Journal: bioRxiv

Article Title: Disrupting IFIT1-STAT2 viral evasion synergy yields live-attenuated flavivirus with enhanced immune priming

doi: 10.64898/2026.06.04.729841

Figure Lengend Snippet: A) Foci images of the DENV2 with ZIKV-equivalent STAT2-targeting mutations. B) Design of bimolecular fluorescence complementation (BiFC) assay for NS5-STAT2 interaction analysis in cells. C) Flow cytometry BiFC readouts of selected NS5 mutants with human STAT2. D) Foci images of DENV2 with single and combined STAT2-targeting mutations.

Article Snippet: Similarly, the human STAT2 gene was sub-cloned from pUNO1-hSTAT2 (InvivoGen), fused with a C-terminal FLAG tag sequence, and cloned into a lentiviral plasmid under the control of the inducible CMVtight promoter, utilizing an mCherry expression cassette as a marker.

Techniques: Bimolecular Fluorescence Complementation Assay, Flow Cytometry

Journal: bioRxiv

Article Title: Disrupting IFIT1-STAT2 viral evasion synergy yields live-attenuated flavivirus with enhanced immune priming

doi: 10.64898/2026.06.04.729841

Figure Lengend Snippet: A) Heatmap of contact analysis of NS5-STAT2 interacting residues from four available NS5-STAT2 complex structures (top and middle, each number represents the numbers of contacts at each residue) with the corresponding mutation fitness data derived from DMS of DENV2 in Vero cells (bottom) . B) Western-blot analysis of STAT2 degradation during infection by DENV2 with selected NS5 mutations at the STAT2-contacting residues. C) Multi-step replication kinetics of the DENV2-WT, -P336G, -N730D, -P336G+N730D, and - P336G+Q845A in Vero cells infected at MOI = 0.1. D) Relative binding of the NS5 mutants with human STAT2 by co-immunoprecipitation assay.

Article Snippet: Similarly, the human STAT2 gene was sub-cloned from pUNO1-hSTAT2 (InvivoGen), fused with a C-terminal FLAG tag sequence, and cloned into a lentiviral plasmid under the control of the inducible CMVtight promoter, utilizing an mCherry expression cassette as a marker.

Techniques: Residue, Mutagenesis, Derivative Assay, Western Blot, Infection, Binding Assay, Co-Immunoprecipitation Assay

Journal: bioRxiv

Article Title: Disrupting IFIT1-STAT2 viral evasion synergy yields live-attenuated flavivirus with enhanced immune priming

doi: 10.64898/2026.06.04.729841

Figure Lengend Snippet: A) Foci images of DENV1-4 and ZIKV with P336G or N730D (DENV2 mutations). B) STAT2 degradation in A549 cells infected by DENV1-4 and ZIKV with P336G or N730D mutations. C) Multi-sequence alignment (MSA) of STAT2-interating residues across DENV1-4 (KDH, 16681, v1043, and H241, respectively) and ZIKV (SV0010) used in A) and B). MSA was performed with full-length NS5 sequences. The STAT2 interacting residues (based on the analysis performed in ) were then extracted from the aligned sequences and displayed using MView .

Article Snippet: Similarly, the human STAT2 gene was sub-cloned from pUNO1-hSTAT2 (InvivoGen), fused with a C-terminal FLAG tag sequence, and cloned into a lentiviral plasmid under the control of the inducible CMVtight promoter, utilizing an mCherry expression cassette as a marker.

Techniques: Infection, Sequencing

Journal: bioRxiv

Article Title: Disrupting IFIT1-STAT2 viral evasion synergy yields live-attenuated flavivirus with enhanced immune priming

