Journal: The Journal of General Physiology

Article Title: Light Dependence of Calcium and Membrane Potential Measured in Blowfly Photoreceptors In Vivo

doi:

Figure Lengend Snippet: Photographs of a dye-injected photoreceptor cell. ( a ) Left eye of a white-eyed mutant blowfly Calliphora vicina photographed with a dry objective. The facet lens overlying the dye-injected cell clearly shines up (scale bar = 0.2 mm). ( b ) Diagram of the photoreceptor organization and the recording situation after optically neutralizing the cornea with a water immersion objective . ( c ) Blue-induced green fluorescence photographed through a water-immersion objective, showing that the rhabdomere and the cell body of the stained cell fluoresce. ( d ) Green-induced red fluorescence of the same eye region showing the characteristic pattern of the rhabdomeres; one rhabdomere, belonging to the stained cell, is brighter than the others ( arrow ), allowing us to identify the stained cell as an R5 photoreceptor (scale bar in c and d = 20 μm).

Article Snippet: A 50% mirror combined the beams, which then passed the microscope's fluorescence cube (Leitz DM 510; i.e., blue excitation causing green emission).

Techniques: Injection, Mutagenesis, Fluorescence, Staining

Journal: The Journal of General Physiology

Article Title: Light Dependence of Calcium and Membrane Potential Measured in Blowfly Photoreceptors In Vivo

doi:

Figure Lengend Snippet: Simultaneous recording of membrane potential ( a and b ) and Ca 2+ -induced fluorescence ( c and d ) from a dark-adapted photoreceptor illuminated with saturating light. The same data are shown with high ( a and c ) and low ( b and d ) temporal resolution. The cell was injected with the low affinity dye OG5N. The fluorescence signal increases very fast after opening the shutter and peaks after ∼100 ms before levelling off towards a plateau. The depolarization of the membrane, however, displays still faster kinetics. All traces are averages of nine recordings.

Article Snippet: A 50% mirror combined the beams, which then passed the microscope's fluorescence cube (Leitz DM 510; i.e., blue excitation causing green emission).

Techniques: Membrane, Fluorescence, Injection

Journal: The Journal of General Physiology

Article Title: Light Dependence of Calcium and Membrane Potential Measured in Blowfly Photoreceptors In Vivo

doi:

Figure Lengend Snippet: The effects of dye filling on the membrane potential. The photoreceptor cell was impaled with an electrode containing OG2 and then filled without applying current. Traces 1–3 were measured 1, 4, and 6 min, respectively, after impalement by a 5-s stimulus of saturating intensity. While the fluorescence signal ( c and d ) increases with time of impalement, indicative for an ongoing dye-loading, the membrane potential response ( a and b ) of the cell during the light stimulus is hardly changed. Only after turning the stimulus off can a distinct prolongation of the depolarizing after-potential be seen, due to an increase in the concentration of the dye ( b ). The fluorescence traces in d are smoothed by adjacent averaging with a window size of 20 sample points (equivalent to 4 ms) and therefore the rising phase that is visible in c is not discernible in d . The traces in e and f were obtained from a different preparation in which no cell had been dye-filled; these traces are averages of five recordings. They show that the tissue autofluorescence is essentially constant, except for a small, transient increase in fluorescence signal (*) that can be attributed to a light-induced change in redox state of mitochondrial flavoproteins .

Article Snippet: A 50% mirror combined the beams, which then passed the microscope's fluorescence cube (Leitz DM 510; i.e., blue excitation causing green emission).

Techniques: Membrane, Fluorescence, Concentration Assay

Journal: The Journal of General Physiology

Article Title: Light Dependence of Calcium and Membrane Potential Measured in Blowfly Photoreceptors In Vivo

doi:

Figure Lengend Snippet: Membrane potential ( a and b ) and fluorescence ( c and d ) signals from two cells injected with the high affinity dyes OG1 ( a and c ) and OG2 ( b and d ), respectively. The dark-adapted cells were illuminated for 5 s with light of saturating intensity. The fluorescence signal increases monotonically upon stimulation and does not display the peak that can be seen when using the low affinity dye OG5N (Fig. ). This indicates that the high affinity dyes OG1 and OG2 are saturated by the levels of Ca i reached under bright illumination. This implies that in these conditions Ca i exceeds 10 μM, the concentration where OG2 saturates . Traces in a and c are averages of five recordings, traces in b and d are averages of seven recordings.

Article Snippet: A 50% mirror combined the beams, which then passed the microscope's fluorescence cube (Leitz DM 510; i.e., blue excitation causing green emission).

Techniques: Membrane, Fluorescence, Injection, Concentration Assay

Journal: The Journal of General Physiology

Article Title: Light Dependence of Calcium and Membrane Potential Measured in Blowfly Photoreceptors In Vivo

doi:

Figure Lengend Snippet: Example of the double pulse experiments used to determine Ca i as a function of light intensity. Membrane potential traces are given in the left hand column. The dye-injected (OG1) and dark-adapted cell was stimulated with an adapting light (5 s), the intensity of which is indicated for each experiment. Intensities are expressed relative to the light intensity that caused a half maximal peak depolarization; this intensity was assigned the value log I = 0. After adapting for 5 s, the level of Ca i was probed with a bright test flash (500 ms). The fluorescence signal measured during this test flash is shown in the right-hand column on an expanded time scale. The horizontal line at the beginning of the fluorescence traces indicates the initial fluorescence value when no adapting light was given. With increasing adapting intensity, the initial value of the fluorescence ( arrows ) increases. At the highest adapting intensities used, no increase in fluorescence can be observed during the test flash due to saturation of the high affinity dye OG1. All traces shown are averages of five recordings.

