Evolutionary rate and expression noise of the static and dynamic modules

<p><b>Copyright information:</b></p><p>Taken from "Revealing static and dynamic modular architecture of the eukaryotic protein interaction network"</p><p></p><p>Molecular Systems Biology 2007;3():110-110.</p><p>Published online 24 Apr 2007</p><p>PMCID:PMC1865589.</p><p>Copyright © 2007, EMBO and Nature Publishing Group</p> Evolutionary rates of yeast proteins derived by were used. () Boxplot of evolutionary rates of family, party and date hubs. Family hubs are static hubs with neighborhood EVs of 0.45 and date hubs are those with neighborhood EV>0.3 and avPC

GADD34 overexpression during prolonged lapatinib treatment allows to overcome ER stress response-mediated inhibition of protein synthesis.

A) Protein levels of GADD34 (PPP1R15A) in the indicated conditions. B-C) Western blot of p-eIF2 (S51) under indicated conditions with and without treatment with guanabenz (GADD34 inhibitor) (B) or GADD34-targeting shRNA (C). D-E) Relative viability of cells at the control (parental) and relapse (2M) stages in response to 72hr treatment with guanabenz (D) or shRNA against GADD34 (E). F-G) Protein synthesis rate measurement with Click-iT Protein synthesis kit in the indicated stages of lapatinib treatment with and without guanabenz (F) or shRNA targeting GADD34 (G). Data is presented as mean fluorescence intensity (MFI). H) A model summarizing the signaling, metabolic and proteostatic changes during the acquisition of resistance to lapatinib in SKBR3 cells. I) mRNA levels of PPP1R15A in the cohort of advanced Her2+ breast cancer patients (n = 53 samples) with short (3 years) duration of response to first-line trastuzumab therapy, from the Long-HER study (33). Statistics: Error bars show standard deviations of 2 (D-E) or 3 (F-G) replicate samples. Data are representative of at least 2 independent experiments (D,F). ***: P S1 Table.</p

List of antibodies.

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RNAseq data.

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The GADD34-eIF2 axis plays an important role in Her3-mediated growth during chronic Her2 inhibition in BT474 cells.

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Raw blot images.

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Metabolomic reprogramming during chronic lapatinib treatment.

A) Heatmap of the most notable altered intracellular metabolites between the different stages of 1μM lapatinib treatment, measured by NMR mass spectrometry. B) A model summarizing the effect of chronic lapatinib treatment on the glucose, energy and amino acid metabolism. Blue lines: reduced flux, red lines: increased flux at the relapse stage (2M). C) Western blot of O-GlcNAc-conjugated protein levels in the indicated conditions. O-GlcNAc levels may serve as a readout of the N-linked hexosamine (GlcNAc) levels. D) Intracellular glucose uptake assay measuring 2-deoxyglucose uptake with and without insulin after lapatinib treatment in SKBR3 cells. Her3 overexpression is unable to rescue the glucose uptake inhibition of lapatinib treatment. E) Overexpression of Her2 or Her3 in the non-transformed MCF10A cells induces similar activation of downstream pathways (p-Akt). F) Glucose uptake rates with control, Her2 or Her3 overexpression in MCF10A cells. Statistics: error bars show standard deviations from 2 replicate samples. Data are representative of 2 independent experiments. ***: P S1 Table.</p

Extracellular metabolomics profiling of SKBr3 cells at different phases of the drug response cycle.

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Her3 overexpression confers tumor cell growth under lapatinib.

A) Total and phospho-Her3 levels at the indicated stages of lapatinib (1μM) treatment. B) Western blot of the indicated signaling proteins with and without shRNA-mediated knock-down of Her3 at the indicated stages of lapatinib treatment. C) Relative viability (compared to control) under the same conditions. D) Her3 was stably silenced or overexpressed in SKBR3 cells, and E) the relative cell growth (relative to control) was calculated after 1μM lapatinib treatment for the indicated time period. Statistics: error bars show standard deviations of 6 (C) or 3 (E) replicate experiments. ***: P S1 Table.</p

Protein homeostasis dynamics during the course of chronic lapatinib treatment.

A) Immunofluorescence images of at the indicated stages of 1μM lapatinib treatment stained for newly synthesized proteins within a 30 min window using the Click-iT Protein Synthesis assay. B) Flow cytometry-based measurement of protein synthesis using the same conditions as in A using the Click-iT assay. Data is presented as mean fluorescence intensity (MFI). C) Protein synthesis rate measurement as in (B) in the indicated conditions with and without Her3 silencing. D) Western blots of the indicated ER stress response markers in the indicated conditions. Statistics: error bars show standard deviations of 2 (B-C) replicates. The data are representative of at least 2 independent experiments. ***: P S1 Table.</p