plasmids encoding hatagged ifitm3  (Addgene inc)

Journal: Molecular Cancer

Article Title: IFITM3 enhances immunosensitivity via MHC-I regulation and is associated with the efficacy of anti-PD-1/-L1 therapy in SCLC

doi: 10.1186/s12943-025-02383-x

Figure Lengend Snippet: IFITM3 is correlated with MHC-I expression in SCLC. (A) UMAP plot displaying 11,718 cancer cells from single-cell RNA sequencing of nine untreated SCLC tumors. (B) Dot plot representing MHC-I gene set scores calculated via the AUCell, UCell, and singscore algorithms. The dot size indicates the percentage of cells expressing the MHC-I gene set, and the color represents the average expression level. (C) UMAP plot categorizing cancer cells on the basis of MHC-I expression levels. (D) Pseudotime trajectory initiated from MHC-I low clusters toward MHC-I high clusters. Pie charts represent the proportion of cells within each category along the trajectory. (E) Heatmap showing 627 genes whose expression significantly changed along the pseudotime trajectory. Cells were ordered by pseudotime (left to right), with gene expression scaled by Z-score (bottom color bar). Clusters represent distinct temporal gene expression patterns: Cluster 1 genes were enriched in early pseudotime states (low-MHC-I cells), and Cluster 2 genes in late pseudotime states (high-MHC-I cells). Sample size: n = 11,718 cells from 9 patients. (F) Bar plot of enriched GO terms for the Cluster 2 gene set from (E). (G) Venn diagram showing overlapping MHC-I-related genes (correlation r > 0.5, P < 0.05) across five SCLC datasets. (H) Correlation matrix depicting IFITM3 expression and key MHC-I genes across the five datasets. Spearman correlation coefficients were calculated for each gene pair. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: Plasmids encoding HA-tagged IFITM3 (Cat# 58397; Addgene) and MYC-tagged NLRC5 (Cat# 37509; Addgene) were used.

Techniques: Expressing, RNA Sequencing, Gene Expression

Journal: Molecular Cancer

Article Title: IFITM3 enhances immunosensitivity via MHC-I regulation and is associated with the efficacy of anti-PD-1/-L1 therapy in SCLC

doi: 10.1186/s12943-025-02383-x

Figure Lengend Snippet: IFITM3 regulates MHC-I expression in SCLC cell lines. (A) Relative mRNA expression of MHC-I-related genes ( HLA-A , HLA-B , HLA-C , TAP1 , TAP2 , and B2M ) in NCI-H446 and NCI-H69 cells after overexpression or knockdown of IFITM3 . (B-C) Western blot analysis of MHC-I protein levels in NCI-H446 and NCI-H69 cells after overexpression or knockdown of IFITM3 . (D-E) Flow cytometry analysis of MHC-I surface expression in NCI-H446 and NCI-H69 cells after overexpression or knockdown of IFITM3 , with or without IFN-γ treatment. The results are presented as the means ± SEMs, n = 3. Statistical comparisons were performed using unpaired two-tailed Student’s t-tests. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: Plasmids encoding HA-tagged IFITM3 (Cat# 58397; Addgene) and MYC-tagged NLRC5 (Cat# 37509; Addgene) were used.

Techniques: Expressing, Over Expression, Knockdown, Western Blot, Flow Cytometry, Two Tailed Test

Journal: Molecular Cancer

Article Title: IFITM3 enhances immunosensitivity via MHC-I regulation and is associated with the efficacy of anti-PD-1/-L1 therapy in SCLC

doi: 10.1186/s12943-025-02383-x

Figure Lengend Snippet: Clinical correlation of IFITM3 expression with immunotherapy outcomes in SCLC. (A) Scatter plot showing the correlation between IFITM3 expression and immune-related parameters via the IPS algorithm. The x-axis represents the spearman correlation coefficient. (B) Kaplan-Meier curves of PFS for SCLC patients from the IMpower133 cohort treated with anti-PD-L1 plus chemotherapy (left) or chemotherapy alone (right), stratified by the optimal IFITM3 expression cutoff. Statistical significance was assessed using the log-rank test. (C) Kaplan-Meier survival curve for PFS in the PH cohort stratified by the optimal IFITM3 expression cutoff. Statistical significance was assessed using the log-rank test. (D) Box plot comparing IFITM3 expression between patients with PD/NE and patients with CR in the Rochester cohort. Statistical significance was assessed using the two-sided Wilcoxon rank-sum test. (E) Schematic of clinical cohort. (F) IFITM3 H-scores across patients with PD, SD, or PR. Representative IHC images show IFITM3, MHC-I, and CD8 staining in PR vs. PD tumors (scale bar: 40 μm). Statistical significance was assessed using the two-sided Wilcoxon rank-sum test. (G) Scatter plot showing correlations between IFITM3 and MHC-I, as well as between IFITM3 and CD8 H-scores. Spearman correlation was used to assess statistical significance. (H) HRs with 95% confidence intervals for PFS among patients with high versus low IFITM3 expression, defined by increasing H-score percentile cutoffs. A range of thresholds (from 15–90%) was tested in the IHC-stained clinical cohort ( n = 42). HRs and P values were calculated using Cox proportional hazards regression models. (I) Kaplan-Meier survival curve for PFS among the indicated groups, with a 75% H-score cutoff for IFITM3 and a 55% cutoff for MHC-I. Survival differences were assessed using the log-rank test. * P < 0.05, ** P < 0.01, n.s., not significant

Article Snippet: Plasmids encoding HA-tagged IFITM3 (Cat# 58397; Addgene) and MYC-tagged NLRC5 (Cat# 37509; Addgene) were used.

