human lung fibroblast cell line hlf 506 05a  (ATCC)

Journal: Molecular cell

Article Title: Increasing cell size remodels the proteome and promotes senescence.

doi: 10.1016/j.molcel.2022.07.017

Figure Lengend Snippet: Figure 7. Cell volume-to-ploidy ratio drives size-dependent proteome changes (A) hTERT RPE-1 cells expressing fluorescent cell- cycle reporters (Cdt1-mKO2, Geminin-mAG) were treated with an Aurora kinase inhibitor barasertib (75 nM, 48 h) to partially inhibit cytokinesis. Cells were then sorted based on ploidy and G1 cell-cycle phase. The attainment of differently sized G1 cells was confirmed using a Coulter counter. The histo- gram shows a representative example of size distri- butions for sorted cells, and the numbers next to it represent mean cell size ± standard error for n = 3 biological replicates. (B) Ploidy-sorted and size-sorted G1 cells were iso- lated by FACS and their proteomes measured using TMT. Ploidy-sorted protein slope values were calcu- lated by plotting the relative protein concentration against the mean cell size in the 2N, 4N, and 8N bins to obtain a slope value (Table S9). (C) Distributions of ploidy-sorted and size-sorted protein slope values. Despite large increases in cell size from 2N to 8N, concentration changes were minimal. (D) Slightly negative correlation between ploidy- sorted and size-sorted protein slope values are consistent with a small increase in DNA-to-cell vol- ume ratio in polyploid G1 cells. x-axis bins are shown in dark blue. Error bars represent the 95% confi- dence interval, and r denotes the Pearson’s correla- tion coefficient. (E) DNA-to-cell-volume-dependent concentration changes for a representative set of proteins. For each protein panel, dotted lines represent unique peptide measurements.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Critical commercial assays Pierce BCA Protein Assay Kit Thermo Scientific Cat#23225 Sep-Pak 50mg C18 Cartridges Waters Cat##054955 High pH Revered-phased Peptide Fractionation Kit Thermo Scientific Cat#84868 Senescence b-Galactosidase Staining Kit Cell Signaling Technology Cat#9860 Direct-zol RNA Microprep Kit Zymo Research Cat#R2063 Click-iT EdU Alexa Fluor 647 Flow Cytometry Assay Kit Thermo Fisher Scientific Cat#C10424 CellTrace CFSE Cell Proliferation Kit Thermo Fisher Scientific Cat#C34554 Deposited data All Proteomics raw files and spectral searches This paper PRIDE: PXD033332 All RNAseq raw files This paper GEO: GSE208348 Experimental models: Cell lines hTERT RPE1 Stearns Lab (Stanford) RRID: CVCL_4388 Primary human fetal lung fibroblasts Cell Applications Cat#506-05f Software and algorithms FlowJo BD Biosciences, https://www.flowjo.com/solutions/flowjo RRID: SCR_008520 FIJI https://imagej.net/software/fiji/ RRID: SCR_002285 MaxQuant Mann Lab N/A Python - Jupyter Notebooks Anaconda N/A

Techniques: Expressing, Protein Concentration, Concentration Assay

Journal: Cell

Article Title: A Dual-Mechanism Antibiotic Kills Gram-Negative Bacteria and Avoids Drug Resistance

doi: 10.1016/j.cell.2020.05.005

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Bacterial and Virus Strains E. coli lptD4213 ΔthyA This paper ZG1599 B. subtilis 168 CRISPRi Library Jason Peters and Carol Gross Peters et al., 2016 Detailed bacterial strain information, including strain identifiers, growth media and temperature, is provided in Table S1 N/A N/A Chemicals, Peptides, and Recombinant Proteins SCH-7979 Tocris Biosciences 1592 Ampicillin MP Biomedicals 190148 Cumene Sigma PHR1210 Daptomycin TCI D4229 Gentamicin sulfate Sigma G1914 Meropenem trihydrate TCI M2279 Nisin MP Biomedicals 155839 Novobiocin sodium salt MP Biomedicals 0215595705 Polymyxin B sulfate salt Sigma P1004 Purified E. coli dihydrofolate reductase (FolA) Genscript N/A DAPI ThermoFisher D1306 SYTOX Green Dead Cell Stain ThermoFisher S34860 FM4–64 Dye ThermoFisher T13320 Critical Commercial Assays BacLight Bacterial Membrane Potential kit ThermoFisher B34950 Colorimetric Dihydrofolate Reductase Assay Kit Sigma CS0340 Deposited Data Proteomics ProteomeXchange Consortium PXD013673 Flow cytometry FlowRespository FR-FCM-Z2JD Raw images and Metabolomics Data This Paper; DataSpace at Princeton University N/A Experimental Models: Cell Lines HEK293 ATCC CRL-1573 HK2 ATCC CRL-2190 HLF Cell Applications 506K-05a PBMC TPCS PB010C Experimental Models: Organisms/Strains Male CD-1 mice Pharmaron N/A Female ovariectomized BALB/c mice Pharmacology Discovery Services Taiwan N/A Galleria mellonella PetCo 2336624 Software and Algorithms MATLAB MathWorks https://www.mathworks.com/products/matlab.html Fiji Eliceiri/LOCI group at University of Washington https://imagej.net/Fiji R Studio R Studio https://rstudio.com/products/rstudio/ FlowJo FlowJo, LLC https://www.flowjo.com/solutions/flowjo/downloads Other Synergy HT microplate reader BioTek N/A InfiniteM200 Pro microplate reader Tecan N/A 27G × 0.5 inch needle BD Biosciences 305109 QuantaMaster 40 Spectrophotometer HORIBA Instruments N/A Open in a separate window KEY RESOURCES TABLE SCH-79797 kills Gram-negative and Gram-positive bacteria with undetectable resistance It works by simultaneously targeting folate metabolism and membrane integrity SCH’s dual-targeting is synergistic, but only when on the same chemical scaffold Irresistin-16, an SCH derivative, effectively treats mouse N. gonorrhoeae infection

Techniques: Virus, Recombinant, Purification, Staining, Membrane, Reductase Assay, Flow Cytometry, Software, Spectrophotometry