human stat1  (Addgene inc)

Journal: bioRxiv

Article Title: Respiratory viruses activate autophagy via the IFN-STAT1/STAT5B-SOCS1 axis

doi: 10.1101/2025.10.28.685013

Figure Lengend Snippet: A, Schematic depiction of the JAK-STAT signaling cascade. First level shows the Type I (red), II (blue) and III (green) IFNs with respective receptors. Second level depicts the receptor-associated kinases (right) Tyrosine kinase 2 (Tyk2), Janus kinase 1 (JAK1), Janus kinase 2 (JAK2) and Janus kinase 3 (JAK) with the respective inhibitors (left) for each kinase. TC JL 37 (TC) inhibits Tyk2, Ruxolitinib (Rux, international nonproprietary name) inhibits JAK1 and JAK2, PF06551600 (PF, international nonproprietary name Ritlecitinib) inhibits JAK3 and CP-690550 (CP, international nonproprietary name Tofacitinib) inhibits JAK1, JAK2 and JAK3. Third level shows exemplary Signal Transducer and Activator of Transcription (STAT) dimer with STAT proteins (STAT1 to STAT6) listed. Fourth level depicts schematic induction of interferon-stimulated gene (ISG) expression in the nucleus. B-D , Quantification of autophagosome levels by flow cytometry in HeLa autophagy reporter cells (HeLa GL) 24 h after treatment with increasing concentrations of IFN-β (B), IFN-γ (C) or IFN-λ1 (D), in presence or absence of TC (100 nM), PF (100 µM), Ruxolitinib (Rux, 100 nM), or CP (100 nM), n = 4 ± SEM (left panels). Area under the curve (AUC) analysis (right panels) of the cumulative autophagy induction. Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test. *, p<0.05, ** p<0.01, *** p<0.001.

Article Snippet: Constructs coding for human STAT1 (eGFP STAT1 WT, Addgene #12301, kindly gifted from Alan Perantoni), STAT3 (pEGFP-N1-STAT3, Addgene #111934, kindly gifted from Geert van den Bogaart) and STAT5B (MAC_N_STA5B, Addgene #167800, kindly gifted from Markku Varjosalo) were obtained from Addgene. pTwist-FLAG was generated by removing the ORF of TRIM3 from pTwist_3x_FLAG_optTRIM (purchased from Twist Biosciences) as described previously (Klute et al.). pTwist-V5 was generated by linearizing pTwist-FLAG by PCR using the pTwist-FLAG fwd and rev primers from , amplifying the V5-gBlock (IDT) and inserting it into the linearized pTwist ‘null’ vector using Gibson Assembly (New England Biolabs).

Techniques: Expressing, Flow Cytometry

Journal: bioRxiv

Article Title: Respiratory viruses activate autophagy via the IFN-STAT1/STAT5B-SOCS1 axis

doi: 10.1101/2025.10.28.685013

Figure Lengend Snippet: A, Quantification of autophagosome levels by flow cytometry in HEK293T autophagy reporter cells (HEK293T GL) transiently expressing Cas9 and at least 2 individual gRNAs against indicated STATs. Treated with 10 pM IFN-β for 24h. n = 4-6± SEM. B, Quantification of autophagosome levels by flow cytometry in HeLa autophagy reporter cells (HeLa GL) transfected with siRNAs targeting STAT1 , STAT3 , STAT5B or ATG5 and treated with 1 nM IFN-β for 24h. BafA1 (625 µM) or Torin-1 (1 µM) for 4h used as controls. n = 4 ± SEM. C , Quantification of autophagosome levels by flow cytometry in HeLa autophagy reporter cells (HeLa GL) 24 h after treatment with increasing concentrations of IFN-β in presence or absence of 1 µM 5,15-DPP (STAT3 Inhibitor, STAT3i), 1 µM AS1517499 (STAT6 Inhibitor, STAT6i), 10 µM Fludarabine (STAT1 Inhibitor, STAT1i) or 100 µM STAT5 Inhibitor (STAT5i). n = 4 ± SEM. D, Area under the curve (AUC) analysis of the data in (C). E , Quantification of autophagosome levels by flow cytometry in HeLa autophagy reporter cells (HeLa GL) 24 h after treatment with increasing concentrations of IFN-γ and STAT5 Inhibitor (STAT5i, 100 µM). n = 4 ± SEM. F , Quantification of autophagosome levels by flow cytometry in HeLa autophagy reporter cells (HeLa GL) 24 h after treatment with increasing concentrations of IFN λ1 and STAT5 Inhibitor (STAT5i, 100 µM). n = 3 ± SEM. Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test. *, p<0.05, ** p<0.01, *** p<0.001.

Article Snippet: Constructs coding for human STAT1 (eGFP STAT1 WT, Addgene #12301, kindly gifted from Alan Perantoni), STAT3 (pEGFP-N1-STAT3, Addgene #111934, kindly gifted from Geert van den Bogaart) and STAT5B (MAC_N_STA5B, Addgene #167800, kindly gifted from Markku Varjosalo) were obtained from Addgene. pTwist-FLAG was generated by removing the ORF of TRIM3 from pTwist_3x_FLAG_optTRIM (purchased from Twist Biosciences) as described previously (Klute et al.). pTwist-V5 was generated by linearizing pTwist-FLAG by PCR using the pTwist-FLAG fwd and rev primers from , amplifying the V5-gBlock (IDT) and inserting it into the linearized pTwist ‘null’ vector using Gibson Assembly (New England Biolabs).

Techniques: Flow Cytometry, Expressing, Transfection

Journal: bioRxiv

Article Title: Respiratory viruses activate autophagy via the IFN-STAT1/STAT5B-SOCS1 axis

doi: 10.1101/2025.10.28.685013

Figure Lengend Snippet: A, Representative confocal immunofluorescence images of HeLa autophagy reporter cells (HeLa GL) transiently expressing FLAG-tagged (red) and V5-tagged (yellow) STAT1, STAT3 or STAT5 in combination or FLAG-tagged TRIM32 (scale bar, 10 μm) (left panel). Quantification of the number of autophagosomes (= eGFP-LC3B positive puncta) per cell in the images in the left panel. n= 9-63 cells ± SEM Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test. B, Co-immunoprecipitation of FLAG-tagged STAT1 and STAT5B from whole cell lysates of HEK293T cells transiently expressing FLAG-tagged and V5-tagged STAT1 and STAT5B or V5-tagged STAT1 or STAT5B alone. Immunoblots stained with anti-V5, anti-FLAG or anti-GAPDH. C, Exemplary images of Proximity Ligation Assay (PLA) of STAT1 and STAT2 or STAT5B (left panel) in HDF hTERT cells with or without treatment with 1 nM IFN-β for 1h. PLA signal (red). DAPI, nuclei (blue). Quantification of the number of PLA spots per nucleus per image of the left panel with single antibody controls (right panel). n = 15-21 tiles ± SEM. Student’s t-test with Welch’s correction. D, Representative confocal immunofluorescence images of NHLF cells treated with 1 nM of IFN-β for 1h and stained with α-STAT1 (green) and α-STAT5B (red), and DAPI (blue, nuclei) (scalebar 25 µm). E, Quantification of the ratio of nuclear to cytosolic intensity of STAT1 and STAT5B in the images in (D). n= 19-20 cells ± SEM. Student’s t-test with Welch’s correction. *, p<0.05, ** p<0.01, *** p<0.001

Article Snippet: Constructs coding for human STAT1 (eGFP STAT1 WT, Addgene #12301, kindly gifted from Alan Perantoni), STAT3 (pEGFP-N1-STAT3, Addgene #111934, kindly gifted from Geert van den Bogaart) and STAT5B (MAC_N_STA5B, Addgene #167800, kindly gifted from Markku Varjosalo) were obtained from Addgene. pTwist-FLAG was generated by removing the ORF of TRIM3 from pTwist_3x_FLAG_optTRIM (purchased from Twist Biosciences) as described previously (Klute et al.). pTwist-V5 was generated by linearizing pTwist-FLAG by PCR using the pTwist-FLAG fwd and rev primers from , amplifying the V5-gBlock (IDT) and inserting it into the linearized pTwist ‘null’ vector using Gibson Assembly (New England Biolabs).

