Journal: Nature Communications

Article Title: Blocking the m 6 Am methyltransferase PCIF1 releases STAT1-mediated Th1 immunity to potentiate cancer immunotherapy

doi: 10.1038/s41467-026-75269-1

Figure Lengend Snippet: a Schematic of m 6 Am-seq in naïve CD4 + T cells. b Metagene profiles of m 6 Am peak distribution along normalized transcripts. c KEGG pathway enrichment analysis of m 6 Am-modified genes in naïve CD4 + T cells. d Schematic of RiboTag-seq in naïve CD4 + T cells. e Volcano plots of differential translation efficiency (TE) between cKO and WT naïve CD4 + T cells based on RiboTag-seq data. TE was calculated as the ratio of Fragments Per Kilobase of exon model per Million mapped fragments (FPKM) in RiboTag-seq to FPKM in input RNA-seq. f Venn diagram of TE-upregulated genes and m 6 Am-modified genes in naïve CD4 + T cells. g RiboTag-qPCR analysis of Stat1 mRNA association with ribosomes in naïve CD4 + T cells ( n = 4). h–j Representative immunoblot ( h ) and corresponding quantitative data ( i ) ( n = 8), along with representative histograms (left) and quantitative analysis (right) in ( j ) ( n = 6) reveal STAT1 expression in naïve CD4 + T cells from WT or cKO mice. k Schematic (left) and the quantification (right) of the dual-luciferase reporter assay used to characterize the effect of A-to-T substitution at the first nucleotide after the m 7 G cap in the 5’-UTR of Stat1 ( n = 12). l Quantification of IFN-γ expression in Th1-differentiated CD4 + T cells treated with a STAT1 inhibitor (WT or cKO Group n = 6, other Group n = 5). Each n represents biologically independent samples ( k ) or independent samples from individual mouse ( a , e , g – j , l ). Error bars represent means ± SEM; two-tailed, unpaired t -test ( g , i–k ); one-way ANOVA ( l ). p -values are shown on the graphs, and p -values < 0.0001 are reported as such.

Article Snippet: During the first 24 h of Th1 differentiation from naïve CD4 + T cells, the STAT1 inhibitor Fludarabine (2.5 μM; MedChemExpress, 21679-14-1) was added.

Techniques: Modification, RNA Sequencing, Western Blot, Expressing, Luciferase, Reporter Assay, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Identifying the therapeutic potential of niclosamide in overcoming IFN-gamma dependent cancer immune evasion in the tumor microenvironment

doi: 10.3389/fimmu.2026.1761715

Figure Lengend Snippet: IFNγ up-regulate PD-L1 expression in MC38 cells through IFN-STAT1 signaling. PD-L1 expression on MC38 cell surface was significantly increased in the presence of IFN-γ analyzed by flow cytometry (A) and by immune fluorescent staining (B-C) at 24 hours. (D) When treated with siRNA of STAT1, the up-regulation of PD-L1 was significantly reduced, while siRNA of STAT3 has an opposite effect. (E, F) The small interfering RNA reaches a sufficient knock down of STAT1 and STAT3, as shown by real-time PCR assay. (G-I) The mRNA of STAT1 target genes such as CXCL10 and IL-15 shown similar trend of regulation by IFN-γ as PD-L1. (J) The mRNA of STAT3 target gene, MCL-1, shown totally different regulation under normal condition and under treatment of siRNAs. (K, L) The expression of STAT1/STAT3 normal and phosphorylated proteins, and PD-L1 proteins in MC38 cell lines were measured by Western blotting with different dose of IFN-γ stimulation (K) or after knocking down of STAT1 (siSTAT1) or STAT3 (siSTAT3) (L ). The number on the image indicates the relative abundance of PD-L1 protein (fold of control). The results are expressed as the mean ± SEM of triplicate measurements in each group. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: We evaluated small molecule STAT1 inhibitor Fludarabine (F-ara-A, NSC 118218) (MedChemExpress, Cat. No.: HY-B0069) and STAT3 inhibitor Niclosamide (BAY2353, MedChemExpress, Cat. No.: HY-B0497).

