Journal: Scientific Reports

Article Title: Metabolic role of dipeptidyl peptidase 4 (DPP4) in primary human (pre)adipocytes

doi: 10.1038/srep23074

Figure Lengend Snippet: Human preadipocytes were stably transduced with shRNA constructs directed against DPP4 mRNA by lentiviral vectors. Unspecific, non-target shRNA was used as a negative control (labeled “SHc” in the figure). Knockdown (KD) of DPP4 expression was confirmed on mRNA Part ( A ) and protein Part ( B ) level by quantitative Real Time PCR and Western blotting, respectively. The picture of the Western blot was cropped for clarity. The entire lanes are shown in . PCR data are presented as mean ∆CP values (normalized to GAPDH, relative to SHc) ± SEM (left y-axis) and calculated fold change values vs. control (right y-axis), n ≥ 5. Statistical analysis was done by one-way ANOVA with Dunnett post-test; **p < 0.01 vs. negative control. Part ( C ) At least 5-fold changes in gene expression resulting from DPP4 knockdown, measured by whole genome DNA array hybridization, are visualized in a heat plot. Hybridization was performed in two-color mode; each line represents the difference between a DPP4 knockdown and sh-control sample. The four lines represent four biological replicates. Up-regulated genes in DPP4 knockdown compared to control are marked in green, down-regulated genes in red. The color intensity indicates the expression level of the respective gene. Part ( D ) Changes in the expression of two representative genes (PPARγC1α and PDK4) over time after infection were followed by quantitative PCR. Data represent mean ∆CP values (vs. GAPDH) ± SEM (left y-axis) and calculated fold change values vs. control (SHc at Day 0) on the right y-axis, n ≥ 3. Statistical analysis was done by t test; *p < 0.05; **p < 0.01 vs. control. Part ( E ) shows a Western blot confirming the up-regulation of PPARγ1Cα on protein level when DPP4 is suppressed. The blot was cropped for clarity. The entire lanes are shown in . The results for DPP4, PPARγ1Cα and the loading control α-actinin from cells infected with sh-control vector (“SHc”) and sh-DPP4 vector (“DPP4 KD”), respectively, are shown as indicated in the figure.

Article Snippet: Primary human preadipocytes from subcutaneous adipose tissue were commercially obtained from PromoCell (Heidelberg, Germany) and Zen-Bio (Durham, NC, USA).

Techniques: Stable Transfection, Transduction, shRNA, Construct, Negative Control, Labeling, Knockdown, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Control, Gene Expression, DNA Array, Hybridization, Infection, Plasmid Preparation

Journal: Scientific Reports

Article Title: Metabolic role of dipeptidyl peptidase 4 (DPP4) in primary human (pre)adipocytes

doi: 10.1038/srep23074

Figure Lengend Snippet: Effects of DPP4 gene expression knockdown in human primary preadipocytes.

Article Snippet: Primary human preadipocytes from subcutaneous adipose tissue were commercially obtained from PromoCell (Heidelberg, Germany) and Zen-Bio (Durham, NC, USA).

Techniques: Gene Expression, Knockdown, Binding Assay, Membrane, Migration, Transduction, Cell Surface Receptor Assay

Journal: Scientific Reports

Article Title: Metabolic role of dipeptidyl peptidase 4 (DPP4) in primary human (pre)adipocytes

doi: 10.1038/srep23074

Figure Lengend Snippet: Part ( A ) Human preadipocytes were stable transduced by lentiviral shRNA constructs against DPP4 or were incubated with a PPARγ agonist (pioglitazone, 10 μM) for 3 days. Incubation of DPP4 knockdown cells with a PPARγ inhibitor (T0070907, 10 μM) is shown on the right. Expression of representative genes was analyzed by quantitative Real Time PCR. Values were normalized to the housekeeping gene GAPDH, and are presented relative to control cells infected with non-targeting shRNA. The left y-axis shows the original ΔCP values, and the calculated fold change vs. Day 0 is displayed on the right y-axis. Part ( B ) shows Oil Red O staining of lipids (red) in human preadipocytes after DPP4 knockdown (“DPP4 KD”, mid panel) or after treatment with pioglitazone (10 μM, right panel) for 10 days. Exemplary lipid vacuoles are marked by arrows. Control cells treated with non-targeting shRNA are shown in the left panel. Part ( C ) Gene expression was measured by RT PCR in human preadipocytes after lentiviral transduction with a shRNA construct against PPARγ (“PPARγ KD”) or after treatment with the PPARγ inhibitor T0070907. Representation of data is the same as in Part ( A ). In Parts ( A ) and ( C ), bars represent mean values + SEM, n ≥ 4, Statistical analysis was done by one-way ANOVA with Dunnett post-test; *p < 0.05; **p < 0.01; ***p < 0.001 vs. control.

