third-generation lentiviral packaging system pmdlg/prre  (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

Journal: bioRxiv

Article Title: The membrane-associated ubiquitin ligase MARCHF8 promotes cancer immune evasion by degrading MHC class I proteins

doi: 10.1101/2024.11.29.626106

Figure Lengend Snippet: HPV+ HNC cells (SCC152) were transduced with five lentiviral shRNAs against MARCHF8 (shR-MARCHF8) or scrambled shRNA (shR-scr). MARCHF8 and HLA-A/B/C protein levels were determined by western blotting ( A - C ). Relative band density was quantified using NIH ImageJ ( B and C ). Cell surface expression of HLA-A/B/C was analyzed by flow cytometry ( D and E ). MFI of three independent experiments ( E ) are shown. Ubiquitinated proteins were pulled down from the lysate of SCC152 cells with shR-scr or shR-MARCHF8 (clone 5) using an anti-ubiquitin antibody and analyzed by western blotting ( F ). HA-tagged wild-type (WT) or mutant MARCHF8 constructs were generated with an amino acid substitution in the RING-CH domain (W114A) and deletion of domain in-between RING and transmembrane (ΔDIRT) ( G ). SCC152 cells with shR-MARCHF8 were stably transduced with HA-tagged MARCHF8. MARCHF8 protein expression was determined by western blot using an anti-HA antibody ( H ). Total ( H and I ) and cell surface ( J and K ) MHC-I levels were measured by western blot and flow cytometry using anti-HLA-A/B/C antibodies. Relative band density was quantified using NIH ImageJ ( I ). The dotted lines are MFI for control ( D and J ). The data shown are means ± SD of three independent experiments. P values were determined by Student’s t -test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: 293T cells (ATCC, CRL-3216) were transfected with each construct and third-generation lentiviral packaging plasmids (Cell BioLabs, VPK-206) using Lipofectamine 3000 (Thermo Fisher) according to the manufacturer’s recommendation.

Techniques: Transduction, shRNA, Western Blot, Expressing, Flow Cytometry, Mutagenesis, Construct, Generated, Stable Transfection, Control

Journal: bioRxiv

Article Title: The membrane-associated ubiquitin ligase MARCHF8 promotes cancer immune evasion by degrading MHC class I proteins

doi: 10.1101/2024.11.29.626106

Figure Lengend Snippet: mEERL cells were transduced with lentiviral Cas9 and one of two sgRNAs against Marchf8 (sgR- Marchf8-2 and sgR- Marchf8-3 ) or scrambled sgRNA (sgR-scr). MARCHF8 protein levels were determined by western blotting ( A ), and relative band density was quantified using NIH ImageJ ( B ). The data shown are means ± SD of three independent experiments. Cell surface levels of H2-Db protein were measured by flow cytometry ( C and D ). The dotted line is MFI for control ( C ). MFI of three independent experiments is shown ( D ). P values were determined by Student’s t -test. *** p < 0.001. mEERL/scr or mEERL/ Marchf8 -/- (sgR- Marchf8-2 and sgR- Marchf8-3 ) cells were injected into the rear right flank of C57BL/6J mice ( n = 10 per group). Tumor volume was measured twice a week ( E - H ). Survival rates of mice were analyzed using a Kaplan-Meier estimator ( G ). The time to event was determined for each group, with the event defined as a tumor size of 2,000 mm 3 . The data shown are means ± SD. P values of mice injected with mEERL/ Marchf8 -/- cells compared with mice injected with mEERL/scr cells were determined for tumor growth ( H ) and survival ( G ) by two-way ANOVA analysis. Shown are representative of two independent experiments.

Article Snippet: 293T cells (ATCC, CRL-3216) were transfected with each construct and third-generation lentiviral packaging plasmids (Cell BioLabs, VPK-206) using Lipofectamine 3000 (Thermo Fisher) according to the manufacturer’s recommendation.

Techniques: Transduction, Western Blot, Flow Cytometry, Control, Injection