Journal:

Article Title: Development of a Primary Tamarin Hepatocyte Culture System for GB Virus-B: a Surrogate Model for Hepatitis C Virus

doi:

Figure Lengend Snippet: Immunofluorescence staining for NS3 protein. Hepatocytes grown on glass coverslips were inoculated with GBV-B 3 days postplating and harvested 3 days postinfection unless noted otherwise. GBV-B NS3 protein was detected by immunofluorescence using a rabbit anti-GST-NS3 antiserum and goat anti-rabbit IgG-fluorescein. (A) Infected hepatocytes stained with anti-NS3 (magnification, ×100); (B) uninfected hepatocytes stained with anti-NS3 (×100); (C) infected hepatocytes stained with normal rabbit (prebleed; ×100); (D) lower magnification of infected hepatocytes stained with anti-NS3 (×50); (E) hepatocytes infected with a low multiplicity and harvested 21 days p.i. (×100); (F) higher magnification of infected hepatocytes stained with anti-NS3 (×200).

Article Snippet: Purified GST-NS3 protein (10 ng per well) was bound to Immulon 2 96-well plates (Dynatech Laboratories, Chantilly, Va.) in borate-buffered saline (145 mM NaCl, 6 mM NaOH, 48 mM H 3 BO 3 , 50 mM KCl [pH 8.2]).

Techniques: Immunofluorescence, Staining, Infection

Journal:

Article Title: Development of a Primary Tamarin Hepatocyte Culture System for GB Virus-B: a Surrogate Model for Hepatitis C Virus

doi:

Figure Lengend Snippet: Specificity of rabbit anti-GST-NS3 antiserum in immunofluorescence staining for NS3 protein. Hepatocytes were inoculated with GBV-B and harvested 3 days p.i. Cells were stained for NS3 protein using rabbit anti-GST-NS3 antiserum (A). To demonstrate specificity, the antiserum was adsorbed for 16 h at 4°C with 5 μg of purified GST (B) or NS3 (C) protein prior to being used for immunofluorescence staining. Adsorption with GST had no effect on staining, while adsorption with NS3 eliminated staining.

Article Snippet: Purified GST-NS3 protein (10 ng per well) was bound to Immulon 2 96-well plates (Dynatech Laboratories, Chantilly, Va.) in borate-buffered saline (145 mM NaCl, 6 mM NaOH, 48 mM H 3 BO 3 , 50 mM KCl [pH 8.2]).

Techniques: Immunofluorescence, Staining, Purification, Adsorption

Journal: Stem Cell Research & Therapy

Article Title: Anti-inflammatory treatment induced regenerative oligodendrogenesis in parkinsonian mice

doi: 10.1186/scrt124

Figure Lengend Snippet: Detrimental effect of chronic TNF-α treatment on mouse neural stem cell cultures . ( A ) Representative images showing NSC immunostaining for Nestin and Ki67 (counterstained with Hoechst); quantified in ( C ). ( B ) Images show representative immunostaining for TuJ1 in differentiated NSC cultures; quantified in ( D ). * P ≤0.05 Mann-Whitney-U-Test, error bars s.e.m.

Article Snippet: Osmotic mini-pumps (Alzet Osmotic Pumps, Cupertino, CA, USA, model 1002, 0.25 µl flow rate, brain infusion kit 3) for 14-day long TNF-α infusion were filled according to the manufacturer's protocol with either sterile saline (0.9% NaCl w/v) or with saline + 10 ng/ml TNF-α (Peprotech) under a sterile hood.

Techniques: Immunostaining, MANN-WHITNEY

Journal: Stem Cell Research & Therapy

Article Title: Anti-inflammatory treatment induced regenerative oligodendrogenesis in parkinsonian mice

doi: 10.1186/scrt124

Figure Lengend Snippet: Chronic ventricular TNF-α infusion negatively affects SVZ neurogenesis . (A ) Schematic showing the experimental setup. ( B ) Representative confocal maximum intensity projections showing TNF-α effect on EdU + and Dcx + cell numbers in SVZ (control: n = 6; TNF-α: n = 11) and pRMS (control: n = 7; TNF-α: n = 11); dotted line indicates ventricle surface; quantified in ( C ) and ( D ). # P ≤0.05 Student's t -test, * P ≤0.05 Mann-Whitney-U-Test, error bars s.e.m.

