent protein loading. Results shown are for one representative experiment of three.

Article Snippet: Membranes were incubated in blocking buffer (1 × TBS, 0.1% Tween 20, and 5% non-fat dry milk) for 1 h at room temperature, followed by hybridization with antip-STAT3[tyr-705] antibody (Cell Signal, 1:1000 dilution), anti-STAT3 antibody (Cell Signal, 1:1000 dilution), anti-MMP-2 antibody (Santa Cruz, 1:500 dilution), anti-VEGF antibody (Santa Cruz, 1:500 dilution) or antiβ-actin antibody (Labvision, Fremont, CA, USA, 1:100 dilution), at 4°C overnight.

Techniques: Expressing, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Transfection, Control, Plasmid Preparation, Western Blot, Membrane

Journal: Cell Death & Disease

Article Title: CUL4B promotes hepatocellular carcinoma progression and oxaliplatin resistance by facilitating FUS degradation

doi: 10.1038/s41419-025-08320-6

Figure Lengend Snippet: A , B Effect of CUL4B overexpression ( A ) or knockdown ( B ) on the proliferation of Huh7 cells treated with or without 2 μM oxaliplatin. Cell proliferation of HCC cells was determined using a CCK8 growth assay for 4 days (mean ± SD, n = 3). C Effect of CUL4B knockdown on IC 50 of oxaliplatin in Huh7 and LM3 cells. IC 50 was determined using a ATP-lite assay on cells treated with oxaliplatin at indicated concentrations for 48 h (mean ± SD, n = 6). D Effect of CUL4B knockdown on cellular morphology of Huh7 cells treated with 40 μΜ oxaliplatin for 48 h. Cell number was counted (mean ± SD, n = 3). E Effect of CUL4B knockdown on apoptosis of Huh7 cells treated with 40 μΜ oxaliplatin for 48 h. Annexin V-FITC/PI double-staining analysis was used to analyze cell apoptosis (mean ± SD, n = 3). Western blotting was used to assess the expression of the apoptosis-related proteins NOXA and cleaved-PARP. F Effect of CUL4B knockdown on the cell cycle of Huh7 cells treated with 40 μΜ oxaliplatin for 48 h. PI staining and FACS analysis were used to analyze the cell cycle profile (mean ± SD, n = 3). Western blotting was used to assess the expression of the cell cycle-related protein Cyclin B. Two-tailed, unpaired t -test was used for ( A ). One-way ANOVA/LSD test was used for ( B − F ). Data are presented as the mean ± standard deviation derived from a minimum of three independent experiments. Statistical significance was assessed using the appropriate testing methods, with * p < 0.05, ** p < 0.01, and *** p < 0.001 denoting levels of significance.

Article Snippet: For apoptosis assays, the Annexin V-FITC Apoptosis Detection Kit (Beyotime, C1062L) was used according to the manufacturer’s instructions.

Techniques: Over Expression, Knockdown, Growth Assay, Double Staining, Western Blot, Expressing, Staining, Two Tailed Test, Standard Deviation, Derivative Assay

Journal: Oncology Letters

Article Title: Chelerythrine chloride induces apoptosis in renal cancer HEK-293 and SW-839 cell lines

doi: 10.3892/ol.2016.4520

Figure Lengend Snippet: CC-induced dose-dependent apoptosis of HEK-293 and human renal cancer SW-839 cells. (A) HEK-293 and (B) SW-839 cells were treated with various concentrations of CC (0, 5 and 10 µM) for 24 h, and stained with annexin V-fluorescein isothiocyanate/propidium iodide. The percentage of early-stage apoptotic cells is shown in the lower right quadrant, while the percentage of late-stage apoptotic cells is shown in the upper right quadrant. (C) Treatment of HEK-293 and SW-839 cells with 5 and 10 µM CC for 24 h induced cell apoptosis. CC, chelerythrine chloride; FL-H, fluorescence line height.

Article Snippet: One Step TUNEL Apoptosis Assay Kit (Beyotime Institute of Biotechnology, Haimen, China) was used to stain the apoptotic tumor cells.

Techniques: Staining, Fluorescence

Journal: Oncology Letters

Article Title: Chelerythrine chloride induces apoptosis in renal cancer HEK-293 and SW-839 cell lines

doi: 10.3892/ol.2016.4520

Figure Lengend Snippet: Tumor growth suppression in a human renal cancer SW-839 xenograft nude mouse model. (A and B) Tumor volume of CC and control tumors. (C) Tumor weight of CC and control tumors. (D) No significant toxicity to mice was observed following treatment with CC (5 mg/kg/day), according to the body weight of the mice. (E) Representative TUNEL staining (red fluorescence) of SW-839 renal cancer xenografts. (F) Quantification of TUNEL + SW-839 cells in tumor xenografts was performed using cellSens Standard software. The data are presented as the mean ± standard deviation of three experiments. **P<0.01 vs. control cells. (G) IHC staining for Bcl-2 and Bcl-2-associated X protein on tumor sections, and (H) quantification of IHC staining using cellSens Standard software. CC, chelerythrine chloride; TUNEL, terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein; IHC, immunohistochemistry.

Article Snippet: One Step TUNEL Apoptosis Assay Kit (Beyotime Institute of Biotechnology, Haimen, China) was used to stain the apoptotic tumor cells.

Techniques: Control, TUNEL Assay, Staining, Fluorescence, Software, Standard Deviation, Immunohistochemistry, End Labeling

Journal: Oncology Letters

Article Title: Chelerythrine chloride induces apoptosis in renal cancer HEK-293 and SW-839 cell lines

doi: 10.3892/ol.2016.4520

Figure Lengend Snippet: (Aa) Western blot analysis of the expression levels of apoptosis-associated proteins in HEK-293 and human renal cancer SW-839 cells following treatment with CC. Quantification of the expression of various proteins in HEK-293 and SW-839 cells, such as (Ab) p53, (Ac) Bax, (Ad) Bcl-2, (Ae) pro-caspase-3, (Af) cleaved caspase 3 and (Ag) PARP using GAPDH as a control. Multiple bands were observed in the cleaved caspase-3 lane due to non-specific binding of antibodies. (Ab-Ag) Quantification of western blotting (*P<0.05 and **P<0.01 vs. controls). (Ba) Western blot analysis of MAPK and Akt pathways after CC treatment in HEK-293 and SW-839 cells. The quantification of protein expression was performed for (Bb) pERK, (Bc) p-p38, (Bd) p-JNK and (Be) p-AKT, with GADPH as a control. The results are representative of ≥3 independent experiments. Multiple bands were observed in the p-ERK, p-JNK and JNK lanes due to non-specific binding of antibodies. *P<0.05 and **P<0.01 vs. controls. CC, chelerythrine chloride; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein; PARP, poly (adenosine diphosphate-ribose) polymerase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; p-, phospho-; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase.

Article Snippet: One Step TUNEL Apoptosis Assay Kit (Beyotime Institute of Biotechnology, Haimen, China) was used to stain the apoptotic tumor cells.

Techniques: Western Blot, Expressing, Control, Binding Assay