Journal: Cancer Cell

Article Title: Hoxa9 and Meis1 Cooperatively Induce Addiction to Syk Signaling by Suppressing miR-146a in Acute Myeloid Leukemia

doi: 10.1016/j.ccell.2017.03.001

Figure Lengend Snippet: Meis1 Increases Syk Protein Levels in Hoxa9-Driven Leukemia (A) Kaplan-Meier survival curves of mice transplanted with either H- or H/M-transformed myeloid progenitor cells (n = 11). The p value is from a Mantel-Cox test. (B) Volcano plot relating q values for differential protein expression to average normalized SILAC ratios from six biological replicates. Blue (higher expression in H cells) and orange (higher expression in H/M cells) dots indicate significantly regulated proteins (q < 0.01). (C) Heatmap of SILAC ratios for significantly differentially expressed proteins in H and H/M cells across the six biological replicates. (D) Syk protein expression in H and H/M cells by immunoblotting. Actin was used as loading control for relative protein quantification. (E) Relative Syk mRNA expression as measured by qPCR, normalized to GAPDH expression (mean ± SD, n = 3); ns, not significant (two-sided unpaired t test). (F and G) Immunohistochemical staining of HOXA9, MEIS1, and SYK in bone marrow biopsies from patients with AML. SYK expression levels were analyzed in 21 AML cases with high HOXA9 expression (F) and 28 cases with high HOXA9/MEIS1 expression (G). Proportions of SYK expression levels as determined by two independent pathologists using a three-stage staining score are shown. See also Figure S1 , , and .

Article Snippet: CD34+ BM-MNCs were isolated using the human CD34 MultiSort Kit (Miltenyi Biotec) following the manufacturer's instructions.

Techniques: Transformation Assay, Expressing, Multiplex sample analysis, Western Blot, Control, Immunohistochemical staining, Staining

Journal: Cancer Cell

Article Title: Hoxa9 and Meis1 Cooperatively Induce Addiction to Syk Signaling by Suppressing miR-146a in Acute Myeloid Leukemia

doi: 10.1016/j.ccell.2017.03.001

Figure Lengend Snippet: Meis1 Sensitizes Hoxa9-Driven Leukemia to Syk Inhibition (A) Syk protein expression in H/M cells transfected with either a control shRNA (GL2) or two shRNAs targeting Syk. Actin was used as loading control for relative protein quantification. (B) Percentage of BFP-positive shRNA-expressing cells relative to BFP-negative shRNA-negative cells at the times indicated (mean ± SD, normalized to day 0, n = 3). (C) Same as (A), before and after 5 days of doxycycline (dox) treatment in vivo. (D) Kaplan-Meier survival curves of mice transplanted with H/M cells and treated with doxycycline for 43 days to express non-specific control and Syk-specific shRNA (n = 8). The p value is from a Mantel-Cox test. (E) Percentage of YFP-positive cells from peripheral blood of mice transplanted with H (left) or H/M (right) cells after treating for 7 days with R788 or placebo. Measurements were taken at the indicated time points. The black line connects median values. (F) Kaplan-Meier survival curves of mice transplanted with either H or H/M cells and treated for 20 days with R788 or placebo (n = 11). The p value is from a Mantel-Cox test. (G) Relative HOXA9 and MEIS1 mRNA expression in MV4-11 and KG1 cell lines, and in patient-derived AML cells as measured by qPCR, normalized to GAPDH expression (mean ± SD, n = 3). (H) (p)SYK expression in the patient-derived AML cells in (G). Actin was used as loading control for relative protein quantification. avg, average. (I) Half maximal inhibitory concentration (IC 50 ) for R406 (left) and PRT062607 (right) in patient-derived AML cells as determined by an Annexin V/7-AAD apoptosis assay. Cells were treated for 24 hr and DMSO was used as a control (n = 3). Representative dose-response curves for AML no. 1 (HOXA9 high, MEIS1 low) and AML no. 5 (HOXA9 high, MEIS1 high) are shown at the top. Ticks correspond to estimated IC 50 values. (J) Relative viability of CD34 + bone marrow cells from healthy donors. Cells were treated with either R406 or PRT062607. Blue lines indicate the IC 50 for both SYK inhibitors in H cells. (K) Kaplan-Meier survival curves of NSG mice transplanted with patient-derived AML cells indicated in (G) and treated for 14 days with R788 or vehicle (n = 6 for AML no. 1 and 5; n = 5 for AML no. 2 and 6). The p values are from a Mantel-Cox test. See also Figure S6 .