Techniques: Expressing, Staining

Journal: Molecular Cancer

Article Title: IFITM3 enhances immunosensitivity via MHC-I regulation and is associated with the efficacy of anti-PD-1/-L1 therapy in SCLC

doi: 10.1186/s12943-025-02383-x

Figure Lengend Snippet: IFITM3 enhances PD-1 inhibitor efficacy and tumor immune responses in SCLC mouse models. (A) A schematic view of the treatment plan. C57BL/6 mice were injected with RPP control or OE-IFITM3 cells, and a PD-1 mAb or vehicle was administered when the tumors reached approximately 100 mm³. (B) Representative images and weight plots of tumors, along with tumor volume plots measured every 3 days. (C) H&E staining and IFITM3 and MHC-I IHC analyses of tumor sections from the indicated treatment groups (scale bar: 100 μm). (D) Flow cytometry analysis of CD3 + CD45 + cells, CD8 + CD3 + cells, and CD69 + , CD44 + , and GZMB + CD8 + cells. (E) A schematic view of the treatment plan. BALB/c nude mice were injected with RPP-control or OE-IFITM3 cells. (F) Representative images and weight plots of tumors harvested after the mice were euthanized, along with tumor volume plots measured every 2 days. The results are presented as the means ± SEMs, n = 4. Statistical comparisons between groups were performed using unpaired two-tailed Student’s t-tests. * P < 0.05, ** P < 0.01, *** P < 0.001, n.s., not significant

Article Snippet: Plasmids encoding HA-tagged IFITM3 (Cat# 58397; Addgene) and MYC-tagged NLRC5 (Cat# 37509; Addgene) were used.

Techniques: Injection, Control, Staining, Flow Cytometry, Two Tailed Test

Journal: Molecular Cancer

Article Title: IFITM3 enhances immunosensitivity via MHC-I regulation and is associated with the efficacy of anti-PD-1/-L1 therapy in SCLC

doi: 10.1186/s12943-025-02383-x

Figure Lengend Snippet: EG enhances the efficacy of PD-1 inhibition and antitumor immune responses in SCLC mouse models. (A) Volcano plots showing differential gene expression following EG treatment in NCI-H446 and NCI-H69 cells. IFITM3 is significantly upregulated upon EG treatment. Vertical dashed lines indicate log2FC = ± 1, and the horizontal dashed line represents the significance threshold at P = 0.05. (B) GSEA plots for the KEGG_ANTIGEN_PROCESSING_AND_PRESENTATION pathway in EG-treated NCI-H446 and NCI-H69 cells. NES and P values are shown. (C) Western blot analysis of IFITM3 and MHC-I protein levels in NCI-H446 and NCI-H69 cells treated with EG or transduced with sh-IFITM3, as indicated. Densitometric quantification is shown. (D) Western blot analysis of IFITM3 and MHC-I protein levels in RPP cells treated with EG (40 μm) for 48 h. (E) Schematic of the BALB/c nude mouse model for subcutaneous injection of RPP cells followed by oral EG treatment. Representative images and weight plots of tumors, along with tumor volume plots measured every 2 days. (F) C57BL/6 mice were implanted with RPP cells and received EG and PD-1 mAb treatment. Representative images and weight plots of tumors along with tumor volume plots measured every 3 days. (G) Flow cytometry analysis of CD3 + CD45 + cells, CD8 + CD3 + cells, and CD69 + , CD44 + , and GZMB + CD8 + cells. The results are presented as the means ± SEMs, n = 4. Statistical comparisons between groups were performed using unpaired two-tailed Student’s t-tests. Statistical comparisons between groups were performed using unpaired two-tailed Student’s t-tests. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, n.s., not significant

Article Snippet: Plasmids encoding HA-tagged IFITM3 (Cat# 58397; Addgene) and MYC-tagged NLRC5 (Cat# 37509; Addgene) were used.