Techniques: Immunofluorescence, Expressing, Immunoprecipitation, Western Blot, Staining, Proximity Ligation Assay

Journal: bioRxiv

Article Title: Respiratory viruses activate autophagy via the IFN-STAT1/STAT5B-SOCS1 axis

doi: 10.1101/2025.10.28.685013

Figure Lengend Snippet: A, Heatmap of variance of the top 40 differentially expressed genes (DEGs) in HeLa GL cells treated with 1 nM IFN-β vs untreated control (mock vs. IFN-β) as assessed by next generation sequencing. Samples treated with 100 μM STAT5 Inhibitor are indicated. n = 3. B, Deming Correlation Analysis of log2-fold changes of DEGs (mock_vs_IFN-β and mock(iSTAT5)_vs_IFN-β (iSTAT5)) in HeLa GL in (A). Deming (Model II) Linear Regression. C , Quantification of Top20 up- and down-regulated genes by STAT5B (=PC2) of data in (B) by Principal Component Analysis. D, Fold changes of normalized counts of SOCS1 and SLFN5 derived from the data in (A and B). n=3± SEM. Student’s t-test with Welch’s correction. E , Quantification of gene expression by qRT-PCR of STAT5 regulated genes or ISGs in human dermal fibroblasts (HDF) cells treated with IFN-β (1 nM), STAT5i (250 μM) or both. Student’s t-test with Welch’s correction. F, Quantification of autophagosome levels by flow cytometry in HeLa autophagy reporter cells (HeLa GL) transfected with siRNAs for RSAD2, SOCS1, ATG5, STAT1 or STAT5B and treated with IFN-β (1 nM, 24h). n = 4 ± SEM. Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test. (G), Representative confocal immunofluorescence images of HeLa autophagy reporter cells (HeLa GL) transiently expressing FLAG- or HA-tagged (red) SOCS1, RSAD2 or TRIM32 (scale bar, 10 μm) (left panel). Quantification of the area of autophagosomes (= px 2 of eGFP-LC3B positive puncta) per cell in the images in the left panel. n= 49-140 cells ± SEM. Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test. H, Quantification of autophagosome levels by flow cytometry in HEK293T autophagy reporter cells (HEL293T GL) expressing HA-SOCS1, TRIM32-FLAG or treated with Torin-1 (1µM, 4h) or Bafilomycin A1 (625nM, 4h) as positive controls. n= 4 ± SEM, Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test. *, p<0.05, ** p<0.01, *** p<0.001.

Article Snippet: Constructs coding for human STAT1 (eGFP STAT1 WT, Addgene #12301, kindly gifted from Alan Perantoni), STAT3 (pEGFP-N1-STAT3, Addgene #111934, kindly gifted from Geert van den Bogaart) and STAT5B (MAC_N_STA5B, Addgene #167800, kindly gifted from Markku Varjosalo) were obtained from Addgene. pTwist-FLAG was generated by removing the ORF of TRIM3 from pTwist_3x_FLAG_optTRIM (purchased from Twist Biosciences) as described previously (Klute et al.). pTwist-V5 was generated by linearizing pTwist-FLAG by PCR using the pTwist-FLAG fwd and rev primers from , amplifying the V5-gBlock (IDT) and inserting it into the linearized pTwist ‘null’ vector using Gibson Assembly (New England Biolabs).

Techniques: Control, Next-Generation Sequencing, Derivative Assay, Gene Expression, Quantitative RT-PCR, Flow Cytometry, Transfection, Immunofluorescence, Expressing

Journal: Communications Biology

Article Title: A feedforward loop between STAT1 and YAP1 stimulates lipid biosynthesis, accelerates tumor growth, and promotes chemotherapy resistance in mutant KRAS colorectal cancer

doi: 10.1038/s42003-025-08740-2

Figure Lengend Snippet: a Evaluation of colony formation efficacy of HCT116 and HK2-8 cells after treatments with either scramble siRNAs or STAT1 siRNAs. The graphs represent data obtained from 3 biological replicates, each of which included 3 technical replicates, and represent ±SEM ( * ) P < 0.05 ( ** ) P < 0.01; ( t -test), NS, non-significant. b Immunoblotting of indicated protein from extracts of siRNA-treated cells. Quantifications show the relative intensity of STAT1 normalized to ACTIN from 3 biological replicates and represent ±SEM * P < 0.05 ( t -test). c Volcano Diagram illustrates the number of genes differentially expressed in STAT1-replete (WT; control) compared to STAT1 -/- HCT116 cells. Essential genes of sterol and lipid biosynthetic pathways are indicated. Dashed horizontal and vertical lines indicate significance thresholds (|FC | > 0.5, P < 0.05). Genes are colored in gray (non-significant), blue ( P -value significant), and red (both P -values and fold change are significant). A positive fold change means that the gene is upregulated by STAT1. All labeled genes exhibit statistically significant upregulation (logFC > 0.5, P < 0.0005), consistent with their role in cholesterol biosynthesis and lipid metabolism. d Bar plot of biological processes (BP) from gene ontology (ON) significantly enriched in STAT1-dependent genes in HCT116 cells. e KEGG pathways under the control of STAT1 in HCT116 cells.

Article Snippet: Plasmids containing the green fluorescence protein (GFP)-tagged forms of wild type STAT1, STAT1 Y701F or STAT1 S727A cDNA were obtained from Addgene (plasmid #12301,12302 and 12304) .

Techniques: Western Blot, Control, Labeling

Journal: Communications Biology

Article Title: A feedforward loop between STAT1 and YAP1 stimulates lipid biosynthesis, accelerates tumor growth, and promotes chemotherapy resistance in mutant KRAS colorectal cancer

doi: 10.1038/s42003-025-08740-2

Figure Lengend Snippet: a Detection of SREBF mRNAs in colon cancer cells with intact or impaired STAT1. SREBF 1 and 2 mRNA levels were normalized to ACTIN and TUBULIN mRNAs used as internal controls. Data obtained from 3 biological replicates each of which contained 3 technical replicates and represent ±SEM ** P < 0.01: *** P < 0.001 ( t -test), NS, non-significant. b Immunoblotting for SREBP1 and 2 in colon cancer cells with intact or downregulated STAT1. Quantifications show the relative intensity of proteins normalized to TUBULIN. FL, full length; M, mature form. c Schematic representation of the mevalonate pathway. Genes in red are SREBP-dependent genes. d ChIP-seq data from ENCODE (UCSC data base) indicating the binding of SREBP1 and 2 to transcriptional regulatory regions of mevalonate pathway genes. Graphs show the expression of ACAT1 , HMGCR and IDI1 mRNAs by qPCR in cells treated with scrambled or STAT1 siRNAs. Gene expressions were normalized to ACTIN and TUBULIN mRNAs used as internal controls. Data were obtained from 3 independent experiments performed in triplicates and represent ±SEM *P < 0.05, **P < 0.01, *** P < 0.001 ( t -test). e Immunoblotting of HCT116 protein extracts replete (control) or deplete ( −/− ) for STAT1 by CRISPR (cell line #1 and #2). Detection of STAT1 and rate-limiting enzymes of sterol and lipid biosynthetic pathway HMGCR and FAS, respectively.