Article Snippet: CD34+ BM-MNCs were isolated using the human CD34 MultiSort Kit (Miltenyi Biotec) following the manufacturer's instructions.

Techniques: Inhibition, Expressing, Transfection, Control, shRNA, In Vivo, Derivative Assay, Concentration Assay, Apoptosis Assay

Journal: Cancer Cell

Article Title: Hoxa9 and Meis1 Cooperatively Induce Addiction to Syk Signaling by Suppressing miR-146a in Acute Myeloid Leukemia

doi: 10.1016/j.ccell.2017.03.001

Figure Lengend Snippet:

Article Snippet: CD34+ BM-MNCs were isolated using the human CD34 MultiSort Kit (Miltenyi Biotec) following the manufacturer's instructions.

Techniques: Recombinant, Blocking Assay, Lysis, SYBR Green Assay, Reporter Assay, Extraction, Bicinchoninic Acid Protein Assay, cDNA Synthesis, Reverse Transcription, TaqMan microRNA Assay, Mass Spectrometry, Microarray, Gene Expression, Retroviral, Negative Control, Plasmid Preparation, Software, Multiplex sample analysis

Journal: Arthritis Research & Therapy

Article Title: Association of circulating microRNAs with prevalent and incident knee osteoarthritis in women: the OFELY study

doi: 10.1186/s13075-019-2086-5

Figure Lengend Snippet: Baseline characteristics of postmenopausal women included in the knee OA study with the localization of OA, if present at other sites than the knee

Article Snippet: Okuhara, 2012 , Trizol reagent (Invitrogen) , Thermocycler (BioRad) , TaqMan miRNA assay kit (Applied) control: U18 , Mini Opticon Real-time PCR System (BioRad) , 146, 155, 181a, 223 upregulated in OA vs CTL ealy stage: 146a and 223 higher than in late stage.

Techniques: Control, Real-time Polymerase Chain Reaction

Journal: Arthritis Research & Therapy

Article Title: Association of circulating microRNAs with prevalent and incident knee osteoarthritis in women: the OFELY study

doi: 10.1186/s13075-019-2086-5

Figure Lengend Snippet: A summary of studies on human miRNAs with dysregulated expression in patients suffering from OA, including the biological fluid tested, the number of patients with their baseline characteristics, the criteria for patient selection, the quantification methods, and the miRNAs significantly dysregulated

Article Snippet: Okuhara, 2012 , Trizol reagent (Invitrogen) , Thermocycler (BioRad) , TaqMan miRNA assay kit (Applied) control: U18 , Mini Opticon Real-time PCR System (BioRad) , 146, 155, 181a, 223 upregulated in OA vs CTL ealy stage: 146a and 223 higher than in late stage.

Techniques: Expressing, Selection, Clinical Proteomics, Biomarker Discovery, Isolation, cDNA Synthesis, Control, Quantitative Proteomics, Diagnostic Assay, Marker, Reverse Transcription, TaqMan microRNA Assay, Real-time Polymerase Chain Reaction, Software, SYBR Green Assay, Gene Expression, Microarray, Labeling, Hybridization, Extraction

Journal: Arthritis Research & Therapy

Article Title: Association of circulating microRNAs with prevalent and incident knee osteoarthritis in women: the OFELY study

doi: 10.1186/s13075-019-2086-5

Figure Lengend Snippet: Differential expression of the miRNA level in the serum from controls and OA patients analyzed by quantitative real-time PCR

Article Snippet: Okuhara, 2012 , Trizol reagent (Invitrogen) , Thermocycler (BioRad) , TaqMan miRNA assay kit (Applied) control: U18 , Mini Opticon Real-time PCR System (BioRad) , 146, 155, 181a, 223 upregulated in OA vs CTL ealy stage: 146a and 223 higher than in late stage.

