Journal: iScience

Article Title: SGLT1/2 inhibition improves glycemic control and multi-organ protection in type 1 diabetes

doi: 10.1016/j.isci.2023.107260

Figure Lengend Snippet: SGLT2 inhibition results in increased luminal SGLT1 expression in renal tissue of diabetic Akimba mice (A and B) Representative SGLT1 immunohistochemistry images of renal tissue of mice treated with (A) vehicle or (B) Empagliflozin [EMPA] via drinking water (25 mg/kg/day) for 8 weeks. (C) Quantitation of SGLT1 intensity; n = 3–4 mice/group; mean ± SEM. Statistical analysis was conducted by two-tailed Student’s t test; ∗∗p = 0.005. Black arrow = luminal SGLT1 expression. Scale bar = 100 μm. Intensity: 0 = absent - 3 = high intensity; FOV, Field of view. See also Figures S1–S3 .

Article Snippet: Real-time PCR to determine the mRNA abundance of mouse Sglt1 and Hprt (house-keeper gene) was performed using a ViiA 7 Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific, Scoresby VIC, Australia) using pre-developed TaqMan probe (FAM-labelled) and primer sets for mouse Sglt1 (Mm00451203_m1), Hprt (Mm01545399_m1) and Taqman Gene Expression Master Mix (Catalog #: 4369016) from Applied Biosystems.

Techniques: Inhibition, Expressing, Immunohistochemistry, Quantitation Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Asthma reduces glioma formation by T cell decorin-mediated inhibition of microglia

doi: 10.1038/s41467-021-27455-6

Figure Lengend Snippet: a Schematic representation of the journey of T cells from the periphery (blood) into the optic nerve and draining through the cervical lymph node. b Increased Ccl4 mRNA expression was observed in optic nerves ( n = 4) of Nf1 OPG mice relative to blood ( n = 5) and draining cervical lymph nodes ( n = 5). One-way ANOVA with Bonferroni post hoc correction. c Schematic representation of the interactions between T cells and microglia in the optic nerve and draining cervical lymph node. d No change in Ccl4 mRNA expression was found in the optic nerves of Nf1 OPG mice treated with OVA ( n = 4) or HDM ( n = 3) relative to vehicle-treated ( n = 7) mice. One-way ANOVA with Bonferroni post hoc correction. e Decreased optic nerve Ccl5 mRNA expression was observed in both OVA- ( n = 4) and HDM- ( n = 3) treated mice relative to PBS-treated controls ( n = 7). One-way ANOVA with Bonferroni post hoc correction. f While Ccl4 gene expression was increased in the cervical lymph nodes of Nf1 OPG mice relative to controls (WT, n = 3; Nf1 OPG , n = 3), Two-tailed Student’s t test. ( g ) No change in cervical lymph node Ccl4 mRNA expression was observed between OVA- or HDM-treated mice (OVA, n = 4; HDM, n = 3) relative to controls ( n = 7). One-way ANOVA with Bonferroni post hoc correction. Data are presented as the means ± SEM. Exact P values are indicated within each panel (ns, not significant). From left to right in each panel: b P < 0.0001, P = 0.0460; d ns, ns; e P = 0.0069, P = 0.0359; f P = 0.0058; g ns.

Article Snippet: Recombinant mouse midkine (R&D Systems, 9760-MD-050) was added, and Ccl4 levels were quantitated by ELISA (R&D Systems, MMB00).

Techniques: Expressing, Gene Expression, Two Tailed Test

Journal: Nature Communications

Article Title: Asthma reduces glioma formation by T cell decorin-mediated inhibition of microglia

