Journal: Nucleic Acids Research

Article Title: A regulatory circuit comprising GATA1/2 switch and microRNA-27a/24 promotes erythropoiesis

doi: 10.1093/nar/gkt848

Figure Lengend Snippet: GATA-1 was located on the upstream of miR-23a∼27a∼24-2 cluster and activated its expression during erythropoiesis. ( A ) Q-PCR analysis of the primary transcript of the miR-23a∼27a∼24-2 cluster in K562s undergoing erythroid differentiation caused by hemin treatment for 0, 24, 48 and 72 h. ( B ) Q-PCR analysis of the mature transcripts of miR-27a, miR-24 and miR-23a in K562s undergoing erythroid differentiation caused by hemin treatment for 0, 24, 48 and 72 h. ( C ) Northern blot analysis of miR-27a, miR-24 and miR-23a in K562s undergoing erythroid differentiation. U6 snRNA was used as a loading control. ( D ) Q-PCR analysis of mature miR-27a and miR-24 expression in CD34+ HPCs undergoing day 4, 7, 11, 15 and 18 of E culture. ( E ) Representation of the human −603 bp miR-23a∼27a∼24 cluster promoter fragments. ( F ) ChIP-PCR and ChIP-qPCR analysis of the GATA-1 hit on the −557 site at the miR-23a∼27a∼24 cluster promoter in K562s. ChIP-q-PCR results are shown as fold enrichment compared with input. Error bars represent the standard deviation obtained from three independent experiments. ** P < 0.01. ( G ) Immunoblot analysis of GATA-1 expression in K562s treated with siRNAs specific to GATA-1 (si_GATA-1) for 48 h or K562s transfected with a construct overexpressing GATA-1 (over_GATA-1) for 48 h, respectively. ( H ) Q-PCR analysis of Pri-27a∼24, mature miR-27a and miR-24 expression in K562s treated as described in (G). ( I ) Immunoblot analysis of GATA-1 expression in K562s undergoing erythroid differentiation. ( J ) ChIP-PCR and ChIP-q-PCR analysis of GATA-1 and pol II occupancy at the −557 site in hemin-treated K562s at 0 and 48 h. ChIP-q-PCR results are shown as fold enrichment compared with input. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05. ( K ) Functional activity of GATA-1 on the wild-type GATA site (WT) or mutant site (MUT) of the miR-23a∼27a∼24 promoter in a luciferase reporter analysis. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05.

Article Snippet: CD34+ cells were enriched from mononuclear cells through positive immunomagnetic selection (CD34 MultiSort kit, Miltenyi Biotec, Bergisch-Glad-bach, Germany).

Techniques: Expressing, Northern Blot, Control, ChIP-qPCR, Standard Deviation, Western Blot, Transfection, Construct, Functional Assay, Activity Assay, Mutagenesis, Luciferase

Journal: Nucleic Acids Research

Article Title: A regulatory circuit comprising GATA1/2 switch and microRNA-27a/24 promotes erythropoiesis

doi: 10.1093/nar/gkt848

Figure Lengend Snippet: MiR-27a and miR-24 promoted erythroid differentiation in CD34+ HPCs. ( A ) Monitoring of the GFP + population (left panel) and the CD235a stained GFP + fraction (medium panel) of Lenti-miRNA-transduced CD34+ HPCs on day 15 of E culture. The morphology (May-Grunwald Giemsa staining) of CD34+ HPCs derivatives on day 15 is shown in the right panel. A 400X magnification of a representative field is shown. ( B ) Detection of the erythroid differentiation degree of CD34+ HPCs transduced with Lenti-27a, Lenti-24 or Lenti-GFP in E culture at the indicated time. Percentages of basophilic (Bas), polychromatophilic (Pol), orthochromatic (Ort) erythroblasts and erythrocytes (Ery) were determined by May-Grunwald/Giemsa staining of cytospin preparations. ( C ) Q-PCR analysis of gamma-globin mRNA expression in CD34+ HPCs transduced with Lenti-27a, Lenti-24 or Lenti-GFP in E culture at the indicated time. ( D ) A comparison of the erythroid colony-forming capacity (CFU-E, BFU-E) of CD34+ HPCs transduced with Lenti-miRNAs. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05; ** P < 0.01. ( E ) FACS monitoring of CD34+ HPCs transduced with Zip-miRNA or Zip-GFP as described in (A). ( F ) Detection of the erythroid differentiation degree of CD34+ HPCs transduced with Zip-27a, Zip-24 or Zip-GFP as described in (B). ( G ) Detection of gamma-globin mRNA level in CD34+ HPCs transduced with Zip-miRNA. ( H ) Colony-forming assay of CD34+ HPCs transduced with Zip-miRNA as described in (D).