Techniques: Quantitative Proteomics

Journal: iScience

Article Title: Prothymosin α accelerates dengue virus-induced thrombocytopenia

doi: 10.1016/j.isci.2023.108422

Figure Lengend Snippet: Overexpression of ProT promotes Nrf2 nuclear translocation and decreases ROS production, leading to reduced PLT production (A) Detection of Nrf2 and viral NS3 in Meg-01 cells infected with DENV-2 at an MOI of 1 at different time points by immunoblotting. Expression of GAPDH served as the loading control. Values shown at the bottom are ratios between the intensity of the bands corresponding to Nrf2 and those corresponding to GAPDH. (B) Nuclear translocation of Nrf2 in ProT-overexpressing cells. Meg-01 cells were transfected with pLAS2w.hProT-HA (ProT-HA), pLAs2w.hProT-Venus (ProT-Venus), or pLAS2w.Ppuro (Vector). After 48 h, nuclear and cytosolic extracts were harvested for immunoblot analysis. Lamin A/C and α-tubulin served as the nuclear and cytoplasmic markers, respectively. Values shown at the bottom are ratios between the intensity of the bands corresponding to Nrf2 and those corresponding to α-tubulin or Lamin A/C. (C and D) Quantification of HO-1 (left) and NQO1 (middle) by RT-qPCR and measurement of ROS production (right) in Meg-01 cells transduced with lentiviral vectors encoding ProT (LV.ProT) or no transgenes (LV.Null) (C) and shRNA specific to ProT (LV.shProT-17 and -20) or LacZ (LV.shLacZ) (D). ROS production in ProT overexpression (C) and knockdown (D) Meg-01 cells or control cells were measured by flow cytometry after stimulation with PMA (10 −6 M) for 7 days and staining with DCFDA. Values shown are means ± SD (n = 3, Student’s t test or one-way ANOVA). (E) Overexpression of miR-126 reduces Nrf2 levels and antioxidant gene expression. Detection and quantification of Nrf2, HO-1, and NQO1 in Meg-01 cells transduced with LV.miR-126 or LV.miR-Ctrl by RT-qPCR. Values shown are mean ± SD (n = 4, Student’s t test). (F) Quantification of GP9 and TUBB1, which are involved in PLT release, in ProT knockdown (shProT-20) or control (shLacZ) Meg-01 cells by RT-qPCR. Values shown are means ± SD (n = 3, Student’s t test). (G) Detection of the PLT activation marker CD42b in ProT knockdown (shProT-20) or control (shLacZ) Meg-01 cells by flow cytometry. Values shown are means ± SD (n = 3, Student’s t test or one-way ANOVA).

Article Snippet: rabbit anti-Nrf2 antibody , Proteintech , Cat #16396-1-AP; RRID: AB_2782956.

Techniques: Over Expression, Translocation Assay, Infection, Western Blot, Expressing, Control, Transfection, Plasmid Preparation, Quantitative RT-PCR, Transduction, shRNA, Knockdown, Flow Cytometry, Staining, Gene Expression, Activation Assay, Marker

Journal: iScience

Article Title: Prothymosin α accelerates dengue virus-induced thrombocytopenia

doi: 10.1016/j.isci.2023.108422

Figure Lengend Snippet:

Article Snippet: rabbit anti-Nrf2 antibody , Proteintech , Cat #16396-1-AP; RRID: AB_2782956.

Techniques: Recombinant, Stripping Membranes, Isolation, Reverse Transcription, TaqMan microRNA Assay, Plasmid Preparation, shRNA, Software, RNA Expression, Knock-Out, Microarray

Journal: Cancer Cell

Article Title: Hoxa9 and Meis1 Cooperatively Induce Addiction to Syk Signaling by Suppressing miR-146a in Acute Myeloid Leukemia

doi: 10.1016/j.ccell.2017.03.001

Figure Lengend Snippet: Meis1 Increases Syk Protein Levels in Hoxa9-Driven Leukemia (A) Kaplan-Meier survival curves of mice transplanted with either H- or H/M-transformed myeloid progenitor cells (n = 11). The p value is from a Mantel-Cox test. (B) Volcano plot relating q values for differential protein expression to average normalized SILAC ratios from six biological replicates. Blue (higher expression in H cells) and orange (higher expression in H/M cells) dots indicate significantly regulated proteins (q < 0.01). (C) Heatmap of SILAC ratios for significantly differentially expressed proteins in H and H/M cells across the six biological replicates. (D) Syk protein expression in H and H/M cells by immunoblotting. Actin was used as loading control for relative protein quantification. (E) Relative Syk mRNA expression as measured by qPCR, normalized to GAPDH expression (mean ± SD, n = 3); ns, not significant (two-sided unpaired t test). (F and G) Immunohistochemical staining of HOXA9, MEIS1, and SYK in bone marrow biopsies from patients with AML. SYK expression levels were analyzed in 21 AML cases with high HOXA9 expression (F) and 28 cases with high HOXA9/MEIS1 expression (G). Proportions of SYK expression levels as determined by two independent pathologists using a three-stage staining score are shown. See also Figure S1 , , and .