doi: 10.1038/s41467-021-27455-6

Figure Lengend Snippet: a Schematic representation of the mouse Nf1 OPG neuron-immune-cancer cell axis in the setting of asthma. b No difference in optic nerve Mdk RNA expression was observed between OVA- ( n = 3) or HDM- ( n = 3) treated mice and controls ( n = 6). Bar graphs represent the means ± SEM of independent biological samples. One-way ANOVA with Bonferroni post hoc correction. c Ccl4 levels are similarly induced with increasing midkine concentrations (0–100 ng/ml) in CD3 + T cells from OVA- and PBS-treated mice. Data are presented as the means ± SEM of n = 3 independent biological samples. One-way ANOVA with Bonferroni post hoc correction. Data are presented as the means ± SEM. d , e Similarly increased CD8 + , relative to CD4 + , T cell content was detected in the optic nerves of Nf1 OPG mice treated with PBS ( n = 8), OVA ( n = 8) or HDM ( n = 8). One-way ANOVA with Bonferroni post hoc correction. Activated T cell medium from ( f ) OVA- ( n = 4) and g HDM- ( n = 4) treated mice induced lower levels of microglial Ccl5 relative to PBS-treated Nf1 OPG mice. Two-tailed Student’s t test. Similar reductions of Ccl5 expression were observed in CD4 + and CD8 + T cells from ( h ) OVA- ( n = 3) and ( i ) HDM- ( n = 3) treated mice relative to PBS-treated controls ( n = 3). One-way ANOVA with Bonferroni post hoc correction. Data are presented as the means ± SEM. d Scale bars, 40 µm. From left to right in each panel: b ns; c ns; ns; ns; ns; e P < 0.0001, P = 0.0022, P = 0.0011; f P = 0.0008; g P = 0.0015; h P = 0.0186, P = 0.0054; i P = 0.0224, P = 0.0007.

Article Snippet: Recombinant mouse midkine (R&D Systems, 9760-MD-050) was added, and Ccl4 levels were quantitated by ELISA (R&D Systems, MMB00).

Techniques: RNA Expression, Two Tailed Test, Expressing

Journal: Nature Communications

Article Title: Asthma reduces glioma formation by T cell decorin-mediated inhibition of microglia

doi: 10.1038/s41467-021-27455-6

Figure Lengend Snippet: a Decorin (800 pg/ml) attenuated activated TCM (act-Tm)-mediated microglia Ccl5 production ( n = 3). Two-tailed Student’s t test. Exact P values are indicated within each panel (ns, not significant). b Decorin reduces microglia Ccl5 production in response to Ccl4 exposure (6 ng/ml) over a physiologic dose range in vitro ( n = 4). One-way ANOVA with Bonferroni post hoc correction. Data are presented as the means ± SEM. c TCM (activated T cell-conditioned media) induced microglial Ccl5 production was significantly attenuated by decorin. The addition of Dcn with CCR5 has the greatest inhibition similar to the combination of CCR5 and CCR8 inhibitors ( n = 6). One-way ANOVA with Bonferroni post hoc correction. Data are presented as the means ± SEM. d Schematic representation of decorin binding to the CCR8 receptor on microglia, culminating is reduced microglial Ccl5 production by inhibiting NFκB activation. e Schematic representation of decorin treatment of Nf1 OPG mice. Nf1 OPG mice were treated between 4 and 6 weeks of age with decorin, while control Nf1 OPG mice received PBS only. Isolated optic nerves were analyzed at 12 weeks of age. f Decorin treatment has no change on optic nerve volume, but ( g ) decreased proliferation (%Ki67 + cells) of Nf1 OPG mice relative to the vehicle-treated Nf1 OPG controls (PBS, n = 8, Decorin, n = 8). No difference in microglia (%Iba1 + cells) or T cell (CD3 + cells) content was observed between decorin-treated mice and their respective controls. Two-tailed Student’s t test (ns, not significant). g Scale bars, 40 µm. From left to right in each panel: a P = 0.0134, b P < 0.0001, P = 0.0035; c P < 0.0001, ns, P = 0.0086, ns; f ns; g P = 0.0010, ns, ns.

Article Snippet: Recombinant mouse midkine (R&D Systems, 9760-MD-050) was added, and Ccl4 levels were quantitated by ELISA (R&D Systems, MMB00).

Techniques: Two Tailed Test, In Vitro, Inhibition, Binding Assay, Activation Assay, Control, Isolation

Journal: Nature Communications

Article Title: Asthma reduces glioma formation by T cell decorin-mediated inhibition of microglia