Article Snippet: Plasmids containing the green fluorescence protein (GFP)-tagged forms of wild type STAT1, STAT1 Y701F or STAT1 S727A cDNA were obtained from Addgene (plasmid #12301,12302 and 12304) .

Techniques: Western Blot, ChIP-sequencing, Binding Assay, Expressing, Control, CRISPR

Journal: Communications Biology

Article Title: A feedforward loop between STAT1 and YAP1 stimulates lipid biosynthesis, accelerates tumor growth, and promotes chemotherapy resistance in mutant KRAS colorectal cancer

doi: 10.1038/s42003-025-08740-2

Figure Lengend Snippet: a ChIP-seq data from ENCODE (UCSC database) indicating STAT1 binding to the regulatory regions of SREBF1 and 2 genes. b ChIP assays of endogenous STAT1 bound to SREBF gene segments containing STAT1 binding sites in HCT116 and HK2-8 cells. IgG, non-specific control antibody. c Expression of GFP (control) and GFP-tagged STAT1 proteins that are either intact (wild type, WT), impaired for phosphorylation (Y701F or S727A) or S727 phosphomimetic (S727E) in HCT116 STAT1 −/− cells. GFP+ cells were sorted by flow cytometry, and extracts were immunoblotted for GFP or ACTIN. d Detection of SREBF-1 and 2 mRNAs by qPCR in HCT116 STAT1 −/− cells expressing either GFP or GFP-STAT1 forms. e ChIP assays of GFP-STAT1 for binding to STAT1 sites of SREBF genes in reconstituted HCT116 STAT1 −/− cells using GFP antibody. b–e Data obtained from 3 biological replicates and represent ±SEM *P < 0.05, **P < 0.01, *** P < 0.001 ( t -test).

Article Snippet: Plasmids containing the green fluorescence protein (GFP)-tagged forms of wild type STAT1, STAT1 Y701F or STAT1 S727A cDNA were obtained from Addgene (plasmid #12301,12302 and 12304) .

Techniques: ChIP-sequencing, Binding Assay, Control, Expressing, Phospho-proteomics, Flow Cytometry

Journal: Communications Biology

Article Title: A feedforward loop between STAT1 and YAP1 stimulates lipid biosynthesis, accelerates tumor growth, and promotes chemotherapy resistance in mutant KRAS colorectal cancer

doi: 10.1038/s42003-025-08740-2

Figure Lengend Snippet: a HCT116 and HK2-8 cells replete or deplete of STAT1 were serum-starved for 18 h (untreated; UT) and stimulated with either 10% fetal bovine serum or 25 μM LPA for 1 h. Cells were subjected to IF analyses of YAP1 (red) along with DAPI staining of DNA (blue). Graphs show the quantification of YAP1 nuclear localization in 300 cells. Scale bar: 25 μm. b , c Cells were subjected to cytoplasmic (C), and nuclear (N) fractionation followed by immunoblotting for the indicated proteins. TUBULIN or THO complex 1 (THOC1) was used as cytoplasmic or nuclear marker, respectively. Quantification in panel b is based on three biological replicates, while panel c is based on two biological replicates. d , e HCT116 STAT1 +/+ and STAT1 −/− cells were transfected with either pGL3-luciferase reporter plasmid (control) or 8xGTIIC plasmid containing the firefly luciferase reporter gene under the control of 8x TEAD binding sites in CTGF minimal promoter. Transfected cells were serum-starved for 18 h followed by stimulation with either 10% fetal bovine serum or 25 μM LPA for 6 h. A plasmid expressing the renilla luciferase gene was used as internal control. f , g HCT116 cells were serum starved for 18 h followed by stimulation of 10% fetal bovine serum in the absence or presence of 2.5 mM cerivastatin (panel f, g) or 10 μM ROCK kinase inhibitor Y-27632 (panel f) for 18 h. Protein extracts were subjected to immunoblotting for the indicated proteins. a , b , d , e Graphs show the quantifications from 3 biological replicates and represent ±SEM *P < 0.05, **P < 0.01, *** P < 0.001 ( t -test), NS, non-significant. In c , data represent the quantification of 2 biological replicates.

Article Snippet: Plasmids containing the green fluorescence protein (GFP)-tagged forms of wild type STAT1, STAT1 Y701F or STAT1 S727A cDNA were obtained from Addgene (plasmid #12301,12302 and 12304) .

Techniques: Staining, Fractionation, Western Blot, Marker, Transfection, Luciferase, Plasmid Preparation, Control, Binding Assay, Expressing

Journal: Communications Biology

Article Title: A feedforward loop between STAT1 and YAP1 stimulates lipid biosynthesis, accelerates tumor growth, and promotes chemotherapy resistance in mutant KRAS colorectal cancer

doi: 10.1038/s42003-025-08740-2

Figure Lengend Snippet: a Volcano diagram showing the number of differentially expressed genes in YAP1-replete (WT; control) compared to YAP1 −/− HCT116 cells. Dashed horizontal and vertical lines indicate significance thresholds (|FC | > 0.5, P < 0.05). A positive fold change means that the gene is upregulated by YAP1. Genes are colored in gray (non-significant), blue ( P -value significant), and red (both P -values and fold change are significant). All labeled genes exhibit statistically significant upregulation (logFC > 0.5, P < 0.0005), consistent with their role in cholesterol biosynthesis and lipid metabolism. b Top KEGG pathways under the control of YAP1 in HCT116 cells. c Graphs assess the expression of SREBF1 and 2 mRNAs by qPCR in cells treated with scrambled or YAP1 siRNAs. Gene expressions were normalized to ACTIN and TUBULIN mRNAs used as internal controls. Data were obtained from 3 independent experiments performed in triplicates and represent ±SEM *P < 0.05, * *P < 0.01, *** P < 0.001 ( t -test). d ChIP-seq data from ENCODE indicating the binding of TEAD4 to transcriptional regulatory regions of SREBF genes. e ChIP assays of YAP1 for binding in complex with TEAD4 to SREBF genes in HCT116 cells, both in the presence and absence of STAT1 and/or YAP1. IgG, non-specific control antibody. f Immunoblotting of SREBP1 and 2 in isogenic pair colon cancer cells prior to and after YAP1 downregulation by siRNAs. FL, full length. g Immunoblotting of SREBP1, 2 and TEAD4 in isogenic pairs of colon cancer cells treated with scrambled or TEAD4 siRNAs. h Immunoblotting of SREBP1 and 2 proteins in HCT116 cells treated with TEAD inhibitor 15 μM VT104 for the indicated time points. f , g , h Quantification of proteins normalized to TUBULIN for each blot is indicated.

Article Snippet: Plasmids containing the green fluorescence protein (GFP)-tagged forms of wild type STAT1, STAT1 Y701F or STAT1 S727A cDNA were obtained from Addgene (plasmid #12301,12302 and 12304) .