Article Snippet: TaqMan® MicroRNA Assay Gapdh , Thermo Fisher Scientific , Cat# 4331182 Assay ID: Mm99999915_g1.

Techniques: Transformation Assay, Expressing, Multiplex sample analysis, Western Blot, Control, Immunohistochemical staining, Staining

Journal: Cancer Cell

Article Title: Hoxa9 and Meis1 Cooperatively Induce Addiction to Syk Signaling by Suppressing miR-146a in Acute Myeloid Leukemia

doi: 10.1016/j.ccell.2017.03.001

Figure Lengend Snippet: Syk Is a Direct Target of miR-146a (A) Schematic workflow of the miRNA expression analysis in H- and H/M-transformed myeloid progenitors. (B) Volcano plot relating q values for differential miRNA expression between H and H/M cells to average miRNA expression fold-changes from three biological replicates. Blue (higher expression in H cells) and orange (higher expression in H/M cells) dots indicate significantly regulated miRNAs (q < 0.01). (C and D) Relative mmu-miR-146a expression (C) and pri-miR-146a expression (D) in H/M versus H cells, measured by qPCR and normalized to sno202 and GAPDH expression, respectively (mean ± SD, n = 3). The p values are from a two-sided unpaired t test. (E) Luciferase assay validating binding of miR-146a to the predicted target sites within the 3′ UTR of Syk (mean ± SD, n = 4); WT, predicted miR-146a target sequence; MUT, mutated version thereof. The p values are from a two-sided unpaired t test. ns, not significant. (F) Luciferase assay validating binding of miR-146a to the full-length Syk 3′ UTR (mean ± SD, n = 4). The p value is from a two-sided unpaired t test. (G) Left, secondary structure of mmu-miR-146 as predicted by RNAfold ( Lorenz et al., 2011 ). The CRISPR/Cas9 cleavage site is indicated. Right, relative expression of miR-146a, measured by qPCR and normalized to sno202 expression, in H cells transduced with either a lentiviral non-specific (nsp) control CRISPR or a CRISPR targeting miR-146 (ΔmiR-146) (mean ± SD, n = 3). The p value is from a two-sided unpaired t test. (H) Corresponding Syk protein expression by immunoblotting. Actin was used as loading control for relative protein quantification. (I) Cell-proliferation curves for H cells transduced with either a lentiviral non-specific (nsp) control CRISPR or a CRISPR targeting miR-146 (ΔmiR-146) (mean ± SD, n = 3). (J) Kaplan-Meier survival curves of mice transplanted with H or H/M cells transduced with a lentiviral non-specific (nsp) control CRISPR, or with H cells transduced with a CRISPR targeting miR-146 (ΔmiR-146) (n = 7). The p value is from a Mantel-Cox test. See also Figure S3 .

Article Snippet: TaqMan® MicroRNA Assay Gapdh , Thermo Fisher Scientific , Cat# 4331182 Assay ID: Mm99999915_g1.

Techniques: Expressing, Transformation Assay, Luciferase, Binding Assay, Sequencing, CRISPR, Transduction, Control, Western Blot