Article Snippet: CD34+ cells were enriched from mononuclear cells through positive immunomagnetic selection (CD34 MultiSort kit, Miltenyi Biotec, Bergisch-Glad-bach, Germany).

Techniques: Staining, Transduction, Expressing, Comparison, Standard Deviation

Journal: Nucleic Acids Research

Article Title: A regulatory circuit comprising GATA1/2 switch and microRNA-27a/24 promotes erythropoiesis

doi: 10.1093/nar/gkt848

Figure Lengend Snippet: GATA-2 was post-transcriptionally regulated by miR-27a and miR-24 during erythropoiesis. ( A ) A computer prediction of conserved and mutated binding sites within the 3′ UTR of GATA-2 mRNA for miR-27a and miR-24. ( B ) Relative luciferase activity of the indicated GATA-2 reporter constructs. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05. ( C ) Immunoblot analysis of GATA-2 in K562s transfected with scramble or miRNA mimics (miR-27a, miR-24) or inhibitors (Anti-27a, Anti-24). ( D ) Immunoblot analysis of GATA-2 in CD34+ HPCs transduced with Lenti-GFP control and lentivirus expressing miR-27a or miR-24 (Lenti-27a or Lenti-24). ( E, F ) ‘Rescue’ assays for miRNAs and GATA-2 during erythroid differentiation. Immunoblot analysis of GATA-2 in K562s treated with scramble or Anti-27a/24 (E) for 24 h. These cells were subsequently treated for another 24 h with control siRNAs or siRNAs specific to GATA-2 and were then treated with hemin treatment for 0, 48 and 72 h. (F) FACS analysis of K562s stained for CD71 and CD235a expression after 48 h of hemin induction as described earlier in the text.

Article Snippet: CD34+ cells were enriched from mononuclear cells through positive immunomagnetic selection (CD34 MultiSort kit, Miltenyi Biotec, Bergisch-Glad-bach, Germany).

Techniques: Binding Assay, Luciferase, Activity Assay, Construct, Standard Deviation, Western Blot, Transfection, Transduction, Control, Expressing, Staining

Journal: Nucleic Acids Research

Article Title: A regulatory circuit comprising GATA1/2 switch and microRNA-27a/24 promotes erythropoiesis

doi: 10.1093/nar/gkt848

Figure Lengend Snippet: The GATA switch regulated miR-27a and miR-24 expression. ( A ) ChIP-PCR and ChIP-q-PCR analysis of GATA-1, GATA-2 and Pol II occupancy at the −557 site in hemin-treated K562s at 0 and 48 h. ChIP-q-PCR results are shown as fold enrichment compared with input. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05; ** P < 0.01. ( B ) Immunoblot analysis of GATA-1 and GATA-2 expression in K562s undergoing erythroid differentiation for 0, 24, 48 and 72 h. ( C ) Functional activity of GATA-1 and GATA-2 on the wild-type GATA site (WT) or mutant site (MUT) of the miR-23a∼27a∼24 promoter in a luciferase reporter analysis. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05. ( D ) Immunoblot analysis of GATA-2 in K562s treated with siRNAs specific to GATA-2 (si_GATA-2) for 48 h or K562s transfected with a construct overexpressing GATA-2 (over_GATA-2) for 48 h, respectively. ( E ) Q-PCR analysis of Pri-27a∼24, mature miR-27a and miR-24 expression in K562s treated as described in (D). ( F ) ChIP-PCR analysis of GATA-1 and GATA-2 occupancy at the −557 site in K562s transfected with control siRNAs or siRNAs specific to GATA-1/GATA-2 or K562s transfected with empty pcDNA3.1 vectors or pcDNA3.1_GATA-1/GATA-2. ( G ) ChIP-PCR and ChIP-q-PCR analysis of GATA-1 and GATA-2 occupancy at the −557 site in CD34+ HPCs of E culture. ( H ) Q-PCR analysis of Pri-27a-24 abundance in CD34+ HPCs of E culture (Top panel). Error bars represent the standard deviation obtained from three independent experiments. An immunoblot analysis of GATA-1 and GATA-2 expression in CD34+ HPCs of E culture (Bottom panel). ( I ) Q-PCR (left panel) and immunoblot (right panel) analysis of GATA-1 and GATA-2 expression in CD34+ HPCs transduced with lentivirus-expressing siRNAs against GATA-1 or control on day 11 of E culture. Error bars represent standard deviation obtained from three independent experiments. * P < 0.05; ** P < 0.01. ( J ) Q-PCR analysis of Pri-27a∼24, mature miR-27a and miR-24 expression in CD34+ HPCs treated as described in (I). Error bars represent the standard deviation obtained from three independent experiments. ** P < 0.01.

Article Snippet: CD34+ cells were enriched from mononuclear cells through positive immunomagnetic selection (CD34 MultiSort kit, Miltenyi Biotec, Bergisch-Glad-bach, Germany).