doi: 10.1038/s41467-021-27455-6

Figure Lengend Snippet: a Nf1 OPG mouse optic nerves ( n = 3) have increased Iκbα phosphorylation relative to WT mice ( n = 3). b The increased Iκbα phosphorylation in PBS-treated Nf1 OPG mouse optic nerves ( n = 3) was reduced by OVA treatment ( n = 3). c Activated TCM (act-Tm) increased Iκbα phosphorylation in microglia, which was reduced following the addition of decorin (800 pg/ml). d Decorin (800 pg/ml) blocks the increased p65-NFκB Ser 536 phosphorylation induced by activated TCM (act-Tm) treatment ( n = 3 ). e Decorin (800 pg/ml) blocks the increased total p65-NFκB expression, ( f ) as well as the nuclear localization of p65-NFκB, induced by activated TCM (act-Tm) treatment ( n = 3). β-actin and HDAC are used as controls for total protein expression and the nuclear fractions, respectively. g Schematic representation of the NFκB inhibitor treatment used. Nf1 OPG mice were treated between 4 and 6 weeks of age with the CAPE NFκB inhibitor ( n = 8), whereas control Nf1 OPG mice received PBS only ( n = 8). Isolated optic nerves were analyzed at 12 weeks of age. h NFκB inhibitor treatment reduced optic glioma volume and ( i ) proliferation (%Ki67 + cells), as well as microglia (%Iba1 + cells) and T-cell (CD3 + ) content within the optic nerves of Nf1 OPG mice relative to vehicle-treated controls. Two-tailed Student’s t test. j Reduced Ccl5 RNA expression was observed in the optic nerves from Nf1 OPG mice treated with the CAPE NFκB inhibitor (NFκB-IN) ( n = 5). Two-tailed Student’s t test. k Proposed model of asthma-induced decorin suppression of the Nf1 OPG neuron-immune-cancer cell axis. Asthma induces T cell production of decorin, which reduces T cell Ccl4-mediated microglia Ccl5 expression through inhibition of NFκB signaling. Data are presented as the means ± SEM. Exact P values are indicated within each panel. i Scale bars 40 µm. From left to right in each panel: h P = 0.0288; i P = 0.0003, P = 0.0196, P = 0.0025; j P = 0.0018.

Article Snippet: Recombinant mouse midkine (R&D Systems, 9760-MD-050) was added, and Ccl4 levels were quantitated by ELISA (R&D Systems, MMB00).

Techniques: Phospho-proteomics, Expressing, Control, Isolation, Two Tailed Test, RNA Expression, Inhibition

Journal: bioRxiv

Article Title: TAK1 blockade as a therapy for retinal neovascularization

doi: 10.1101/2021.01.29.428701

Figure Lengend Snippet: (A) Schematic diagram of the Sprague-Dawley rat model of oxygen-induced retinopathy (OIR). Neonatal rats were exposed to 80% of oxygen for 21 hours each day from postnatal day 0 (P0) to P14. Rats were returned to room air from P14 to P18. Vessel loss was induced during oxygen exposure and retinal angiogenesis could be induced after rats returned to room air. (B) qPCR analysis of TAK1 expression in OIR at P18 as compared with the normoxic control (Nor) group (n = 6-7). (C) Western blot analysis of TAK1 expression in OIR at P18 compared with normoxic control group (n = 10-12). Representative immunoblotting from 4 independent samples were shown for normoxic control (lanes 1-4) and OIR group (lanes 5-8). (D) Immunobiological staining indicated expression of TAK1 in the pathological vessel. Vasculature were visualized by isolectin GS-IB4 labelling (green) and counterstained with TAK1 (red) in retinal whole mount. Arrowheads indicate vessel tufts. Scale bar: 100 µm. (E) GSEA indicated that most of TAK1 upstream pathways including TNF, IL1, TCR, BCR, TLR, and VEGF pathways (from Reactome Pathway Database) were positively enriched in OIR at P18. Engagement of TAK1-activated NFκB, p38 MAPK and JNK pathways as well as negative impact on retinal neural functions were also identified. Group data are shown as means ± SEM. Statistical analysis was undertaken with two-tailed unpaired t-test; *** P < 0.001, **** P < 0.0001. NSE: normalized enrichment score; FDR: false discovery rate.

Article Snippet: The expression levels of target genes were normalized to the levels of rat beta-actin (Rn00821065_g1) or human GAPDH (Hs99999905_m1).

Techniques: Expressing, Control, Western Blot, Staining, Two Tailed Test

Journal: Molecular metabolism

Article Title: Bitter-tasting drugs tune GDF15 and GLP-1 expression via bitter taste or motilin receptors in the intestine of patients with obesity.

doi: 10.1016/j.molmet.2024.102002

Figure Lengend Snippet: Figure 1: Effect of oral intake of Plaquenil on GDF15 and ghrelin plasma levels, and hunger scores in healthy volunteers. A) Time-dependent changes in plasma GDF15 levels after oral ingestion of Plaquenil or placebo in healthy volunteers (n ¼ 10; *P < 0.05: versus baseline [10 min], ##P < 0.01: versus the placebo condition; ANCOVA mixed model). Dashed line: trendline in the fasted state; dotted line: extrapolation from trendline in the fasted state. B and C) Oral intake of Plaquenil, but not placebo, resulted in a negative correlation between GDF15 plasma levels and hungers scores measured between 0 and 90 min after administration. (n ¼ 10; Pearson correlation coefficient within individuals was transformed into Fisher z to calculate the average coefficient [r] and P values). D and E) Oral intake of Plaquenil, but not placebo, resulted in a negative correlation between GDF15 plasma levels and ghrelin plasma levels measured between 0 and 90 min after administration. (n ¼ 10; Pearson correlation coefficient within individuals was transformed into Fisher z to calculate the average coefficient [r] and P values).All data were presented as mean SEM.

