stat3 luciferase reporter assays  (BPS Bioscience)

Journal: bioRxiv

Article Title: Aberrant expression of the testis kinase TSSK6activates FAK–STAT3 signaling to promote tumorigenic growth

doi: 10.64898/2026.03.10.710807

Figure Lengend Snippet: A. HCT116 (n= 10) or B. DLD-1 ± TSSK6-V5 (n=9) were transfected with siCTRL or siSTAT3 for 48 hours then plated into soft agar. 10-14 days later, samples were stained and quantitated. Bars represent mean ± s.d. C. 293Ts were transfected with STAT3 reporter ± TSSK6-V5 (n=9). After 48 hours, vehicle control or 75 ng/uL Il-6 was added, and cells were incubated overnight. Dual Glo Luciferase assays were used to read STAT3 reporter firefly luminescence normalized to constitutively active renilla luminescence. Samples with IL-6 were analyzed with a paired t-test. D. Confocal images of DLD-1 ± TSSK6-V5 cells plated onto glass coverslips for 1 day prior to staining with STAT3. The scale bar is 20 µm. Nuclear STAT3 signal normalized to total STAT3 signal per cell was quantified using ImageJ. Data points represent individual cells from 3 fields of view across 4 coverslips. Paired t-test used for statistical analysis.

Article Snippet: STAT3 Luciferase Reporter Assays were performed in 293T cells using a STAT3 Reporter Kit (Cat#79730, BPS Bioscience) as described by the manufacturer.

Techniques: Transfection, Staining, Control, Incubation, Luciferase

Journal: bioRxiv

Article Title: Aberrant expression of the testis kinase TSSK6activates FAK–STAT3 signaling to promote tumorigenic growth

doi: 10.64898/2026.03.10.710807

Figure Lengend Snippet: A. Volcano plots from differential gene expression analysis from loss-of-function (HCT116) and gain-of-function (HCEC) RNA-sequencing. Vertical dotted lines indicate log2(fold change) equals -0.5 or 0.5 and horizontal dotted lines indicate –log10(p-value) equals 2. Data points below this horizontal dotted line are not shown. B. Datasets returned in the Top Ten of Enrichr’s Pathways suite of databases as being enriched for in gain-of-function (HCEC, log2(fold change)>0.5 and p-value<0.01) and loss-of-function (HCT116, log2(fold change) <-0.5 and p-value<0.01) differential gene expression analysis. C. STAT3 datasests returned from Enrichr Transcription suite of databases as being enriched for in gain-of-function (HCEC, log2(fold change)>0.5 and p-value<0.01) and loss-of-function (HCT116, log2(fold change) <-0.5 and p-value<0.01) differential gene expression analysis. D. HCT116 were transfected with indicated siRNA for 48 hours then plated into soft agar. 10-14 days later, samples were stained and quantitated.

Article Snippet: STAT3 Luciferase Reporter Assays were performed in 293T cells using a STAT3 Reporter Kit (Cat#79730, BPS Bioscience) as described by the manufacturer.

Techniques: Gene Expression, RNA Sequencing, Transfection, Staining

Journal: bioRxiv

Article Title: Aberrant expression of the testis kinase TSSK6activates FAK–STAT3 signaling to promote tumorigenic growth

doi: 10.64898/2026.03.10.710807

Figure Lengend Snippet: A. HCT116 (n= 10) or B. DLD-1 ± TSSK6-V5 (n=9) were transfected with siCTRL or siSTAT3 for 48 hours then plated into soft agar. 10-14 days later, samples were stained and quantitated. Bars represent mean ± s.d. C. 293Ts were transfected with STAT3 reporter ± TSSK6-V5 (n=9). After 48 hours, vehicle control or 75 ng/uL Il-6 was added, and cells were incubated overnight. Dual Glo Luciferase assays were used to read STAT3 reporter firefly luminescence normalized to constitutively active renilla luminescence. Samples with IL-6 were analyzed with a paired t-test. D. Confocal images of DLD-1 ± TSSK6-V5 cells plated onto glass coverslips for 1 day prior to staining with STAT3. The scale bar is 20 µm. Nuclear STAT3 signal normalized to total STAT3 signal per cell was quantified using ImageJ. Data points represent individual cells from 3 fields of view across 4 coverslips. Paired t-test used for statistical analysis.

