plasmid pcmv6 xl5mod il4 nm 000589  (OriGene)

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