doi: 10.64898/2026.06.04.729841

Figure Lengend Snippet: A) Type-I IFN sensitivity of DENV2 with MTase, STAT2-targeting, and combined mutations (MTase+STAT2) in A549-WT and A549-IFIT1-KO, STAT2-KO, and PAF1-KO. The A549 cells were treated with IFN-α2 at 500U/ml for 4 hours before virus infection at MOI = 0.1. The virus titers (FFU/ml) for calculating IFN sensitivity were measured at 2 dpi. B) Multi-step replication kinetics of the DENV2 panel in WT A549 (left plot) and IFIT1-KO A549 (right plot). The A549 cells were infected with DENV2 at MOI = 0.1. C) Western-blot analysis of STAT2 expression level from the multi-step replication kinetics experiment in B). D) Flow cytometry measurement of STAT2 levels (as mean fluorescence intensity, MFI) in the infected and the bystander WT- and -IFIT1-KO A549 at 1 and 2 dpi (left and middle plot) and in the WT- and KO cells treated with type-I IFN at 500U/ml (right plot). E) Proteomic analysis showing differential human protein levels between WT- and IFIT1KO A549 at 2 dpi infected with either DENV2-E217A or DENV2-S150T+P336G+N730D as in B) (left volcano plot). The right heatmap shows the expression profile of ISGs with similar profile to STAT2 in different A549 cells (x-axis). F) Measurement of secreted IFN-β and TNF-α (pg/ml) of A549-WT and -IFIT1-KO cells infected with one of the three viruses, DENV2-WT, - E217A, or S150T+P336G+N730D (MOI = 1) at 1 and 2 dpi. The data points in the top bar plot from each virus were aggregated by cell types. The bottom paired line plots were matched by virus, with the data points in the IFIT1-KO being an average of the measurements from three IFIT1-KO clones. Statistical significance is indicated by brackets above the data sets. Exact p value is provided for each comparison (* ; ns, not significant). Statistical analysis was performed using an unpaired Student’s t-test for the top bar plots and a paired t-test for the bottom line plots.

Article Snippet: Similarly, the human STAT2 gene was sub-cloned from pUNO1-hSTAT2 (InvivoGen), fused with a C-terminal FLAG tag sequence, and cloned into a lentiviral plasmid under the control of the inducible CMVtight promoter, utilizing an mCherry expression cassette as a marker.

Techniques: Virus, Infection, Western Blot, Expressing, Flow Cytometry, Fluorescence, Clone Assay, Comparison

Journal: bioRxiv

Article Title: Disrupting IFIT1-STAT2 viral evasion synergy yields live-attenuated flavivirus with enhanced immune priming

doi: 10.64898/2026.06.04.729841

Figure Lengend Snippet: A) Multi-step replication kinetics of the mutant DENV2 in STAT2-KO and PAF1-KO A549 cells. A549 cells were infected with DENV2 at MOI = 0.1. B) Western-blot analysis of IFIT1 and dengue NS3 expression in wild-type A549 cells during multi-step replication kinetics analysis in A). C) Flow cytometry of DENV2 infection by anti-NS3 in WT and IFIT1-KO A549 cells. D) Volcano plots illustrating the distribution of quantified proteins. The x-axis represents the fold change (LFC), and the y-axis represents the statistical significance as (-value). Proteins with a fold change > 2 and a p-value < 0.01 were considered significantly differentially expressed. Selected ISGs upregulated during DENV infections in WT- and IFIT1-KO A549 are labeled by their gene symbols. Aggregate data of DENV2-E217A and DENV2-S150T+P336G+N730D were used to represent the data of infected cells. E-F) Comparison between the cytokine concentrations (pg/ml) secreted from WT-A549 and those from IFIT1-KO-A549 infected with one of the three viruses, DENV2-WT, -E217A, or S150T+P336G+N730D (MOI = 1) at 1 and 2 dpi. The data points in the top bar plots (E) from each virus were aggregated by cell types. The bottom paired line plots (F) were matched by virus, with the data points in the IFIT1-KO being an average of the measurement from three IFIT1-KO clones. Statistical significance is indicated by brackets above the data sets. Exact values are provided for each comparison (* ; ns, not significant). Statistical analysis was performed using an unpaired Student’s t-test for the top bar plots and a paired t-test for the bottom line plots. G) Dose-response analysis of IFN-α2 and STAT2 level in A549 cells. A549 cells were treated with IFN-α2 48 hours before measurement by flow cytometry.

