retroviral vector pmigr1  (Addgene inc)

Journal: The Journal of Experimental Medicine

Article Title: Targeting Mettl8-Tcf1 axis promotes CD8 + T PEX differentiation and antitumor immunity

doi: 10.1084/jem.20250424

Figure Lengend Snippet: GA enhances the antitumor response of CD8 + T cells. (A) Schematic diagram of detecting Mettl8 expression in vitro : HEK293T cells were transfected with pMigR1-Mettl8-Flag plasmid, and Flag-tagged Mettl8 was detected by western blotting. (B) The detection included GA-treated HEK293T cells after transfection (top) or treated protein extracted from transfected HEK293T cells (bottom). (C) Schematic diagram of GA treatment to B16F10 tumor–bearing Mettl8 -tdTomato-Flag mice: Mice were subcutaneously injected with 2 × 10 5 B16F10 cells, followed by GA treatment every 2 days from day 6 to day 12. Mice were harvested at day 13. (D) Tumor growth of the mice in C. n = 7 per group. (E) Tumor growth of the mice in C displayed in each replicate. (F and G) Tumor weight (F) and the absolute number of tumor-infiltrating CD44 + CD8 + T cells (G). n = 7 per group. (H) Representative flow cytometry plots (left) and cumulative data (right) show the MFI of tdTomato and Ly108 in tumor-infiltrating CD8 + T cells. n = 5–6 per group. (I and J) Representative flow cytometry plots and cumulative data show the frequency and absolute number of CX3CR1 + Tcf1 − Int-T EX (I), Tcf1 + Tim3 − T PEX , and Tim3 + Tcf1 − T EX cells (J) gated on tumor-infiltrating CD44 + CD8 + T cells. n = 7 per group. (K) Representative flow cytometry plots (left) and cumulative data (right) show the frequency of GzmB, IFN-γ, and perforin gated on tumor-infiltrating CD44 + CD8 + T cells. n = 6–8 per group. (L and M) Cumulative data show the absolute number (L) and MFI (M) of IFN-γ, GzmB, and perforin gated on tumor-infiltrating CD44 + CD8 + T cells. n = 6–8 per group. Data are representative of three independent experiments. P value was calculated by two-way ANOVA (D) and two-tailed Student’s t test (F–M); *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Source data are available for this figure: .

Article Snippet: Mettl8 cDNA was cloned into a GFP-expressing retroviral vector pMigR1 (cat. no. #27490; Addgene).

Techniques: Expressing, In Vitro, Transfection, Plasmid Preparation, Western Blot, Injection, Flow Cytometry, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Targeting Mettl8-Tcf1 axis promotes CD8 + T PEX differentiation and antitumor immunity

doi: 10.1084/jem.20250424

Figure Lengend Snippet: Mettl8 inhibition promotes CD8 + T cell antitumor immunity and synergistically enhances PD-1 blockade. (A) Tumor growth of the mice in GA-treated adoptive-transferred model displayed in each replicate. n = 8 per group. (B) Western blot analysis of Mettl8-Flag and GAPDH in lysates from HEK293T cells transfected with pMIGR1-Empty, pMIGR1-Mettl8-WT, and pMIGR1-Mettl8-Mut plasmid. (C) Tumor growth curves for individual mice in the Mettl8-mutated mouse model. n = 4–6 per group. (D) Representative flow cytometry plots and cumulative data show the frequency of GzmB, IFN-γ, and perforin gated on tumor-infiltrating OT-I cells from the mice in C. n = 4–6 per group. (E) Tumor growth of the mice from the combined Mettl8 KO and anti–PD-1 treatment model displayed in each replicate. n = 8 per group. (F) Representative flow cytometry plots and cumulative data show the frequency of IFN-γ, GzmB, and perforin gated on tumor-infiltrating OT-I cells from the mice in E. n = 6 per group. (G) Tumor growth of the mice from GA and anti–PD-1 combined treatment model displayed in each replicate. n = 9 per group. (H) Cumulative data show the absolute number of tumor-infiltrating OT-I cells from the mice in G. (I) Representative flow cytometry plots cumulative data show the frequency and absolute number of CX3CR1 + Tcf1 − Int-T EX cells gated on tumor-infiltrating OT-I cells from the mice in G. n = 7 mice per group. P value was calculated by two-tailed Student’s t test; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Source data are available for this figure: .

