Journal: Scientific Reports

Article Title: Prenatal Betamethasone interferes with immune system development and alters target cells in autoimmune diabetes

doi: 10.1038/s41598-018-37878-9

Figure Lengend Snippet: Betamethasone exposure alters gene expression in NIT-1 cells, islets of Langerhans and whole pancreas. ( A ) Relative gene expression of 13 selected genes in NIT-1 cells after betamethasone treatment (0.1 μM betamethasone, n ≥ 8) analysed by qRT-PCR. Gene expression was normalized to Gapdh . Bars show the mean ± s.d. of the Log2 of Fold Change (FC) using basal (complete medium-cultured NIT-1) transcription as standard value ( + p ≤ 0.05 and ++ p < 0.01, Wilcoxon test). ( B ) Relative gene expression of 13 selected genes in purified islets of Langerhans from 4 mice after culture with betamethasone (0.1 μM betamethasone, n = 4) analysed by qRT-PCR. Gene expression was normalized to Gapdh . Bars show the mean ± s.d. of the Log2 of FC using basal (complete medium-cultured islets) transcription as standard value (Wilcoxon test); n.d., not detected. ( C ) Histogram of relative gene expression of 8 selected genes in pancreases from newborn pups prenatally treated with 0.1 mg betamethasone (grey bars) or vehicle control (black bars) (n = 4 pancreases/group) analysed by qRT-PCR. Gene expression was normalized to Gapdh . Bars show the mean ± s.d. of gene expression (Mann-Whitney test).

Article Snippet: Relative quantification was performed by normalising the expression for each gene to that of the housekeeping gene GAPDH (Mm99999915_g1), as described in the 2 −ΔCt method .

Techniques: Gene Expression, Quantitative RT-PCR, Cell Culture, Purification, Control, MANN-WHITNEY

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Indoxyl Sulfate Upregulates Liver P-Glycoprotein Expression and Activity through Aryl Hydrocarbon Receptor Signaling

doi: 10.1681/ASN.2017030361

Figure Lengend Snippet: IS levels and abcb1 expression are increased in two mice models of CKD. Kidney function in mice was assessed using (A) plasma creatinine and (B) BUN, which indicates a reduction in GFR in adenine-treated mice and 5/6-nephrectomized (5/6 nx) mice. (C) Serum concentrations of IS in mice were increased in the two models of CKD. (D and E) mRNA expression levels of two genes, abcb1a and abcb1b, that encode to P-gp in mice. *P<0.05; **P<0.01; ***P<0.001.

Article Snippet: Primers and MGB‐Taqman probes were purchased from Life Technologies ( abcb1a : Mm00440761_m1; abcb1b : Mm00440736_m1; and the housekeeping gene Gusb : Mm01197698_m1).

Techniques: Expressing, Clinical Proteomics

Journal: Frontiers in Physiology

Article Title: Protonation of Piezo1 Impairs Cell-Matrix Interactions of Pancreatic Stellate Cells

doi: 10.3389/fphys.2020.00089

Figure Lengend Snippet: Yoda-1 modifies myosin light chain phosphorylation (P-MYL9) in PSCs. (A) Immunocytochemistry images of PSCs treated with vehicle (0.2% DMSO; Ctrl) or 5 μM Yoda1 for 1 h, indicating P-MYL9 (red) and NMIIA (green) of different cells. Scatter plot indicates the total cellular fluorescence intensity of P-MYL9 normalized to NMIIA fluorescence ( n = 90, N = 3). (B) Representative Western blots of P-MYL2 and MYL-2, compared to GAPDH, respectively, without or with 5 μM Yoda1 treatment. Scatter plot shows the results for individual PSC lysates (Ctrl: n = 5, N = 5; Yoda1 n = 4, N = 4). * p < 0.05.