Article Snippet: GDF15 protein levels in the cell culture supernatant and cell lysate were measured using a Human GDF-15 ELISA Kit (Boster Biological Technology), following the manufacturer’s protocol.

Techniques: Clinical Proteomics, Transformation Assay

Journal: Molecular metabolism

Article Title: Bitter-tasting drugs tune GDF15 and GLP-1 expression via bitter taste or motilin receptors in the intestine of patients with obesity.

doi: 10.1016/j.molmet.2024.102002

Figure Lengend Snippet: Figure 2: Distribution and characterization of GDF15 expressing epithelial cells in the proximal gut of normal-weight individuals and patients with obesity. A) Relative mRNA expression (efficiencyDDCt method) of GDF15 in resection specimens from the fundus (nNW ¼ 7, nOB ¼ 10), corpus (nNW ¼ 5, nOB ¼ 8), antrum (nNW ¼ 5, nOB ¼ 8), and jejunum (nNW ¼ 5, nOB ¼ 7) of normal-weight individuals and patients with obesity. (Proc Mixed Model with Sidák correction for multiple comparisons). B) Representative single-immunofluorescence staining for GDF15 in jejunal sections (10 mm) of a normal-weight individual and a patient with obesity. GDF15þ cells were stained with Cy3 (green) and nuclei were stained with DAPI (blue). Scale bars: 25 mm. C) Average number and intensity of GDF15þ cells in jejunal tissue sections from both normal-weight individuals and patients with obesity (nNW ¼ 5, nOB ¼ 5; two-tailed unpaired Student’s t-test). DeH) Representative double-immunofluorescence staining for GDF15 (green [Cy3 or Alexa594]) with markers for (D) goblet (MUC2: red [Cy5]), (E) Paneth (a-defensin 6: red [Cy5]) or (FeG) enteroendocrine (CHGA: red [Alexa488]) and ghrelin (red [Alexa488]) cells in jejunal sections (10 mm) from normal-weight individuals. Arrows indicate co-localization. Normal rabbit serum was used as negative control. Nuclei were labeled with DAPI (blue). Scale bars: 25 mm. NW: normal-weight, OB: obese. Data of figure A, C represent mean SEM and single values are plotted. *P < 0.05: versus jejunum in normal-weight individuals, $$$P < 0.001: versus jejunum in patients with obesity, #P < 0.05, ###P < 0.001: versus normal-weight individuals.

Article Snippet: GDF15 protein levels in the cell culture supernatant and cell lysate were measured using a Human GDF-15 ELISA Kit (Boster Biological Technology), following the manufacturer’s protocol.

Techniques: Expressing, Staining, Two Tailed Test, Negative Control, Labeling

Journal: Molecular metabolism

Article Title: Bitter-tasting drugs tune GDF15 and GLP-1 expression via bitter taste or motilin receptors in the intestine of patients with obesity.

doi: 10.1016/j.molmet.2024.102002

Figure Lengend Snippet: Figure 3: Effects of bitter generalists and intermediates on GDF15 or GLP-1 expression in jejunal crypts from patients with obesity. A) Effect of 4-hour stimulation with 100 mM HCQS on relative GDF15 mRNA expression (efficiencyDDCt method) (n ¼ 5; two-tailed paired Student’s t-test). B) Effect of 4-hour stimulation with 100 mM HCQS on relative GLP-1 mRNA expression (efficiencyDDCt method) (n ¼ 5; two-tailed paired Student’s t-test). C) Effect of bitter generalists on fold change in relative GDF15 mRNA expression after 4 h of stimulation (n ¼ 6e9; Proc Mixed Model with Sidák correction for multiple comparisons). D) Effect of bitter intermediates on fold change in relative GDF15 mRNA expression after 4 h of stimulation (n ¼ 4e6; Proc Mixed Model with Sidák correction for multiple comparisons). E) Relative GDF15 secretion in the supernatant of primary crypts from patients with obesity stimulated with 1 mM azithromycin. Azithromycin was removed after 4 h for the 24-hour timepoint (n ¼ 3e6; one-tailed paired Student’s t-test). F) Relative GLP-1 secretion in the supernatant of primary crypts from patients with obesity stimulated with 1 mM azithromycin for 6 h (n ¼ 3; two-tailed paired Student’s t-test). All data are present as mean SEM and single values are plotted. *P < 0.05, **P < 0.01, ***P < 0.001: versus the vehicle group.