Article Snippet: Osmotic mini-pumps (Alzet Osmotic Pumps, Cupertino, CA, USA, model 1002, 0.25 µl flow rate, brain infusion kit 3) for 14-day long TNF-α infusion were filled according to the manufacturer's protocol with either sterile saline (0.9% NaCl w/v) or with saline + 10 ng/ml TNF-α (Peprotech) under a sterile hood.

Techniques: MANN-WHITNEY

Journal: PLoS ONE

Article Title: PPARγ Regulates Expression of Carbohydrate Sulfotransferase 11 ( CHST11/C4ST1 ), a Regulator of LPL Cell Surface Binding

doi: 10.1371/journal.pone.0064284

Figure Lengend Snippet: (A) Chip-Seq data of PPARγ and RXRα occupancy on the Chst11 gene according to Nielssen et al. . UCSC Genome Browser tracks at day 0, 1, 2 4 and 6 of differentiation are shown. Please note differences in y-axes. Two intronic PPARy-RXRα binding sites were designated site 1 and 2. (B) ChIP-PCR on 3T3-L1 preadipocytes and adipocytes. Chromatin was prepared on day 0 and day 6 of differentiation and subjected to immunoprecipitation using antibodies against PPARγ. Enriched DNA was analysed using quantitative PCR with primers located at site 1 and 2 in the Chst11 gene (dark gray and blacks bars, respectively). As a positive control, primers located at the Fabp4 PPREs (∼5500 bp from transcription start site; light gray bars) were used, primers located in the globin locus were used as a negative control (white bars). Results are shown relative to normalized ChIP recovery data of day 0 and results are representative of at least 3 independent experiments. (C and D) U2OS cells were cotransfected with a reporter construct (TKpGL3) containing Chst11 site 1 or site 2 sequences, or the parental reporter construct, together with empty (pCDNA) or PPARγ encoding expression vectors. Activation of the luciferase reporter in the absence or presence of 1 µM rosiglitazone is expressed as fold induction over that with empty reporter cotransfected with pCDNA in the absence of rosiglitazone after normalisation for Renilla luciferase activity. The error bars display SEM and significance is shown by the asterisks (p<0.05), n = 3 (E) Chst11 mRNA expression in 3T3-L1 adipocytes that had been treated with control or PPARy siRNA from the start of differentiation and analyzed at day 5. Relative mRNA expression levels were related to control siRNA treated cells and normalized for the TFIIb reference gene. The error bars display SEM and significance is shown by the asterisks (p<0.05), n = 3 (F) Chst11 mRNA expression in 3T3-L1 adipocytes and the effect of rosiglitazone treatment (1 µM, 24 h). Relative mRNA expression levels were normalized for the TFIIb reference gene. (G) As in panel F, but after treatment with the PPARγ antagonist GW9662 (10 µM, 24 h).

Article Snippet: In short, cells were transfected in 24-well plates with 1 µg reporter plasmid, 10 ng PPARy expression construct, and 2 ng pCMV-Renilla reporter plasmid (Promega).

Techniques: ChIP-sequencing, Binding Assay, Immunoprecipitation, Real-time Polymerase Chain Reaction, Positive Control, Negative Control, Construct, Expressing, Activation Assay, Luciferase, Activity Assay