Techniques: Control, Labeling, Expressing, ChIP-sequencing, Binding Assay, Western Blot

Journal: Communications Biology

Article Title: A feedforward loop between STAT1 and YAP1 stimulates lipid biosynthesis, accelerates tumor growth, and promotes chemotherapy resistance in mutant KRAS colorectal cancer

doi: 10.1038/s42003-025-08740-2

Figure Lengend Snippet: Venn diagram ( a ) and Volcano diagram ( b ) of genes that are commonly upregulated by STAT1 and YAP1 in HCT116 cells. Essential genes of sterol and lipid biosynthetic pathways are indicated in the Volcano diagram. b Dashed horizontal and vertical lines indicate significance thresholds (|FC | > 0.5, P < 0.05). A positive fold change means that the gene is upregulated by STAT1 and YAP1. Genes are colored in gray (non-significant), blue ( P -value significant), and Red (both P -values and fold change are significant). All labeled genes exhibit statistically significant upregulation (logFC > 0.5, P < 0.0005), consistent with their role in cholesterol biosynthesis and lipid metabolism. c Bar plot of biological processes (BP) from gene ontology (GO) significantly enriched in the common set of genes under the control of both YAP1 and STAT1 identified by gene expression profile analysis. d KEGG pathways under the control of STAT1 and YAP1 in HCT116 cells. e This schematic illustrates the cooperative role of STAT1 and YAP1 in promoting SREBP expression and activating the mevalonate pathway in mutant KRAS CRCs. The STAT1–YAP1 axis functions as a feedforward autoregulatory loop that sustains sterol biosynthesis. STAT1, phosphorylated at S727, directly induces the transcription of SREBF1 and SREBF2 genes. Elevated SREBP levels, in turn, enhance mevalonate pathway activity, leading to the prenylation, plasma membrane anchoring and activation of RHO GTPases. This activation promotes further phosphorylation of STAT1 at S727 and stimulates YAP1 nuclear localization and activation. Although YAP1 acts downstream of STAT1, it also reinforces the loop by cooperating with TEAD4 to transcriptionally upregulate SREBF genes. Created in BioRender. Koromilas, A. (2025) https://BioRender.com/nz5wlhr .

Article Snippet: Plasmids containing the green fluorescence protein (GFP)-tagged forms of wild type STAT1, STAT1 Y701F or STAT1 S727A cDNA were obtained from Addgene (plasmid #12301,12302 and 12304) .

Techniques: Labeling, Control, Gene Expression, Expressing, Mutagenesis, Activity Assay, Clinical Proteomics, Membrane, Activation Assay, Phospho-proteomics

Journal: Communications Biology

Article Title: A feedforward loop between STAT1 and YAP1 stimulates lipid biosynthesis, accelerates tumor growth, and promotes chemotherapy resistance in mutant KRAS colorectal cancer

doi: 10.1038/s42003-025-08740-2

Figure Lengend Snippet: a , b HCT116 cells with intact or depleted STAT1 and/or YAP1 expression were subcutaneously transplanted into female nu/nu mice (n = 10 per group). Tumor volume (mm³) was measured over the indicated time course. At the end of the study, tumor tissues from 3 mice were analyzed by IHC to assess H&E staining and the subcellular localization of YAP1 ( b ). c , d Similarly, HCT116 cells with combined YAP1 and STAT1 deletion or expression were transplanted into nu/nu mice (n = 5 per group). When tumors reached ~200 mm 3 , mice were treated by oral gavage with either vehicle or cerivastatin (CERI). Tumor growth was monitored over time. Red and blue arrows indicate the start point of treatment of YAP1 +/+ STAT1 +/+ and YAP1 −/− STAT1 −/− tumors, respectively. At the endpoint, tumors from 3 mice were subjected to IHC for H&E and YAP1 detection ( d ). a–d Data represent mean ± SEM. Statistical significance was determined by t -test: * P < 0.05, ** P < 0.01, *** P < 0.001. Scale bar in panels b and d: 50 μm and 25 μm (insert image). Quantification graphs panels b and d show Histo (H)-scores for nuclear and cytoplasmic YAP1 staining. e HCT116 xenografts were established in nu/nu mice (n = 5 per group). Upon tumor growth to ~200 mm 3 (red arrow), mice received either vehicle, cerivastatin (oral), afatinib (intraperitoneal), or the combination of both. Tumor volume was tracked for the indicated duration. Data represent mean ± SEM. *** P < 0.001 (t-test). f In a similar setup, mice bearing HCT116 xenografts (~200 mm 3 tumors) were treated with vehicle, cerivastatin (oral), VT104 (oral), or their combination. Tumor growth was monitored throughout the experiment. Data represent mean ± SEM. ** P < 0.01 (t-test). g This schematic model illustrates how the STAT1–YAP1 signaling axis enhances the mevalonate pathway, thereby supporting tumor growth and chemoresistance in mutant KRAS CRC. Inhibiting YAP1-TEAD4 activity (e.g., using VT104) disrupts the SREBP-driven feedback loop that sustains mevalonate pathway activation. Additionally, pharmacological blockade of the mevalonate pathway with statins increases tumor sensitivity to YAP1–TEAD4 inhibition in xenograft models of mutant KRAS CRC.

Article Snippet: Plasmids containing the green fluorescence protein (GFP)-tagged forms of wild type STAT1, STAT1 Y701F or STAT1 S727A cDNA were obtained from Addgene (plasmid #12301,12302 and 12304) .

Techniques: Expressing, Staining, Mutagenesis, Activity Assay, Activation Assay, Inhibition

Journal: Communications Biology

Article Title: A feedforward loop between STAT1 and YAP1 stimulates lipid biosynthesis, accelerates tumor growth, and promotes chemotherapy resistance in mutant KRAS colorectal cancer

doi: 10.1038/s42003-025-08740-2

Figure Lengend Snippet: a Evaluation of colony formation efficacy of HCT116 and HK2-8 cells after treatments with either scramble siRNAs or STAT1 siRNAs. The graphs represent data obtained from 3 biological replicates, each of which included 3 technical replicates, and represent ±SEM ( * ) P < 0.05 ( ** ) P < 0.01; ( t -test), NS, non-significant. b Immunoblotting of indicated protein from extracts of siRNA-treated cells. Quantifications show the relative intensity of STAT1 normalized to ACTIN from 3 biological replicates and represent ±SEM * P < 0.05 ( t -test). c Volcano Diagram illustrates the number of genes differentially expressed in STAT1-replete (WT; control) compared to STAT1 -/- HCT116 cells. Essential genes of sterol and lipid biosynthetic pathways are indicated. Dashed horizontal and vertical lines indicate significance thresholds (|FC | > 0.5, P < 0.05). Genes are colored in gray (non-significant), blue ( P -value significant), and red (both P -values and fold change are significant). A positive fold change means that the gene is upregulated by STAT1. All labeled genes exhibit statistically significant upregulation (logFC > 0.5, P < 0.0005), consistent with their role in cholesterol biosynthesis and lipid metabolism. d Bar plot of biological processes (BP) from gene ontology (ON) significantly enriched in STAT1-dependent genes in HCT116 cells. e KEGG pathways under the control of STAT1 in HCT116 cells.

Article Snippet: Plasmids containing the green fluorescence protein (GFP)-tagged forms of wild type STAT1, STAT1 Y701F or STAT1 S727A cDNA were obtained from Addgene (plasmid #12301,12302 and 12304) .

