lentiviral plko 1 shrna construct shgfp  (Addgene inc)

Journal: Nature Communications

Article Title: Polycomb deficiency drives a FOXP2-high aggressive state targetable by epigenetic inhibitors

doi: 10.1038/s41467-023-35784-x

Figure Lengend Snippet: a FOXP2 protein expression in HBEC3KT, BEAS2B, H460, and H2030 cells transduced with FOXP2 lentivirus was examined by immunoblot. EV is the empty vector control line. b Average diameters and organoid counts of BEAS2B 3D organoids with or without FOXP2 overexpression plotted as mean values +/− SEM. * indicates P = 0.034 by two-tailed t test. n = 3 individual cell cultures per group. c Representative images, scale bar = 100 µm, and d average numbers of migrated cells normalized to growth rates in HBEC3KT, BEAS2B, H460, and H2030 cells with or without FOXP2 overexpression plotted as mean +/− SEM. * indicates P < 0.047, ** indicates P = 0.0041 by two-tailed t test. n = 3, 4, 5, 5 for HBEC3KT, BEAS2B, H460, H2030 individual cell cultures per group, respectively. e Rank-ordered gene lists were queried against the MSigDB databases and enrichment scores of selected gene signatures enriched in FOXP2 overexpressing cell lines relative to EV lines were plotted. Dot size estimates FDR. Data are summarized in Supplementary Table . f FOXP2 protein expression in 2D H460 cells transduced with the indicated small hairpins was examined by immunoblot and representative of n = 4 blotting experiments from one cell culture per group. shGFP is the control cell line. g Crystal violet growth assays were performed on 2D H460 cells with or without FOXP2 knockdown at the indicated days of culture and mean values +/− SEM were graphed. ** indicates P < 0.0024, *** indicates P < 0.001 shFOXP2 compared to shGFP by two-tailed t test. n = 6 individual cell cultures per group. h Average diameters of 3D H460 spheroids with or without FOXP2 knockdown plotted as mean values +/− SEM. *** indicates P = 0.000017 shGFP vs shFOXP2.1 and P = 0.00000038 shGFP vs . shFOXP2.2 tumoroids by two-tailed t test. n = 6 individual cell cultures per group. i Average percentages of migrated cells normalized to shGFP and relative cell growth rates graphed as mean values +/− SEM. *** indicates P = 0.000014 compared to shGFP cells by two-tailed t test. n = 4 individual cell cultures per group. j Representative images of migrated cells in H460 with or without FOXP2 knockdown. Source Data are provided for this figure.

Article Snippet: The pLKO.1 EZH2 shRNA (TRCN0000040076 and TRCN0000040073) and FOXP2 shRNA (TRCN0000426742, TRCN0000426178) construct clones were purchased from SIGMA and the shGFP plasmid #12273 and pENTR- FOXP2 plasmid #47053 were available on Addgene .

Techniques: Expressing, Transduction, Western Blot, Plasmid Preparation, Control, Over Expression, Two Tailed Test, Cell Culture, Knockdown