third generation packaging system  (Addgene inc)

Journal: Genes

Article Title: Screening Methods to Discover the FDA-Approved Cancer Drug Encorafenib as Optimally Selective for Metallothionein Gene Loss Ovarian Cancer.

doi: 10.3390/genes16010042

Figure Lengend Snippet: Figure 2. Zinc and cadmium metal toxicity in MT2A knockdown. (A) Amino acid alignment of the 11 expressed human metallothioneins. Note that cysteine residues, which chelate metals, are highly conserved among the proteins and represent a third of all the amino acids in metallothioneins. (B) Nuclear magnetic resonance structure of metallothionein MT2A alpha (PDB ID: 2MRB) and beta (PDB ID:1MRB) domains, with bound Cd2+ ions shown. Zn2+ ions are predicted to be similarly located within the protein in the absence of Cd2+. (C) CAOV3 cells, which only express MT2A (higher) and MT1X (lower), were knocked down by lentiviral shMT2A and validated by RT-qPCR as reduced in expression relative to scrambled shRNA control (shScr). (D) ZnCl2 toxicity was assessed by Hoechst-33342-stained nuclei counts relative to control treated cells, with 2-day (2 d) or 3 d exposure. (E) CdCl2 toxicity was assessed as in (D). Error bars are standard error of the mean. * p < 0.05, ** p < 0.01, *** p < 0.001 by t-test of technical replicates relative to shScr control from a representative experiment.

Article Snippet: Fluorescent protein nls-GFP (Addgene, #126688) or nls-BFP (Addgene, #36085) was stably added via lentivirus using third generation lentiviral packaging vectors (Addgene, #12251, 12253, 12259) packaged in HEK293T cells (ATCC, #CRL-3216).

Techniques: Knockdown, Nuclear Magnetic Resonance, Quantitative RT-PCR, Expressing, shRNA, Control, Staining