Article Snippet: GDF15 protein levels in the cell culture supernatant and cell lysate were measured using a Human GDF-15 ELISA Kit (Boster Biological Technology), following the manufacturer’s protocol.

Techniques: Expressing, Two Tailed Test, One-tailed Test

Journal: Molecular metabolism

Article Title: Bitter-tasting drugs tune GDF15 and GLP-1 expression via bitter taste or motilin receptors in the intestine of patients with obesity.

doi: 10.1016/j.molmet.2024.102002

Figure Lengend Snippet: Figure 4: Effects of bitter specialists on GDF15 mRNA or protein expression in primary jejunal crypts from patients with obesity. A) Effect of specialists on relative GDF15 mRNA expression after 4 h of stimulation (n ¼ 4e7; Proc Mixed Model with Sidák correction for multiple comparisons). B) Representative single-immunofluorescence staining for GDF15 (green [Cy3]) in primary crypts treated with 1 mM gallic acid or vehicle after 24 h with 4 h stimulation. Nuclei were labeled with DAPI (blue). Scale bars: 20 mm. C) Average intensity of GDF15þ cells in primary crypts treated with 1 mM gallic acid or vehicle. Gallic acid was removed after 4 h of stimulation and staining was performed 20 h later (n ¼ 4; two-tailed paired Student’s t-test). D) Relative GDF15 secretion in the supernatant of primary crypts stimulated with 3 mM acetaminophen for 6 h (n ¼ 3; one-tailed paired Student’s t-test). E) Relative GDF15 secretion in the supernatant of primary crypts stimulated with 3 mM acetaminophen. Acetaminophen was removed after 4 h for the 24-hour timepoint (n ¼ 3; one-tailed paired Student’s t-test). F) Venn diagram of bitter compounds that affected GDF15 and/or GLP-1 mRNA expression after stimulation or primary crypts for 4 h. Asterisk (*) indicates an inhibitory effect on mRNA expression; the use of hashtags (#) signifies potential variations in the effects on GDF15 or GLP-1 mRNA, depending on the genotype of the patients. Data of figure A, C, D, E are present as mean SEM and single values are plotted. **P < 0.01, ***P < 0.001: versus the vehicle group.

Article Snippet: GDF15 protein levels in the cell culture supernatant and cell lysate were measured using a Human GDF-15 ELISA Kit (Boster Biological Technology), following the manufacturer’s protocol.

Techniques: Expressing, Staining, Labeling, Two Tailed Test, One-tailed Test

Journal: Molecular metabolism

Article Title: Bitter-tasting drugs tune GDF15 and GLP-1 expression via bitter taste or motilin receptors in the intestine of patients with obesity.