Techniques: Expressing, Standard Deviation, Western Blot, Functional Assay, Activity Assay, Mutagenesis, Luciferase, Transfection, Construct, Control, Transduction

Journal: Nucleic Acids Research

Article Title: A regulatory circuit comprising GATA1/2 switch and microRNA-27a/24 promotes erythropoiesis

doi: 10.1093/nar/gkt848

Figure Lengend Snippet: A regulatory circuit involving GATA-1, GATA-2 and miR-27a/24 in erythropoiesis. ( A ) A schematic representation of the regulatory circuit comprised GATA-1, GATA-2, miR-27a and miR-24 in erythroid differentiation. ( B , C ) ChIP-PCR and ChIP-q-PCR analysis of GATA-1 and GATA-2 occupancy at the −557 site in K562s transfected with scramble or miRNA mimics and inhibitors (B, r miR-27a and C, miR-24). ( D ) Q-PCR analysis of Pri-27a∼24 abundance in K562s treated as described in (B) and (C). Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05; ** P < 0.01. ( E ) Q-PCR analysis of Pri-27a∼24 abundance in CD34+ HPCs transduced with Lenti-miR-27a/24 or Lenti-GFP control for 11 days. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05. ( F ) Q-PCR analysis of Pri-27a∼24 abundance in CD34+ HPCs transduced with Zip-miRNA or Zip-GFP for 11 days. Error bars represent the standard deviation obtained from three independent experiments. ** P < 0.01.

Article Snippet: CD34+ cells were enriched from mononuclear cells through positive immunomagnetic selection (CD34 MultiSort kit, Miltenyi Biotec, Bergisch-Glad-bach, Germany).

Techniques: Transfection, Standard Deviation, Transduction, Control

Journal: Cancer Cell

Article Title: Metabolic Imaging Detects Resistance to PI3Kα Inhibition Mediated by Persistent FOXM1 Expression in ER + Breast Cancer

doi: 10.1016/j.ccell.2020.08.016

Figure Lengend Snippet: Persistent FOXM1 Expression in Breast Cancer PDXs That Have Acquired Resistance to PI3Kα Inhibition (A) Relapse of an HCI-011 PDX after treatment with GDC-0032 (HCI-011R). (B) Mean tumor volumes (cm 3 ) ± SEM of HCI011R xenografts receiving drug vehicle (n = 5) or GDC-0032 (n = 8). p values were defined using two-sided Wald t tests. (C) Immunoblots of lysates from (B) and from an HCI-011 tumor. (D) Mean tumor volumes (cm 3 ) ± SEM of HCI011R xenografts treated with drug vehicle (n = 2), drug vehicle plus tamoxifen (n = 3), and tamoxifen plus GDC-0032 (n = 5). p values were defined using two-sided Wald t tests. (E) Immunoblots of lysates from (D). Mean ± standard deviation (n = 2 to 4). p values were calculated using two-sided Welch's t tests. (F) Gene expression (Log2) enrichment (heatmap: red, positive; white, neutral; blue, negative) of FOXM1 gene targets in HCI-011 (n = 4) and in HCI-011R xenografts (n = 8). The top row shows false discovery rate (FDR)-adjusted p values. (G) Gene expression (Log2) enrichment of genes mediating resistance to BYL-719 in HCI-011 (n = 4 for vehicle and drug) and in HCI-011R (n = 5 vehicle, n = 8 GDC-0032). FDR-adjusted p values of drug-resistant versus drug-sensitive differential expression are shown in the top row. (H) Mean tumor volume (cm 3 ) ± SEM of drug-resistant T47DR tumors after long-term treatment with GDC-0032. p values were defined using two-sided Wald t tests. (I) Immunoblot of tumors lysates from (H). See also Figure S5 .

Article Snippet: PDTCs from HCI-001, HCI-011, HCI-011R tumors and cells from T47DR tumors were obtained by dissociating them using a kit (human 130-095-929, Miltenyi Biotec), as described previously ( ).

Techniques: Expressing, Inhibition, Western Blot, Standard Deviation, Gene Expression, Quantitative Proteomics

Journal: Cancer Cell

Article Title: Metabolic Imaging Detects Resistance to PI3Kα Inhibition Mediated by Persistent FOXM1 Expression in ER + Breast Cancer

