Journal: eLife

Article Title: Time-resolved mapping of genetic interactions to model rewiring of signaling pathways

doi: 10.7554/eLife.40174

Figure Lengend Snippet: ( A ) Quality control of the co-RNAi screen. Scatter plots showing all replicate measurements of normalized and processed phenotypic feature data for cell count (PCC = 0.95) and cell eccentricity ( B , PCC = 0.92). Plates for which positive (RasGAP1) and negative (Diap1) controls could not be distinguished (Z’-factor < 0.3) were excluded. Data was normalized for plate and batch effects using median normalization and scaled and centered using Z-score analysis. ( C ) RNAi reagents ranked by dsRNA design correlation. dsRNAs were paired by gene to calculate pairwise correlations along 16 selected features. All dsRNAs together showed an average design correlation of PCC = 0.77. Figure C and D show data 96 hr after MEK inhibitor treatment representatively. ( D ) Assay performance assessed by statistical effect size. The multi-variate Z’-factors between the positive control knockdown of RasGAP1 and a negative control knockdown of Pvr were calculated for the selected 16 phenotypic features for the MEKi screen after 96 hr.

Article Snippet: Chemical compound, drug , PD-0325901; MEKi; MEK inhibitor , Cayman Chemical Cat# 13034 , Cayman Chemical Cat# 13034; CAS: 391210-10-9 , 1.5 nM.

Techniques: Cell Counting, Positive Control, Negative Control

Journal: eLife

Article Title: Time-resolved mapping of genetic interactions to model rewiring of signaling pathways

doi: 10.7554/eLife.40174

Figure Lengend Snippet: ( A ) S2 cells were treated for either 24 hr, 48 hr, 72 hr and 96 hr using 150 nM PD-0325901 in 0.5% DMSO. Samples were lysed and subjected to western blot analysis. The DMSO only control was treated the same way in the same experiment and blotted on the same membrane, however initially not right next to the other samples. Membranes were stripped and re-probed for total-rolled and α-tubulin after each development. ( B ) Correlation analysis of compound (MEKi) vs. RNAi perturbation of Dsor1. Data was taken from B-Score normalized genome wide chemo-genetic screens. Thirty-eight features were analysed between MEK inhibitor and anti-RLUC treated controls (MEKi) and either Dsor1 or RasGAP1 knockdown. Correlation of median feature vectors is given as Pearson correlation coefficient (PCC). ( C ) Image based illustration of the concentration depended decline in cellular fitness (Scale bar = 140 µm). ( D ) Dose-response curve under co-perturbation with a dsRNA against GFP (not expressed in these cells). ( E ) Dose-response curve of the same cells after the drug containing medium was washed out and cells were left to grow for another 72 hr. ( F ) Dose-response curve after treatment of freshly seeded cells with the remainder medium of the treated cells. A horizontal blue line demarks the ED50 as estimated by four-parameter logistic regression (blue curve fit). All values are mean ±s.e.m. (red, n = 32). ( G ) Dose-Response curves were measured using a fluorescent microcopy-based read-out. Cells were segmented and extracted features summarized per well. Multiple features show a distinctive reactivity towards chemical inhibition of Dsor1 and demonstrate the power of multi-parametric phenotyping for measuring cellular reactions to small molecule treatment. The blue line indicates the ED 50 estimate in this experiment. Data were taken from the ‘used compound’ treatment ( E ).

Article Snippet: Chemical compound, drug , PD-0325901; MEKi; MEK inhibitor , Cayman Chemical Cat# 13034 , Cayman Chemical Cat# 13034; CAS: 391210-10-9 , 1.5 nM.

Techniques: Western Blot, Genome Wide, Concentration Assay, Inhibition

Journal: eLife

Article Title: Time-resolved mapping of genetic interactions to model rewiring of signaling pathways

doi: 10.7554/eLife.40174

Figure Lengend Snippet: ( A ) Upon Rel or Rel / pnt knockdown cells behave normally, growth is inhibited upon pnt knockdown alone. ( B ) Dsor1 inhibitor treatment attenuated this alleviating interaction. Scale bar = 30 µm. Images are pseudo colored, DNA/DAPI = blue, FITC/α-tubulin = green. ( C ) Quantified negative treatment-sensitive interaction between Rel and pnt . The trajectory of the MEK inhibitor treatment is lower than the solvent control condition for cell count interaction indicating synthetic lethality under MEK inhibition. ( C’ ) Actin major axis shows a strong positive interaction (cells are enlarged like under pnt knockdown). Error of fit is shown as 95% confidence interval. Dashed lines show trendlines of a treatment wise model fit. ( D, E ) Expression of candidate and marker genes assessed by qPCR (3 days RNAi treatment, n = 3, log 2 fold RLUC , mean ±s.e.m., t-test) on S2 cells. ( D ) pnt expression is reduced upon pnt and Pvr knockdown. Rel knockdown does not rescue pnt expression. Rel expression is increased upon pnt and Pvr knockdown and decreased upon Rel knockdown. Upon pnt and Rel knock down, Rel expression is rescued to normal levels. ( E ) Pvf2 expression is induced only upon Rel / pnt double knockdown. This leads to increased expression of sty and RasGAP1. RasGAP1 knockdown increases sty expression and decreases RasGAP1 expression. ( D–E ) *=p < 0.05, **=p < 0.01. ( F ) A model summarizes the qPCR results in context of the Ras signaling cascade. Dashed lines are transcriptional interactions, solid lines are protein-protein interactions. All black interactions are known, while the green interaction is inferred from the data. Blue arrows indicate that Pvf2 , sty and RasGAP1 were upregulated upon Rel / pnt co-knockdown and by that Ras pathway activity was restored. A similar pattern could be observed upon RasGAP1 knockdown, which causes intrinsic hyper-activation of Ras signaling by constitutive Ras activation (measured by upregulation of sty , red arrows).

Article Snippet: Chemical compound, drug , PD-0325901; MEKi; MEK inhibitor , Cayman Chemical Cat# 13034 , Cayman Chemical Cat# 13034; CAS: 391210-10-9 , 1.5 nM.

Techniques: Cell Counting, Inhibition, Expressing, Marker, Activity Assay, Activation Assay

Journal: eLife

Article Title: Time-resolved mapping of genetic interactions to model rewiring of signaling pathways

doi: 10.7554/eLife.40174

Figure Lengend Snippet:

Article Snippet: Chemical compound, drug , PD-0325901; MEKi; MEK inhibitor , Cayman Chemical Cat# 13034 , Cayman Chemical Cat# 13034; CAS: 391210-10-9 , 1.5 nM.

Techniques: Recombinant, Sequencing, Synthesized, Software