Article Snippet: A 50% mirror combined the beams, which then passed the microscope's fluorescence cube (Leitz DM 510; i.e., blue excitation causing green emission).

Techniques: Membrane, Injection, Fluorescence

Journal: The Journal of General Physiology

Article Title: Light Dependence of Calcium and Membrane Potential Measured in Blowfly Photoreceptors In Vivo

doi:

Figure Lengend Snippet: Summary of the double pulse experiments. ( a ) Normalized depolarization of the peak ( filled symbols ) and plateau ( open symbols ) of the membrane potential are given as a function of adapting light intensity. The smooth curves are obtained by fitting the logistic function V = V max * I n /( I n + 1) to the experimental data (exponent n = 0.45 for peak values and 0.47 for plateau values; the normalized V max for plateau values was fitted to be 0.43). The light intensity I is taken relative to the light intensity that causes a half maximal peak depolarization; this intensity was assigned the value log I = 0. ( b ) Normalized fluorescence increase caused by the adapting light for the three dyes used ( symbols connected by thin lines ); data from three different cells are shown for each dye. Normalization procedures for all panels are explained in materials and methods . The membrane potential and fluorescence data obtained with a specific dye are indicated by the same symbol. The fluorescence increase reported by OG1 and OG2 rises with increasing adapting light intensity and saturates at bright light. The signal that was obtained with OG5N continues to increase up to the highest intensities. This shows that Ca i is regulated in a graded fashion over the whole intensity range and that Ca i levels exceed 10 μM at high light intensities. The bold lines are fits to the experimental data obtained by calculating the fluorescence ( F ) as a function of Ca i according to F (Ca i ) = Ca i h /(Ca i h + K d h ) and normalizing, as detailed in materials and methods . Ca i was assumed to increase with a simple power function of adaptation light intensity yielding the curve shown in c (see results ).

Article Snippet: A 50% mirror combined the beams, which then passed the microscope's fluorescence cube (Leitz DM 510; i.e., blue excitation causing green emission).

Techniques: Membrane, Fluorescence

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: Human TMEM30a partially reconstitutes phospholipid import in ⊗Lem3 S. cerevisiae

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques:

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) ΔLem3 S. cerevisiae transformed with empty vector or two isolates transformed with human TMEM30a were grown on glucose or galactose to induce TMEM30a expression. NBD-phosphatidylcholine uptake was determined by flow cytometry. (B) Concentration dependent effect of Edelfosine on colony growth of serially diluted wild-type S. cerevisiae or ΔLem3 transformed with empty vector or two ΔLem3 isolates transformed with human TMEM30a.

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Transformation Assay, Plasmid Preparation, Expressing, Flow Cytometry, Concentration Assay

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) NBD-phosphatidylcholine uptake determined by flow cytometry for wild-type S. cerevisiae transformed with empty vector or ΔLem3 transformed with Lem3, TMEM30a or a chimera (Table 1) of Lem3 and TMEM30a. (B) Quantitation (n=3) of NBD-phosphatidylcholine uptake by ΔLem3 transformed with Lem3-TMEM30a (LT; see Table 1 for sequence), TMEM30a-Lem3 (TL), or TMEM30a-Lem3-TMEM30a (TLT) chimeras. Western blot (top) for V5 antigen contained in sequences encoding TMEM30a and its chimeras isolated from protein extracts of S. cerevisiae grown in galactose to induce insert expression or non-inducing glucose. (C) Concentration dependent effect of Edelfosine on colony formation on glucose or galactose plates for wild-type S. cerevisiae or ΔLem3 transformed with galactose induced human, yeast or chimeric constructs. (D) Effect of Edelfosine on ΔLem3 viability after introduction of human TMEM30a, yeast Lem3p, or chimeras formed from them. Cell number (OD600) in liquid culture of wildtype or ΔLem3 transformed with the stated vectors at defined concentrations (left) or 12.5 μg/ml (right).

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Flow Cytometry, Transformation Assay, Plasmid Preparation, Quantitation Assay, Sequencing, Western Blot, Isolation, Expressing, Concentration Assay, Construct

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) CHO cells stably transfected with TMEM30a-GFP and then stained with CellMask™ Orange Plasma Membrane to mark the plasma membrane (top) then imaged by confocal microscopy. Co-expression of the appropriate orange fluorescent protein Organelle Light defined endoplasmic reticulum (row 2), or Golgi (row 3). TMEM30a-GFP expressing CHO cells were labeled with MitoTracker Red to identify polarized mitochondria (bottom). (B) Western blot for GFP or plasma membrane Na/K ATPase in density gradient fractions from HepG2 cells stably expressing TMEM30a-GFP. (C) Fluorescent intensity of TMEM30a-Jurkat cells during flow cytometry after 10 min incubation in the presence of NBD-phosphatidylcholine (1 μM) alone or additionally with 5 μM Az-LPAF or Edelfosine.