Article Snippet: Primary human preadipocytes from subcutaneous adipose tissue were commercially obtained from PromoCell (Heidelberg, Germany) and Zen-Bio (Durham, NC, USA).

Techniques: shRNA, Construct, Incubation, Knockdown, Expressing, Real-time Polymerase Chain Reaction, Control, Infection, Staining, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Transduction

Journal: Scientific Reports

Article Title: Metabolic role of dipeptidyl peptidase 4 (DPP4) in primary human (pre)adipocytes

doi: 10.1038/srep23074

Figure Lengend Snippet: Human preadipocytes were stable transduced by lentiviral shRNA constructs against DPP4; infected cells were selected with puromycin and differentiated for up to 12 days. Gene expression was measured by Real Time PCR and was normalized to the housekeeping gene GAPDH. Full lines represent the time course of gene expression during differentiation in DPP4 knockdown (“DPP4 KD”) cells, broken lines refer to cells transduced with non-targeting shRNA (sh-control, “SHc”). Data are displayed as mean ∆CP values ± SEM (left y-axis). For better understanding, calculated values of fold change vs. control (SHc at Day 0) are indicated on the right y-axis. Statistical analysis (n ≥ 3) was done by t test; *p < 0.05; ***p < 0.001 vs. control.

Article Snippet: Primary human preadipocytes from subcutaneous adipose tissue were commercially obtained from PromoCell (Heidelberg, Germany) and Zen-Bio (Durham, NC, USA).

Techniques: shRNA, Construct, Infection, Gene Expression, Real-time Polymerase Chain Reaction, Knockdown, Transduction, Control

Journal: Scientific Reports

Article Title: Metabolic role of dipeptidyl peptidase 4 (DPP4) in primary human (pre)adipocytes

doi: 10.1038/srep23074

Figure Lengend Snippet: Human preadipocytes were transduced by lentiviral shRNA directed against DPP4 (labeled “DPP4-KD” in the figure) or, as control, by non-targeting shRNA (labeled “SHc” in the figure. Part ( A ) Activation of signaling pathways was analyzed by Western blotting with antibodies directed against the phosphorylated (active) form of the respective signaling protein, pERK (phospho-Extracellular-signal Regulated Kinase) or pAkt (phospho-Akt1). The effect of DPP4 knockdown on insulin (ins) signaling via the pAkt and the pERK pathway is shown. The insulin concentration used was 100 nM, incubation time was 10 min. Detection of α-actinin served as loading control. Insulin receptor expression was also detected in the preadipocytes (lower panel of Part A) and was not affected by treatment. The blots were cropped for clarity. Uncropped pictures are shown in . The densitometric quantification of the phosphoproteins is shown in Part ( B ). Statistical analysis was done by one-way ANOVA with Dunnett post-test; *p < 0.05; **p < 0.01 vs. insulin-treated sh-control (SHc). Part ( C ) Proliferation of the preadipocytes after DPP4 knockdown vs. SHc was assessed by cell counting at various time points as indicated.

Article Snippet: Primary human preadipocytes from subcutaneous adipose tissue were commercially obtained from PromoCell (Heidelberg, Germany) and Zen-Bio (Durham, NC, USA).

Techniques: shRNA, Labeling, Control, Activation Assay, Protein-Protein interactions, Western Blot, Knockdown, Concentration Assay, Incubation, Expressing, Cell Counting

Journal: Cancers

Article Title: MYC Regulates a DNA Repair Gene Expression Program in Small Cell Carcinoma of the Ovary, Hypercalcemic Type

doi: 10.3390/cancers17132255

Figure Lengend Snippet: Depletion of MYC in BIN-67 cells. ( a ) Engineered BIN-67 (clone 1) cells were treated with DMSO, 500 nM dTAG V -1-NEG (NEG), or 500 nM dTAG V -1 (V1) for 24 h to deplete MYC. GAPDH is included as a loading control. SNF5 and BAF155 are SWI/SNF subunits. Uncropped Western blots are included in . ( b ) Engineered BIN-67 (clone 9) cells were treated as in ( a ) for 24 h to deplete MYC. Similar controls are included. Uncropped Western blots are included in . ( c ) Engineered BIN-67 (clone 1) cells were plated with 500 nM NEG, 500 nM V1, or DMSO and then allowed to grow for five days before recounting. The fold change in cell number was determined using the total cell number obtained on day five as compared to the day of plating. (The error bars are the standard error of the mean, unpaired student’s t -test ** p = 0.0019, n = 3 biological replicates). ( d ) The engineered BIN-67 (clone 9) cells were plated as in ( c ), and the fold change was calculated and graphed. (The error bars are the standard error of the mean, unpaired student t -test ** p = 0.0089, n = 4 biological replicates). ( e ) The engineered BIN-67 (clone 1) cells were plated with 500 nM NEG, 500 nM V1, or DMSO. After five days, a cell cycle analysis was performed, and the data were quantified for each phase, as shown. (The error bars are the standard error of the mean, one-way ANOVA analysis Sub-G1 ** p = 0.0030, G2/M ** p = 0.0018, n.s. = not significant, n = 4 biological replicates).