Article Snippet: STAT3 Luciferase Reporter Assays were performed in 293T cells using a STAT3 Reporter Kit (Cat#79730, BPS Bioscience) as described by the manufacturer.

Techniques: Transfection, Staining, Control, Incubation, Luciferase

Journal: bioRxiv

Article Title: Aberrant expression of the testis kinase TSSK6activates FAK–STAT3 signaling to promote tumorigenic growth

doi: 10.64898/2026.03.10.710807

Figure Lengend Snippet: A. Volcano plots from differential gene expression analysis from loss-of-function (HCT116) and gain-of-function (HCEC) RNA-sequencing. Vertical dotted lines indicate log2(fold change) equals -0.5 or 0.5 and horizontal dotted lines indicate –log10(p-value) equals 2. Data points below this horizontal dotted line are not shown. B. Datasets returned in the Top Ten of Enrichr’s Pathways suite of databases as being enriched for in gain-of-function (HCEC, log2(fold change)>0.5 and p-value<0.01) and loss-of-function (HCT116, log2(fold change) <-0.5 and p-value<0.01) differential gene expression analysis. C. STAT3 datasests returned from Enrichr Transcription suite of databases as being enriched for in gain-of-function (HCEC, log2(fold change)>0.5 and p-value<0.01) and loss-of-function (HCT116, log2(fold change) <-0.5 and p-value<0.01) differential gene expression analysis. D. HCT116 were transfected with indicated siRNA for 48 hours then plated into soft agar. 10-14 days later, samples were stained and quantitated.

Article Snippet: STAT3 Luciferase Reporter Assays were performed in 293T cells using a STAT3 Reporter Kit (Cat#79730, BPS Bioscience) as described by the manufacturer.

Techniques: Gene Expression, RNA Sequencing, Transfection, Staining

Journal: bioRxiv

Article Title: Microbial metabolism of methotrexate produces a STAT3 signaling molecule that alleviates gut inflammation

doi: 10.64898/2026.03.11.711168

Figure Lengend Snippet: DAMPA binds folate receptor to promote cytoplasmic STAT3 localization. (A) In silico docking of DAMPA with human Folate Receptor Alpha (FRα; PDB: 4LRH) using DiffDock-L. Lower SMINA affinity indicates stronger predicted binding. Lower Minimized RMSD minimized affinity indicates more stable and energetically favorable binding. (B) Competitive binding assay of DAMPA to folate receptor using membrane fractions isolated from HT29 cells. Folic acid-d2 (15 nM) was used as a competitor. Binding curves were generated by nonlinear regression of peak area under the curve (AUC) measured by LC-MS (n = 5 per group). (C) STAT3 activation measured by a dual-luciferase reporter assay in HT29 cells treated with DAMPA or MTX treatment in the absence and presence of TNFα (DMSO without TNFα; n = 6, all other groups; n = 9 per group, two-way ANOVA). (D and E) Phosphorylated STAT3 at Tyr705 (pY-STAT3) staining in HT29 cells treated with DAMPA or MTX in the presence or absence of TNFα. (D) Representative images. Scale bar, 20 µm. (E) Ratio of cytoplasmic to nuclear pY-STAT3 fluorescence intensity quantified by ImageJ (n = 5 per group, two-way ANOVA). (F and G) Co-staining of pY-STAT3 and mitochondria in HT29 cells. (F) Representative images showing pY-STAT3 and MitoTracker colocalization. Scale bar, 10 µm. (G) Quantification of pY-STAT3–mitochondria colocalization area by Image J. Colocalization area was normalized to cell number (DAPI) (n = 5 per group, two-way ANOVA). (H and I) Co-staining of pY-STAT3 and the endoplasmic reticulum (ER) in HT29 cells. (H) Representative images showing pY-STAT3 and ER-Tracker colocalization. Scale bar, 10 µm. (I) Colocalization area was quantified using Image J and normalized to total cell number based on DAPI staining (n = 10 per group, two-way ANOVA). (J) LC3B-II expression in HT29 cells treated with the STAT3 inhibitor stattic (5 µM), 1 h prior to DAMPA exposure under TNFα stimulation. LC3B-II levels were normalized to β-actin (n = 5 per group). All scatter dot plots are represented as mean ± SEM, with each data point representing an individual biological replicate. Exact p values are indicated in each graph.