Techniques: Western Blot, Control, Labeling

Journal: Communications Biology

Article Title: A feedforward loop between STAT1 and YAP1 stimulates lipid biosynthesis, accelerates tumor growth, and promotes chemotherapy resistance in mutant KRAS colorectal cancer

doi: 10.1038/s42003-025-08740-2

Figure Lengend Snippet: a Detection of SREBF mRNAs in colon cancer cells with intact or impaired STAT1. SREBF 1 and 2 mRNA levels were normalized to ACTIN and TUBULIN mRNAs used as internal controls. Data obtained from 3 biological replicates each of which contained 3 technical replicates and represent ±SEM ** P < 0.01: *** P < 0.001 ( t -test), NS, non-significant. b Immunoblotting for SREBP1 and 2 in colon cancer cells with intact or downregulated STAT1. Quantifications show the relative intensity of proteins normalized to TUBULIN. FL, full length; M, mature form. c Schematic representation of the mevalonate pathway. Genes in red are SREBP-dependent genes. d ChIP-seq data from ENCODE (UCSC data base) indicating the binding of SREBP1 and 2 to transcriptional regulatory regions of mevalonate pathway genes. Graphs show the expression of ACAT1 , HMGCR and IDI1 mRNAs by qPCR in cells treated with scrambled or STAT1 siRNAs. Gene expressions were normalized to ACTIN and TUBULIN mRNAs used as internal controls. Data were obtained from 3 independent experiments performed in triplicates and represent ±SEM *P < 0.05, **P < 0.01, *** P < 0.001 ( t -test). e Immunoblotting of HCT116 protein extracts replete (control) or deplete ( −/− ) for STAT1 by CRISPR (cell line #1 and #2). Detection of STAT1 and rate-limiting enzymes of sterol and lipid biosynthetic pathway HMGCR and FAS, respectively.

Article Snippet: Plasmids containing the green fluorescence protein (GFP)-tagged forms of wild type STAT1, STAT1 Y701F or STAT1 S727A cDNA were obtained from Addgene (plasmid #12301,12302 and 12304) .

Techniques: Western Blot, ChIP-sequencing, Binding Assay, Expressing, Control, CRISPR

Journal: Communications Biology

Article Title: A feedforward loop between STAT1 and YAP1 stimulates lipid biosynthesis, accelerates tumor growth, and promotes chemotherapy resistance in mutant KRAS colorectal cancer

doi: 10.1038/s42003-025-08740-2

Figure Lengend Snippet: a ChIP-seq data from ENCODE (UCSC database) indicating STAT1 binding to the regulatory regions of SREBF1 and 2 genes. b ChIP assays of endogenous STAT1 bound to SREBF gene segments containing STAT1 binding sites in HCT116 and HK2-8 cells. IgG, non-specific control antibody. c Expression of GFP (control) and GFP-tagged STAT1 proteins that are either intact (wild type, WT), impaired for phosphorylation (Y701F or S727A) or S727 phosphomimetic (S727E) in HCT116 STAT1 −/− cells. GFP+ cells were sorted by flow cytometry, and extracts were immunoblotted for GFP or ACTIN. d Detection of SREBF-1 and 2 mRNAs by qPCR in HCT116 STAT1 −/− cells expressing either GFP or GFP-STAT1 forms. e ChIP assays of GFP-STAT1 for binding to STAT1 sites of SREBF genes in reconstituted HCT116 STAT1 −/− cells using GFP antibody. b–e Data obtained from 3 biological replicates and represent ±SEM *P < 0.05, **P < 0.01, *** P < 0.001 ( t -test).

Article Snippet: Plasmids containing the green fluorescence protein (GFP)-tagged forms of wild type STAT1, STAT1 Y701F or STAT1 S727A cDNA were obtained from Addgene (plasmid #12301,12302 and 12304) .

Techniques: ChIP-sequencing, Binding Assay, Control, Expressing, Phospho-proteomics, Flow Cytometry

Journal: Communications Biology

Article Title: A feedforward loop between STAT1 and YAP1 stimulates lipid biosynthesis, accelerates tumor growth, and promotes chemotherapy resistance in mutant KRAS colorectal cancer

doi: 10.1038/s42003-025-08740-2

Figure Lengend Snippet: a HCT116 and HK2-8 cells replete or deplete of STAT1 were serum-starved for 18 h (untreated; UT) and stimulated with either 10% fetal bovine serum or 25 μM LPA for 1 h. Cells were subjected to IF analyses of YAP1 (red) along with DAPI staining of DNA (blue). Graphs show the quantification of YAP1 nuclear localization in 300 cells. Scale bar: 25 μm. b , c Cells were subjected to cytoplasmic (C), and nuclear (N) fractionation followed by immunoblotting for the indicated proteins. TUBULIN or THO complex 1 (THOC1) was used as cytoplasmic or nuclear marker, respectively. Quantification in panel b is based on three biological replicates, while panel c is based on two biological replicates. d , e HCT116 STAT1 +/+ and STAT1 −/− cells were transfected with either pGL3-luciferase reporter plasmid (control) or 8xGTIIC plasmid containing the firefly luciferase reporter gene under the control of 8x TEAD binding sites in CTGF minimal promoter. Transfected cells were serum-starved for 18 h followed by stimulation with either 10% fetal bovine serum or 25 μM LPA for 6 h. A plasmid expressing the renilla luciferase gene was used as internal control. f , g HCT116 cells were serum starved for 18 h followed by stimulation of 10% fetal bovine serum in the absence or presence of 2.5 mM cerivastatin (panel f, g) or 10 μM ROCK kinase inhibitor Y-27632 (panel f) for 18 h. Protein extracts were subjected to immunoblotting for the indicated proteins. a , b , d , e Graphs show the quantifications from 3 biological replicates and represent ±SEM *P < 0.05, **P < 0.01, *** P < 0.001 ( t -test), NS, non-significant. In c , data represent the quantification of 2 biological replicates.

Article Snippet: Plasmids containing the green fluorescence protein (GFP)-tagged forms of wild type STAT1, STAT1 Y701F or STAT1 S727A cDNA were obtained from Addgene (plasmid #12301,12302 and 12304) .

Techniques: Staining, Fractionation, Western Blot, Marker, Transfection, Luciferase, Plasmid Preparation, Control, Binding Assay, Expressing

Journal: Communications Biology

Article Title: A feedforward loop between STAT1 and YAP1 stimulates lipid biosynthesis, accelerates tumor growth, and promotes chemotherapy resistance in mutant KRAS colorectal cancer

doi: 10.1038/s42003-025-08740-2

Figure Lengend Snippet: a Volcano diagram showing the number of differentially expressed genes in YAP1-replete (WT; control) compared to YAP1 −/− HCT116 cells. Dashed horizontal and vertical lines indicate significance thresholds (|FC | > 0.5, P < 0.05). A positive fold change means that the gene is upregulated by YAP1. Genes are colored in gray (non-significant), blue ( P -value significant), and red (both P -values and fold change are significant). All labeled genes exhibit statistically significant upregulation (logFC > 0.5, P < 0.0005), consistent with their role in cholesterol biosynthesis and lipid metabolism. b Top KEGG pathways under the control of YAP1 in HCT116 cells. c Graphs assess the expression of SREBF1 and 2 mRNAs by qPCR in cells treated with scrambled or YAP1 siRNAs. Gene expressions were normalized to ACTIN and TUBULIN mRNAs used as internal controls. Data were obtained from 3 independent experiments performed in triplicates and represent ±SEM *P < 0.05, * *P < 0.01, *** P < 0.001 ( t -test). d ChIP-seq data from ENCODE indicating the binding of TEAD4 to transcriptional regulatory regions of SREBF genes. e ChIP assays of YAP1 for binding in complex with TEAD4 to SREBF genes in HCT116 cells, both in the presence and absence of STAT1 and/or YAP1. IgG, non-specific control antibody. f Immunoblotting of SREBP1 and 2 in isogenic pair colon cancer cells prior to and after YAP1 downregulation by siRNAs. FL, full length. g Immunoblotting of SREBP1, 2 and TEAD4 in isogenic pairs of colon cancer cells treated with scrambled or TEAD4 siRNAs. h Immunoblotting of SREBP1 and 2 proteins in HCT116 cells treated with TEAD inhibitor 15 μM VT104 for the indicated time points. f , g , h Quantification of proteins normalized to TUBULIN for each blot is indicated.

