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: Fig. 3 Asthma reduces T cell induction of Ccl5 in microglia. a Schematic representation of the mouse Nf1OPG 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 Nf1OPG 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 Nf1OPG 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: 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