Article Snippet: HT29 cells were seeded in 48-well plates and transfected at 70–80% confluence with the STAT3 Reporter Kit plasmids (BPS Bioscience, #79730) using Lipofectamine 3000 (Invitrogen, #L3000150) according to the manufacturer’s instructions.

Techniques: In Silico, Binding Assay, Competitive Binding Assay, Membrane, Isolation, Generated, Liquid Chromatography with Mass Spectroscopy, Activation Assay, Luciferase, Reporter Assay, Staining, Fluorescence, Expressing

Journal: bioRxiv

Article Title: Microbial metabolism of methotrexate produces a STAT3 signaling molecule that alleviates gut inflammation

doi: 10.64898/2026.03.11.711168

Figure Lengend Snippet: DAMPA administration attenuates intestinal inflammation in IL10KO mice. (A) Schematic of a daily oral gavage of DAMPA (4 mg/kg) administered to IL10KO mice (14-16 weeks old) for 50 days. (B-E) Histological analysis of colonic tissues at day 50. (B) Representative hematoxylin and eosin (H&E)-stained colon sections. Scale bar, 200 µm. (C) Crypt length quantification (DMSO, n = 25; DAMPA, n = 19, two-tailed Welch’s t test), (D) the number of goblet cells per crypt (DMSO, n = 30; DAMPA, n = 24, two-tailed Welch’s t test), and (E) neutrophil infiltration normalized to crypt area and expressed relative to the DMSO control (DMSO, n = 25; DAMPA, n = 19, Mann-Whitney test). Each n represents one crypt. 4-6 crypts per mouse were analyzed. (F) Fecal lipocalin-2 (LCN-2) levels measured by ELISA in samples collected at day 35 and day 50. Each n represents one mouse (DMSO, n = 5; DAMPA, n = 4, two-way ANOVA). (G) Serum pro-inflammatory cytokine (GM-CSF, IFN-α, and IL-6) levels measured by multiplex bead-based immunoassay. Each n represents one mouse (DMSO, n = 5; DAMPA, n = 4, two-way ANOVA). (H) Volcano plot showing differentially expressed proteins (DEPs) from the coon tissue proteomics analysis. The colored background indicates proteins with p < 0.05. (I) Western blot analysis of FUNDC1 protein levels in colon tissues (DMSO, n = 3; DAMPA, n = 3, two-tailed Welch’s t test). (J-M) 16S rRNA sequencing of fecal samples collected at day 50 (DMSO, n = 5; DAMPA, n = 4). (J) Alpha diversity (Chao1 index). (K) Principal coordinate analysis (PCoA). (L and M) Average relative abundance of microbial composition at (L) the phylum and (M) the class levels. (N) Proposed model of DAMPA-induced FR–STAT3 signaling. DAMPA binding to folate receptor promotes STAT3 localization to cytoplasmic compartments, including mitochondria and the endoplasmic reticulum, which may support mitochondrial function and mitophagy under inflammatory conditions. All scatter dot plots are represented as mean ± SEM. Violin plots depict the distribution of the data. The central line indicates the median, and dotted lines indicate the interquartile range (25th–75th percentiles). Each data point represents one crypt (C-E) or individual biological replicate (F, G, and I-K). Exact p values are indicated in each graph.

Article Snippet: HT29 cells were seeded in 48-well plates and transfected at 70–80% confluence with the STAT3 Reporter Kit plasmids (BPS Bioscience, #79730) using Lipofectamine 3000 (Invitrogen, #L3000150) according to the manufacturer’s instructions.

Techniques: Staining, Two Tailed Test, Control, MANN-WHITNEY, Enzyme-linked Immunosorbent Assay, Multiplex Assay, Bead-based Assay, Western Blot, Sequencing, Binding Assay