Techniques: Expressing, Flow Cytometry, Staining, Small Interfering RNA, Knockdown, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: Frontiers in Immunology

Article Title: Identifying the therapeutic potential of niclosamide in overcoming IFN-gamma dependent cancer immune evasion in the tumor microenvironment

doi: 10.3389/fimmu.2026.1761715

Figure Lengend Snippet: IFN-γ modulate cancer cell stemness in MC38 cells indirectly through IFN-STAT3 pathway. (A-C) Ki-67 expression in the MC38 tumor spheres was significantly reduced in the presence of IFN-γ analyzed by flow cytometry and immune fluorescence. (D, E) The tumor sphere forming unit induced in the MC38 cells were also reduced in the presence of IFN-γ (Arrowhead: tumor spheroid. Scale bar: 200μm). (F) When MC38 cells were pre-treated with siRNA of STAT1/STAT3, the SFU show different changes. (G) When MC38 cells were pre-treated with siRNA of STAT1/STAT3, both the CD44 hi CD133- and CD44 hi CD133+ cell population show different trend of regulation by IFN-γ. (H-K) The mRNA of cancer stem cell markers was measured by real-time PCR. (L) The expression of cancer stem cell marker proteins in cell nucleus was measured by Western blot. The results are expressed as the mean ± SEM of triplicate measurements in each group. *p<0.05, **p<0.01, nd, no significant difference.

Article Snippet: We evaluated small molecule STAT1 inhibitor Fludarabine (F-ara-A, NSC 118218) (MedChemExpress, Cat. No.: HY-B0069) and STAT3 inhibitor Niclosamide (BAY2353, MedChemExpress, Cat. No.: HY-B0497).

Techniques: Expressing, Flow Cytometry, Fluorescence, Real-time Polymerase Chain Reaction, Marker, Western Blot

Journal: Frontiers in Immunology

Article Title: Identifying the therapeutic potential of niclosamide in overcoming IFN-gamma dependent cancer immune evasion in the tumor microenvironment

doi: 10.3389/fimmu.2026.1761715

Figure Lengend Snippet: Niclosamide has inhibition effect on both STAT1 and STAT3, which blocks IFN-γ-induced PD-L1 up-regulation in MC38 cells while also reduces CSCs formation. (A) The expression of STAT1/STAT3 signaling pathway and PD-L1 proteins in MC38 cell lines with Fludarabine and Niclosamide treatment was measured by Western blot. (B) The surface PDL1 expression level was measured by FACS with IFN-γ and Niclosamide treatment. (C) The cell viability of MC38 when co-cultured with T cells and pre-treated with Fludarabine (F-ara-A) or Niclosamide were measured by CCK8 assay. (D) The sphere forming units induced in MC38 cells was measured with or without Niclosamide treatment. (E) The cell population of CD44 hi CD133+ in MC38 treated with Niclosamide was measured by FACS. (F) The expression of cancer stem cell marker proteins in cell nucleus was measured by Western blot. The results are expressed as the mean ± SEM of triplicate measurements in each group. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: We evaluated small molecule STAT1 inhibitor Fludarabine (F-ara-A, NSC 118218) (MedChemExpress, Cat. No.: HY-B0069) and STAT3 inhibitor Niclosamide (BAY2353, MedChemExpress, Cat. No.: HY-B0497).

Techniques: Inhibition, Expressing, Western Blot, Cell Culture, CCK-8 Assay, Marker

Journal: Frontiers in Immunology

Article Title: Identifying the therapeutic potential of niclosamide in overcoming IFN-gamma dependent cancer immune evasion in the tumor microenvironment

doi: 10.3389/fimmu.2026.1761715

Figure Lengend Snippet: Hypoxia enhances PD-L1 upregulation by IFN-γ, while Niclosamide down-regulates Hif1α under hypoxic conditions. (A, B) The expression level of STAT1/STAT3 signaling pathway and PD-L1 proteins in MC38 cell lines with siRNA (A) or Fludarabine and Niclosamide treatment (B) was measured by Western blot. (C) Cell viability of MC38 cells pre-treated with different dose of IFN-γ and co-cultured with primary T cells were measured by CCK8 assay, under normoxia or hypoxic conditions. (D) The Cell viability of MC38 cells treated with different dose of Fludarabine or Niclosamide were measured by CCK8 assay, under normoxia or hypoxic conditions. The results are expressed as the mean ± SEM of triplicate measurements in each group. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: We evaluated small molecule STAT1 inhibitor Fludarabine (F-ara-A, NSC 118218) (MedChemExpress, Cat. No.: HY-B0069) and STAT3 inhibitor Niclosamide (BAY2353, MedChemExpress, Cat. No.: HY-B0497).