Article Snippet: Plasmids containing the green fluorescence protein (GFP)-tagged forms of wild type STAT1, STAT1 Y701F or STAT1 S727A cDNA were obtained from Addgene (plasmid #12301,12302 and 12304) .

Techniques: Control, Labeling, Expressing, ChIP-sequencing, Binding Assay, Western Blot

Journal: Communications Biology

Article Title: A feedforward loop between STAT1 and YAP1 stimulates lipid biosynthesis, accelerates tumor growth, and promotes chemotherapy resistance in mutant KRAS colorectal cancer

doi: 10.1038/s42003-025-08740-2

Figure Lengend Snippet: Venn diagram ( a ) and Volcano diagram ( b ) of genes that are commonly upregulated by STAT1 and YAP1 in HCT116 cells. Essential genes of sterol and lipid biosynthetic pathways are indicated in the Volcano diagram. b Dashed horizontal and vertical lines indicate significance thresholds (|FC | > 0.5, P < 0.05). A positive fold change means that the gene is upregulated by STAT1 and YAP1. Genes are colored in gray (non-significant), blue ( P -value significant), and Red (both P -values and fold change are significant). All labeled genes exhibit statistically significant upregulation (logFC > 0.5, P < 0.0005), consistent with their role in cholesterol biosynthesis and lipid metabolism. c Bar plot of biological processes (BP) from gene ontology (GO) significantly enriched in the common set of genes under the control of both YAP1 and STAT1 identified by gene expression profile analysis. d KEGG pathways under the control of STAT1 and YAP1 in HCT116 cells. e This schematic illustrates the cooperative role of STAT1 and YAP1 in promoting SREBP expression and activating the mevalonate pathway in mutant KRAS CRCs. The STAT1–YAP1 axis functions as a feedforward autoregulatory loop that sustains sterol biosynthesis. STAT1, phosphorylated at S727, directly induces the transcription of SREBF1 and SREBF2 genes. Elevated SREBP levels, in turn, enhance mevalonate pathway activity, leading to the prenylation, plasma membrane anchoring and activation of RHO GTPases. This activation promotes further phosphorylation of STAT1 at S727 and stimulates YAP1 nuclear localization and activation. Although YAP1 acts downstream of STAT1, it also reinforces the loop by cooperating with TEAD4 to transcriptionally upregulate SREBF genes. Created in BioRender. Koromilas, A. (2025) https://BioRender.com/nz5wlhr .

Article Snippet: Plasmids containing the green fluorescence protein (GFP)-tagged forms of wild type STAT1, STAT1 Y701F or STAT1 S727A cDNA were obtained from Addgene (plasmid #12301,12302 and 12304) .

Techniques: Labeling, Control, Gene Expression, Expressing, Mutagenesis, Activity Assay, Clinical Proteomics, Membrane, Activation Assay, Phospho-proteomics

Journal: Communications Biology

Article Title: A feedforward loop between STAT1 and YAP1 stimulates lipid biosynthesis, accelerates tumor growth, and promotes chemotherapy resistance in mutant KRAS colorectal cancer

doi: 10.1038/s42003-025-08740-2

Figure Lengend Snippet: a , b HCT116 cells with intact or depleted STAT1 and/or YAP1 expression were subcutaneously transplanted into female nu/nu mice (n = 10 per group). Tumor volume (mm³) was measured over the indicated time course. At the end of the study, tumor tissues from 3 mice were analyzed by IHC to assess H&E staining and the subcellular localization of YAP1 ( b ). c , d Similarly, HCT116 cells with combined YAP1 and STAT1 deletion or expression were transplanted into nu/nu mice (n = 5 per group). When tumors reached ~200 mm 3 , mice were treated by oral gavage with either vehicle or cerivastatin (CERI). Tumor growth was monitored over time. Red and blue arrows indicate the start point of treatment of YAP1 +/+ STAT1 +/+ and YAP1 −/− STAT1 −/− tumors, respectively. At the endpoint, tumors from 3 mice were subjected to IHC for H&E and YAP1 detection ( d ). a–d Data represent mean ± SEM. Statistical significance was determined by t -test: * P < 0.05, ** P < 0.01, *** P < 0.001. Scale bar in panels b and d: 50 μm and 25 μm (insert image). Quantification graphs panels b and d show Histo (H)-scores for nuclear and cytoplasmic YAP1 staining. e HCT116 xenografts were established in nu/nu mice (n = 5 per group). Upon tumor growth to ~200 mm 3 (red arrow), mice received either vehicle, cerivastatin (oral), afatinib (intraperitoneal), or the combination of both. Tumor volume was tracked for the indicated duration. Data represent mean ± SEM. *** P < 0.001 (t-test). f In a similar setup, mice bearing HCT116 xenografts (~200 mm 3 tumors) were treated with vehicle, cerivastatin (oral), VT104 (oral), or their combination. Tumor growth was monitored throughout the experiment. Data represent mean ± SEM. ** P < 0.01 (t-test). g This schematic model illustrates how the STAT1–YAP1 signaling axis enhances the mevalonate pathway, thereby supporting tumor growth and chemoresistance in mutant KRAS CRC. Inhibiting YAP1-TEAD4 activity (e.g., using VT104) disrupts the SREBP-driven feedback loop that sustains mevalonate pathway activation. Additionally, pharmacological blockade of the mevalonate pathway with statins increases tumor sensitivity to YAP1–TEAD4 inhibition in xenograft models of mutant KRAS CRC.

Article Snippet: Plasmids containing the green fluorescence protein (GFP)-tagged forms of wild type STAT1, STAT1 Y701F or STAT1 S727A cDNA were obtained from Addgene (plasmid #12301,12302 and 12304) .

Techniques: Expressing, Staining, Mutagenesis, Activity Assay, Activation Assay, Inhibition

Journal: Communications Biology

Article Title: A feedforward loop between STAT1 and YAP1 stimulates lipid biosynthesis, accelerates tumor growth, and promotes chemotherapy resistance in mutant KRAS colorectal cancer

doi: 10.1038/s42003-025-08740-2

Figure Lengend Snippet: a Evaluation of colony formation efficacy of HCT116 and HK2-8 cells after treatments with either scramble siRNAs or STAT1 siRNAs. The graphs represent data obtained from 3 biological replicates, each of which included 3 technical replicates, and represent ±SEM ( * ) P < 0.05 ( ** ) P < 0.01; ( t -test), NS, non-significant. b Immunoblotting of indicated protein from extracts of siRNA-treated cells. Quantifications show the relative intensity of STAT1 normalized to ACTIN from 3 biological replicates and represent ±SEM * P < 0.05 ( t -test). c Volcano Diagram illustrates the number of genes differentially expressed in STAT1-replete (WT; control) compared to STAT1 -/- HCT116 cells. Essential genes of sterol and lipid biosynthetic pathways are indicated. Dashed horizontal and vertical lines indicate significance thresholds (|FC | > 0.5, P < 0.05). Genes are colored in gray (non-significant), blue ( P -value significant), and red (both P -values and fold change are significant). A positive fold change means that the gene is upregulated by STAT1. All labeled genes exhibit statistically significant upregulation (logFC > 0.5, P < 0.0005), consistent with their role in cholesterol biosynthesis and lipid metabolism. d Bar plot of biological processes (BP) from gene ontology (ON) significantly enriched in STAT1-dependent genes in HCT116 cells. e KEGG pathways under the control of STAT1 in HCT116 cells.