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Stable Transfection, Transfection, Staining, Confocal Microscopy, Expressing, Labeling, Western Blot, Flow Cytometry, Incubation

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) NBD-phosphatidylcholine uptake by CHO cells transfected with empty vector or a TMEM30a vector assessed by confocal microscopy (40X). Inset, 60X. (B) Uptake of [3H]PAF by CHO cells expressing TMEM30a containing a GFP or Lumio tag (n=3). (C) Phosphatidylserine surface expression is not reduced in TMEM30a transfected CHO cells. Surface phosphatidylserine was detected (n=3) by flow cytometry with annexin V conjugated with Alexa647 as described in “Methods.”

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Transfection, Plasmid Preparation, Confocal Microscopy, Expressing, Flow Cytometry

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) Quantitative PCR for TMEM30a mRNA after transfection by empty vector or one containing TMEM30a shRNA (n=3). (B) Jurkat viability to Edelfosine exposure after transfection with an empty vector or TMEM30a shRNA (n=3). (C) Jurkat cell uptake of fluorescent NBD-phosphatidylcholine (upper) or NBD-phosphatidylethanolamine (lower) by cells expressing TMEM30a shRNA or its vector (n=3). (D) Quantitation of NBD-phosphatidylcholine accumulation by Jurkat cells expressing TMEM30a shRNA or empty vector (n=3). (E) Uptake of [3H]PAF by Jurkat cells is reduced by TMEM30a shRNA knockdown (n=4). All quantitative measures used triplicate determinations in each experiment.

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, shRNA, Expressing, Quantitation Assay

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) Flow cytometric analysis of JC-1 green fluorescence (FL1, x axis) and orange/red fluorescence (FL2, y axis) in the presence of the stated azelaoyl lysoPAF concentration in vector and TMEM30a shRNA transfected Jurkat cells. The cationic dye JC1 in functional, polarized mitochondria is aggregated and fluoresces red/orange, while monomeric dye free in the cytoplasm fluoresces green. (B) Flow cytometric analysis of JC-1 fluorescence in the stated concentration of Edelfosine.

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Fluorescence, Concentration Assay, Plasmid Preparation, shRNA, Transfection, Functional Assay

Journal: ACS biomaterials science & engineering

Article Title: Templated Pluripotent Stem Cell Differentiation via Substratum-Guided Artificial Signaling.

doi: 10.1021/acsbiomaterials.4c00885

Figure Lengend Snippet: Figure 1. PSC-MATRIX design and summary of properties of PSC-MATRIX investigated. (A) Schematic of the PSC-MATRIX platform. The defined substratum is composed of adhesion peptides derived from vitronectin (GAG Binding Peptide, GBP) and fibronectin (cyclic RGD, cRGD) in addition to the GFP-affinity motif (GFP-TRAP). This customized substratum supports PSC adhesion and facilitates the conversion of a soluble orthogonal ligand, GFP, into an immobilized input competent to activate downstream transgene expression via synNotch signaling. (B) PSC- MATRIX was used with mCherry reporter synNotch-hESCs with activating ligand, GFP, dosed at different time points to demonstrate temporal control of artificial signaling events. Then, PSC-MATRIX was used to selectively functionalize regions of a cell culture plate to enable the activation of synNotch-hESCs in a spatially constrained manner. PSC-MATRIX was also implemented to guide mesendoderm/early peri-gastrulation differentiation and to produce midbrain dopaminergic (mDa) neuron progenitors with WNT transgene expression via artificial signaling by the orthogonal ligand nonfluorescent GFP. Figure created with BioRender.

Article Snippet: To generate the peptide-presenting surfaces, we used combinations of biotinylated adhesion peptides�glycosaminoglycan binding peptide (GBP) (GenScript Express, biotin-Ahx-GKKQRFRHRNRKG), cyclic RGD (cRGD) (Carbosynth, cyclo[Arg-Gly-Asp-D-Phe-Lys(Biotin-PEGPEG)), and a GFP capturing motif�GFP-TRAP40,41 which was conjugated to a biotinylated polyethylene glycol (PEG) linker (PEG GFP-TRAP).

Techniques: Derivative Assay, Binding Assay, Expressing, Control, Cell Culture, Activation Assay

Journal: ACS biomaterials science & engineering

Article Title: Templated Pluripotent Stem Cell Differentiation via Substratum-Guided Artificial Signaling.