Article Snippet: After overnight transfer to PVDF membranes at 4°C, we blocked the membrane with PBS containing 5% skim milk for 1 h, then incubated the blots with primary antibodies against mPiezo1 (Proteintech, Manchester, UK, #15939-1-AP, 1:100), P-MYL2 (1:500, MYL-Ser19 mouse mAb, #3675 Cell Signaling Technology, Danvers, Ma, USA), or MYL2 (1:500, MYL2 rabbit Ab, #3672 Cell Signaling Technology, Danvers, Ma, USA), and for housekeeping control GAPDH (1:2,000, GAPDH mouse mAb, #ab125247, Abcam, Cambridge, UK) overnight at 4°C.

Techniques: Phospho-proteomics, Immunocytochemistry, Fluorescence, Western Blot

Journal: Cell Death & Disease

Article Title: Exosomes from CD99-deprived Ewing sarcoma cells reverse tumor malignancy by inhibiting cell migration and promoting neural differentiation

doi: 10.1038/s41419-019-1675-1

Figure Lengend Snippet: a Representative western blotting showing CD99 secreted in the supernatants of eight EWS cell lines; CD99 protein expression in the corresponding EWS cells is shown for comparison. GAPDH was used as a loading control. b Evaluation of cell growth in serum-free condition in EWS cells receiving CD99pos- or CD99neg EXOs isolated from the TC-71 (purple bars) and IOR/CAR (green bars) experimental models. Columns show the mean values of at least two independent biological experiments with two replicates/each, and the bars represent the SE (* p < 0.05, ** p < 0.01, Student's t- test, n = 2–4)

Article Snippet: Relative quantification as performed with the ΔΔCT method, and the expression levels of the target genes were normalized to those of the housekeeping gene GAPDH (Hs99999905_m1), RNU6b (Hs001093) or miR-16 (Hs000391).

Techniques: Western Blot, Expressing, Comparison, Control, Isolation

Journal: Cell Death & Disease

Article Title: Exosomes from CD99-deprived Ewing sarcoma cells reverse tumor malignancy by inhibiting cell migration and promoting neural differentiation

doi: 10.1038/s41419-019-1675-1

Figure Lengend Snippet: a Cluster analysis of TC-71 cells with or without CD99neg EXOs performed using a list of differentially expressed Affymetrix transcriptional clusters (adjusted p < 0.05). Genes (columns) and samples (rows) were grouped by hierarchical clustering (Pearson’s correlation). High expression and low expression were normalized to the average expression across all samples. b Top-scored map (the map with the the lowest p value) based on MetaCore pathway enrichment analysis of 1839 transcriptional clusters (corresponding to 508 annotated genes). Changes in gene expression after CD99neg EXOs addition are depicted on the map as a thermometer-like figures. Upregulated genes have an upward thermometer and a red color, whereas downward (blue) thermometers indicate downregulated genes in TC-71+TC-CD99neg EXOs vs TC-71 cells. c AP-1 transcriptional activity as assessed by luciferase assay and c-Fos expression as assessed by western blotting in TC-71 cells receiving or not receiving (CTR) CD99neg EXOs. For the luciferase assay, the data are represented as RLU and are expressed as the fold induction over the control value. The values were normalized to Renilla luciferase activity. The data are shown as the mean ± SE of three independent biological experiments, with three replicates/each (** p < 0.01, Student’s t -test, n = 6). For western blotting, GAPDH was used as a loading control. d The mRNA expression levels of CCND1, MMP9, and MMP1 were evaluated by qPCR in TC-71 cells treated or not treated (−) with CD99neg EXOs. The results were quantified by the 2 −ΔΔCt method, and untreated cells were used as calibrators (2 −ΔΔCt = 1). GAPDH was used as a housekeeping gene. The data are shown as the mean ± SE of three independent experiments, with two replicates/each (** p < 0.01, Student’s t -test, n = 6)

Article Snippet: Relative quantification as performed with the ΔΔCT method, and the expression levels of the target genes were normalized to those of the housekeeping gene GAPDH (Hs99999905_m1), RNU6b (Hs001093) or miR-16 (Hs000391).

