Journal: SLAS discovery : advancing life sciences R & D

Article Title: A novel flow-cytometric assay to identify inhibitors of RBPJ-DNA interactions

doi: 10.1177/2472555220932552

Figure Lengend Snippet: (A) The GST-RBPJ protein used in this study. (B) Specific binding of RBPJ to the 19-bp Cy5-DNA probe containing an RBPJ-binding consensus. Median fluorescence of bead-associated Cy5-DNA probe is plotted vs Cy5-DNA probe concentration. (C) Experimental scheme. GST-RBPJ immobilized glutathione-coated particles were added to 384-well plates containing small molecules. Plates were incubated for 30 minutes at room temperature with rotation. The Cy-5 DNA probe was then added and the plates were incubated for an additional 60 minutes at room temperature with rotation. Plates were sampled using the HyperCyt high-throughput flow cytometry platform connected with a BD Accuri flow cytometer. (D) Scatter plot showing screening data from a Prestwick Chemical Library plate. Individual well values were determined using HyperSip software (RFU = relative fluorescence units).

Article Snippet: Plates were sampled using the HyperCyt high-throughput flow cytometry platform connected with a BD Accuri flow cytometer. ( ) Scatter plot showing screening data from a Prestwick Chemical Library plate.

Techniques: Binding Assay, Fluorescence, Concentration Assay, Incubation, High Throughput Screening Assay, Flow Cytometry, Software

Journal: Molecular Systems Biology

Article Title: Condition-specific genetic interaction maps reveal crosstalk between the cAMP/PKA and the HOG MAPK pathways in the activation of the general stress response

doi: 10.15252/msb.20156451

Figure Lengend Snippet: Dissecting pathways regulating the general stress response in five conditions The transcription factors Msn2/4 are the main regulators of the induced branch of yeast general stress response. Msn2/4 are regulated by multiple pathways that respond to a large range of environmental conditions. The motivation for this work was to systematically dissect the contribution of the pathways to the general stress response and reconstruct the regulatory network upstream to Msn2/4. Experimental design. Strains containing genetic perturbations and genomically integrated HSP12-GFP reporter were stimulated. The GFP levels of each strain were measured in 2–3 repeats using flow cytometry under three stress and two growth conditions (Materials and Methods). Examples of target gene deletions that affect the levels of Hsp12-GFP under various stress conditions. The median levels of Hsp12-GFP relative to WT are presented. The knockouts can increase/decrease the GFP levels significantly and this effect can be general or stress specific. The median levels of Hsp12-GFP in 68 mutant strains under five conditions, shown relative to the matching WT levels (log ratio, note color scale; Δ marks are omitted for succinctness). The rows and the columns are clustered hierarchically. The clustering highlights the strong activators/repressors of HSP12-GFP and highlights groups with condition-specific effects. Knockouts were classified according to their condition specificity (Materials and Methods). (left) Venn diagram showing the specificity/commonality of knockouts to stress conditions. (right) Venn diagram comparing stress-dependent effects (in one or more stress conditions) to growth effects.

Article Snippet: All samples were analyzed by high-throughput flow cytometry (BD FACSCalibur with CyTek upgrade) using the HyperCyt automated sampler (IntelliCyt).

Techniques: Flow Cytometry, Mutagenesis

Journal: Molecular Systems Biology

Article Title: Condition-specific genetic interaction maps reveal crosstalk between the cAMP/PKA and the HOG MAPK pathways in the activation of the general stress response

doi: 10.15252/msb.20156451

Figure Lengend Snippet: Validity of reporter assay HSP12-GFP is induced during late logarithmic growth phase. WT and Δ msn2 Δ msn4 strains carrying the HSP12-GFP reporter were grown overnight and diluted to OD ˜0.2 at the beginning of the experiment. The strains were grown for 21 h in 96-well microtiter plate. The fluorescence (top panel) and optical density (OD, bottom panel) of each well were measured every ˜30 min in a fluorescent plate reader (BioTek, Synergy S1). HSP12-GFP expression is mostly Msn2/4 dependent. Median Hsp12-GFP levels of WT and Δ msn2 Δ msn4 strains were measured using flow cytometry in different stress and growth conditions (Materials and Methods). Fluorescence levels of strains carrying HSP12-GFP reporter were measured using a flow cytometer, 90 min after the exposure to KCl stress. The histograms of the GFP levels in populations of different mutants are shown.

Article Snippet: All samples were analyzed by high-throughput flow cytometry (BD FACSCalibur with CyTek upgrade) using the HyperCyt automated sampler (IntelliCyt).

Techniques: Reporter Assay, Fluorescence, Expressing, Flow Cytometry

Journal: Molecular Systems Biology

Article Title: Condition-specific genetic interaction maps reveal crosstalk between the cAMP/PKA and the HOG MAPK pathways in the activation of the general stress response

doi: 10.15252/msb.20156451

Figure Lengend Snippet: Reproducibility of flow cytometry data A–C Comparison of two independent repeats of the different growth and stress conditions (Materials and Methods). Scatter plot of the median Hsp12-GFP levels (log scale) of 1,566 mutant strains in the two repeats.

Article Snippet: All samples were analyzed by high-throughput flow cytometry (BD FACSCalibur with CyTek upgrade) using the HyperCyt automated sampler (IntelliCyt).

Techniques: Flow Cytometry, Mutagenesis