Journal: Cancer Cell

Article Title: Hoxa9 and Meis1 Cooperatively Induce Addiction to Syk Signaling by Suppressing miR-146a in Acute Myeloid Leukemia

doi: 10.1016/j.ccell.2017.03.001

Figure Lengend Snippet: Meis1 Downregulates miR-146a through PU.1 (A) Fold enrichment of PU.1 binding over IgG control as measured by ChIP-qPCR in H and H/M cells (mean ± SD, n = 3). The miR-146a −10 kb region spans the transcription start site of the miR-146a host gene; ns, not significant. (B) PU.1 protein expression in H and H/M cells by immunoblotting. Histone H3 was used as loading control for relative protein quantification. (C) Relative PU.1 mRNA expression in H versus H/M cells measured by qPCR and normalized to GAPDH expression (mean ± SD, n = 3). (D and E) Immunohistochemical staining of PU.1 in bone marrow biopsies from patients with AML. PU.1 expression levels were analyzed in 21 AML cases with high HOXA9 expression (D) and 28 cases with high HOXA9/MEIS1 expression (E). Proportions of PU.1 expression levels as determined by two independent pathologists using a three-stage staining score are shown. (F) PU.1 and SYK protein expression by immunoblotting in H cells transfected with either a control shRNA (nsp) or an shRNA targeting PU.1 (KD). Tubulin was used as loading control for relative protein quantification. (G) mmu-miR-146a and pri-miR-146a expression as measured by qPCR after PU.1 knockdown (KD) relative to control shRNA (nsp) (mean ± SD, n = 4). The p values are from a two-sided unpaired t test. See also Figure S4 and .

Article Snippet: TaqMan® MicroRNA Assay Gapdh , Thermo Fisher Scientific , Cat# 4331182 Assay ID: Mm99999915_g1.

Techniques: Binding Assay, Control, ChIP-qPCR, Expressing, Western Blot, Immunohistochemical staining, Staining, Transfection, shRNA, Knockdown

Journal: Cancer Cell

Article Title: Hoxa9 and Meis1 Cooperatively Induce Addiction to Syk Signaling by Suppressing miR-146a in Acute Myeloid Leukemia

doi: 10.1016/j.ccell.2017.03.001

Figure Lengend Snippet: Meis1 Sensitizes Hoxa9-Driven Leukemia to Syk Inhibition (A) Syk protein expression in H/M cells transfected with either a control shRNA (GL2) or two shRNAs targeting Syk. Actin was used as loading control for relative protein quantification. (B) Percentage of BFP-positive shRNA-expressing cells relative to BFP-negative shRNA-negative cells at the times indicated (mean ± SD, normalized to day 0, n = 3). (C) Same as (A), before and after 5 days of doxycycline (dox) treatment in vivo. (D) Kaplan-Meier survival curves of mice transplanted with H/M cells and treated with doxycycline for 43 days to express non-specific control and Syk-specific shRNA (n = 8). The p value is from a Mantel-Cox test. (E) Percentage of YFP-positive cells from peripheral blood of mice transplanted with H (left) or H/M (right) cells after treating for 7 days with R788 or placebo. Measurements were taken at the indicated time points. The black line connects median values. (F) Kaplan-Meier survival curves of mice transplanted with either H or H/M cells and treated for 20 days with R788 or placebo (n = 11). The p value is from a Mantel-Cox test. (G) Relative HOXA9 and MEIS1 mRNA expression in MV4-11 and KG1 cell lines, and in patient-derived AML cells as measured by qPCR, normalized to GAPDH expression (mean ± SD, n = 3). (H) (p)SYK expression in the patient-derived AML cells in (G). Actin was used as loading control for relative protein quantification. avg, average. (I) Half maximal inhibitory concentration (IC 50 ) for R406 (left) and PRT062607 (right) in patient-derived AML cells as determined by an Annexin V/7-AAD apoptosis assay. Cells were treated for 24 hr and DMSO was used as a control (n = 3). Representative dose-response curves for AML no. 1 (HOXA9 high, MEIS1 low) and AML no. 5 (HOXA9 high, MEIS1 high) are shown at the top. Ticks correspond to estimated IC 50 values. (J) Relative viability of CD34 + bone marrow cells from healthy donors. Cells were treated with either R406 or PRT062607. Blue lines indicate the IC 50 for both SYK inhibitors in H cells. (K) Kaplan-Meier survival curves of NSG mice transplanted with patient-derived AML cells indicated in (G) and treated for 14 days with R788 or vehicle (n = 6 for AML no. 1 and 5; n = 5 for AML no. 2 and 6). The p values are from a Mantel-Cox test. See also Figure S6 .

Article Snippet: TaqMan® MicroRNA Assay Gapdh , Thermo Fisher Scientific , Cat# 4331182 Assay ID: Mm99999915_g1.