doi: 10.1021/acsbiomaterials.4c00885

Figure Lengend Snippet: Figure 2. Assessment of synNotch-PSC adhesion and activation on varied substrata. (A) Representative phase contrast and fluorescence microscopy images of GFP-responsive reporter-synNotch H9 hESCs dosed with 5 nM GFP after being plated on various functionalized substrata for 4 days. Cells were plated at high density to simulate PSC-differentiation protocols. Single peptide substrata were composed of 5 μM GBP, 5 μM cRGD, or 0.8 μM GFP-TRAP. Dual peptide surfaces were composed of 5 μM GBP or cRGD and 0.4 μM GFP-TRAP. The tri-peptide substratum was composed of 5 μM GBP, 2.15 μM cRGD, and 0.4 μM GFP-TRAP. (B) Mean mCherry fluorescence intensity of GFP-responsive reporter- synNotch H9 hESCs dosed with 5 nM GFP on different substrata combinations after 4 days. One-way ANOVA with Tukey’s multiple comparisons post hoc. ****p < 0.0001. (C) Mean mCherry fluorescence intensity of GFP-responsive reporter-synNotch H9 hESCs on the tri-peptide surface dosed with 5 nM GFP at various GFP-TRAP concentrations on day 2 of culture. One-way ANOVA with Tukey’s multiple comparisons post hoc: **** p < 0.0001. (D) Mean mCherry fluorescence intensity of GFP-responsive reporter-synNotch H9 hESCs plated on the tri-peptide substratum or a Geltrex-coated surface mixed with GFP-TRAP with and without 5 nM GFP supplementation. Two-way ANOVA with Tukey’s multiple comparisons post hoc: ****p < 0.0001. In all plots, n = 3 replicate wells; error bars indicate SEM. Scale bars = 200 μm.

Article Snippet: To generate the peptide-presenting surfaces, we used combinations of biotinylated adhesion peptides�glycosaminoglycan binding peptide (GBP) (GenScript Express, biotin-Ahx-GKKQRFRHRNRKG), cyclic RGD (cRGD) (Carbosynth, cyclo[Arg-Gly-Asp-D-Phe-Lys(Biotin-PEGPEG)), and a GFP capturing motif�GFP-TRAP40,41 which was conjugated to a biotinylated polyethylene glycol (PEG) linker (PEG GFP-TRAP).

Techniques: Activation Assay, Fluorescence, Microscopy

Journal: ACS biomaterials science & engineering

Article Title: Templated Pluripotent Stem Cell Differentiation via Substratum-Guided Artificial Signaling.

doi: 10.1021/acsbiomaterials.4c00885

Figure Lengend Snippet: Figure 5. Demonstration of the capacity of PSC-MATRIX to facilitate both stem cell micropatterning and spatially constrained artificial signaling. (A) Experimental configuration of micropatterned cell culture with the functionalized surface in HUESM-CM differentiation medium with or without GFP. (B) Representative day 2 phase contrast and fluorescence microscopy of inducible WNT3a synNotch-hESCs depicts ligand- dependent synNotch activation via mCherry expression. Scale bars = 500 μm. (C) Quantification of average disc diameter calculated using Leica LAS X software and mean pixel intensity of mCherry fluorescence of each disc calculated using ImageJ software (+GFP n = 22; -GFP n = 18). Unpaired t test for statistical significance: ***p < 0.001, ****p < 0.0001; error bars indicate SEM. (D) Experimental configuration illustrating the functionalization of a 2-panel insert; the left panel was only functionalized with 5 μM GBP and 2.15 μM cRGD whereas the right panel was

Article Snippet: To generate the peptide-presenting surfaces, we used combinations of biotinylated adhesion peptides�glycosaminoglycan binding peptide (GBP) (GenScript Express, biotin-Ahx-GKKQRFRHRNRKG), cyclic RGD (cRGD) (Carbosynth, cyclo[Arg-Gly-Asp-D-Phe-Lys(Biotin-PEGPEG)), and a GFP capturing motif�GFP-TRAP40,41 which was conjugated to a biotinylated polyethylene glycol (PEG) linker (PEG GFP-TRAP).

Techniques: Cell Culture, Fluorescence, Microscopy, Activation Assay, Expressing, Software

Journal: bioRxiv

Article Title: Integration of Patient-Derived Organoids and Organ-on-Chip Systems: Investigating Colorectal Cancer Invasion within the Mechanical and GABAergic Tumor Microenvironment

