Construction and Screening of a Lentiviral Secretome Library Tao Liu, Panpan Jia, Huailei Ma, Sean A. Reed, Xiaozhou Luo, H. Benjamin Larman, Peter G. Schultz  Cell Chemical Biology  Volume 24, Issue 6, Pages 767-771.e3 (June 2017) DOI: 10.1016/j.chembiol.2017.05.017 Copyright © 2017 Elsevier Ltd Terms and Conditions

Cell Chemical Biology 2017 24, 767-771. e3DOI: (10. 1016/j. chembiol Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 Construction of a Secretome ORF-Enriched Library (A) Cloning of a lentiviral construct pLV-ORF-ΔCD4. (B) A schematic representation of virus production and transduction into 293F cells. (C) A schematic representation of cells transduced with ORFs encoding either secreted proteins or non-secreted proteins, and the separation method. See also Figure S1. Cell Chemical Biology 2017 24, 767-771.e3DOI: (10.1016/j.chembiol.2017.05.017) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 Next-Generation Sequencing Analysis of the Secretome ORF-Enriched Library (A) Number of ORFs present in the library, categorized by cellular location of their encoding proteins. (B) The percentage of each ORF category in the library pool. See also Table S3. Cell Chemical Biology 2017 24, 767-771.e3DOI: (10.1016/j.chembiol.2017.05.017) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 A Phenotypic Autocrine System for Lentiviral Secretome Library Screening (A) Plasmid map for the first-generation library design. SP, secreted protein. (B) A schematic illustration of the first-generation library screen. Cells containing library ORFs were grown in a semi-solid medium and acted on by a high local concentration of the secreted protein (C) Plasmid map for the second-generation library design. (D) A schematic illustration of the second-generation library screening protocol. Secreted proteins are tethered to the membrane of the producing cells. See also Figure S3. Cell Chemical Biology 2017 24, 767-771.e3DOI: (10.1016/j.chembiol.2017.05.017) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 Hit Validation First-generation (A) and second-generation (B) pLV library vectors containing ORFs encoding selected hit GMCSF and three other cytokines (known to stimulate TF1 proliferation); IL3, IL6, and EPO ORFs were used to produce viruses. pLV vector containing the dsRed gene was used as a negative control. Transduction was performed under OGOC conditions and transduced cells were seeded at densities of 10,000 cells/well (A) and 30,000 cells/well (B). Cells were quantified by the addition of AlamarBlue reagent after 72 hr of culture. Fluorescence was recorded on a Tecan Infinite M200 fluorescence plate reader at 610 nm with 540 nm excitation. Experiments were performed in quadruplicate. Data represent average values ± SD. Cell Chemical Biology 2017 24, 767-771.e3DOI: (10.1016/j.chembiol.2017.05.017) Copyright © 2017 Elsevier Ltd Terms and Conditions