Journal: Cancers

Article Title: Molecular Atlas of HER2+ Breast Cancer Cells Treated with Endogenous Ligands: Temporal Insights into Mechanisms of Trastuzumab Resistance

doi: 10.3390/cancers16030553

Figure Lengend Snippet: Study design and summary of multiomic measurements. ( a ) Study design—500,000 cells/well were plated in 6-well plates and cultured for 48 h in complete media with one media change after 24 h. Cells were serum starved using media containing 0.1% FBS for 14–16 h. After serum starvation, cells were treated with 50 ng/mL of epidermal growth factor (EGF), 10 ng/mL of heregulin (HRG), or vehicle (water) in freshly made serum starvation media for 30 min (for RPPA), 1 h, 12 h, or 24 h (for ATAC/RNAseq). Time-zero (T0) samples were collected after serum starvation. All treatments were performed in duplicate. ( b ) An infographic that highlights the types of comparisons performed within this manuscript. Differential expression (fold change) for each omics data modality (RNAseq, ATACseq, and RPPA) was computed as pairwise comparisons between treatment and their respective control for each time point. The results were then compared between the two cell lines to identify functional differences (functional comparisons). ( c ) Differentially expressed proteins (DEPs, adj p < 0.05) detected from RPPA measurements for 30 min, 1 h, 12 h, and 24 h for BT474R (R30, R1, R12, and R24, left panel) and sensitive cell line BT474 (S30, S1, S12, and S24, right panel) showed distinct and increased response in BT474 after treatment in contrast to BT474R. ( d ) A comparison of phosphorylated DEPs, which alludes to a highly active cell state, shows a more robust response in BT474 after both HRG and EGF. ( e ) A comparison of the differentially accessible regions (DARs) in BT474 and BT474R assessed from the ATACseq data reflected trends similar to DEPs, with BT474 overall being more accessible across time after treatments. ( f ) The proportion of DARs (as percentage of total DARs) that were observed in the promoter (upstream 1 kb downstream 1 kb of transcription start site (TSS)) and non-promoter regions of annotated genes (adj p < 0.05) for BT474R (left panel) and BT474 (right panel). BT474 clearly showed increased accessibility in its promoter region (compared to their respective control, “+”), especially after EGF. ( g ) Differentially expressed genes ( p adj < 0.05) identified from RNAseq data reflected trends observed in RPPA and ATACseq with both EGF and HRG treated BT474 (right panel) in a transcriptionally more active state compared to BT474R (left panel) across time. Of note, BT474 seems to be most sensitive to treatment at 12 h, as observed.

Article Snippet: For SMLM, BT474, BT474R, and MDA-MB-468 cells were seeded on clean coverslips for 36 h and switched to starvation media (phenol red-free DMEM supplemented with 0.1% FBS, 1 mM of sodium pyruvate, and 4 mM of GlutaMAX) for 14 h. For BT474 and BT474R cells, the media was aspirated, and fresh starvation media containing specific ligands were added and placed in the cell culture incubator for 30 min, 1 h, and 12 h. Concentrations used for ligand experiments were 50 ng/mL for EGF (GenScript Biotech, Piscataway, NJ, USA) and 10 ng/mL for HRG (GenScript Biotech).

Techniques: Cell Culture, Quantitative Proteomics, Control, Functional Assay, Comparison

Journal: Cancers

Article Title: Molecular Atlas of HER2+ Breast Cancer Cells Treated with Endogenous Ligands: Temporal Insights into Mechanisms of Trastuzumab Resistance

doi: 10.3390/cancers16030553

Figure Lengend Snippet: Membrane HER2 clustering, as detected through qSMLM in BT474 and BT474R after EGF and HRG treatment. ( a ) HER2 was detected using trastuzumab-AF647 as probe in BT474 and BT474R. Cells were imaged with SMLM after 14 h starvation (t = 0 h) or after 14 h starvation, followed by treatment with ligands for the indicated time. Red points are the (x,y) coordinates of localizations detected using SMLM in representative cells. Scale bars are 5 µm. ( b , c ) Average detected HER2 densities and clustering parameters after EGF treatment and HRG treatment are shown for each cell line (BT474 in ( b ) and BT474R in ( c )). A total of 10–20 cells (36–77 ROIs) were analyzed for trastuzumab detection. Clustering parameters were calculated from clustered ROIs; the total number of these ROIs ranged from 19 to 53, depending on the ligand and time point. Graphs are plotted for each individual time point using the mean value. Error bars are represented by standard error of the mean (SEM). For clarity, only significant p values between EGF and HRG treatment for each cell are shown ( p value indicated as * <0.05; ** <0.01; *** <0.001.); all other numerical details and additional clustering parameters are provided in .

