blunt end cloning vector pjet1 2  (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: iScience

Article Title: Horizontal and vertical gene transfer shape the plasmid host range in surface-associated microbial systems

doi: 10.1016/j.isci.2026.115299

Figure Lengend Snippet: Hypothesis and experiment system (A) We hypothesize that vertical and horizontal gene transfer (VGT and HGT) are influenced by the characteristics of the potential recipient cell types and determine the proliferation and diversity of transconjugant cells. Because the potential recipient community comprises multiple cell types with varying growth traits and conjugation probabilities, we expect the resulting composition of transconjugant cells to be shaped by these cell type-specific traits. (B) Our experimental system consists of E . coli MG1655 lacI q -pLpp-mCherry as the plasmid donor strain and pB10 as the focal plasmid. pB10 donor cells express RFP from the chromosome and transconjugants express GFP from pB10.

Article Snippet: MBP- mCherry expression plasmid (Amp R ) , Addgene , Plasmid# 29747.

Techniques: Conjugation Assay, Plasmid Preparation

Journal: iScience

Article Title: Horizontal and vertical gene transfer shape the plasmid host range in surface-associated microbial systems

doi: 10.1016/j.isci.2026.115299

Figure Lengend Snippet: Transconjugant proportions and diversities after surface-associated conjugation assays for different environmental conditions (A) Proportion of transconjugant cells relative to total cells after surface-associated conjugation assays using the WWTP community as the potential recipient cell population. We conducted conjugation assays on 1×SWW, 10×SWW, or LB agar plates using E . coli MG1655 lacI q -pLpp-mCherry as the pB10 donor strain. (B) Relative abundances of bacterial class in the total potential recipient cell population (T) and the transconjugant cell population (TC) as identified by 16S rRNA gene sequencing. We separated and identified TC cells using FC-FACS-sorting of GFP-positive cells. (C) Normalized Shannon index of the transconjugant populations after surface-associated conjugation assays on 1×SWW, 10×SWW, or LB agar plates. We normalized the Shannon index of the TC populations to their corresponding T populations. (D) Principal coordinate analysis (PCoA) based on weighted UniFrac distances of T and TC populations after surface-associated conjugation assays on 1×SWW, 10×SWW, or LB agar plates. (E) Phylogenetic tree of transconjugant ASVs detected after surface-associated conjugation assays on 1×SWW, 10×SWW, or LB agar plates. The outer colored box denotes the bacterial phylum of each ASV, corresponding to the phylum-level groupings shown in panel (B). The inner heatmap box aligned with each tip shows the log 10 fold-changes in ASV abundance (TC relative to T) across the three conditions. For (A and C), each point is an independent biological replicate ( n = 3), horizontal bars are the means, error bars are ±1 standard deviation, and asterisks indicate statistically significant differences between the means based on two-way ANOVA with Holm correction (∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, ns = not significant). For (D), each point is an independent biological replicate ( n = 3).

Article Snippet: MBP- mCherry expression plasmid (Amp R ) , Addgene , Plasmid# 29747.

Techniques: Conjugation Assay, Sequencing, Standard Deviation

Journal: iScience

Article Title: Horizontal and vertical gene transfer shape the plasmid host range in surface-associated microbial systems

doi: 10.1016/j.isci.2026.115299

Figure Lengend Snippet: Transconjugant growth during surface-associated conjugation assays for different environmental conditions (A) Representative fluorescence microscopy images of transconjugant cells during surface-associated conjugation assays on LB agar plates. E . coli MG1655 lacI q -pLpp-mCherry is the pB10 donor strain and show red fluorescence. Transconjugant cells are green. The time indicated in the images refers to the point at which transconjugant cells first became detectable. (B) Normalized microcolony area ( A / a 0 ) plotted as a function of time during the surface-associated conjugation assays on LB agar plates. A is the total microcolony area and a 0 is the initial transconjugant area. Connected data points are for individual colonies ( n = 12). (C) Microcolony area at the endpoint of the mating assay (t = 24 h) for different environmental conditions. The half-violin and scatterplots present the sample distribution and individual microcolony measurements for surface-associated conjugation assays on different medium (n 1xSWW = 880, n 10xSWW = 664, n LB = 1,070, for microcolony number). We performed each experiment at least three independent experiments. Horizontal bars are the mean microcolony areas, error bars are the 99% confidence intervals, and asterisks indicate statistically significant differences between the means based on two-way ANOVA with Holm correction (∗∗ p < 0.01, ∗∗∗∗ p < 0.0001, ns = not significant).

Article Snippet: MBP- mCherry expression plasmid (Amp R ) , Addgene , Plasmid# 29747.

Techniques: Conjugation Assay, Fluorescence, Microscopy