Journal: Journal of Cell Science

Article Title: The AHR target gene scinderin activates the WNT pathway by facilitating the nuclear translocation of β-catenin

doi: 10.1242/jcs.260028

Figure Lengend Snippet: RNA-seq identifies transcriptional signatures regulated by TCDD, Kyn and the AHR inhibitor CH223191. (A) RT-qPCR analyses for AHR normalized to the housekeeping gene RPS18 in a panel of colon cancer cell lines. (B–D) Relative proliferation determined by absorbance at 460 nm of DLD1 (B), HT29 (C) and HCT116 (D) cells after transfection with siRNA to knockdown AHR. These experiments were repeated twice. Data show the mean±s.e.m. *P<0.0001 (two-tailed unpaired t-test). (E–G) Volcano plots showing the differentially expressed genes upon incubation with TCDD (1 nM) (E), Kyn (10 µM) (F) and CH223191 (10 µM) (G). (H–J) Gene Ontology analyses showing the major pathways that are regulated upon incubation with TCDD (H), Kyn (I) and CH223191 (J). (K) Venn diagram showing the overlap of genes regulated by TCDD, Kyn and CH223191. (L) Heatmap of genes regulated by Kyn, TCDD and CH223191 after 8 h of treatment. For all analyses, log2FC≤0.585 and adjusted P-value≤0.05 were applied. (M) Venn diagram showing the overlap of genes regulated by TCDD and Kyn. (N) Heatmap of genes regulated by TCDD, Kyn and CH223191 after 8 h of incubation. (O) Molecular functions of the genes that are differentially expressed.

Article Snippet: DLD1 colon cancer cells incubated with DMSO, TCDD (Cambridge Isotope Laboratories) or Kyn (Sigma-Aldrich) for 1 h were harvested and subjected to RNA-seq as previously described ( Lafita-Navarro et al., 2018 ).

Techniques: RNA Sequencing, Quantitative RT-PCR, Transfection, Knockdown, Two Tailed Test, Incubation

Journal: Journal of Cell Science

Article Title: The AHR target gene scinderin activates the WNT pathway by facilitating the nuclear translocation of β-catenin

doi: 10.1242/jcs.260028

Figure Lengend Snippet: Activation of AHR by TCDD and Kyn promotes the transcription of a common signature of genes. (A) Molecular structures of the different ligands of AHR (Kyn, TCDD, FICZ and quercetin). (B) Western blot analysis showing the translocation of AHR upon treatment with DMSO, 1 nM TCDD, 20 µM Kyn, 1 µM FICZ and 10 µM quercetin for 30 min in DLD1 cells. Images are representative of three experiments. (C–H) RT-qPCR validation of the expression of the genes regulated by Kyn. DLD1 cells were treated with DMSO, 1 nM TCDD, 20 µM Kyn, 1 µM FICZ and 10 µM quercetin for 4 h and 8 h. Expression of the specified genes was normalized to RPS18. *P≤0.05 for comparison between DMSO and each ligand at 4 h or 8 h (two-tailed unpaired Student's t-test). (I–N) RT-qPCR analyses for the expression of the indicated genes in colon cancer cell lines. HCT116, HCT15 and HT29 cells were incubated with DMSO or 20 mM Kyn for 8 h. Expression of the specified genes was normalized to RPS18. *P≤0.05 comparison between DMSO and Kyn for each cell line (Student's t-test). Data show the mean±s.e.m.

Article Snippet: DLD1 colon cancer cells incubated with DMSO, TCDD (Cambridge Isotope Laboratories) or Kyn (Sigma-Aldrich) for 1 h were harvested and subjected to RNA-seq as previously described ( Lafita-Navarro et al., 2018 ).

Techniques: Activation Assay, Western Blot, Translocation Assay, Quantitative RT-PCR, Biomarker Discovery, Expressing, Comparison, Two Tailed Test, Incubation

Journal: Journal of Cell Science

Article Title: The AHR target gene scinderin activates the WNT pathway by facilitating the nuclear translocation of β-catenin

doi: 10.1242/jcs.260028

Figure Lengend Snippet: Modulation of AHR activity by shAHR, TCDD and Kyn regulates the transcription of a common signature of genes in colon cancer cells. (A) RT-qPCR for AHR in DLD1 cells stably expressing empty vector or shRNA for AHR in the presence or absence of TCDD. (B) Western blot for AHR in DLD1 cells stably expressing empty vector or shRNA for AHR in the presence or absence of TCDD. (C–H) RT-qPCR of genes regulated by AHR. DLD1 cells transduced with empty vector or a shRNA targeting AHR were incubated with DMSO or 1 nM TCDD for 24 h. Expression of the specified genes was normalized to RPS18. Fold change values were obtained by normalizing to pLKO-expressing control cells treated with DMSO. *P≤0.05 for comparison between DMSO and TCDD; *P≤0.05 for comparison between pLKO and shAHR (two-tailed unpaired Student's t-test). (I) Western blot for AHR, CYP1A1, SCIN, ABCG2, ALDH1A3, β-catenin and tubulin in AHR CRISPR KO DLD1 cells. (J) Relative proliferation of DLD1 control and AHR CRISPR KO cells with Crystal Violet staining 4 days after seeding. **P<0.0065; ****P<0.0001 (ordinary one-way ANOVA, Tukey's multiple comparisons test, with a single pooled variance, and two-tailed unpaired Student's t-test). Data show the mean±s.e.m. Images are representative of three experiments.

Article Snippet: DLD1 colon cancer cells incubated with DMSO, TCDD (Cambridge Isotope Laboratories) or Kyn (Sigma-Aldrich) for 1 h were harvested and subjected to RNA-seq as previously described ( Lafita-Navarro et al., 2018 ).