Article Snippet: For SMLM, BT474, BT474R, and MDA-MB-468 cells were seeded on clean coverslips for 36 h and switched to starvation media (phenol red-free DMEM supplemented with 0.1% FBS, 1 mM of sodium pyruvate, and 4 mM of GlutaMAX) for 14 h. For BT474 and BT474R cells, the media was aspirated, and fresh starvation media containing specific ligands were added and placed in the cell culture incubator for 30 min, 1 h, and 12 h. Concentrations used for ligand experiments were 50 ng/mL for EGF (GenScript Biotech, Piscataway, NJ, USA) and 10 ng/mL for HRG (GenScript Biotech).

Techniques: Membrane

Journal: Cancers

Article Title: Molecular Atlas of HER2+ Breast Cancer Cells Treated with Endogenous Ligands: Temporal Insights into Mechanisms of Trastuzumab Resistance

doi: 10.3390/cancers16030553

Figure Lengend Snippet: HER2 clustering and downstream signaling. ( a ) Heatmaps highlight the fold changes observed for genes that are known to affect HER2 organization at the membrane. HRG-treated BT474R shows an absence of significant regulation of several of the transcripts within our data, alluding to altered HER2 membrane organization. ( b ) Transcription factor (TF)-target activity, as ascertained by DoRothEA, highlights the increased activity of IRF TFs (red) and HIF1A in EGF-treated BT474R, particularly at the later time points. * indicate TF, which were also identified as DEGs at least one time point. ( c ) A similar DoRothEA TF-target activity heatmap for BT474 treated with EGF and HRG. ( d ) A comparative dotplot highlighting the Hallmark enrichment identified for BT474R (R1, R12, and R24) and BT474 (S1, S12, and S24) after EGF (upper panels) and HRG (lower panels) treatments. Both ( b , c ) consistently showed activation of PI3K/AKT signaling in BT474 (adj p < 10 −3 ) and its downstream mTORC1, particularly at early time points.

Article Snippet: For SMLM, BT474, BT474R, and MDA-MB-468 cells were seeded on clean coverslips for 36 h and switched to starvation media (phenol red-free DMEM supplemented with 0.1% FBS, 1 mM of sodium pyruvate, and 4 mM of GlutaMAX) for 14 h. For BT474 and BT474R cells, the media was aspirated, and fresh starvation media containing specific ligands were added and placed in the cell culture incubator for 30 min, 1 h, and 12 h. Concentrations used for ligand experiments were 50 ng/mL for EGF (GenScript Biotech, Piscataway, NJ, USA) and 10 ng/mL for HRG (GenScript Biotech).

Techniques: Membrane, Activity Assay, Activation Assay

Journal: Cancers

Article Title: Molecular Atlas of HER2+ Breast Cancer Cells Treated with Endogenous Ligands: Temporal Insights into Mechanisms of Trastuzumab Resistance

doi: 10.3390/cancers16030553

Figure Lengend Snippet: Differential bioenergetics in BT474 and BT474R. ( a ) Heatmaps highlight the fold changes observed in glycolysis-associated gene products in BT474, with both HRG and EGF-BT474, specifically at the later time points. ( b ) Glucose (2-NBDG) uptake in BT474R at different time points (R1, R12, and R24) and in BT474 (across time, S1, S12, and S24) after EGF and HRG treatment show a fold change over control. Flow cytometry analysis of serum-starved BT474 incubated with 2-NBDG after treatment for 1 h, 12 h, and 24 h. Error bars represent the SEM of three independent experiments (n = 9). ( c ) Active mitochondria exhibit brighter red fluorescence signal compared to mitochondria with lower membrane potential, which fluoresce green. Changes in the red/green fluorescence signal ratio can be used to determine healthy versus depolarized mitochondria (mitochondria membrane potential (Δψm)). Bar plot represents the Δψm (JC-1 red/green median fluorescence intensity (MFI)) of serum-starved BT474 and BT474R cells stained with MitoProbe JC-1 ( p indicated as **** < 0.0001). ( d ) Bar plot shows the JC-1 red/green MFI after EGF and HRG treatment for 1 h, 12 h, and 24 h in BT474R (fold change over vehicle). Error bars represent the SEM of three independent experiments (n = 9). ( e ) RNA and RPPA measurements of AR across all treatment and time point combinations are shown for BT474R. ( f ) RNA and RPPA measurements of AR across all treatment and time point combinations are shown for BT474.