Techniques: Expressing, Gene Expression, Activity Assay, Luciferase, Western Blot, Control

Journal: Cell Death & Disease

Article Title: Exosomes from CD99-deprived Ewing sarcoma cells reverse tumor malignancy by inhibiting cell migration and promoting neural differentiation

doi: 10.1038/s41419-019-1675-1

Figure Lengend Snippet: a Validation of miR-199a-3p expression by qPCR in CD99neg- and CD99pos EXOs and their respective producing cells. The relative miRNA levels were normalized to the expression of miR-16 (for EXOs) or RNU6b (for cells). The data are shown as the mean ± SE of three independent experiments, with two replicates/each (** p < 0.01, *** p < 0.001, one-way ANOVA, n = 4). b miR-199a-3p mimic (30 n m ) reduced cell growth (left; trypan blue vital count) and cell migration (right; transwell chambers) in TC-71 cells. The data are shown as the mean ± SE of three independent biological experiments with two duplicates/each (* p < 0.05, one-way ANOVA, n = 6). c miR-199a-3p mimic (30 n m ) inhibited AP-1 transcriptional activity (as evaluated by luciferase assay) and c-Fos expression (as evaluated by western blotting) in TC-71 cells. The data are shown as the mean ± SE of three independent experiments with three replicates/each (** p < 0.01, Student’s t -test, n = 9). SCR, nonspecific control miRNAs. GAPDH was used as a loading control. d After transfection of miR-199a-3p mimic in TC-71, EXOs enriched for this miRNA were harvested (CD99pos EXOs [miR-199a-3p]) and used to treat parental EWS cells, TC-71. These enriched vesicles inhibited AP-1 transcriptional activity (as evaluated by luciferase assay) and c-Fos expression (as evaluated by western blotting) in TC-71 cells. The data are shown as the mean ± SE of at least three independent experiments (*** p < 0.001, Student’s t -test). SCR, nonspecific control miRNAs. GAPDH was used as a loading control. e miR-199a-3p mimic (30 n m ) induced neural differentiation. Representative immunofluorescence images from one experiment representative of two, showing β-III Tubulin expression (green) and H-NF expression (green) in TC-71 cells after exposure to miR-199a-3p. The nuclei were labeled with Hoechst 33258 (blue). Scale bars: 50 μm

Article Snippet: Relative quantification as performed with the ΔΔCT method, and the expression levels of the target genes were normalized to those of the housekeeping gene GAPDH (Hs99999905_m1), RNU6b (Hs001093) or miR-16 (Hs000391).

Techniques: Biomarker Discovery, Expressing, Migration, Activity Assay, Luciferase, Western Blot, Control, Transfection, Immunofluorescence, Labeling

Journal: Frontiers in Physiology

Article Title: N-acetylcysteine Counteracts Adipose Tissue Macrophage Infiltration and Insulin Resistance Elicited by Advanced Glycated Albumin in Healthy Rats

doi: 10.3389/fphys.2017.00723

Figure Lengend Snippet: Gene expression in periepididymal adipose tissue. Ager (A) , Ddost (B) , Cd36 (C) , Nfkb1 (D) , Il6 (E) , Tnf (F) Adipoq (G) , Retn (H) , Slc2a4 (I) , Ppara (J) , Ccl2 (K) , Mrc (L) , Itgam (M) , Arg1 (N) , Il12 (O) mRNA were evaluated in periepididymal adipose tissue from animals treated with C-albumin ( n = 8), C-albumin + NAC ( n = 7), AGE-albumin ( n = 8) and AGE-albumin + NAC ( n = 7), by real time quantitative PCR (RT-qPCR; Applied Biosystems, Foster City, CA, USA). The relative expression of each gene was normalized to the housekeeping Hprt1 gene and relative quantification analysis was performed using the comparative cycle threshold (Ct) (2− ΔΔCt ) method. Results (mean ± SEM) were compared by One-way ANOVA with Newman-Keuls post-test or Kruskal-Wallis followed by multiple Dunns test.

Article Snippet: The relative expression of each gene was normalized to the housekeeping gene Hprt1 (hypoxanthine phosphoribosyltransferase 1; Rn01527840_m1) and relative quantification analysis was performed with StepOne Software 2.0 (Applied Biosystems) using the comparative cycle threshold (Ct) (2− ΔΔCt ) method (Livak and Schmittgen, ).