Article Snippet: Approximately 2.0 × 10 6 cells were treated with either dimethyl sulfoxide (DMSO), 500 nM dTAG V -1-NEG (Tocris, 6915, Minneapolis, MN, USA), or 500 nM dTAG V -1 (Tocris, 6914) for 24 h before the cells were washed twice with phosphate-buffered saline (PBS), containing 137 mM NaCl, 2.7 mM KCl, and 10 mM phosphate (VWR, 76371-734, Radnor, PA, USA) and pelleted.

Techniques: Control, Western Blot, Cell Cycle Assay

Journal: Cancers

Article Title: MYC Regulates a DNA Repair Gene Expression Program in Small Cell Carcinoma of the Ovary, Hypercalcemic Type

doi: 10.3390/cancers17132255

Figure Lengend Snippet: Depletion of MYC impacts the expression of diverse sets of genes. ( a ) The magnitude and significance of gene expression changes resulting from an RNA-seq analysis of engineered BIN-67 cells treated with DMSO or 500 nM dTAG V -1-NEG (NEG) for 24 h. The grey lines denote a false discovery rate (FDR) of 0.05 and a fold change of 1.5. ( b ) A volcano plot showing the magnitude and significance of gene expression changes from an RNA-seq analysis of engineered BIN-67 cells treated with 500 nM NEG or dTAG V -1 (V1). The blue dots indicate genes that decrease significantly in expression (FDR < 0.05) with a fold change less than −1.5. The red dots indicate genes that increase significantly (FDR < 0.05) in expression and have a fold change that is higher than 1.5. ( c ) A heatmap depicting z-score-normalized read counts for genes that significantly changed in expression (FDR < 0.05) following treatment with V1. The data for each replicate is included. ( d ) A gene ontology analysis was performed using genes that decrease in expression following MYC depletion, as determined in the V1 vs. NEG analysis (FDR < 0.05, fold change < −1.5). The analysis was performed using ShinyGO 0.82 and “GO Biological Process”. ( e ) A gene set enrichment analysis (GSEA) was performed by testing the differentially expressed genes (V1 vs. NEG) against MSigDB hallmark datasets [ , ]. The loss of MYC causes an upregulation of genes related to coagulation and epithelial mesenchymal transition. ( f ) GSEA was performed as in ( e ). The loss of MYC downregulates genes related to E2F targets, DNA repair, G2/M checkpoint, and oxidative phosphorylation. The complete GSEA results are in .

Article Snippet: Approximately 2.0 × 10 6 cells were treated with either dimethyl sulfoxide (DMSO), 500 nM dTAG V -1-NEG (Tocris, 6915, Minneapolis, MN, USA), or 500 nM dTAG V -1 (Tocris, 6914) for 24 h before the cells were washed twice with phosphate-buffered saline (PBS), containing 137 mM NaCl, 2.7 mM KCl, and 10 mM phosphate (VWR, 76371-734, Radnor, PA, USA) and pelleted.

Techniques: Expressing, Gene Expression, RNA Sequencing, Coagulation, Phospho-proteomics

Journal: Cancers

Article Title: MYC Regulates a DNA Repair Gene Expression Program in Small Cell Carcinoma of the Ovary, Hypercalcemic Type