doi: 10.1016/j.molmet.2024.102002

Figure Lengend Snippet: Figure 5: Role of TAS2Rs and the motilin receptor in the effect of bitter agonists on GDF15 or GLP-1 expression in primary crypts from patients with obesity. A) The TAS2R antagonist, GIV3727 (110 mM) blocked the effect of gallic acid (1 mM) on GDF15 mRNA expression (efficiencyDDCt method) (n ¼ 4; Proc Mixed Model with Sidák correction for multiple comparisons). B) GIV3727 (110 mM) did not block the effect of gallic acid (1 mM) on GLP-1 mRNA expression (efficiencyDDCt method) (n ¼ 4; Proc Mixed Model with Sidák correction for multiple comparisons). C) GIV3727 (110 mM) did not block the effect of azithromycin (75 mM) on GDF15 mRNA expression (efficiencyDDCt method) (n ¼ 3; Proc Mixed Model with Sidák correction for multiple comparisons). D) The motilin receptor antagonist MA-2029 blocked the effect of azithromycin (75 mM) on GDF15 mRNA expression (efficiencyDDCt method) (n ¼ 4; Proc Mixed Model with Sidák correction for multiple comparisons). E) C12-O-AHL (0.15 mM) increased relative GDF15 mRNA expression in primary crypts from obese patients with TAS2R4(GG/CG) (n ¼ 9) but not TAS2R4(CC) (n ¼ 4) genotype (Proc Mixed Model with Sidák correction for multiple comparisons). F) C12-O-AHL (0.15 mM) decreased GLP-1 mRNA expression in primary crypts from obese patients with the TAS2R4(GG/CG) (n ¼ 9) but not the TAS2R4(CC) genotype (n ¼ 4). (Proc Mixed Model with Sidák correction for multiple comparisons). G) Aloin at 30 mM (white dots) and 100 mM (black dots) decreased relative GDF15 mRNA expression in primary crypts from obese patients with the TAS2R43(þ)GG/CG (n ¼ 9) but not the TAS2R43(þ)CC (n ¼ 6) or TAS2R43() (n ¼ 6) genotype (Proc Mixed Model with Sidák correction for multiple comparisons). H) Aloin at 30 mM (white dots) and 100 mM (black dots) did not affect the relative GLP-1 mRNA expression in primary crypts from obese patients with the TAS2R43(þ) GG/GC (n ¼ 9), TAS2R43(þ) CC (n ¼ 6), or TAS2R43() (n ¼ 4) genotype (Proc Mixed Model with Sidák correction for multiple comparisons). IeK) Representative double-immunofluorescence images for GDF15 (green [Cy3]) and TAS2R4 (red [Cy5]), TAS2R43 (red [Cy5]) or TAS2R10 (red [Cy5]) in jejunal sections (10 mm thickness) from normal-weight populations. Arrows indicate co-localization. Nuclei were labeled with DAPI (blue). Scale bars: 20 mm. MeO) Representative double-immunofluorescence images for GDF15 (green [Cy3]) and TAS2R4 (red [Cy5]), TAS2R43 (red [Cy5]) or TAS2R10 (red [Cy5]) in primary jejunal crypt from patients with obesity. Arrows indicate co-localization. Nuclei were labeled with DAPI (blue). Scale bars: 20 mm. (L and P) Normal rabbit serum as a negative control. Nuclei were labeled with DAPI (blue). Scale bars: 20 mm. Data of figure AeH are presented as mean SEM and single values are plotted. *P < 0.05, **P < 0.01, ***P < 0.001: versus vehicle group, ##P < 0.01, ###P < 0.001: bitter treatment antagonist, $P < 0.05: versus TAS2R4(CC) population, &P < 0.05: versus TAS2R43() population.

Article Snippet: GDF15 protein levels in the cell culture supernatant and cell lysate were measured using a Human GDF-15 ELISA Kit (Boster Biological Technology), following the manufacturer’s protocol.

Techniques: Expressing, Blocking Assay, Labeling, Negative Control

Journal: Molecular metabolism

Article Title: Bitter-tasting drugs tune GDF15 and GLP-1 expression via bitter taste or motilin receptors in the intestine of patients with obesity.

doi: 10.1016/j.molmet.2024.102002

Figure Lengend Snippet: Figure 6: Role of the unfolded protein response pathway in the effect of bitter agonists on GDF15 and GLP1 mRNA expression in primary crypts from patients with obesity. A) Positive correlation between Log2 fold change of GDF15 and DDIT3 mRNA expression induced by bitter agonists (bitter: DB, AZI, GA, PHE, EM, ACE, BERB; n ¼ 4e5/bitter treatment; Pearson correlation coefficient [r]; P value on graph). B) Positive correlation between Log2 fold change of GDF15 and ATF4 mRNA expression induced by bitter agonists from figure A (n ¼ 4e5/bitter treatment; Pearson correlation coefficient [r]; P value on graph). C) Positive correlation between Log2 fold change of DDIT3 and ATF4 mRNA expression induced by bitter agonist from figure A (n ¼ 4e5/bitter treatment; Pearson correlation coefficient [r]; P value on graph). D) ISRIB (5 mM) blocked the effect of gallic acid (1 mM) on GDF15 mRNA expression (n ¼ 3; Proc Mixed Model with Sidák correction for multiple comparisons). E) ISRIB (5 mM) did not affect the effects of gallic acid (1 mM) on GLP-1 mRNA expression (n ¼ 2; Proc Mixed Model with Sidák correction for multiple comparisons). Data are presented as mean SEM and single values are plotted. *P < 0.05, **P < 0.01, ***P < 0.001: versus vehicle group, ##P < 0.01: bitter treatment antagonist.

Article Snippet: GDF15 protein levels in the cell culture supernatant and cell lysate were measured using a Human GDF-15 ELISA Kit (Boster Biological Technology), following the manufacturer’s protocol.