Techniques: Inhibition, Expressing, Transfection, Control, shRNA, In Vivo, Derivative Assay, Concentration Assay, Apoptosis Assay

Journal: Cancer Cell

Article Title: Hoxa9 and Meis1 Cooperatively Induce Addiction to Syk Signaling by Suppressing miR-146a in Acute Myeloid Leukemia

doi: 10.1016/j.ccell.2017.03.001

Figure Lengend Snippet:

Article Snippet: TaqMan® MicroRNA Assay Gapdh , Thermo Fisher Scientific , Cat# 4331182 Assay ID: Mm99999915_g1.

Techniques: Recombinant, Blocking Assay, Lysis, SYBR Green Assay, Reporter Assay, Extraction, Bicinchoninic Acid Protein Assay, cDNA Synthesis, Reverse Transcription, TaqMan microRNA Assay, Mass Spectrometry, Microarray, Gene Expression, Retroviral, Negative Control, Plasmid Preparation, Software, Multiplex sample analysis

Journal: Frontiers in Immunology

Article Title: Hsa-miR-31 Governs T-Cell Homeostasis in HIV Protection via IFN-γ-Stat1-T-Bet Axis

doi: 10.3389/fimmu.2021.771279

Figure Lengend Snippet: miR-31 is correlated with disease progression during both acute and chronic HIV-1 infection. (A) Unsupervised clustering of the 251 miRNAs. After normalization and filtering of the microarray data, 251 miRNAs were retained for further analysis. Average linkage hierarchical clustering was performed using a centered correlation metric. Twenty-three samples from the FBD study were clustered into 2 groups: the left cluster was a mixture of elite controllers, viremic controllers and progressors; the right was mainly progressors with one exception. (B) Venn diagram showing the numbers of candidate miRNAs filtered with different criteria. The miRNAs in the lower left and right circles were generated by significance analysis of microarrays (SAM) of participants stratified by the CD4+ T cell count (<250 cells/μL vs. >450 cells/μL) and viral load (<2000 copies/mL vs. >10000 copies/mL), respectively. The identified 15 miRNA candidates were marked in red in (A) . (C) Correlation between expression levels of miR-31 and CD4+ T cell counts in HIV-1 infected individuals (FBD, former blood donor cohort). miR-31 expression was quantified by quantitative RT-PCR, and the relationship between relative level of miR-31 and CD4+ T cell count was examined by Spearman correlation (n = 50). Red dots represent patients that eventually reached the defined endpoints. (D) Kaplan-Meier survival curves of FDB patients stratified by median whole blood miR-31 level during the late phase of chronic infection. (E–G) Kaplan-Meier survival curves of another HIV patient cohort (an acute-phase prospective men who have sex with men (MSM) cohort) stratified by plasma miR-31 levels before and after infection. Absolute CD4+ T cell count below 350 cells/μL, initiation of long-term ART, progression to AIDS and death were defined as endpoints of the study. Patients were separated into two groups stratified by the median miR-31 level in plasma collected before infection (E) , during acute infection phase (F) , during early phase of chronic infection (G) .

Article Snippet: PAXgene Blood miRNA Kit (QIAGEN, Hilden, Germany) was used for extraction and purification of total RNA, including miRNA, from whole blood stabilized in PAXgene Blood RNA Tubes (PreAnalytix, BD, UK).

Techniques: Biomarker Discovery, Infection, Microarray, Generated, Cell Counting, Expressing, Quantitative RT-PCR, Clinical Proteomics

Journal: Frontiers in Immunology

Article Title: Hsa-miR-31 Governs T-Cell Homeostasis in HIV Protection via IFN-γ-Stat1-T-Bet Axis