Techniques: Activity Assay, Quantitative RT-PCR, Stable Transfection, Expressing, Plasmid Preparation, shRNA, Western Blot, Transduction, Incubation, Control, Comparison, Two Tailed Test, CRISPR, Staining

Journal: Journal of Cell Science

Article Title: The AHR target gene scinderin activates the WNT pathway by facilitating the nuclear translocation of β-catenin

doi: 10.1242/jcs.260028

Figure Lengend Snippet: SCIN is necessary for colon cancer cell viability. (A–E) RT-qPCR analyses of DLD1 cells transfected with siControl or siRNAs targeting each of the indicated genes. Cells were harvested 3 days after siRNA transfection. Experiments were repeated three times. (F–J) Relative proliferation of DLD1 cells quantified by Crystal Violet staining 6 days after transfection with control siRNA or siRNAs targeting the indicated genes. Experiments were repeated three times. (K) RT-qPCR for SCIN in colon cancer cell lines. (L) Relative proliferation of HCT116 and HT29 cells 5 days after transfection with siControl or siRNA targeting SCIN. Experiments were repeated twice. (M) Western blots of DLD1 cells stably expressing SCIN compared with control cells (Vector). Images are representative of three experiments. (N) Relative proliferation of DLD1 cells stably expressing SCIN compared with control cells. *P≤0.05 for comparison between siControl and siRNAs targeting the indicated genes (ordinary one-way ANOVA, and Tukey's multiple comparisons test, with a single pooled variance). Data show the mean±s.e.m.

Article Snippet: DLD1 colon cancer cells incubated with DMSO, TCDD (Cambridge Isotope Laboratories) or Kyn (Sigma-Aldrich) for 1 h were harvested and subjected to RNA-seq as previously described ( Lafita-Navarro et al., 2018 ).

Techniques: Quantitative RT-PCR, Transfection, Staining, Control, Western Blot, Stable Transfection, Expressing, Plasmid Preparation, Comparison

Journal: Journal of Cell Science

Article Title: The AHR target gene scinderin activates the WNT pathway by facilitating the nuclear translocation of β-catenin

doi: 10.1242/jcs.260028

Figure Lengend Snippet: SCIN expression promotes nuclear translocation of β-catenin. (A) Immunofluorescence analyses showing SCIN (V5 tag) and the nucleus (DAPI staining). (B) Immunofluorescence analyses for actin and β-catenin in DLD1 cells expressing empty vector or SCIN. Scale bars: 10 μm. (C) Western blots for β-catenin and tubulin in lysates of DLD1 cells stably expressing vector (empty), SCIN, pLKO (vector for shRNA), shSCIN#22 and shSCIN#23; showing that total β-catenin levels are not affected by SCIN expression. (D) Western blots of nuclear (‘N’) and cytoplasmic (‘C’) fractions of DLD1 cells stably expressing vector or SCIN, immunoblotted for β-catenin, tubulin and histone H3. (E) Western blot of nuclear (‘N’) and cytoplasmic (‘C’) fractions of DLD1 cells stably expressing pLKO, shSCIN#22 or shSCIN#23 immunoblotted for β-catenin, tubulin and histone H3. (F) Western blot of nuclear (N) and cytoplasmic (C) fractions of DLD1 cells control and CRISPR AHR KO cells immunoblotted for β-catenin, tubulin and histone H3. (G) Schematic representation of the β-catenin responsive reporter TOPFlash and its negative control FOPFlash. (H) DLD1 cells stably expressing vector or SCIN were transfected with TOPFlash or FOPFlash and the luciferase reporter activity was measured 48 h later. Experiments were repeated three times. (I) DLD1 cells stably expressing empty vector (pLKO) and shRNA for SCIN were transfected with a TOPFlash or FOPFlash and processed as in H. Experiments were repeated three times. (J) DLD1 cells stably expressing vector or SCIN were transfected with TOPFlash or FOPFlash and treated with 10 µM ICG-001, and luciferase reporter activity was measured 48 h later. Results were normalized by FOPflash levels. (K) Western blots of control (vector) and SCIN-expressing DLD1 cells analyzed for SCIN, MYC, cyclin D1, axin 2 and tubulin. (L) Western blots of control (pLKO) and shSCIN #22 DLD1 cells analyzed for SCIN, MYC, cyclin D1, axin 2 and tubulin. (M) Growth curve of control (vector) DLD1 cells after treatment with DMSO, or 5 or 10 µM ICG-001. (N) Growth curve of SCIN DLD1 cells after treatment with DMSO, or 5 or 10 µM ICG-001. (O) Quantification of SCIN expression in colon cancer patients comparing normal and tumor samples. (P) Example of tumor sample displaying low levels of SCIN expression measured by immunohistochemistry for SCIN. (Q) Representative immunohistochemistry for SCIN and β-catenin in biopsies from a patient with colon cancer containing normal and tumor tissues of the same patient. Scale bars: 10 μm. Data show the mean±s.e.m. Images are representative of n=3. *P<0.05 (one-way ANOVA multiple comparison test).

Article Snippet: DLD1 colon cancer cells incubated with DMSO, TCDD (Cambridge Isotope Laboratories) or Kyn (Sigma-Aldrich) for 1 h were harvested and subjected to RNA-seq as previously described ( Lafita-Navarro et al., 2018 ).

Techniques: Expressing, Translocation Assay, Immunofluorescence, Staining, Plasmid Preparation, Western Blot, Stable Transfection, shRNA, Control, CRISPR, Negative Control, Transfection, Luciferase, Activity Assay, Immunohistochemistry, Comparison