Article Snippet: For SMLM, BT474, BT474R, and MDA-MB-468 cells were seeded on clean coverslips for 36 h and switched to starvation media (phenol red-free DMEM supplemented with 0.1% FBS, 1 mM of sodium pyruvate, and 4 mM of GlutaMAX) for 14 h. For BT474 and BT474R cells, the media was aspirated, and fresh starvation media containing specific ligands were added and placed in the cell culture incubator for 30 min, 1 h, and 12 h. Concentrations used for ligand experiments were 50 ng/mL for EGF (GenScript Biotech, Piscataway, NJ, USA) and 10 ng/mL for HRG (GenScript Biotech).

Techniques: Control, Flow Cytometry, Incubation, Fluorescence, Membrane, Staining

Journal: Cancers

Article Title: Molecular Atlas of HER2+ Breast Cancer Cells Treated with Endogenous Ligands: Temporal Insights into Mechanisms of Trastuzumab Resistance

doi: 10.3390/cancers16030553

Figure Lengend Snippet: Mechanistic underpinnings of BT474 and BT474R. The top panel represents the major mechanisms observed via our multimodal measurements as being prominent in BT474, irrespective of treatment. We observed an active PI3K/AKT/mTORC1 cascade, evidence of both aerobic and anaerobic glycolysis, increased lipogenesis mediated by AR, and HIF1A-mediated stress response, which also contributes to the metabolic reprogramming in BT474. BT474 shows a mix of substrate utilization for bioenergetics across time and treatments. The bottom panel captures the findings of this study as it pertains to BT474R, particularly after treatment with EGF. BT474R showed inherently high mitochondrial bioenergetics for both ligands but showed a ligand-dependent activation/absence of certain signaling cascades. Only EGF-BT474R displayed increased IFN-independent, IRF1-mediated signaling, which has been reported to be a consequence of EGFR activation. The self-amplification circuit using STAT2, IRFF9, and STAT1 likely contributes to the sustained IRF1 activation across time. The downstream activation of interferon-stimulated genes (ISGs) and interferon-sensitive response element genes could impact cellular bioenergetics through their activation of other signaling cascades, such as NF-κB. STAT1 repression of HIF1A could further contribute to the “flux state” in cellular bioenergetics within EGF-BT474R. As evidenced through our differential co-expression network analysis, an increased correlation of genes in EGF-BT474R associated with hypoxia, NF-κB signaling, and metabolic reprogramming further alludes to the differential bioenergetics with respect to BT474. HRG-BT474R (with respect to control) showed a distinct lack of FASN-mediated lipid metabolism-associated genes, likely mediated by early AR dynamics, and warrants further research.

Article Snippet: For SMLM, BT474, BT474R, and MDA-MB-468 cells were seeded on clean coverslips for 36 h and switched to starvation media (phenol red-free DMEM supplemented with 0.1% FBS, 1 mM of sodium pyruvate, and 4 mM of GlutaMAX) for 14 h. For BT474 and BT474R cells, the media was aspirated, and fresh starvation media containing specific ligands were added and placed in the cell culture incubator for 30 min, 1 h, and 12 h. Concentrations used for ligand experiments were 50 ng/mL for EGF (GenScript Biotech, Piscataway, NJ, USA) and 10 ng/mL for HRG (GenScript Biotech).

Techniques: Activation Assay, Amplification, Expressing, Control