doi: 10.1016/j.ccell.2020.08.016

Figure Lengend Snippet: Persistent FOXM1 Expression Contributes to Drug Resistance and Can Be Explained by Cytoplasmic Localization of FOXO3a (A) Representative confocal microscopy images of T47D PTEN wt (Ctrl) and PTEN KO cells drug-treated for 72 h. FOXO-3a staining (green), DAPI staining (blue). (B) Relative number of red fluorescent MCF7 and T47D cells (mStrawberry-positive cells, co-expressing FOXM1C) in mixtures with parental controls following treatment for 120 h. p values were calculated using two-sided Welch's t tests. (C) Viability of HCI-011R PDTCs treated with the indicated drugs for 120 h. Left: viability of HCI-011R PDTCs treated for 120 h. Mean ± standard deviation (five technical replicates, n = 3 experiments). Right: mean ± standard deviation (five technical replicates, n = 4 experiments). p values were calculated using two-sided Welch's t tests on the averages of replicates. (D) Viability of cells dissociated from a T47DR tumor treated with the indicated drugs for 120 h. Left: mean ± standard deviation (five technical replicates, n = 4 experiments). Right: mean ± standard deviation (five technical replicates, n = 3 experiments). p values were calculated using two-sided Welch's t tests on the averages of replicates. (E) Viability of T47D PTEN KO cells expressing FOXM1 targeting sequences (shFOXM1 [sequence 1 or 2]) or a non-targeting control sequence (shCtrl) treated for 120 h. Mean ± standard deviation (n = 5, five technical replicates). p values were calculated using two-sided Welch's t tests on the averages of replicates. (F) Viability of HCI-011R PDTCs expressing doxycycline-inducible shRNA sequences targeting FOXM1 (sequence 1) or a control shRNA and treated for 120 h. Mean ± standard deviation (three technical replicates). p values were calculated using two-sided Welch's t tests on the averages of the replicates. (G) LDH activity in the cells used in (E) and treated for 96 h. Figure shows the mean ± standard deviation (n = 3 experiments). p values were calculated using two-sided Welch's t tests on the averages of replicates. (H) Immunoblots of lysates from cells used in (G). (I) Mean tumor volume (cm 3 ) ± SEM of T47D PTEN KO xenografts expressing doxycycline-inducible shFOXM1 (sequence 1) or shCtrl, following prolonged GDC-0032 treatment. One cohort received standard food (shCtrl n = 3 and shFOXM1 n = 3) and the other cohort food plus doxycycline (shCtrl n = 3 and shFOXM1 n = 4; 0.2 g/kg food pellet Harlan D.98186). p values were defined using two-sided Wald t tests. (J) Immunoblot of lysates from tumors in (I). p values were calculated using two-sided Welch's t tests. See also Figure S6 .

Article Snippet: PDTCs from HCI-001, HCI-011, HCI-011R tumors and cells from T47DR tumors were obtained by dissociating them using a kit (human 130-095-929, Miltenyi Biotec), as described previously ( ).

Techniques: Expressing, Confocal Microscopy, Staining, Standard Deviation, Sequencing, Control, shRNA, Activity Assay, Western Blot

Journal: bioRxiv

Article Title: Tuning of granulopoietic signaling by de novo designed agonists

doi: 10.1101/2023.11.25.568662

Figure Lengend Snippet: Representative western blot images of intracellular levels of phospho-STAT3 (Tyr705) and STAT3 (A) and phospho-STAT5 (Tyr694) and STAT5 (B) proteins after treatment of NFS-60 cells with 1 nM of different designs (saturating conditions) for 5 min (left pane) or 30 min (right pane). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) staining was used as a loading control. Three biological replicates of this experiment showed the same trend . (C, D) The pSTAT3 and pSTAT5 levels were quantified after 5 min and 30 min from those replicates and are represented in (C) and (D), respectively. Shown is the fold change to the GAPDH normalized rhG-CSF signal. (E, F) To probe the effect of the agonist designs (100 ng/ml) and rhG-CSF (10 ng/ml) on healthy donoŕs CD34 + HSPCs, proliferation assays were performed without (E) or with the addition of 50 ng/mL SCF and 20 ng/mL IL-3 (F). Ori0 without SCF and IL-3 was tested at three concentrations 100 ng/mL (diamond), 10 ng/mL (triangle-right), and 1 ng/mL (triangle-left). Shown is the mean (points) and standard deviation (shades) of three parallel replicates. (G, H, I) Additionally, we performed CFU assays of healthy donoŕs CD34 + HSPCs incubated on semi-solid medium supplemented with corresponding cytokines and design agonists. Shown is the fold change to rhG-CSF of the quantified colony-forming units (CFU) of granulocytes (G-CFU) (G), granulocyte-macrophages (GM-CFU) (H), and macrophages (M-CFU) (I). The circles represent the obtained values for each condition of three independent experiments with two parallel replicates each. The indicated p-values were estimated by an ordinary one-way ANOVA followed by a Tukey HSD test.

Article Snippet: Human CD34 + cells were isolated from the bone marrow mononuclear cell fraction of two healthy donors by Ficoll density gradient centrifugation with subsequent magnetic bead separation using the Human CD34 Progenitor Cell Isolation Kit (Miltenyi Biotech Germany; #130-046-703).

Techniques: Western Blot, Staining, Control, Standard Deviation, Incubation

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: 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: 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