Article Snippet: Plasmids containing the green fluorescence protein (GFP)-tagged forms of wild type STAT1, STAT1 Y701F or STAT1 S727A cDNA were obtained from Addgene (plasmid #12301,12302 and 12304) .

Techniques: Western Blot, Control, Labeling

Journal: Communications Biology

Article Title: A feedforward loop between STAT1 and YAP1 stimulates lipid biosynthesis, accelerates tumor growth, and promotes chemotherapy resistance in mutant KRAS colorectal cancer

doi: 10.1038/s42003-025-08740-2

Figure Lengend Snippet: a Detection of SREBF mRNAs in colon cancer cells with intact or impaired STAT1. SREBF 1 and 2 mRNA levels were normalized to ACTIN and TUBULIN mRNAs used as internal controls. Data obtained from 3 biological replicates each of which contained 3 technical replicates and represent ±SEM ** P < 0.01: *** P < 0.001 ( t -test), NS, non-significant. b Immunoblotting for SREBP1 and 2 in colon cancer cells with intact or downregulated STAT1. Quantifications show the relative intensity of proteins normalized to TUBULIN. FL, full length; M, mature form. c Schematic representation of the mevalonate pathway. Genes in red are SREBP-dependent genes. d ChIP-seq data from ENCODE (UCSC data base) indicating the binding of SREBP1 and 2 to transcriptional regulatory regions of mevalonate pathway genes. Graphs show the expression of ACAT1 , HMGCR and IDI1 mRNAs by qPCR in cells treated with scrambled or STAT1 siRNAs. Gene expressions were normalized to ACTIN and TUBULIN mRNAs used as internal controls. Data were obtained from 3 independent experiments performed in triplicates and represent ±SEM *P < 0.05, **P < 0.01, *** P < 0.001 ( t -test). e Immunoblotting of HCT116 protein extracts replete (control) or deplete ( −/− ) for STAT1 by CRISPR (cell line #1 and #2). Detection of STAT1 and rate-limiting enzymes of sterol and lipid biosynthetic pathway HMGCR and FAS, respectively.

Article Snippet: Plasmids containing the green fluorescence protein (GFP)-tagged forms of wild type STAT1, STAT1 Y701F or STAT1 S727A cDNA were obtained from Addgene (plasmid #12301,12302 and 12304) .

Techniques: Western Blot, ChIP-sequencing, Binding Assay, Expressing, Control, CRISPR

Journal: Communications Biology

Article Title: A feedforward loop between STAT1 and YAP1 stimulates lipid biosynthesis, accelerates tumor growth, and promotes chemotherapy resistance in mutant KRAS colorectal cancer

doi: 10.1038/s42003-025-08740-2

Figure Lengend Snippet: a ChIP-seq data from ENCODE (UCSC database) indicating STAT1 binding to the regulatory regions of SREBF1 and 2 genes. b ChIP assays of endogenous STAT1 bound to SREBF gene segments containing STAT1 binding sites in HCT116 and HK2-8 cells. IgG, non-specific control antibody. c Expression of GFP (control) and GFP-tagged STAT1 proteins that are either intact (wild type, WT), impaired for phosphorylation (Y701F or S727A) or S727 phosphomimetic (S727E) in HCT116 STAT1 −/− cells. GFP+ cells were sorted by flow cytometry, and extracts were immunoblotted for GFP or ACTIN. d Detection of SREBF-1 and 2 mRNAs by qPCR in HCT116 STAT1 −/− cells expressing either GFP or GFP-STAT1 forms. e ChIP assays of GFP-STAT1 for binding to STAT1 sites of SREBF genes in reconstituted HCT116 STAT1 −/− cells using GFP antibody. b–e Data obtained from 3 biological replicates and represent ±SEM *P < 0.05, **P < 0.01, *** P < 0.001 ( t -test).

Article Snippet: Plasmids containing the green fluorescence protein (GFP)-tagged forms of wild type STAT1, STAT1 Y701F or STAT1 S727A cDNA were obtained from Addgene (plasmid #12301,12302 and 12304) .

Techniques: ChIP-sequencing, Binding Assay, Control, Expressing, Phospho-proteomics, Flow Cytometry

Journal: Communications Biology

Article Title: A feedforward loop between STAT1 and YAP1 stimulates lipid biosynthesis, accelerates tumor growth, and promotes chemotherapy resistance in mutant KRAS colorectal cancer

doi: 10.1038/s42003-025-08740-2

Figure Lengend Snippet: a HCT116 and HK2-8 cells replete or deplete of STAT1 were serum-starved for 18 h (untreated; UT) and stimulated with either 10% fetal bovine serum or 25 μM LPA for 1 h. Cells were subjected to IF analyses of YAP1 (red) along with DAPI staining of DNA (blue). Graphs show the quantification of YAP1 nuclear localization in 300 cells. Scale bar: 25 μm. b , c Cells were subjected to cytoplasmic (C), and nuclear (N) fractionation followed by immunoblotting for the indicated proteins. TUBULIN or THO complex 1 (THOC1) was used as cytoplasmic or nuclear marker, respectively. Quantification in panel b is based on three biological replicates, while panel c is based on two biological replicates. d , e HCT116 STAT1 +/+ and STAT1 −/− cells were transfected with either pGL3-luciferase reporter plasmid (control) or 8xGTIIC plasmid containing the firefly luciferase reporter gene under the control of 8x TEAD binding sites in CTGF minimal promoter. Transfected cells were serum-starved for 18 h followed by stimulation with either 10% fetal bovine serum or 25 μM LPA for 6 h. A plasmid expressing the renilla luciferase gene was used as internal control. f , g HCT116 cells were serum starved for 18 h followed by stimulation of 10% fetal bovine serum in the absence or presence of 2.5 mM cerivastatin (panel f, g) or 10 μM ROCK kinase inhibitor Y-27632 (panel f) for 18 h. Protein extracts were subjected to immunoblotting for the indicated proteins. a , b , d , e Graphs show the quantifications from 3 biological replicates and represent ±SEM *P < 0.05, **P < 0.01, *** P < 0.001 ( t -test), NS, non-significant. In c , data represent the quantification of 2 biological replicates.

Article Snippet: Plasmids containing the green fluorescence protein (GFP)-tagged forms of wild type STAT1, STAT1 Y701F or STAT1 S727A cDNA were obtained from Addgene (plasmid #12301,12302 and 12304) .