doi: 10.3390/cancers17132255

Figure Lengend Snippet: Gene regulation by chromatin-bound MYC. ( a ) Magnitude of change in expression for genes bound by MYC that go up (red) or down (blue) following depletion of MYC in BIN-67 cells. MYC-bound genes are defined as a gene with an MYC binding site localized within 1 kb of the annotated TSS. ( b ) Gene ontology analysis of MYC-bound genes that decrease in expression following the depletion of MYC using DAVID Bioinformatics [ , ]. The number next to each bar signifies the number of genes in that category. ( c ) A KEGG pathway analysis was performed on MYC-bound genes that decrease in expression following MYC depletion. An analysis was performed using ShinyGO 0.82 and the “KEGG pathway database” . ( d ) A Venn diagram comparing the number of significantly downregulated genes in the V1 vs. NEG RNA-seq analysis with genes annotated to MYC binding sites and a curated list of DNA repair genes from . The Venn diagram was generated using https://molbiotools.com/listcompare.php (Accessed on 3 March 2025). ( e ) Top: heatmap showing the magnitude of gene expression changes for the 27 MYC-bound DNA repair genes from ( d ). Genes marked with solid yellow are bound by MYC but have no detectable change in gene expression. Bottom: heatmap showing the magnitude of gene expression changes for the 84 non-MYC-bound DNA repair genes from ( d ). ( f ) Western blot showing the levels of MYC, ATR, and RAD51 in BIN-67 (clone 1) cells treated with DMSO, 500 nM NEG, or 500 nM V1 for 24 h. GAPDH serves as a loading control. ( g ) Western blot as described in ( f ) for BIN-67 (clone 9) cells treated similarly. ( h ) Western blot as described in ( f ) for engineered SNF5-null G401 cells treated similarly. Uncropped Western blots are included in .

Article Snippet: Approximately 2.0 × 10 6 cells were treated with either dimethyl sulfoxide (DMSO), 500 nM dTAG V -1-NEG (Tocris, 6915, Minneapolis, MN, USA), or 500 nM dTAG V -1 (Tocris, 6914) for 24 h before the cells were washed twice with phosphate-buffered saline (PBS), containing 137 mM NaCl, 2.7 mM KCl, and 10 mM phosphate (VWR, 76371-734, Radnor, PA, USA) and pelleted.

Techniques: Expressing, Binding Assay, RNA Sequencing, Generated, Gene Expression, Western Blot, Control

Journal: iScience

Article Title: Pharmacological inhibition of RAS overcomes FLT3 inhibitor resistance in FLT3-ITD+ AML through AP-1 and RUNX1

doi: 10.1016/j.isci.2024.109576

Figure Lengend Snippet: Pharmaceutical inhibition of RAS signaling is unaffected by cytokine treatment (A) Chemical structures of RAS small molecule inhibitors. (B) Western blot of extracts from MOLM14 cells treated with 10 nM Gilteritinib or 15 μM Ch-3 in the presence and absence of 10 ng/mL IL-3. A representative western blot and densitometry of signals from phospho-ERK, ERK and GAPDH are shown (n = 3), error bars show standard deviation, p values of significance vs. the untreated sample were calculated using Student’s t test are shown in the table. (C and D) Dose-response curve depicting the viability of MV4-11 cells (C) and ITD18 primary cells (D) treated with Ch-3 cultured in the presence of various mixtures of cytokines at 10 ng/mL. Tables of IC50 ± standard deviation (n = 3) are shown in bottom panels. (E) Dose-response curves depicting the viability of primary cells ITD18 and ITD16 relapse sample after FLT3i treated with RAS inhibitors in cultures with low, high or high+IL-3 concentrations. The table of IC50s ± standard deviation (n = 3) is included in the bottom panel. (F) Dose-response curves depicting the viability of FLT3-ITD+ AML (ITD19), FLT3 WT AML and heathy CD34 + cells treated with Ch-3. The table of IC50 ± standard deviation (n = 3) is shown. (G) Histogram of EDU+ cells from the ITD16_G (n = 3) and ITD18 (n = 2) LSC or Blast populations treated with Gilteritinib or Ch-3 in the presence or absence of 100 ng/mL IL-3. Significant differences are indicated by p values calculated using Student’s t test comparing populations treated with and without IL-3, error bars show standard deviation. (H) Relative signal of phosphorylated signaling proteins detected by CYTOF in ITD18 cells treated with 100 nM Gilteritinib or 20 μM Ch-3 in the presence or absence of 100 ng/mL IL-3. All IC50 values show the mean IC50 ± standard deviation (n = 3).

Article Snippet: Human Mobilized Peripheral Blood CD34 + Cells, used as healthy controls, were purchased from AMS Biotechnology (Europe) Limited.

Techniques: Inhibition, Western Blot, Standard Deviation, Cell Culture

Journal: iScience

Article Title: Pharmacological inhibition of RAS overcomes FLT3 inhibitor resistance in FLT3-ITD+ AML through AP-1 and RUNX1

doi: 10.1016/j.isci.2024.109576

Figure Lengend Snippet:

Article Snippet: Human Mobilized Peripheral Blood CD34 + Cells, used as healthy controls, were purchased from AMS Biotechnology (Europe) Limited.

Techniques: Control, Virus, Recombinant, Modification, Saline, Stripping Membranes, Protease Inhibitor, Reverse Transcription, Membrane, Labeling, Gel Extraction, Plasmid Preparation, Software