doi: 10.1101/2023.09.14.557797

Figure Lengend Snippet: (A.) Schematic representing the initial events of the metastatic cascade that can be measured using the CRC-OOC. Tumor cells in the top channel (1) can be visualized and analyzed separately from tumor cells that have invaded and adhered in the endothelial compartment (2). Additionally, tumor cells that are found in the endothelial effluent (3; circulating tumor cells (“CTC-like” cells)) can also be collected and analyzed. (B.) Circulating tumor cells were collected from the endothelial effluent of stretched and not stretched HCT116 CRC-Chips and RNAseq was performed. GO Pathway analysis was performed on a subset of genes with either a 2-fold difference between stretched and not stretched CTCs or an FDR-adjusted p-value <0.1. N=2 biological replicates with 3 pooled chips in each replicate. (C.) Gene expression of neurotransmitter-related genes were measured by a neurotransmitter-specific PCR array. HCT116 tumor cells of stretched and not stretched chips were harvested on day 6 from the top epithelial channel and isolated via FACs. Data is displayed as the gene expression fold change of stretch versus not stretch conditions Expression was normalized to the average of 5 housekeeping genes. Genes that had a 1.5-fold increase or decrease in the stretched condition are displayed. N=3 biological replicates with 3 chips pooled per biological replicate. (D.) Schematic of the production of GABA from glutamate by the enzyme GAD1. In this figure, experiments related to GABA are indicated in purple and experiments related to GAD1 are indicated in light blue. (E.) Representative confocal immunofluorescent images of the epithelial (top; 1) or endothelial (bottom; 2) channel of the CRC-Chips stained for GABA (purple) on day 6. Invaded HCT116 H2B-GFP stain positive for GABA, while HCT116 H2B-GFP tumor cells that are in the top channel stain weakly for GABA. DAPI stains the nuclei of Caco2 C2BBe1 cells in the top channel and endothelial cells in the bottom channel. Scale bars represent 200 μm in the top channel image and 100 μm in the bottom channel images. Top channel images are maximum projections that span a 35 μm Z-height with a 5 μm step size. Bottom channel images are maximum projections that span a 10 μm Z-height with a 5 μm step size. (F.) RNAseq analysis was performed on CRC organoids and normalized GAD1 expression is shown. N=5 independent donors with 2-3 replicates each. Individual data points are shown and mean ± SEM is displayed. Analysis between US and UP data was performed using an unpaired t-test; ***p<0.001 ( G.) CRC organoids were isolated from stretched chips and qPCR analysis of GAD1 gene expression was performed. N=5 independent doners with 3 replicates each. Individual data points are shown and mean ± SEM is displayed. Analysis between US and UP data was performed using an unpaired t-test p<0.05. ( H.) GAD1 mRNA expression from TCGA in KRAS, NRAS, or BRAF mutant primary colon cancer tumors. N=196 patients with KRAS, NRAS, or BRAF mutant tumors; N=201 patients with KRAS, NRAS, or BRAF wildtype tumors. Individual data points are shown and median with interquartile range is represented. Data was analyzed with an unpaired t-test; ****p<0.0001. ( I.) Kaplan-Meier curve with univariate analysis of the survival of patients with KRAS, NRAS, or BRAF mutated CRC tumors based on high versus low expression of GAD1 (defined as above or below the median GAD1 mRNA expression z-score of 0.3). Data was extracted from the TCGA. N=254 patients. Data was analyzed using a log-rank (Mantel-Cox test). ( J.) Effluent from the epithelial channel of the patient-derived organoids was collected on day 0 (D0) and day 6 (D6). GABA intensity was analyzed from extracted metabolites N=6 chips per timepoint per patient; n=4 on D0 and 2 on D6 for UK. Data was analyzed using a two-way ANOVA; ***p<0.001; ****p<0.0001. ( K .) US-H2B-GFP (top) and UP-H2B-GFP (bottom) stretched tumor-chips were stained for GABA (purple). Scale bars represent 200 μm. L. Representative 10x immunofluorescence images of the 5 tumors stained for EpCAM (green), CK20 (red), and GABA (purple). Scale bars represent 500 μm and 200 μm for UK. All schematics were made in or are from BioRender.

Article Snippet: ABAT human shRNA lentiviral particles (Santa Cruz Biotechnology, Inc., #sc-93288-V) and control shRNA lentiviral particles (Santa Cruz Biotechnology, Inc., #sc-108080) were used to produce HCT116 H2B-GFP ABAT knock down cells at a multiplicity of infection (MOI) of 5 and 2 mg/mL of puromycin (Santa Cruz Biotechnology, #sc-108071) was used to select for knocked down cells.

Techniques: Gene Expression, Isolation, Expressing, Staining, Mutagenesis, Derivative Assay, Immunofluorescence

Journal: bioRxiv

Article Title: Integration of Patient-Derived Organoids and Organ-on-Chip Systems: Investigating Colorectal Cancer Invasion within the Mechanical and GABAergic Tumor Microenvironment