doi: 10.3389/fimmu.2021.771279

Figure Lengend Snippet: Loss of miR-31 triggers CD4+ T cell activation. (A) miR-31 levels in different immune cell subtypes. Data were obtained from a miRNA RTqPCR data from Rossi et al ’s work (see the text for reference). (B) Comparison of absolute naïve CD4+ T cell counts in blood of HIV-1 infected individuals (FBD, n = 50) stratified by miR-31 expression. (C, D) Correlation between miR-31 levels and frequencies of CD38+ T cells (C) or HLA-DR+ T cells in blood of HIV infected individuals (FBD, n=44) (E) Gene set enrichment analysis (GSEA) of “naïve” signature in antagomiR-31- versus antagoNC- treated naïve CD4+ T cells. NES, normalized enrichment score. (F) Heatmap of representative genes associated with activation versus naïve state of T cells. Shown is log2 fold changes of gene expression in antagomiR-31-treated naïve CD4+ T cells relative to that in antagoNC-treated cells (n=3). (G) Effects of antagomiR-31 treatment on CD25 expression of naïve CD4+ T cells, assessed by frequency of CD25+ cells and median fluorescent intensity (MFI) of CD25. Average fold changes were 4.45 and 2.25, respectively (n = 8). Blue, antagomir-31 treated group; red, antagoNC-treated group. Representative FACS data for CD25 were shown in the left panel. (H) Schema of the in vitro assay used for examining the role of miR-31 in HIV-1 infection. CD4+ T cells were sorted, followed by transfection with antagomiR-31 or antagoNC. After 48 hours, cells were stimulated with a mix of anti-CD3 and anti-CD28 antibodies and infected with HIV-1 IIIB 5 days later. (I, J) Cells and supernatants were collected on days 5 and 11 post infection and respectively subjected to flow cytometry for determination of P24-expressing CD4+ T cells (I) and ELISA for quantification of released P24 proteins (J) (n = 3).

Article Snippet: PAXgene Blood miRNA Kit (QIAGEN, Hilden, Germany) was used for extraction and purification of total RNA, including miRNA, from whole blood stabilized in PAXgene Blood RNA Tubes (PreAnalytix, BD, UK).

Techniques: Activation Assay, Comparison, Infection, Expressing, Gene Expression, In Vitro, Transfection, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Toxicology Reports

Article Title: Placental microRNAs: Responders to environmental chemicals and mediators of pathophysiology of the human placenta

doi: 10.1016/j.toxrep.2020.08.002

Figure Lengend Snippet: miRNAs in the placenta that are responsive to exposure to environmental toxicants.

Article Snippet: Toxic Metals , Placenta, JEG-3 cells , N = 32 , 861 , miR-26a, miR-155 , AllPrep DNA/RNA/miRNA Univerisal Kit (Qiagen) , Human miRNA Oligo microarray (Agilent); qRT-PCR , miRNA U6 , (Brooks, Martin et al. 2016).

Techniques: Extraction, Microarray, Isolation

Journal: Physiological Genomics

Article Title: Microarray analysis of aging-associated immune system alterations in the rostral ventrolateral medulla of F344 rats

doi: 10.1152/physiolgenomics.00131.2016

Figure Lengend Snippet: List of immune system genes used for quantitative PCR validation of microarray analysis and their TaqMan assay IDs

Article Snippet: Actb, Hprt1, Ldha , and Rplp1 genes were used as endogenous controls and the geometric average of their Ct values was used to calculate each gene’s ΔCt value ( 1 ). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Functional Category Gene Symbol TaqMan Assay ID Complement system C1qa Rn01519903_m1 C1qc(C1qg) Rn01516757_m1 Cd93(C1qr1) Rn00584525_g1 C3 Rn00584525_g1 C4a Rn00566466_m1 Cfd Rn00709527_m1 Cfh Rn01535436_g1 Vwf Rn00590326_m1 Klkb1 Rn01488161_m1 Microglial cells Cd14 Rn00572656_g1 Cd68 Rn01495634_g1 Tlr2 Rn02133647_s1 Cx3cr1 Rn02134446_s1 Trem2 Rn01512170_m1 Fcrl2 Rn01455191_m1 B2m Rn00560865_m1 Endogenous controls Actb Rn00667869_m1 Hprt1 Rn01527840_m1 Ldha Rn00820751_g1 Rplp1 Rn03467157_gH Open in a separate window List of immune system genes used for quantitative PCR validation of microarray analysis and their TaqMan assay IDs Microarray Data Analysis Hybridization signal intensities from microarrays were extracted with Agilent Feature Extraction Software, version 9.5.1.1. (Agilent Technologies) (Gene Expression Omnibus accession number {"type":"entrez-geo","attrs":{"text":"GSE90956","term_id":"90956"}} GSE90956 ).

Techniques: Real-time Polymerase Chain Reaction, Biomarker Discovery, Microarray, TaqMan Assay