Techniques: Inhibition, Gene Expression, Western Blot, Transduction, Injection, Flow Cytometry, Two Tailed Test

Journal: Molecular Cancer

Article Title: IFITM3 enhances immunosensitivity via MHC-I regulation and is associated with the efficacy of anti-PD-1/-L1 therapy in SCLC

doi: 10.1186/s12943-025-02383-x

Figure Lengend Snippet: IFITM3 enhances MHC-I expression via NLRC5 upregulation and nuclear trafficking in SCLC cells. (A) Volcano plots showing DEGs between IFITM3-overexpressing NCI-H446 and NCI-H69 cells and control cells. Vertical dashed lines indicate log2FC = ± 1, and the horizontal dashed line represents the significance threshold at P = 0.05. (B) GSEA showing activated antigen processing and presentation pathways caused by IFITM3 overexpression in NCI-H446 and NCI-H69 cells. Enrichment significance was determined using the NES and nominal P values, as calculated by the GSEA algorithm. (C) Venn diagram of overlapping DEGs between NCI-H446 and NCI-H69 cells, accompanied by a bar plot of enriched GO terms. (D) Box plots displaying NLRC5 expression levels in NCI-H446 and NCI-H69 cells following IFITM3 overexpression. (E) Immunofluorescence images of NCI-H69 and NCI-H446 cells overexpressing MYC-NLRC5 (green) and/or HA-IFITM3 (red). Nuclei were counterstained with DAPI (blue). Scale bar: 20 μm. (F) Subcellular fractionation assays showing NLRC5 expression in cytoplasmic and nuclear fractions following IFITM3 overexpression. (G) Co-IP of IFITM3 with NLRC5 in NCI-H446 and NCI-H69 cells. The results are presented as the means ± SEMs. Statistical comparisons between groups were performed using unpaired two-tailed Student’s t-tests. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: Plasmids encoding HA-tagged IFITM3 (Cat# 58397; Addgene) and MYC-tagged NLRC5 (Cat# 37509; Addgene) were used.

Techniques: Expressing, Control, Over Expression, Immunofluorescence, Fractionation, Co-Immunoprecipitation Assay, Two Tailed Test

Journal: Molecular Cancer

Article Title: IFITM3 enhances immunosensitivity via MHC-I regulation and is associated with the efficacy of anti-PD-1/-L1 therapy in SCLC

doi: 10.1186/s12943-025-02383-x

Figure Lengend Snippet: Mechanism of action of IFITM3 in regulating MHC-I expression to enhance immunotherapy sensitivity in SCLC

Article Snippet: Plasmids encoding HA-tagged IFITM3 (Cat# 58397; Addgene) and MYC-tagged NLRC5 (Cat# 37509; Addgene) were used.

Techniques: Expressing

Journal: Frontiers in Molecular Neuroscience

Article Title: Mutually Dependent Clustering of SynDIG4/PRRT1 and AMPA Receptor Subunits GluA1 and GluA2 in Heterologous Cells and Primary Neurons

doi: 10.3389/fnmol.2022.788620

Figure Lengend Snippet: Synapse differentiation-induced gene 4 (SD4)/IFITM3 chimeras.

Article Snippet: Full length mouse IFITM3 was obtained from AddGene (#58389).

Techniques:

Journal: Frontiers in Molecular Neuroscience

Article Title: Mutually Dependent Clustering of SynDIG4/PRRT1 and AMPA Receptor Subunits GluA1 and GluA2 in Heterologous Cells and Primary Neurons

doi: 10.3389/fnmol.2022.788620

Figure Lengend Snippet: The proline rich N-terminus of SD4 is dispensable for clustering with GluA1 and GluA2. (A) Schematic depicting the chimeric protein structures. Chimeras were generated expressing either (i) the N-terminus of SD4 and membrane domain of IFITM3 (SD4-NTD/IF-M) or (ii) the N-terminus of IFITM3 and membrane domain of SD4 (IF-NTD/SD4-M) (as shown in of Methods). (B) Immunoblot of COS cell lysates transfected with SD4 and IFITM3 chimeras. β-tubulin was used as the loading control. (C) Representative confocal images depict either GluA1 expressed alone or co-expressed with either IF/SD4 chimera. Scale bar = 20 μm. (D,E) Graph depicts the mean cluster size of GluA1 puncta from the stratification of GluA1 co-localized or not co-localized with SD4-NTD/IF-M chimeras (D) or IF-NTD/SD4-M chimeras (E) compared with GluA1 alone. GluA1 alone ( n = 10), GluA1 + SD4-NTD/IF-M ( n = 12), and GluA1 + IF-NTD/SD4-M ( n = 12). (F) Representative confocal images of GluA2 alone or co-expressed with IF/SD4 chimeras. Scale bar = 20 μm. (G,H) Graph depicts the mean cluster size of GluA2 puncta from the stratification of GluA2 co-localized or not co-localized with SD4-NTD/IF-M chimeras (G) or IF-NTD/SD4-M chimeras (H) compared with GluA2 alone. GluA2 alone ( n = 13), GluA2 + SD4-NTD/IF-M ( n = 12), and GluA2 + IF-NTD/SD4-M ( n = 16). Data are represented as mean cluster size ± SEM; n.s. not significant; * p < 0.05; ** p < 0.01; **** p < 0.0001; one-way ANOVA with post-hoc Tukey’s test.

Article Snippet: Full length mouse IFITM3 was obtained from AddGene (#58389).

Techniques: Generated, Expressing, Membrane, Western Blot, Transfection, Control