doi: 10.1101/2023.09.14.557797

Figure Lengend Snippet: (A.) Invasion of HCT116 tumor-chips in the presence or absence of exogenous GABA (flowed through the epithelial channel) was measured on day 6 (D6) of the experiment and normalized to day 0 (D0) invasion. N=6 chips. Individual data are shown, with mean ± SEM represented and analyzed using a one-way ANOVA; ****p<0.0001. (B.) Intracellular [ 13 C 4 ]GABA or unlabeled GABA was measured via mass spectrometry-based metabolomics in the HCT116 tumor-chips after the addition of exogenous GABA for six days. N=3 chips. (C.) Schematic of GABA catabolism by ABAT, subsequent entry into the TCA cycle, and inhibition of ABAT activity by vigabatrin. In this figure, experiments related to GABA are indicated as purple, and experiments related to ABAT are indicated as teal. (D.) Western blot analysis of ABAT in shRNA control or ABAT shRNA HCT116 tumor cells. Cropped western blot (left) and quantification (right) confirm knockdown of ABAT. (E.) Growth rate of ABAT-knockdown or control HCT116 tumor cells when grown in traditional cell culture methods. N=3. Individual data are shown and mean ± SEM are represented. Data was analyzed using a t-test; **p<0.01. (F.) Numbers of ABAT-knockdown or control HCT116 tumor cells in the top channel on-chip as measured via fluorescence microscopy and quantified on day 0 (D0) and day 6 (D6). N=5-6 chips. Individual data are shown and mean ± SEM are represented. Data was analyzed using a two-way ANOVA; *p<0.05;***p<0.001. (G.) Invasion of ABAT knockdown (KD) or control shRNA HCT116 tumor-chips in the presence or absence of stretching was measured on day 6 (D6) of the experiment and normalized to day 0 (D0) invasion. N=5-6 chips. Individual data points are shown and mean ± SEM are represented. Data was analyzed using a one-way ANOVA; *p<0.05. (H.) Numbers of HCT116 tumor cells in the top channel on-chip in the presence or absence of stretching, with or without vigabatrin was measured via fluorescence microscopy and quantified on day 0 (D0) and day 6 (Day 6). Individual data are shown and mean ± SEM are represented. N=4 chips. Data was analyzed using a two-way ANOVA; **p<0.01. (I.) Invasion of HCT116 tumor-chips in the presence or absence of stretching, with or without vigabatrin was measured on day 6 (D6) of the experiment and normalized to day 0 (D0) invasion. N=4 chips. Individual data points are shown and mean ± SEM are represented. Data was analyzed using a one-way ANOVA; ***p<0.001. (J.) Numbers of US-H2B-GFP (red) or UP-H2B-GFP (blue) tumor cells in the top channel on-chip in the presence or absence of stretching, with or without vigabatrin was measured via fluorescence microscopy and quantified on day 0 (D0) and day 6 (Day 6). N=4-5 chips. Individual data are shown and mean ± SEM are represented. Data was analyzed using a two-way ANOVA; ns=p>0.05. (K.) Invasion of US-H2B-GFP or UP-H2B-GFP organoid-tumor chips in the presence or absence of stretching, with or without vigabatrin was measured on day 6 (D6) of the experiment and normalized to day 0 (D0) invasion. N=4-5 chips. Individual data are shown and mean ± SEM are represented. Data was analyzed using a one-way ANOVA; **p<0.01. All schematics were made in or are from BioRender.

Article Snippet: ABAT human shRNA lentiviral particles (Santa Cruz Biotechnology, Inc., #sc-93288-V) and control shRNA lentiviral particles (Santa Cruz Biotechnology, Inc., #sc-108080) were used to produce HCT116 H2B-GFP ABAT knock down cells at a multiplicity of infection (MOI) of 5 and 2 mg/mL of puromycin (Santa Cruz Biotechnology, #sc-108071) was used to select for knocked down cells.

Techniques: Mass Spectrometry, Inhibition, Activity Assay, Western Blot, shRNA, Control, Knockdown, Cell Culture, Fluorescence, Microscopy

Journal: The Journal of Cell Biology

Article Title: Chk2 prevents mitotic exit when the majority of kinetochores are unattached

doi: 10.1083/jcb.201310071

Figure Lengend Snippet: Chk2-deficient cells exit mitosis when the majority of kinetochores are unattached. (A and B) Mitotic index analysis. Cells transfected with negative siRNA (control), Chk2 siRNA (siChk2), Mps1 siRNA (siMps1), or treated with Chk2 inhibitor II were incubated with 3.32 µM nocodazole (A) or taxol (B). ***, P < 0.001. (C) Wild-type (WT) or Chk2 −/− DT40 cells were treated with 3.32 µM nocodazole (nocod) or taxol. Error bars show the SD from the means from three independent experiments. ***, P < 0.001 compared with WT plus nocodazole. (D) An example of mitotic exit. Cells expressing H2B-GFP were treated with 3.32 µM nocodazole in the absence or presence of Chk2 inhibitor II (inh II) and analyzed by time-lapse microscopy. Phase-contrast (right) and fluorescence images (left) of a cell arrested with condensed chromatin (top row) or a cell that exited mitosis and formed micronuclei (bottom row). Time is from the start of chromatin condensation. See also Video 1 and Video 2 . (E) Microtubule–kinetochore attachments in CHO cells. Values in parentheses indicate the frequency of unattached kinetochores from n cells. A 4× magnification of kinetochores is shown. Arrows indicate unattached kinetochores. (F) Cdk1-associated histone H1 kinase activity and Western blot analysis of immunoprecipitated (IP) Cdk1. Cells were treated with 3.32 µM nocodazole in the absence (control) or presence of Chk2 inhibitor II or AZ3146 (AZ). Phosphorylated H1 (pH1) values at 0 h were taken as 1. (G) Western blot analysis of total Cyclin B and actin in cells treated as in F. Values at 0 h were taken as 1. (H) Cyclin B fluorescence in cells treated as in F. Values show mean Cyclin B fluorescence intensity from n cells ± SDs. Bars, 5 µm.

Article Snippet: These vectors were then transfected into CHO cells (T-REx; Invitrogen) stably expressing the tetracycline repressor together with the pTK-Hyg selection vector (Agilent Technologies) conferring resistance to hygromycin (Invitrogen).