Techniques: Expressing

Journal: Oncotarget

Article Title: DNA-demethylating and anti-tumor activity of synthetic miR-29b mimics in multiple myeloma

doi:

Figure Lengend Snippet: Differential expression of DNMT3A (A) and DNMT3B (B) in controls, MM and PCL patients. DNMT3A and DNMT3B mRNA levels were obtained by cDNA microarray and reported as raw expression values. The statistical significance of differences among the groups was assessed using Kruskal-Wallis test (P=0,0018 for DNMT3A; P=0,05 for DNMT3B). (C). Quantitative RT-PCR of DNMT3A or DNMT3B in KMS12 (left panel) and NCI-H929 (right panel) cells co-cultured with MM patient-derived BMSCs and then immunopurified by immunomagnetic sorting with anti-CD138 beads. Raw Ct values were normalized to GAPDH and expressed as ΔΔCt values calculated using the comparative cross threshold method. DNMT3A or DNMT3B levels in cells cultured in absence of BMSCs were set as internal reference. Data are the average of two independent co-culture experiments performed in triplicate. P values were obtained using two-tailed t test. * P<0,01.

Article Snippet: For mRNA dosage studies, oligo-dT-primed cDNA was obtained using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) and then used as template to quantify DNMT3A (Hs01027166_m1) and DNMT3B (Hs00171876_m1) levels by TaqMan assay (Applied Biosystems); normalization was performed with GAPDH (Hs03929097_g1).

Techniques: Quantitative Proteomics, Microarray, Expressing, Quantitative RT-PCR, Cell Culture, Derivative Assay, Co-Culture Assay, Two Tailed Test

Journal: Oncotarget

Article Title: DNA-demethylating and anti-tumor activity of synthetic miR-29b mimics in multiple myeloma

doi:

Figure Lengend Snippet: (A). Quantitative RT-PCR of miR-29b levels in INA-6 cells transfected with synthetic miR-29b mimics or scrambled oligonucleotides (NC). Raw Ct values were normalized to RNU44 housekeeping snoRNA and expressed as ΔΔCt values. MiR-29b levels in cells transfected with NC were set as internal reference. Data are the average of two independent transfection experiments performed in triplicate.(B). Dual luciferase assay of INA-6 cells co-transfected with firefly luciferase constructs containing the 3'UTR of DNMT3A or DNMT3B and miR-29b or scrambled oligonucleotides (NC) as indicated. The firefly luciferase activity was normalized to renilla luciferase activity. The data are shown as relative luciferase activity of miR-29b-transfected cells as compared to the control (NC) of a total of six experiments from three independent transfections. °P<0,01. (C). Quantitative RT-PCR of DNMT3A and DNMT3B 24 hours after transfection with synthetic miR-29b or scrambled oligonucleotides (NC) in INA-6 cells. The results are shown as average mRNA expression, in three independent experiments, after normalization with GAPDH and ΔΔCt calculations *P<0,05. (D). Immunoblot of DNMT3A and DNMT3B 24 hours after transfection of INA-6 with synthetic miR-29b or scrambled oligonucleotides (left panel) or in SKMM1 cells transduced with antagomiR-29b (anti-miR-29b) or the empty vector (right panel). GAPDH was used as loading control. (E). GDMi values of U266 and NCI-H929 cells transfected with synthetic miR-29b mimics or scrambled oligonucleotides (NC). The values represent the main of three independent triplicate experiments with standard error mean. °P<0,01.

Article Snippet: For mRNA dosage studies, oligo-dT-primed cDNA was obtained using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) and then used as template to quantify DNMT3A (Hs01027166_m1) and DNMT3B (Hs00171876_m1) levels by TaqMan assay (Applied Biosystems); normalization was performed with GAPDH (Hs03929097_g1).

Techniques: Quantitative RT-PCR, Transfection, Luciferase, Construct, Activity Assay, Control, Expressing, Western Blot, Transduction, Plasmid Preparation

Journal: Oncotarget

Article Title: DNA-demethylating and anti-tumor activity of synthetic miR-29b mimics in multiple myeloma

doi:

Figure Lengend Snippet: Correlation of endogenous miR-29b levels with DNMT3A (A) and DNMT3B (B) mRNA levels determined by high density microarray of mRNA and miRNA expression in a panel of 17 MM cell lines. Log values of raw data are reported in graph. R= regression coefficient.

Article Snippet: For mRNA dosage studies, oligo-dT-primed cDNA was obtained using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) and then used as template to quantify DNMT3A (Hs01027166_m1) and DNMT3B (Hs00171876_m1) levels by TaqMan assay (Applied Biosystems); normalization was performed with GAPDH (Hs03929097_g1).