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Quantitative Proteomics

Journal: Frontiers in Physiology

Article Title: N-acetylcysteine Counteracts Adipose Tissue Macrophage Infiltration and Insulin Resistance Elicited by Advanced Glycated Albumin in Healthy Rats

doi: 10.3389/fphys.2017.00723

Figure Lengend Snippet: RNA-seq of the periepididymal adipose tissue. Col4a 1 (A) , Col4a2 (B) , Col5a1 (C) , Col5a3 (D) , Itgb8 (E) , Col12a1 (F) mRNA were validated in periepididymal adipose tissue from animals treated with C ( n = 7) or AGE- albumin ( n = 8) by real time quantitative PCR (RT-qPCR; Applied Biosystems, Foster City, CA, USA). The relative expression of each gene was normalized to the housekeeping Hprt1 and relative quantification analysis was performed using the comparative cycle threshold (Ct) (2− ΔΔCt ) method. Results (mean ± SEM) were compared by One-way ANOVA with Newman-Keuls post-test or Kruskal-Wallis followed by multiple Dunns test.

Article Snippet: The relative expression of each gene was normalized to the housekeeping gene Hprt1 (hypoxanthine phosphoribosyltransferase 1; Rn01527840_m1) and relative quantification analysis was performed with StepOne Software 2.0 (Applied Biosystems) using the comparative cycle threshold (Ct) (2− ΔΔCt ) method (Livak and Schmittgen, ).

Techniques: RNA Sequencing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Quantitative Proteomics

Journal: Frontiers in Immunology

Article Title: Phosphatidylserine-Liposomes Promote Tolerogenic Features on Dendritic Cells in Human Type 1 Diabetes by Apoptotic Mimicry

doi: 10.3389/fimmu.2018.00253

Figure Lengend Snippet: Quantitative RT-PCR validates the RNA-seq results. Relative gene expression of 8 selected targets in immature dendritic cells (iDCs), after phagocytosis of PSA-liposomes and PSB-liposomes (PSAB-DCs), and in mature dendritic cells (mDCs), analyzed by quantitative RT-PCR. Gene expression signals were normalized to GAPDH . Bars show the mean ± SD of gene expression in control subjects (white bars, n ≥ 3) and patients with type 1 diabetes (T1D) (black bars, n ≥ 4). Statistically significant differences were found when comparing the different conditions in the same group of subjects (* p < 0.05, Wilcoxon test), and differences were not found when comparing the same culture conditions between patients with T1D and control subjects (Mann–Whitney test).

Article Snippet: Relative quantification was performed by normalizing the expression for each gene of interest to that of the housekeeping gene GAPDH (Hs02758991_g1), as described in the 2 –ΔCt method ( ).

Techniques: Quantitative RT-PCR, RNA Sequencing, Gene Expression, Liposomes, Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: IL-32 is a metabolic regulator promoting survival and proliferation of malignant plasma cells

doi: 10.1101/2021.02.22.431638

Figure Lengend Snippet: (A) Overall survival of IL-32 expressing patients (10 th percentile) compared to non-expressing patients (90 th percentile) in the IA13 CoMMpass dataset P= 8.9e-5, using Cox proportional-hazards regression model. (B) IL-32 expression in individual patients at diagnosis and first relapse in RNA-sequenced CD138+ cells from CoMMpass IA13. Significance was determined by Wilcoxon signed-rank test. (C) GO-analysis of the differentially expressed genes (Benjamini-Hochberg-adjusted P-value < 0.05; log2 fold change >0 and <0 for up-and-down-regulated genes, respectively) between IL-32-expressing patients (10 th percentile) and IL-32 non-expressing patients (90 th percentile). Top significantly enriched biological processes upregulated in IL-32 expressing patients are shown. The GO terms are ordered by the Benjamini-hochberg adjusted p-values. (D) GO-analysis of the differentially expressed genes (Benjamini-Hochberg-adjusted P-value < 0.05; log2 fold change >0 and <0 for up-and-down-regulated genes, respectively) between IL-32-expressing patients (10 th percentile) and IL-32 non-expressing patients (90 th percentile). Top significantly enriched biological processes downregulated in IL-32 expressing patients are shown. The GO terms are ordered by the Benjamini-hochberg adjusted P-values. (E) Correlation between IL32 and a proliferative index gene signature (calculated as the sum of expression values of the gene set as described in ). (F) Heatmap showing unsupervised hierarchical clustering of genes in the GO-term oxidative phosphorylation for IL-32-expressing patients (10 th percentile) and patients without IL-32 (90 th percentile).