Article Snippet: Similarly, the human STAT2 gene was sub-cloned from pUNO1-hSTAT2 (InvivoGen), fused with a C-terminal FLAG tag sequence, and cloned into a lentiviral plasmid under the control of the inducible CMVtight promoter, utilizing an mCherry expression cassette as a marker.

Techniques: Mutagenesis, Infection, Western Blot, Expressing, Flow Cytometry, Labeling, Comparison, Virus, Clone Assay

Journal: Cancer Medicine

Article Title: Linking Targeted Pancreatic Cancer Genes With Metabolic Disorders: A Cross‐Species Translational Pathway

doi: 10.1002/cam4.71775

Figure Lengend Snippet: Overall workflow of the study. Targeted* genes are ITGAM, PECAM1, CCL5, STAT1, STAT2, and CD44.

Article Snippet: The quality and quantity of RNA were determined by Nanodrop One (Thermofisher); all isolated RNA samples had A260/A280 ratios of 1.8–2.0. mRNA levels of ITGAM, PECAM, CCL5, CXCL10, STAT1, STAT2, and GZMB were determined by RT‐qPCR, relative to the housekeeping gene GAPDH, using the iTaq Universal One‐Step RT‐qPCR Kit (Bio‐Rad) and gene‐specific primers (Thermofisher; assay IDs: ITGAM Hs00167304_m1, PECAM1 Hs01065279_m1, CCL5 Hs00982282_m1, CXCL10 Hs00171042_m1, STAT1 Hs01013996_m1, STAT2 Hs01013115_g1, GZMB Hs00188051_m1).

Techniques:

Journal: Cancer Medicine

Article Title: Linking Targeted Pancreatic Cancer Genes With Metabolic Disorders: A Cross‐Species Translational Pathway

doi: 10.1002/cam4.71775

Figure Lengend Snippet: (A–F): Targeted gene (ITGAM, PECAM1, CCL5, STAT1, STAT2, and CD44) expression plots across different samples showing comparatively higher expression levels in metabolically disordered (obese) mice and humans compared to healthy individuals. Plots with p < 0.05 are only considered. Overlapping jittered points reflect individual sample variability and were retained to preserve consistent visualisation across datasets.

Article Snippet: The quality and quantity of RNA were determined by Nanodrop One (Thermofisher); all isolated RNA samples had A260/A280 ratios of 1.8–2.0. mRNA levels of ITGAM, PECAM, CCL5, CXCL10, STAT1, STAT2, and GZMB were determined by RT‐qPCR, relative to the housekeeping gene GAPDH, using the iTaq Universal One‐Step RT‐qPCR Kit (Bio‐Rad) and gene‐specific primers (Thermofisher; assay IDs: ITGAM Hs00167304_m1, PECAM1 Hs01065279_m1, CCL5 Hs00982282_m1, CXCL10 Hs00171042_m1, STAT1 Hs01013996_m1, STAT2 Hs01013115_g1, GZMB Hs00188051_m1).

Techniques: Expressing, Metabolic Labelling

Journal: Cancer Medicine

Article Title: Linking Targeted Pancreatic Cancer Genes With Metabolic Disorders: A Cross‐Species Translational Pathway

doi: 10.1002/cam4.71775

Figure Lengend Snippet: Violin plots showing the expression levels of genes of interest (ITGAM, PECAM1, CCL5, STAT1, STAT2, and CD44) across different clusters in the human pancreatic tissue, (A) non‐cancerous (control) and (B) cancerous. STAT1, STAT2, and CD44 have shown quite consistent expressions across all clusters. However, genes ITGAM, PECAM1, and CCL5 are highly expressed in clusters 8, 9, and 0 in cancerous compared to noncancerous. Identity signifies Cluster identities on the horizontal axis. Dataset used: GSE212966 .

Article Snippet: The quality and quantity of RNA were determined by Nanodrop One (Thermofisher); all isolated RNA samples had A260/A280 ratios of 1.8–2.0. mRNA levels of ITGAM, PECAM, CCL5, CXCL10, STAT1, STAT2, and GZMB were determined by RT‐qPCR, relative to the housekeeping gene GAPDH, using the iTaq Universal One‐Step RT‐qPCR Kit (Bio‐Rad) and gene‐specific primers (Thermofisher; assay IDs: ITGAM Hs00167304_m1, PECAM1 Hs01065279_m1, CCL5 Hs00982282_m1, CXCL10 Hs00171042_m1, STAT1 Hs01013996_m1, STAT2 Hs01013115_g1, GZMB Hs00188051_m1).