Techniques: Microarray, Expressing

Journal: Oncotarget

Article Title: DNA-demethylating and anti-tumor activity of synthetic miR-29b mimics in multiple myeloma

doi:

Figure Lengend Snippet: (A). Cell cycle analysis of NCI-H929 cells transduced with two different shRNAs against DNMT3A or DNMT3B or with a vector carrying a scrambled sequence (SCR). At least 20000 events for each point were analyzed in three independent experiments. Results are representative of one out of three experiments. (B). Cell growth curves of NCI-H929 cells transfected with synthetic miR-29b (miR-29b) or scrambled oligonucleotides (NC) with 5μM azacitidine (5-AZA) or vehicle (RPMI medium). Averaged values of three independent experiments are plotted including ±SD. P values 72hours after electroporation were obtained using two-tailed t test (P= 0,0039 for NC vs miR-29b; P=0,0028 for NC+AZA vs miR-29b+AZA). (C). Cell cycle analysis of NCI-H929 cells transfected with synthetic miR-29b mimics or scrambled oligonucleotides (NC) and then treated with 5μM azacitidine (5-AZA) or vehicle for 24, 48 or 72 hours. Results are representative of one out of three independent experiments.

Article Snippet: For mRNA dosage studies, oligo-dT-primed cDNA was obtained using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) and then used as template to quantify DNMT3A (Hs01027166_m1) and DNMT3B (Hs00171876_m1) levels by TaqMan assay (Applied Biosystems); normalization was performed with GAPDH (Hs03929097_g1).

Techniques: Cell Cycle Assay, Transduction, Plasmid Preparation, Sequencing, Transfection, Electroporation, Two Tailed Test

Journal: Oncotarget

Article Title: DNA-demethylating and anti-tumor activity of synthetic miR-29b mimics in multiple myeloma

doi:

Figure Lengend Snippet: (A). In vivo tumor growth of SKMM1 xenografts intratumorally-treated with NLE (MaxSuppressor TM In Vivo RNA-LANCER II)-miR-29b or controls. Palpable subcutaneous tumor xenografts were treated every 3 days (indicated by arrows) for a total of 4 injections, with 20 μg of formulated miR-29b or miR-NC (NC). As control 2 separate groups of tumor–bearing animals were injected with vehicle alone or NLE-formulated scrambled oligonucleotides (NC). Tumors were measured with an electronic caliper every 3 days, averaged tumor volume of each group ±SD are shown. P values were calculated for miR-29b versus miR-NC. *P<0.001. (B). Quantitative RT-PCR of miR-29b levels in retrieved xenografts after intratumor injection of miR-29b mimics or scrambled oligonucleotides. Raw Ct values were normalized to RNU44 housekeeping snoRNA and expressed as ΔΔCt values. Data are the average of two independent triplicate experiments performed on two NC and two miR-29b injected animals °P<0,01. (C). In vivo tumor growth of OPM1 xenografts after systemic delivery of miR-29b or scrambled oligonucleotides (NC). Mice carrying palpable subcutaneous OPM1 tumor xenografts were treated with 20 μg of NLE-formulated miR-29b or scrambled oligonucleotides (NC) by intravenous tail vein injections (arrows indicate the day of injection). Caliper measurement of tumors were taken every day from the day of first treatment. Averaged tumor volumes of mice are reported ± SE. *P<0.05). (D). Quantitative RT-PCR of miR-29b levels in retrieved xenografts after system injection of miR-29b mimics or scrambled oligonucleotides (NC). Data are the average of two triplicate experiments performed on two NC and two miR-29b injected animals. °P<0,01. (E). Quantitative RT-PCR of DNMT3A or DNMT3B in retrieved xenografts after system injection of miR-29b mimics or scrambled oligonucleotides (NC). Raw Ct values were normalized to GAPDH and expressed as ΔΔCt values calculated using the comparative cross threshold method. DNMT3A or DNMT3B levels in NC-tumors were set as internal reference. Data are the average of two independent triplicate experiments performed on two NC and two miR-29b injected animals. °P<0,01.

Article Snippet: For mRNA dosage studies, oligo-dT-primed cDNA was obtained using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) and then used as template to quantify DNMT3A (Hs01027166_m1) and DNMT3B (Hs00171876_m1) levels by TaqMan assay (Applied Biosystems); normalization was performed with GAPDH (Hs03929097_g1).

Techniques: In Vivo, Control, Injection, Quantitative RT-PCR