Journal: Cell reports

Article Title: A co-opted endogenous retroviral envelope promotes cell survival by controlling CTR1-mediated copper transport and homeostasis.

doi: 10.1016/j.celrep.2023.113065

Figure Lengend Snippet: Figure 3. Refrex1 interacts with CTR1 (A) Graphical representation of the CTR1 copper transporter. (B) Representative immunoblot of HA-tagged CTR1 in lysates from Chinese hamster ovary (CHO) cells stably transduced with murine leukemia virus (MLV)-based LXSN retroviral vector38 either empty or carrying the human (hu) or cat CTR1 cDNA. (C) CHO cells from (B) were evaluated for DC16RBD and FLVCRBD binding by flow cytometry. Numbers indicate the specific change in mean fluorescence intensity compared with non-specific mock staining (gray histogram) of a representative experiment (n = 3 biological replicates). (D) Detection of CTR1 by immunoblot in cell lysates of 293T cells either parental (WT) or KO for CTR1 (CTR1 KO) as generated by CRISPR-Cas9 technology. (E) Cells from (D) were evaluated as in (C) using the indicated RBD ligands or an anti-CTR1 mAb. Representative experiment (n = 3 biological replicates). Numbers indicate the specific change in mean fluorescence intensity compared with non-specific mock staining (gray histogram) of a representative experiment (n = 3 biological replicates). (F) 293T cells grown in 100 mM CuCl2 or MgCl2 overnight were evaluated for CTR1 (DC16RBD), SLC20A2 (AmphoRBD), and FLVCR1 (FLVCRBD) cell-surface expression by flow cytometry using the indicated RBD ligands. Numbers indicate the specific change in mean fluorescence intensity compared with non-specific mock staining (gray histogram) of a representative experiment (n = 3 biological replicates).

Article Snippet: RBD ligands were produced and used as previously described.70 Human CTR1 was also detected using a mouse anti-CTR1 monoclonal antibody (Proteintech).

Techniques: Western Blot, Stable Transfection, Transduction, Virus, Retroviral, Binding Assay, Cytometry, Staining, Generated, CRISPR, Expressing

Journal: Cell reports

Article Title: A co-opted endogenous retroviral envelope promotes cell survival by controlling CTR1-mediated copper transport and homeostasis.

doi: 10.1016/j.celrep.2023.113065

Figure Lengend Snippet: Figure 4. Altered copper homeostasis in the absence of Refrex1 in cat cells (A) Detection of HA-tagged Refrex1 by immunoblot in cell lysates and supernatants of 293T cells overexpressing empty vector pCHIX or DC7 or DC16 alone or in combination (DC7 + DC16). (B) Sensitivity of cells from (A) to infection by EGFP lentiviral vectors pseudotyped with ERV-DC14 or VSV-G Env. Data are means ± SEM from n = 3 independent experiments. (C) Cells from (A) were evaluated for DC14RBD binding by flow cytometry. Numbers indicate the specific change in mean fluorescence intensity compared with non-specific mock staining (gray histogram) of a representative experiment (n = 3 biological replicates). (D) Total amount of copper (Cu) or magnesium (Mg) was evaluated 48 h post-transfection by inductively coupled plasma mass spectrometry (ICP-MS) in 293T cells from (A). Data are means ± SEM from n = 3 independent experiments. One-way ANOVA with Dunnett’s multiple comparisons test, *p % 0.05, **p % 0.01. (E) Total amount of copper (Cu) or magnesium (Mg) was evaluated by ICP-MS 48 h after transfecting feline FEA and CRFK cells with siLuc or siDC16 (n = 3). Data are means ± SEM from n = 3 independent experiments. Mann-Whitney test, *p % 0.05, **p % 0.01. (F) mRNA expression of several genes involved in copper homeostasis (CTR1, ATP7A, ATP7B, COMMD1, and MT1/2) was evaluated by qRT-PCR in feline FEA and CRFK cells transfected with siDC16 or control siLuc and normalized to cat GAPDH. Graphs represent the ratio of normalized expression of indicated genes in siDC16-transfected cells to normalized expression in siLuc-transfected cells. Data are means ± SEM from at least n = 3 independent experiments. Two-way ANOVA with Sidak’s multiple comparisons test, *p % 0.05, **p % 0.01, ****p % 0.0001.

Article Snippet: RBD ligands were produced and used as previously described.70 Human CTR1 was also detected using a mouse anti-CTR1 monoclonal antibody (Proteintech).

Techniques: Western Blot, Plasmid Preparation, Infection, Binding Assay, Cytometry, Staining, Transfection, Clinical Proteomics, Mass Spectrometry, MANN-WHITNEY, Expressing, Quantitative RT-PCR, Control

Journal: Cell reports

Article Title: A co-opted endogenous retroviral envelope promotes cell survival by controlling CTR1-mediated copper transport and homeostasis.

doi: 10.1016/j.celrep.2023.113065

Figure Lengend Snippet: Figure 7. Refrex1 is present in cat serum (A) 293T cells were incubated overnight with various dilutions of human and cat sera and evaluated for their sensitivity to infection by EGFP lentiviral vector pseudotyped with ERV-DC14 or VSV-G Env. Data are means ± SEM from n = 3 independent experiments. (B) Evaluation of cell-surface expression of FLVCR1 and CTR1 on 293T cells incubated overnight with human and cat serum dilutions (1:50) by flow cytometry using the indicated RBD ligands. Numbers indicate the specific change in mean fluorescence intensity compared with non-specific mock staining (gray his- togram) of a representative experiment (n = 3 biological replicates). (C) Evaluation of cell-surface expression of FLVCR1, GLUT1, and CTR1 on human and cat red blood cells by flow cytometry using RBD ligands. Binding on 293T is shown as a control. Numbers indicate the specific change in mean fluorescence intensity compared with non-specific mock staining (gray histogram) of a representative experiment (n = 3 biological replicates). (D) Evaluation by qRT-PCR of ERV-DC16 env expression normalized with GAPDH in cat PBMCs isolated from whole blood of three independent male cats and incubated overnight in 250 mM CuCl2.

Article Snippet: RBD ligands were produced and used as previously described.70 Human CTR1 was also detected using a mouse anti-CTR1 monoclonal antibody (Proteintech).

Techniques: Incubation, Infection, Plasmid Preparation, Expressing, Cytometry, Staining, Binding Assay, Control, Quantitative RT-PCR, Isolation