Techniques: Transfection, Control, Incubation, Expressing, Time-lapse Microscopy, Fluorescence, Activity Assay, Western Blot, Immunoprecipitation

Journal: The Journal of Cell Biology

Article Title: Chk2 prevents mitotic exit when the majority of kinetochores are unattached

doi: 10.1083/jcb.201310071

Figure Lengend Snippet: Chk2 is required for Aurora B–S331 phosphorylation in high nocodazole. (A) Aurora B–phospho-S331 (pS331) staining. Cells transfected with negative siRNA (control) or Chk2 siRNA (siChk2) were treated with 3.32 µM nocodazole (nocod) and MG132 for 4 h in the absence or presence of AZ3146 (AZ). (B) Aurora B localization in cells treated as in A. (C) Mitotic index analysis. Tetracycline-induced CHO cells expressing WT, S331A, or S331E Aurora B (AurB) were treated with 3.32 µM nocodazole in the absence or presence of Chk2 inhibitor II (inh II). ***, P < 0.001 compared with cells without Chk2 inhibitor. (D) Tetracycline-induced S331E Aurora B cells expressing Mad2-GFP were treated with 3.32 µM nocodazole and MG132 in the absence or presence of Chk2 inhibitor II for 4 h. (E) Mitotic index analysis. Tetracycline-induced cells expressing WT, S331A, or S331E Aurora B were treated with 3.32 µM nocodazole and AZ3146 for 8 h. Error bars show the SD from the means of three independent experiments. ***, P < 0.001. (F) Tetracycline-induced WT, S331A, or S331E Aurora B cells expressing Mad2-GFP were treated with 3.32 µM nocodazole, AZ3146, and MG132 for 4 h. Boxed values show mean green/red fluorescence intensity ± SDs. Values in square brackets show kinetochore pairs and number of cells analyzed. Bars, 5 µm. The insets in A, B, D, and F show 1.7× magnification of kinetochores.

Article Snippet: These vectors were then transfected into CHO cells (T-REx; Invitrogen) stably expressing the tetracycline repressor together with the pTK-Hyg selection vector (Agilent Technologies) conferring resistance to hygromycin (Invitrogen).

Techniques: Phospho-proteomics, Staining, Transfection, Control, Expressing, Fluorescence

Journal: The Journal of Cell Biology

Article Title: Chk2 prevents mitotic exit when the majority of kinetochores are unattached

doi: 10.1083/jcb.201310071

Figure Lengend Snippet: Chk2 is required for Mps1 localization and stability in nocodazole. (A) Cells expressing Mps1-GFP transfected with negative siRNA (control) or Chk2 siRNA (siChk2) were treated with 3.32 µM nocodazole (nocod) in the presence of AZ3146 (AZ) and MG132 for 4 h. Boxed values show mean Mps1-GFP/Hec1 fluorescence intensity ± SDs. Values in square brackets show kinetochore pairs and number of cells analyzed. Bars, 5 µm. Insets show 1.7× magnification of kinetochores. (B) Western blot analysis of total GFP and actin in cells expressing WT Mps1-GFP and treated with 3.32 µM nocodazole for 8 h. Values at control were taken as 1. (C) Cells expressing WT, T288A Mps1-GFP, or untransfected were treated as in B. (top) Western blot analysis of GFP-associated phosphothreonine (pThr) and Mps1 after GFP immunoprecipitation (IP). (bottom) Western blot (WB) analysis of total GFP and actin. Values at WT were taken as 1. (D) Western blot analysis of total GFP and actin in cells expressing T288A or T288E Mps1-GFP and treated as in B. Values at control T288E were taken as 1. (E) Western blot analysis of total phosphorylated Y15 (pY 15 ), Cdk1, and actin. Tetracycline-induced CHO WT Aurora B cells expressing WT or T288E Mps1-GFP were treated with 3.32 µM nocodazole in the absence or presence of Chk2 inhibitor II for 8 h followed by shake-off. Values at samples without Chk2 inhibitor were taken as 1. (F and G) Mitotic index analysis. Tetracycline-induced WT, S331A, or S331E Aurora B cells expressing GFP or T288E Mps1-GFP were treated with 3.32 µM nocodazole (and AZ3146; G) in the absence (control) or presence of Chk2 inhibitor II (inh II). Mitotic index shows the percentage of mitotic green cells/total green cells. Error bars show the SD from the means of three independent experiments. ***, P < 0.001.

Article Snippet: These vectors were then transfected into CHO cells (T-REx; Invitrogen) stably expressing the tetracycline repressor together with the pTK-Hyg selection vector (Agilent Technologies) conferring resistance to hygromycin (Invitrogen).

Techniques: Expressing, Transfection, Control, Fluorescence, Western Blot, Immunoprecipitation

Journal: The Journal of Cell Biology

Article Title: Chk2 prevents mitotic exit when the majority of kinetochores are unattached

doi: 10.1083/jcb.201310071

Figure Lengend Snippet: Chk2 is required for spindle checkpoint signaling in unperturbed early prometaphase. (A) Phosphorylated Aurora B–S331 (pS331). Cells were transfected with negative siRNA (control) or Chk2 siRNA (siChk2). (B) Cyclin B fluorescence in cells transfected as in A. ***, P < 0.001. A minimum of 20 cells per treatment was analyzed. Error bars show SDs. (C) Cells expressing Mad2-GFP were treated as in A. (D) Cells expressing Mps1-GFP were transfected as in A and treated with 2 µM AZ3146 for 1 h. (E) Tetracycline-induced CHO WT, S331A, or S331E Aurora B cells expressing Mps1-GFP were treated with 2 µM AZ3146 for 1 h. (F) WT or S331A Aurora B cells expressing Mad2-GFP were induced with tetracycline. Boxed values show mean green/red fluorescence intensity ± SDs. Values in square brackets show kinetochore pairs and number of cells analyzed. Bars, 5 µm. The insets in A, C, and D–F show 1.7× magnification of kinetochores.