Article Snippet: Mettl8 cDNA was cloned into a GFP-expressing retroviral vector pMigR1 (cat. no. #27490; Addgene).

Techniques: Inhibition, Western Blot, Transfection, Plasmid Preparation, Flow Cytometry, Two Tailed Test

Journal: Frontiers in immunology

Article Title: Nanoscale Colocalization of NK Cell Activating and Inhibitory Receptors Controls Signal Integration.

doi: 10.3389/fimmu.2022.868496

Figure Lengend Snippet: FIGURE 1 | Generation and expression of NKG2D(S/L)-GFP and Ly49A-RFP-expressing natural killer (NK) cells in vivo. (A) Schematic representation of the constructs of mouse NKG2D-GFP and Ly49A-RFP used for functional and colocalization studies. All constructs were subcloned into the pMIGR1 retroviral expression vector. (B) Schematic of NKG2D-GFP and Ly49A-RFP at the NK cell surface. Both molecules form homodimers. Image representation is not to scale. (C) NK cells expressing NKG2D-GFP and Ly49A-RFP generated in vivo. Retroviral-mediated expression of NKG2D-GFP (S/L) and Ly49A in murine B3Z CD8+ T cell line (top), NK cells differentiated in vitro from hematopoietic progenitor cells (HPCs, middle) and NK cells obtained from retrogenic (RT) mice (bottom, see also Supplementary Figure S1). Experiments with NK cells differentiated in vitro were performed by transducing HPCs from NKG2D-deficient C57BL/6 mice (Supplementary Figure S2), and the data shown are gated on NK1.1+, NKp46+, and CD3- cells. Splenic NK cells from RT mice were gated as CD3-, CD8-, NK1.1+, and CD49+ (DX5) cells. The data are representative of more than three experiments.

Article Snippet: To subclone the fusion protein construct GFPNKG2D-S/L into the retroviral stem cell vector pMIGR1 (Addgene, plasmid 27490), forward 5′-TAGTAGGAA TTCGCCACCATGAGCGGGGGCGAGGAC-3′ and reverse 5′-TAGAGGTCGACCTTACACCGCCCTTTTCATGCAG-3′ primers were used.

Techniques: Expressing, In Vivo, Construct, Functional Assay, Retroviral, Plasmid Preparation, Generated, In Vitro

Journal: The Journal of Biological Chemistry

Article Title: DHHC7 Palmitoylates Glucose Transporter 4 (Glut4) and Regulates Glut4 Membrane Translocation *

doi: 10.1074/jbc.M116.747139

Figure Lengend Snippet: Regulation of insulin (Ins)-dependent Glut4 membrane translocation by DHHC7. A, CHO cells co-transduced with AAV HA-Glut4 and either scrambled (SCR) shRNA or DHHC3 shRNA or DHHC7 shRNA were serum-deprived and treated with or without insulin (10 nm, 15 min). PM fractions and total lysates (Tcl) were analyzed by Western blotting with anti-Glut4 (i, iii, and iv). In addition, immunoblots of total cell lysates were probed for phospho-Akt (S373) (v) and total Akt (vi), whereas PM fractions were probed for Glut1 as loading control (iii). B, summary statistics of PM Glut4 data illustrated in A. The data were normalized to PM Glut4 of insulin-untreated scrambled RNA-transfected cells and to PM Glut1 levels of each sample. C and D, analyses analogous to A and B except that the cells were, in addition, infected with pMIGR1 retrovirus vector expressing GFP or shRNA-resistant FLAG-DHHC7. PM Glut4 levels were normalized to PM Glut4 in scrambled shRNA-expressing cells without insulin treatment and total levels of Glut4. Note that shRNA-resistant FLAG-DHHC7 restores PM association of Glut4 in DHHC7 shRNA-treated cells. Bar graphs and error bars represent means ± S.D. (n = 3).

Article Snippet: Then DHHC3 shRNA-resistant FLAG-tagged DHHC7 cDNA was cloned into pMIGR1 retroviral vector (addgene) between BglII and EcoR1 sites.

Techniques: Translocation Assay, Transduction, shRNA, Western Blot, Transfection, Infection, Plasmid Preparation, Expressing