Techniques: Expressing, Control

Journal: Cancer Medicine

Article Title: Linking Targeted Pancreatic Cancer Genes With Metabolic Disorders: A Cross‐Species Translational Pathway

doi: 10.1002/cam4.71775

Figure Lengend Snippet: Synovial tissue gene expression profiles in healthy controls and osteoarthritis (OA) patients. Box and whisker plots display ΔCt values (y‐axis; normalised to GAPDH) for (A) ITGAM, (B) PECAM, (C) CCL5, (D) CXCL10, (E) STAT1, (F) STAT2, and (G) GZMB, quantified by qPCR. The x‐axis compares groups: Healthy Controls (synovium from neck‐of‐femur fracture patients), OA Normal‐Weight, and OA Obese. Lower ΔCt reflects higher gene expression. Whiskers span the full data range, boxes represent the IQR (25th–75th percentiles), and the central line marks the mean. Significance thresholds: “*” p < 0.05, “**” p < 0.01, “***” p < 0.001 (One‐way ANOVA with Tukey post hoc test for normally distributed data; Kruskal–Wallis with Dunn's post hoc test for not‐normally distributed data).

Article Snippet: The quality and quantity of RNA were determined by Nanodrop One (Thermofisher); all isolated RNA samples had A260/A280 ratios of 1.8–2.0. mRNA levels of ITGAM, PECAM, CCL5, CXCL10, STAT1, STAT2, and GZMB were determined by RT‐qPCR, relative to the housekeeping gene GAPDH, using the iTaq Universal One‐Step RT‐qPCR Kit (Bio‐Rad) and gene‐specific primers (Thermofisher; assay IDs: ITGAM Hs00167304_m1, PECAM1 Hs01065279_m1, CCL5 Hs00982282_m1, CXCL10 Hs00171042_m1, STAT1 Hs01013996_m1, STAT2 Hs01013115_g1, GZMB Hs00188051_m1).

Techniques: Gene Expression, Whisker Assay

Journal: Cancer Medicine

Article Title: Linking Targeted Pancreatic Cancer Genes With Metabolic Disorders: A Cross‐Species Translational Pathway

doi: 10.1002/cam4.71775

Figure Lengend Snippet: Adipose tissue gene expression profiles in osteoarthritis (OA) patients stratified by BMI. Box‐and‐whisker plots display ΔCt values (y‐axis; normalised to GAPDH) for qPCR‐measured genes: (A) ITGAM, (B) PECAM, (C) CCL5, (D) CXCL10, (E) STAT1, (F) STAT2, and (G) GZMB. The x‐axis compares the OA Normal‐Weight and OA Obese groups. Lower ΔCt values indicate higher gene expression. Whiskers show the full data range, boxes span the IQR (25th–75th percentiles), and the central line denotes the median. Significance thresholds: p < 0.05, p < 0.01, p < 0.001 ( t ‐tests or Mann–Whitney tests if not normally distributed).

Article Snippet: The quality and quantity of RNA were determined by Nanodrop One (Thermofisher); all isolated RNA samples had A260/A280 ratios of 1.8–2.0. mRNA levels of ITGAM, PECAM, CCL5, CXCL10, STAT1, STAT2, and GZMB were determined by RT‐qPCR, relative to the housekeeping gene GAPDH, using the iTaq Universal One‐Step RT‐qPCR Kit (Bio‐Rad) and gene‐specific primers (Thermofisher; assay IDs: ITGAM Hs00167304_m1, PECAM1 Hs01065279_m1, CCL5 Hs00982282_m1, CXCL10 Hs00171042_m1, STAT1 Hs01013996_m1, STAT2 Hs01013115_g1, GZMB Hs00188051_m1).

Techniques: Gene Expression, Whisker Assay, MANN-WHITNEY