Article Snippet: These vectors were then transfected into CHO cells (T-REx; Invitrogen) stably expressing the tetracycline repressor together with the pTK-Hyg selection vector (Agilent Technologies) conferring resistance to hygromycin (Invitrogen).

Techniques: Transfection, Control, Fluorescence, Expressing

Journal: The Journal of Cell Biology

Article Title: Chk2 prevents mitotic exit when the majority of kinetochores are unattached

doi: 10.1083/jcb.201310071

Figure Lengend Snippet: Expression of S331E Aurora B rescues chromosome alignment or segregation after Chk2 inhibition. (A) Western blot analysis of total Mps1 and actin in cells transfected with negative siRNA (control) or Chk2 siRNA (siChk2) followed by shake-off. Values at control were taken as 1. MI, mitotic index. (B) Tetracycline-induced CHO S331E Aurora B cells expressing Mps1-GFP were untreated or treated with Chk2 inhibitor II (inh II) in the presence of 2 µM AZ3146 for 3 h. Boxed values show mean Mps1-GFP/CENP-B fluorescence intensity ± SDs. Values in square brackets show kinetochore pairs and number of cells analyzed. Insets show 1.7× magnification of kinetochores. (C and D) Tetracycline-induced WT or S331E Aurora B cells were treated with Chk2 inhibitor II for 2 h (C) or with Chk2 inhibitor and MG132 for 1 h (D). (C, left) Anaphase with lagging chromatin (indicated by an arrow). (C, right) Normal anaphase. (D, top) Metaphase with misaligned chromosomes. (D, bottom) Normal metaphase. The frequency of cells exhibiting the respective phenotype is shown in parentheses. (E) Model for the role of Chk2 in mitosis. p indicates phosphorylation. Dotted lines indicate prolonged checkpoint activation. Bars, 5 µm.

Article Snippet: These vectors were then transfected into CHO cells (T-REx; Invitrogen) stably expressing the tetracycline repressor together with the pTK-Hyg selection vector (Agilent Technologies) conferring resistance to hygromycin (Invitrogen).

Techniques: Expressing, Inhibition, Western Blot, Transfection, Control, Fluorescence, Phospho-proteomics, Activation Assay

Journal: Cell death & disease

Article Title: Caspase-dependent cell death-associated release of nucleosome and damage-associated molecular patterns.

doi: 10.1038/cddis.2014.450

Figure Lengend Snippet: Figure 7 Calpain activity is a prerequisite for the release of nucleosomes and DAMPs in dying cells. HeLa cells were incubated in amino acid-depleted medium (HBSS) or staurosporine-containing medium (Stau) (1 μg/ml) for 24 or 8 h, respectively, in the presence of solvent control, BAPTA-AM (50 μM) or A23187 (1 μg/ml). Released DNA was quantified with PicoGreen (a). Amount of intracellular free Ca2+ was measured in cells treated with solvent control or staurosporine at the indicated time periods by eFluor 514 (b). Cells were treated with staurosporine for 8 h in the presence of control solvent, ALLN (20 μM), or PD150606 (20 μM), and released DNA or protein was detected by PicoGreen staining (c) or western blot for histones and DAMPs (d), respectively. Genomic DNA was separated in agarose gel electrophoresis from staurosporine-treated cells with or without PD150606 (e). The cells treated with staurosporine and PD150606 for 8 h, were stained for lamin A/C and histone H1, and examined by confocal microscopy; the arrows indicate nuclear fragmentations (f). Cells treated with control solvent, zVAD-fmk (20 μM), or PD150606 with or without staurosporine for 8 h were measured for viabilities with Calcein assay (g). Cells treated with staurosporine were measured for calpain activity, caspases 3 and 7 activity, or released DNAs at the indicated time periods (h). HeLa cells transfected with non- targeting shRNA (NT), calpastatin shRNA, calpain S1 shRNA, or overexpressing calpain 1, calpain 2, or calpastatin were incubated with staurosporine for 8 h and the released DNAs were measured by PicoGreen staining (i). Cells expressing calpain 1 or calpain 2 tagged with Flag at the N-terminus and HA at the C-terminus were treated with staurosporine and western-blotted with anti-Flag antibody or anti-HA antibody (j). HeLa cells expressing halide-sensitive YFP were treated with staurosporine and/or PD150606 for 4 h, and their chloride ion currents were measured by fluorometry by detecting quenching of YFP fluorescence (k). Data performed in triplicate are presented as mean± S.D. (a–c and g–i)

Article Snippet: Cl-amidine was purchased from Cayman Chemical (Ann Arbor, MI, USA). zVAD-fmk, zDEVD-fmk, Necrostatin-1, BAPTA-AM, diphenyliodonium, PD150606, ALLN and A23187 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Activity Assay, Incubation, Solvent, Control, Staining, Western Blot, Agarose Gel Electrophoresis, Confocal Microscopy, Transfection, shRNA, Expressing, Fluorescence