Techniques: Staining, Fractionation, Western Blot, Marker, Transfection, Luciferase, Plasmid Preparation, Control, Binding Assay, Expressing

Journal: Communications Biology

Article Title: A feedforward loop between STAT1 and YAP1 stimulates lipid biosynthesis, accelerates tumor growth, and promotes chemotherapy resistance in mutant KRAS colorectal cancer

doi: 10.1038/s42003-025-08740-2

Figure Lengend Snippet: a Volcano diagram showing the number of differentially expressed genes in YAP1-replete (WT; control) compared to YAP1 −/− HCT116 cells. Dashed horizontal and vertical lines indicate significance thresholds (|FC | > 0.5, P < 0.05). A positive fold change means that the gene is upregulated by YAP1. Genes are colored in gray (non-significant), blue ( P -value significant), and red (both P -values and fold change are significant). All labeled genes exhibit statistically significant upregulation (logFC > 0.5, P < 0.0005), consistent with their role in cholesterol biosynthesis and lipid metabolism. b Top KEGG pathways under the control of YAP1 in HCT116 cells. c Graphs assess the expression of SREBF1 and 2 mRNAs by qPCR in cells treated with scrambled or YAP1 siRNAs. Gene expressions were normalized to ACTIN and TUBULIN mRNAs used as internal controls. Data were obtained from 3 independent experiments performed in triplicates and represent ±SEM *P < 0.05, * *P < 0.01, *** P < 0.001 ( t -test). d ChIP-seq data from ENCODE indicating the binding of TEAD4 to transcriptional regulatory regions of SREBF genes. e ChIP assays of YAP1 for binding in complex with TEAD4 to SREBF genes in HCT116 cells, both in the presence and absence of STAT1 and/or YAP1. IgG, non-specific control antibody. f Immunoblotting of SREBP1 and 2 in isogenic pair colon cancer cells prior to and after YAP1 downregulation by siRNAs. FL, full length. g Immunoblotting of SREBP1, 2 and TEAD4 in isogenic pairs of colon cancer cells treated with scrambled or TEAD4 siRNAs. h Immunoblotting of SREBP1 and 2 proteins in HCT116 cells treated with TEAD inhibitor 15 μM VT104 for the indicated time points. f , g , h Quantification of proteins normalized to TUBULIN for each blot is indicated.

Article Snippet: Plasmids containing the green fluorescence protein (GFP)-tagged forms of wild type STAT1, STAT1 Y701F or STAT1 S727A cDNA were obtained from Addgene (plasmid #12301,12302 and 12304) .

Techniques: Control, Labeling, Expressing, ChIP-sequencing, Binding Assay, Western Blot

Journal: Communications Biology

Article Title: A feedforward loop between STAT1 and YAP1 stimulates lipid biosynthesis, accelerates tumor growth, and promotes chemotherapy resistance in mutant KRAS colorectal cancer

doi: 10.1038/s42003-025-08740-2

Figure Lengend Snippet: Venn diagram ( a ) and Volcano diagram ( b ) of genes that are commonly upregulated by STAT1 and YAP1 in HCT116 cells. Essential genes of sterol and lipid biosynthetic pathways are indicated in the Volcano diagram. b Dashed horizontal and vertical lines indicate significance thresholds (|FC | > 0.5, P < 0.05). A positive fold change means that the gene is upregulated by STAT1 and YAP1. Genes are colored in gray (non-significant), blue ( P -value significant), and Red (both P -values and fold change are significant). All labeled genes exhibit statistically significant upregulation (logFC > 0.5, P < 0.0005), consistent with their role in cholesterol biosynthesis and lipid metabolism. c Bar plot of biological processes (BP) from gene ontology (GO) significantly enriched in the common set of genes under the control of both YAP1 and STAT1 identified by gene expression profile analysis. d KEGG pathways under the control of STAT1 and YAP1 in HCT116 cells. e This schematic illustrates the cooperative role of STAT1 and YAP1 in promoting SREBP expression and activating the mevalonate pathway in mutant KRAS CRCs. The STAT1–YAP1 axis functions as a feedforward autoregulatory loop that sustains sterol biosynthesis. STAT1, phosphorylated at S727, directly induces the transcription of SREBF1 and SREBF2 genes. Elevated SREBP levels, in turn, enhance mevalonate pathway activity, leading to the prenylation, plasma membrane anchoring and activation of RHO GTPases. This activation promotes further phosphorylation of STAT1 at S727 and stimulates YAP1 nuclear localization and activation. Although YAP1 acts downstream of STAT1, it also reinforces the loop by cooperating with TEAD4 to transcriptionally upregulate SREBF genes. Created in BioRender. Koromilas, A. (2025) https://BioRender.com/nz5wlhr .

Article Snippet: Plasmids containing the green fluorescence protein (GFP)-tagged forms of wild type STAT1, STAT1 Y701F or STAT1 S727A cDNA were obtained from Addgene (plasmid #12301,12302 and 12304) .

Techniques: Labeling, Control, Gene Expression, Expressing, Mutagenesis, Activity Assay, Clinical Proteomics, Membrane, Activation Assay, Phospho-proteomics

Journal: Communications Biology

Article Title: A feedforward loop between STAT1 and YAP1 stimulates lipid biosynthesis, accelerates tumor growth, and promotes chemotherapy resistance in mutant KRAS colorectal cancer

doi: 10.1038/s42003-025-08740-2

Figure Lengend Snippet: a , b HCT116 cells with intact or depleted STAT1 and/or YAP1 expression were subcutaneously transplanted into female nu/nu mice (n = 10 per group). Tumor volume (mm³) was measured over the indicated time course. At the end of the study, tumor tissues from 3 mice were analyzed by IHC to assess H&E staining and the subcellular localization of YAP1 ( b ). c , d Similarly, HCT116 cells with combined YAP1 and STAT1 deletion or expression were transplanted into nu/nu mice (n = 5 per group). When tumors reached ~200 mm 3 , mice were treated by oral gavage with either vehicle or cerivastatin (CERI). Tumor growth was monitored over time. Red and blue arrows indicate the start point of treatment of YAP1 +/+ STAT1 +/+ and YAP1 −/− STAT1 −/− tumors, respectively. At the endpoint, tumors from 3 mice were subjected to IHC for H&E and YAP1 detection ( d ). a–d Data represent mean ± SEM. Statistical significance was determined by t -test: * P < 0.05, ** P < 0.01, *** P < 0.001. Scale bar in panels b and d: 50 μm and 25 μm (insert image). Quantification graphs panels b and d show Histo (H)-scores for nuclear and cytoplasmic YAP1 staining. e HCT116 xenografts were established in nu/nu mice (n = 5 per group). Upon tumor growth to ~200 mm 3 (red arrow), mice received either vehicle, cerivastatin (oral), afatinib (intraperitoneal), or the combination of both. Tumor volume was tracked for the indicated duration. Data represent mean ± SEM. *** P < 0.001 (t-test). f In a similar setup, mice bearing HCT116 xenografts (~200 mm 3 tumors) were treated with vehicle, cerivastatin (oral), VT104 (oral), or their combination. Tumor growth was monitored throughout the experiment. Data represent mean ± SEM. ** P < 0.01 (t-test). g This schematic model illustrates how the STAT1–YAP1 signaling axis enhances the mevalonate pathway, thereby supporting tumor growth and chemoresistance in mutant KRAS CRC. Inhibiting YAP1-TEAD4 activity (e.g., using VT104) disrupts the SREBP-driven feedback loop that sustains mevalonate pathway activation. Additionally, pharmacological blockade of the mevalonate pathway with statins increases tumor sensitivity to YAP1–TEAD4 inhibition in xenograft models of mutant KRAS CRC.

Article Snippet: Plasmids containing the green fluorescence protein (GFP)-tagged forms of wild type STAT1, STAT1 Y701F or STAT1 S727A cDNA were obtained from Addgene (plasmid #12301,12302 and 12304) .

Techniques: Expressing, Staining, Mutagenesis, Activity Assay, Activation Assay, Inhibition