Article Snippet: Probes were as follows: human IL-32 (Hs00992441_m1) housekeeping gene TATA-binding protein ( TBP ; Hs00427620_m1) or GAPDH (Hs99999905_m1).

Techniques: Expressing, Biomarker Discovery, Phospho-proteomics

Journal: bioRxiv

Article Title: IL-32 is a metabolic regulator promoting survival and proliferation of malignant plasma cells

doi: 10.1101/2021.02.22.431638

Figure Lengend Snippet: A) UMAP plot colored by the identified clusters. B) UMAP plot colored by the level of IL32-expression per cell. C) UMAP plot colored by patient sample. D) Top 20 gene ontology terms (biological processes) for genes enriched in IL-32 expressing patient cells. The GO terms are ordered by the Benjamini-hochberg adjusted p-values. The data were obtained from Ryu et al. .

Article Snippet: Probes were as follows: human IL-32 (Hs00992441_m1) housekeeping gene TATA-binding protein ( TBP ; Hs00427620_m1) or GAPDH (Hs99999905_m1).

Techniques: Expressing

Journal: bioRxiv

Article Title: IL-32 is a metabolic regulator promoting survival and proliferation of malignant plasma cells

doi: 10.1101/2021.02.22.431638

Figure Lengend Snippet: ( A ) Venn-diagram of overlapping significant genes (p>0.01) that were downregulated in KOs (comparing two INA-6 KO clones (KO1, KO2) with WT mock cells) and upregulated in IL-32 patients (comparing IL-32-expressing patients vs non-expressing patients). ( B ) Gene expression of MME , IRF8 and SORL1 in patients expressing IL-32 (10 th percentile) compared to non-expressing (90 th percentile) patients. Significance determined by Wilcoxon signed-rank test in R . ( C ) Gene expression of MME , IRF8 and SORL1 in INA-6 IL-32 KO1, KO2 and WT mock cells. Significance determined by limma in R with Benjamini-Hochberg-adjusted P-values. ( D ) Evaluation of gene expression of markers associated with less differentiated stages of B-cell maturation in CoMMpass IA13, comparing IL-32 expressing patients (upper 10 th percentile) to non-expressing patients (lower 90 th percentile). Significance determined by Wilcoxon signed-rank test in R . ( E ) Scatterplot of same genes as in (D) in single cells with (N=142) and without (N=346) IL32-expression (from single cell transcriptomics). P-values were calculated from a two-sided Wilcoxon signed-rank test in R . ( F ) Surface expression of CD45 and CD38 in INA-6 KO and WT cells. Data is presented as median fluorescence intensity (MFI) from 3 independent experiments and significance determined by unpaired student’s T-test. ( G ) Concentration of kappa light chain/cell detected in conditioned media from WT and KO cells as indicated. P-values were calculated by the ratio paired t-test.

Article Snippet: Probes were as follows: human IL-32 (Hs00992441_m1) housekeeping gene TATA-binding protein ( TBP ; Hs00427620_m1) or GAPDH (Hs99999905_m1).

Techniques: Clone Assay, Expressing, Gene Expression, Single-cell Transcriptomics, Fluorescence, Concentration Assay

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Large-scale isolation of human skeletal muscle satellite cells from post-mortem tissue and development of quantitative assays to evaluate modulators of myogenesis

doi: 10.1007/s13539-012-0097-z

Figure Lengend Snippet: Summary of genes analysed using TaqMan-PCR low-density assays (TLDA)

Article Snippet: HPRT1 , HPRT1 , HPRT , Housekeeper , Hs99999909_m1.

Techniques: Functional Assay, TLDA Assay, Marker, Binding Assay, Ubiquitin Proteomics