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: NPJ Precision Oncology

Article Title: Loss of MicroRNA-29b promotes DNMT3b-mediated STING downregulation to attenuate radiotherapy-induced antitumor immunity in KRAS-mutated colorectal cancer

doi: 10.1038/s41698-026-01290-8

Figure Lengend Snippet: A WiDr cells were infected with lentivirus carrying pBabe-puro-vector (Vec.) or pBabe-puro-KRAS G12D and selected for three days. WiDr-Vec. and WiDr-KRAS G12D cells were irradiated (5 Gy), and then harvested for qRT-PCR analysis after 24 hr ( n = 3). One-way ANOVA t test. * p < 0.05. B CoLo320 cells were infected with lentivirus carrying pBabe-puro-vector (Vec.) or pBabe-puro-KRAS G12D and selected for three days. CoLo320-Vec. and CoLo320-KRAS G12D cells were irradiated (5 Gy), and then harvested for qRT-PCR analysis after 24 hr ( n = 3). One-way ANOVA t test. * p < 0.05. C HCT116 cells were infected with lentivirus carrying pLKO-scramble shRNA (shNC) or pLKO-shKRAS (shKRAS) and selected for three days. HCT116 shNC and HCT116 shKRAS cells were irradiated (5 Gy), and then harvested for qRT-PCR analysis after 24 hr ( n = 3). One-way ANOVA t test. * p < 0.05 and ** p < 0.01. D SW620 shNC and SW620 shKRAS cells were irradiated (5 Gy), and then harvested for qRT-PCR analysis after 24 hr ( n = 3). One-way ANOVA t test. * p < 0.05 and ** p < 0.01. E WiDr and Colo320DM (endogenous wild-type KRAS) cells were individually infected with vector (Vec.) and KRAS G12D . Cells were selected by puromycin for three days. The level of DNMT1, DNMT3a and DNMT3b was evaluated by immunoblotting ( n = 3). One-way ANOVA t test. ** p < 0.01. F CT26, HCT116 and SW620 (endogenous mutant KRAS) cells individually infected with lentivirus carry shNC and shKRAS. Cells were selected by puromycin for three days. The level of DNMT1, DNMT3a and DNMT3b was evaluated by immunoblotting ( n = 3). One-way ANOVA t test. ** p < 0.01. G KRAS G12D -tranduced WiDr (endogenous wild-type KRAS) cells were treated with 2.5 μmol/L and 5.0 μmol/L AZA for two days. The level of STING was evaluated by immunoblotting ( n = 3). One-way ANOVA t test. * p < 0.05 and ** p < 0.01. H . KRAS G12D -tranduced CoLo320 (endogenous wild-type KRAS) cells were treated with 2.5 μmol/L and 5.0 μmol/L AZA for two days. The level of STING was evaluated by immunoblotting ( n = 3). One-way ANOVA t test. * p < 0.05 and ** p < 0.01. I HCT116 and SW620 (endogenous mutant KRAS) cells individually infected with lentivirus carry shDNMT1, shDNMT3a and shDNMT3b. Cells were selected by puromycin for three days. The mRNA level of STING was evaluated by qRT-PCR ( n = 3). One-way ANOVA t test. * p < 0.05. J The protein level of STING was evaluated by immunoblotting ( n = 3).

Article Snippet: The retroviral pBabe-puro vector (#1764) and pBabe-puro-KRAS G12D (#58902) were purchased from Addgene.

Techniques: Infection, Plasmid Preparation, Irradiation, Quantitative RT-PCR, shRNA, Western Blot, Mutagenesis