Techniques: Expressing, Western Blot, Cell Culture, CCK-8 Assay

Journal: Cells

Article Title: Pathologic Th1–Treg Cells Exacerbate Acute Lung Injury and Lethality in Sepsis

doi: 10.3390/cells15060521

Figure Lengend Snippet: eCIRP activates STAT1 and STAT5 via TLR4 to induce Th1-Treg cells. ( A , B ) CD4 + T cells from WT and TLR4 −/− mice were cultured with PBS or 2.5 μg/mL of eCIRP for 48 h. The frequency of Th1-Treg cells of PBS-treated CD4 + T cells and eCIRP-treated CD4 + T cells was evaluated by flow cytometry. ( C , D ) CD4 + T cells from WT and TLR4 −/− mice were cultured with 2.5 μg/mL of eCIRP for 2 h. The frequency of pSTAT1 + pSTAT5 + cells of PBS-treated CD4 + T cells and eCIRP-treated CD4 + T cells was evaluated by flow cytometry. Experiments were performed 2 times, and all data were used for analysis. Data are expressed as mean ± SEM (n = 6, 7 samples/group) and compared by one-way ANOVA and Tukey’s multiple comparison test for multiple groups. * p < 0.05 vs. WT PBS, # p < 0.05 vs. WT eCIRP. ( E , F ) CD4 + T cells from WT mice were cultured with 2.5 μg/mL of eCIRP for 48 h in the presence and absence of 1 μM of STAT1 inhibitor and 10 μM of STAT5 inhibitor. The frequency of Th1-Treg cells was analyzed by flow cytometry. Experiments were performed 2 times, and all data were used for analysis. Data are expressed as mean ± SEM (n = 6 samples/group) and compared by one-way ANOVA and Tukey’s multiple comparison test for multiple groups. * p < 0.05 vs. PBS, # p < 0.05 vs. eCIRP alone.

Article Snippet: CD4 + T cells were treated with 1.0, 2.5 μg/mL of eCIRP for the indicated time periods on CD3/CD28 coated plates (Ultra-LEAF Purified anti-mouse CD3ε, Cat. No.: 100340; Ultra-LEAF Purified anti-mouse CD28, Cat. No.: 102116, Biolegend, San Diego, CA, USA) in the presence and absence of 1 μM of STAT1 inhibitor (Fludarabine; Cat. No.: HY-B0069, MedChemExpress, Monmouth Junction, NJ, USA) and/or 10 μM of STAT5 inhibitor (Cat. No.: HY-101853, MedChemExpress), followed by stimulation with Cell Activation Cocktail (with Brefeldin A) (Cat. No.: 423304, Biolegend) for 4 h for cytokine evaluation.

Techniques: Cell Culture, Flow Cytometry, Comparison

Journal: Cells

Article Title: Pathologic Th1–Treg Cells Exacerbate Acute Lung Injury and Lethality in Sepsis

doi: 10.3390/cells15060521

Figure Lengend Snippet: Finding summary. During sepsis, eCIRP signals through TLR4 to activate STAT1 and STAT5, driving the differentiation of pathogenic Th1-Treg cells. These cells accumulate in the lungs, exacerbate acute lung injury, and ultimately increase mortality in sepsis.

Article Snippet: CD4 + T cells were treated with 1.0, 2.5 μg/mL of eCIRP for the indicated time periods on CD3/CD28 coated plates (Ultra-LEAF Purified anti-mouse CD3ε, Cat. No.: 100340; Ultra-LEAF Purified anti-mouse CD28, Cat. No.: 102116, Biolegend, San Diego, CA, USA) in the presence and absence of 1 μM of STAT1 inhibitor (Fludarabine; Cat. No.: HY-B0069, MedChemExpress, Monmouth Junction, NJ, USA) and/or 10 μM of STAT5 inhibitor (Cat. No.: HY-101853, MedChemExpress), followed by stimulation with Cell Activation Cocktail (with Brefeldin A) (Cat. No.: 423304, Biolegend) for 4 h for cytokine evaluation.

Techniques: