Journal: Cells

Article Title: Regulation of Ketogenic Enzyme HMGCS2 by Wnt/β-catenin/PPARγ Pathway in Intestinal Cells

doi: 10.3390/cells8091106

Figure Lengend Snippet: Inhibition of Wnt/β-catenin signaling increased 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) expression in human intestinal cancer cells. ( A , B ) LS174T or Caco2 cells transfected with non-target control (NTC) siRNA or β-catenin (β-cat) siRNA were incubated for 48 h. ( A ) Western blot analysis was performed using the antibodies as indicated. HMGCS2 expression from three separate western blots was quantitated densitometrically and is expressed as fold change with respect to β-actin ( n = 3, data represent mean ± SD; * p < 0.05 vs. NTC). ( B ) The level of HMGCS2 mRNA was assessed by real-time RT-PCR ( n = 3, data represent mean ± SD; * p < 0.05 vs. NTC). ( C , D ) Inhibition of Wnt/β-catenin signaling increased the expression of HMGCS2 in LS174T and Caco2 cells. LS174T or Caco2 cells were treated with iCRT3 for 24 h (LS174T) or 48 h (Caco2). ( C ) Western blot analysis was performed using the antibodies as indicated. ( D ) HMGCS2 mRNA expression was assessed by real time RT-PCR ( n = 3, data represent mean ± SD; * p < 0.05 vs. 0 μM iCRT3).

Article Snippet: The human colon cancer cell lines used in these studies, LS174T and Caco2, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Inhibition, Expressing, Transfection, Control, Incubation, Western Blot, Quantitative RT-PCR

Journal: Cells

Article Title: Regulation of Ketogenic Enzyme HMGCS2 by Wnt/β-catenin/PPARγ Pathway in Intestinal Cells

doi: 10.3390/cells8091106

Figure Lengend Snippet: Activation of Wnt/β-catenin pathway suppressed HMGCS2 expression. ( A , B ) LS174T and Caco2 cells were treated with 200 ng/mL Wnt3a for 24 h. ( A ) Western blot analysis was performed using the antibodies as indicated. Densitometric quantification from three separate experiments was performed and is represented as fold change with respect to β-actin ( n = 3, data represent mean ± SD; * p < 0.05 vs. 0 ng/mL Wnt3a). ( B ) The level of HMGCS2 mRNA was determined by real-time RT-PCR. ( n = 3, data represent mean ± SD; * p < 0.05 vs. 0 ng/mL Wnt3a). ( C , D ) LS174T and Caco2 cells were treated with various dosages of LiCl or 40 mM of NaCl as control for 24 h. ( C ) Western blot analysis was performed using the antibodies as indicated. ( D ) The level of HMGCS2 mRNA was determined by real-time RT-PCR. ( n = 3, data represent mean ± SD; * p < 0.05 vs. 40 mM NaCl). ( E ). Mouse small intestinal organoids were treated with 100 ng/mL Wnt3a for 3 days. HMGCS2 protein expression was determined by western blotting. Densitometric analysis from three independent experiments was performed and is represented as fold change with respect to β-actin ( n = 3, data represent mean ± SD; * p < 0.05 vs. 0 ng/mL Wnt3a).

Article Snippet: The human colon cancer cell lines used in these studies, LS174T and Caco2, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Activation Assay, Expressing, Western Blot, Quantitative RT-PCR, Control

Journal: Cells

Article Title: Regulation of Ketogenic Enzyme HMGCS2 by Wnt/β-catenin/PPARγ Pathway in Intestinal Cells

doi: 10.3390/cells8091106

Figure Lengend Snippet: Regulation of β-hydroxybutyrate (βHB) production by Wnt/β-catenin signaling in intestinal cells. ( A ) LS174T and Caco2 cells, transfected with NTC or β-catenin siRNA (β-cat) and incubated for 48 h or treated with iCRT3 for 24 h (LS174T) or 48 h (Caco2), were lysed and βHB content was measured using a βHB assay kit ( n = 3, data represents mean ± SD; * p < 0.05 vs. NTC or 0 μM iCRT3). ( B ) LS174T and Caco2 cells were treated with 200 ng/mL Wnt3a or 40 mM LiCl (40 mM NaCl as control) for 24 h. Cell lysates were used for measurement of βHB content using a βHB assay kit ( n = 3, data represents mean ± SD; * p < 0.05 vs. 0 ng/mL Wnt3a or 40 mM NaCl). ( C ) Mouse small intestinal organoids were treated with 100 ng/mL Wnt3a for 3 days. βHB content in cell lysates and conditioned media was determined using a βHB assay kit ( n = 3, data represents mean ± SD; * p < 0.05 vs. 0 ng/mL Wnt3a).

Article Snippet: The human colon cancer cell lines used in these studies, LS174T and Caco2, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Transfection, Incubation, Control

Journal: Cells

Article Title: Regulation of Ketogenic Enzyme HMGCS2 by Wnt/β-catenin/PPARγ Pathway in Intestinal Cells

doi: 10.3390/cells8091106

Figure Lengend Snippet: The expression and activity of PPARγ was regulated by the Wnt/β-catenin pathway in intestinal cancer cell lines. ( A , B ) LS174T or Caco2 cells were transfected with NTC or β-catenin siRNA and incubated for 48 h. ( A ) Western blot analysis was performed using the antibodies as indicated. PPARγ expression from three independent experiments was quantitated densitometrically and is expressed as fold change with respect to β-actin ( n = 3, data represent mean ± SD; * p < 0.05 vs. NTC). ( B ) PPAR γ mRNA was assessed by real-time RT-PCR ( n = 3, data represent mean ± SD; * p < 0.05 vs. NTC). ( C , D ) LS174T or Caco2 cells were treated with NaCl (40 mM) as control or LiCl (40 mM) for 24 h. ( C ) Western blotting was performed using the antibodies as indicated. Densitometric analysis from three separate experiments was performed and PPARγ expression is represented as fold change with respect to β-actin ( n = 3, data represent mean ± SD; * p < 0.05 vs. 40 mM/mL NaCl). ( D ) PPAR γ mRNA was assessed by real-time RT-PCR ( n = 3, data represents mean ± SD; * p < 0.05 vs. 40 mM/mL NaCl control). ( E ) LS174T cells were transfected with NTC or β-catenin siRNA for 48 h or treated with 40 mM NaCl or 40 mM LiCl for 24 h and PPARγ DNA-binding activity was determined as described under Materials and Methods ( n = 3, data represents mean ± SD; * p < 0.05 vs. NTC or 40 mM NaCl). ( F ) Expression of FABP2 and PLIN2 mRNA was assessed by real-time RT-PCR. ( n = 3, data represents mean ± SD; * p < 0.05 vs. NTC or 40 mM/mL NaCl).

Article Snippet: The human colon cancer cell lines used in these studies, LS174T and Caco2, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Activity Assay, Transfection, Incubation, Western Blot, Quantitative RT-PCR, Control, Binding Assay

Journal: Cells

Article Title: Regulation of Ketogenic Enzyme HMGCS2 by Wnt/β-catenin/PPARγ Pathway in Intestinal Cells

doi: 10.3390/cells8091106

Figure Lengend Snippet: PPARγ regulated HMGCS2 expression in intestinal cancer cell lines. ( A ). LS174T cells were transfected with NTC or PPARγ siRNA. Expression of PPARγ and HMGCS2 was determined by western blot analysis. HMGCS2 expression from three separate western blots was quantitated densitometrically and is expressed as fold change with respect to β-actin ( n = 3, data represent mean ± SD; * p < 0.05 vs. NTC). ( B , C ). ( B ) LS174T cells were treated with rosiglitazone (RGZ), an agonist of PPARγ for 24 h; ( C ) LS174T cells were treated with iCRT3 in the presence or absence of T 0070907 (T007), an antagonist of PPARγ for 24 h. Protein expression of HMGCS2 was measured by western blot analysis. Densitometric quantification from three independent experiments was performed and is represented as fold change with respect to β-actin ( n = 3, data represent mean ± SD; * p < 0.05 vs. 40 mM NaCl or vehicle control). The level of HMGCS2 mRNA was assessed by real-time RT-PCR ( n = 3, data represents mean ± SD; * p < 0.05 vs. control). ( D ). LS174T cells were transfected with NTC or β-catenin siRNA. DNA was extracted and ChIP-qPCR was performed using PPARγ or IgG antibody. The binding efficiency of PPARγ to the promoter regions of HMGCS2 or IFN-λ1 (as non-regulated control (NRC)) was analyzed and is represented as a percent of input ( n = 3, data represents mean ± SD; * p < 0.05 vs. NTC). ( E ). LS174T cells were treated with RGZ for 24 h, or transfected with NTC or β-catenin siRNA; Caco2 cells were treated with RGZ or T007 for 24 h. Cells were lysed and βHB content was determined using a βHB assay kit. ( n = 3, data represents mean ± SD; * p < 0.05 vs. control or NTC).

Article Snippet: The human colon cancer cell lines used in these studies, LS174T and Caco2, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Transfection, Western Blot, Control, Quantitative RT-PCR, ChIP-qPCR, Binding Assay

Journal: Cells

Article Title: Regulation of Ketogenic Enzyme HMGCS2 by Wnt/β-catenin/PPARγ Pathway in Intestinal Cells

doi: 10.3390/cells8091106

Figure Lengend Snippet: Regulation of glycolysis by Wnt/β-catenin/HMGCS2 pathway. ( A ) LS174T cells transfected with NTC siRNA or siRNA targeting HMGCS2 or β-catenin were subjected to Seahorse Extracellular Flux analysis (left) ( n = 9, data represent mean ± SD; * p < 0.05 vs. NTC). Knockdown of HMGCS2 and β-catenin was confirmed by western blot analysis (right). ( B , C ) LS174T cells were treated with 2-DG, a glycolysis inhibitor for 24 h. Caco2 cells were treated with 2-DG for 24 h (RNA) or 48 h (IAP activity assay). ( B ) IAP activity was measured ( n = 3, data represents mean ± SD; * p < 0.05 vs. 0 mM 2-DG). ( C ) Expression of SI , p21 Waf1 and CDX2 mRNA was assessed by real-time RT-PCR. ( n = 3, data represents mean ± SD; * p < 0.05 vs. 0 mM 2-DG). ( D ) Inhibition of the Wnt/β-catenin pathway resulted in the increased expression and activation of PPARγ, and thus increased ketogenesis by induction of HMGCS2. Ketogenesis contributes to intestinal cell differentiation via the inhibition of glycolysis.

Article Snippet: The human colon cancer cell lines used in these studies, LS174T and Caco2, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Transfection, Knockdown, Western Blot, Activity Assay, Expressing, Quantitative RT-PCR, Inhibition, Activation Assay, Cell Differentiation

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: A Nucleotide Analog Prevents Colitis-Associated Cancer via Beta-Catenin Independently of Inflammation and Autophagy

doi: 10.1016/j.jcmgh.2020.05.012

Figure Lengend Snippet: ( A ) Thiopurine pro-drug metabolism . TG and MP are purine analogue bases. Azathioprine (AZA) is non-enzymatically converted to MP. TGMP, TGDP, and TGTP are TGN. ( B ) TG, but not MP, inhibited β-catenin activity in vitro. β-catenin–driven transcriptional activity in HCT116 or Caco2 cells treated for 16 hours with either TG or MP in presence or absence of 1 μmol/L CHIR-99021 (highly selective inhibitor of glycogen synthase kinase 3, which activates β-catenin by promotion of Wnt signalling), IC50 TG ∼2 μmol/L. N = 4. ( C ) Immunofluorescence staining of HCT116 cells for β-catenin ( green ) and nuclei (DAPI, blue ) ± 5 μmol/L TG treatment ± 1 μmol/L CHIR for 16 hours. Data are representative of N = 4 independent experiments. ( D ) Real-time imaging to 60 hours of HCT116 cells seeded into 96-well plates (7500 cells/well) by using a phase Cyte FLR live cell imager. ( E ) TG inhibited β-catenin in spheroid cells derived from an ulcerative colitis cancer, treated with vehicle control or TG (10 μmol/L) for 4 days. ( F ) TG conversion to TGN in Caco 2 and HCT116 cell lysates. MP was not converted to TGN in the acute in vitro experiments (N = 4 at each concentration).

Article Snippet: Stable HCT116 and Caco2 pooled clones overexpressing reporter construct were treated with 1 μmol/L CHIR 99021(Selleckchem, Houston, TX) for 16 hours.

Techniques: Activity Assay, In Vitro, Immunofluorescence, Staining, Imaging, Derivative Assay, Control, Concentration Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: A Nucleotide Analog Prevents Colitis-Associated Cancer via Beta-Catenin Independently of Inflammation and Autophagy

doi: 10.1016/j.jcmgh.2020.05.012

Figure Lengend Snippet: TG inhibits β-catenin activity in vitro independently of autophagy. ( A ) Knockdown of ATG7 in Caco2 and HCT116 cells 48 hours after transfection with ATG7 (#3) or ATG7 (#4) or control (-) siRNA was assessed by Western blotting. ( B ) Basal β-catenin–driven transcriptional activity in Caco2 and HCT116 cells transfected with ATG7 or control siRNA for 48 hours was increased with inhibition of the autophagy gene. (N = 4). ( C ) Treatment of the same cells in presence of 1 μmol/L CHIR-99021 and indicated dose of TG for 16 hours inhibited β-catenin–driven transcriptional activity. Data are representative of 4 independent experiments. RLU, relative light units.

Article Snippet: Stable HCT116 and Caco2 pooled clones overexpressing reporter construct were treated with 1 μmol/L CHIR 99021(Selleckchem, Houston, TX) for 16 hours.

Techniques: Activity Assay, In Vitro, Knockdown, Transfection, Control, Western Blot, Inhibition

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: A Nucleotide Analog Prevents Colitis-Associated Cancer via Beta-Catenin Independently of Inflammation and Autophagy

doi: 10.1016/j.jcmgh.2020.05.012

Figure Lengend Snippet: TG inhibits β-catenin activity via TGN. ( A ) Knockdown of HPRT in Caco2 and HCT116 cells 48 hours after transfection with HPRT (#1) or HPRT (#2) or control (-) siRNA, assessed by Western blotting. ( B ) TG in presence of 1 μmol/L CHIR-99021versus β-catenin–driven transcriptional activity in cells silenced with HPRT siRNA. ( C ) WT and Hprt -/- derived primary murine fibroblasts treated with 5 μmol/L CHIR ± 10 μmol/L TG or vehicle for 16 hours. Immunofluorescence staining for β-catenin ( green ) and nuclei (DAPI, blue ). Data are representative of 4 independent experiments. ( D ) Western blotting of cytosol and nuclear compartments of WT and Hprt -/- derived primary murine fibroblasts treated with 5 μmol/L CHIR ± 10 μmol/L TG or vehicle for 16 hours showing TG inhibiting β-catenin activity and nuclear translocation with CHIR only in WT cells.

Article Snippet: Stable HCT116 and Caco2 pooled clones overexpressing reporter construct were treated with 1 μmol/L CHIR 99021(Selleckchem, Houston, TX) for 16 hours.

Techniques: Activity Assay, Knockdown, Transfection, Control, Western Blot, Derivative Assay, Immunofluorescence, Staining, Translocation Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: A Nucleotide Analog Prevents Colitis-Associated Cancer via Beta-Catenin Independently of Inflammation and Autophagy

doi: 10.1016/j.jcmgh.2020.05.012

Figure Lengend Snippet: TG at low concentrations suppressed β-catenin activation via inhibition of Rac1. ( A ) Knockdown of Rac1 in Caco2 and HCT116 cells 48 hours after transfection with Rac1 siRNA #6 or Rac1 siRNA #7 versus control (-) siRNA, assessed by Western blotting. ( B ) [ TG] versus β-catenin–driven transcriptional activity in control cells versus Rac1 -siRNA silenced cells. ( C ) Cartoon showing role of Rac1 in β-catenin–driven transcription of Wnt target gene: C1 , normal physiology with GTP; C2 , TG with WT Rac1; C3 , TG with genetically inactivated GTPase Q61L Rac1. ( D ) Left panel : Western blotting for Flag in HCT116 and Caco2 cells confirms overexpression of Rac1 WT and Rac1 mutant in these cells. Right panels : [TG] versus β-catenin–driven transcriptional activity in WT and Q61L (ie, constitutively active Rac1). Caco2 and HCT116 cells. Data are representative of 3 independent experiments. ( E ) Modelling experiments showing TGTP docked with β-catenin protein crystal structure ( left panel ) and docked in the conserved Rac1 nucleotide binding site ( right panel ). RLU, relative light units.

Article Snippet: Stable HCT116 and Caco2 pooled clones overexpressing reporter construct were treated with 1 μmol/L CHIR 99021(Selleckchem, Houston, TX) for 16 hours.

Techniques: Activation Assay, Inhibition, Knockdown, Transfection, Control, Western Blot, Activity Assay, Over Expression, Mutagenesis, Binding Assay

Journal: Cancer letters

Article Title: PDP1 promotes KRAS mutant colorectal cancer progression by serving as a scaffold for BRAF and MEK1.

doi: 10.1016/j.canlet.2024.217007

Figure Lengend Snippet: Fig. 1. Elevated PDP1 expression in KRAS mutant CRC. (A) Western blot analysis showing PDP1 and MAPK signal mediators’ expression in KRASG13D/- and KRAS ±

Article Snippet: The human KRAS mutant CRC cell lines (HCT116, SW480, DLD1, HCT8, SW620), human KRAS wild type CRC cell lines (SW48, Caco2, RKO, HT29), human normal colon epithelial cell line (NCM460), mouse KRAS mutant CRC cell line (CT26), and human embryonic kidney epithelial cell line 293T (HEK293T) were obtained from American Type Culture Collection (ATCC).

Techniques: Expressing, Mutagenesis, Western Blot

Journal: Cancer letters

Article Title: PDP1 promotes KRAS mutant colorectal cancer progression by serving as a scaffold for BRAF and MEK1.

doi: 10.1016/j.canlet.2024.217007

Figure Lengend Snippet: Fig. 2. PDP1 enhances proliferation and metastasis ability of KRAS mutant CRC cells in vitro. (A) Cell proliferation ability of KRAS mutant CRC cells transfected with control or PDP1 knockdown plasmids, assessed using CCK8 assays. (B) Colony formation assays of KRAS mutant CRC cells transfected with control or PDP1 knockdown plasmids. (C) Sphere formation capacity of KRAS mutant CRC cells transfected with control or PDP1 knockdown plasmids. Scale bar = 500 μm. (D) Wound-healing assays evaluating migration in KRAS mutant CRC cells transfected with control or PDP1 knockdown plasmids. Scale bar = 50 μm. (E) Transwell experiment showing the migration and invasion capabilities of KRAS mutant CRC cells transfected with control or PDP1 knockdown plasmids. Scale bar = 50 μm. (F) Cell proliferation ability of KRAS mutant CRC cells transfected with control or PDP1 overexpression plasmids, evaluated by CCK8 assays. (G) Colony formation assays of KRAS mutant CRC cells transfected with control or PDP1 overexpression plasmids. (H) Sphere formation capacity of KRAS mutant CRC cells transfected with control or PDP1 overexpression plasmids. Scale bar = 500 μm. (I) Wound-healing assays evaluating migration in KRAS mutant CRC transfected with control or PDP1 overexpression plasmids, assessing migration. Scale bar = 50 μm. (J) Transwell experiment showing the migration and invasion capabilities of KRAS mutant CRC cells transfected with control or PDP1 overexpression plasmids. Scale bar = 50 μm. These data were derived from three independent repetitions. Values are rep resented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, determined by one-way ANOVA (A-E) and two-tailed Student’s t-test (F-J).

Article Snippet: The human KRAS mutant CRC cell lines (HCT116, SW480, DLD1, HCT8, SW620), human KRAS wild type CRC cell lines (SW48, Caco2, RKO, HT29), human normal colon epithelial cell line (NCM460), mouse KRAS mutant CRC cell line (CT26), and human embryonic kidney epithelial cell line 293T (HEK293T) were obtained from American Type Culture Collection (ATCC).

Techniques: Mutagenesis, In Vitro, Transfection, Control, Knockdown, Migration, Over Expression, Derivative Assay, Two Tailed Test

Journal: Cancer letters

Article Title: PDP1 promotes KRAS mutant colorectal cancer progression by serving as a scaffold for BRAF and MEK1.

doi: 10.1016/j.canlet.2024.217007

Figure Lengend Snippet: Fig. 3. PDP1 facilitates proliferation and metastasis ability of KRAS mutant CRC cells in vivo. (A-C) PDP1 knockdown inhibits subcutaneous tumor growth. (A) Representative images of tumors from HCT8 cells with various transfections (n = 9). (B) Growth curves of tumors from transfected HCT8 cells. (C) Weights of tumors from transfected HCT8 cells. (D-F) PDP1 overexpression promotes subcutaneous tumor growth. (D) Representative images of tumors from DLD1 cells with various transfections (n = 8). (E) Growth curves of tumors from transfected DLD1 cells. (F) Weights of tumors from transfected DLD1 cells. (G-H) PDP1 knockdown inhibits pulmonary metastasis. (G) Representative images and (H) count of lung metastases in HCT8 cells with indicated transfections. Red arrows highlight metastatic nodes. Scale bar = 1 mm (original) and 250 μm (magnified). (I-J) PDP1 overexpression promotes pulmonary metastasis. (I) Representative images and (J) count of lung metastases in DLD1 cells with indicated transfections. Red arrows highlight metastatic nodes. Scale bar = 1 mm (original) and 250 μm (magnified). (K) Gross images and representative HE staining of mouse colon orthotopic tumors and liver metastasis nodes (n = 5). Scale bar = 50 μm (HE staining). (L) Volumes of orthotopic tumors in specified groups. (M) Comparing analysis of human HPRT1 mRNA expression levels in livers of colon orthotopic CRC mouse models (normalized to mouse 18S rRNA expression levels), detected by qRT-PCR. (N) Analysis of formation and liver metastasis in orthotopic xenograft tumors. Values are mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, determined by two-tailed Student’s t-test.

Article Snippet: The human KRAS mutant CRC cell lines (HCT116, SW480, DLD1, HCT8, SW620), human KRAS wild type CRC cell lines (SW48, Caco2, RKO, HT29), human normal colon epithelial cell line (NCM460), mouse KRAS mutant CRC cell line (CT26), and human embryonic kidney epithelial cell line 293T (HEK293T) were obtained from American Type Culture Collection (ATCC).

Techniques: Mutagenesis, In Vivo, Knockdown, Transfection, Over Expression, Staining, Expressing, Quantitative RT-PCR, Two Tailed Test

Journal: Cancer letters

Article Title: PDP1 promotes KRAS mutant colorectal cancer progression by serving as a scaffold for BRAF and MEK1.

doi: 10.1016/j.canlet.2024.217007

Figure Lengend Snippet: Fig. 4. KRAS mutation elevates PDP1 expression in a KLF5-dependent manner. (A) Workflow for screening transcription factors that regulate PDP1. (B) Correlation analysis between PDP1 and KLF5 expression in CRC, using TIMER2.0 database. (C) Relative mRNA levels of PDP1 in KRAS mutant CRC cells transfected with control or KLF5 overexpression plasmids. (D) Western blot analysis of PDP1 and KLF5 in KRAS mutant CRC cells transfected with control or KLF5 overexpression plasmids. (E) Relative mRNA levels of PDP1 in KRAS mutant CRC cells transfected with control or KLF5 knockdown plasmids. (F) Western blot analysis of PDP1 and KLF5 in KRAS mutant CRC cells transfected with control or KLF5 knockdown plasmids. (G) Relative mRNA levels of PDP1 in RKO cells (KRAS wild type) with control or KLF5 knockdown, transfected with KRAS expression plasmids. (H) Western blot analysis of PDP1 and KLF5 in RKO cells with control or KLF5 knockdown, transfected with KRAS expression plasmids. (I) Increased PDP1 transcriptional activity in KRAS mutant CRC cells with KLF5 overexpression. (J) Decreased PDP1 transcriptional activity in KRAS mutant CRC cells with KLF5 knockdown. (K) Dual-luciferase promoter activity analysis of PDP1 in HEK293T cells transfected with KRAS plasmids. (L) Schematic of the PDP1 promoter with predicted KLF5 binding regions (JASPAR database). (M) ChIP-qPCR analysis of KLF5 occupancy on the PDP1 promoter in DLD1 and HCT116 cells. (N) Strategy for mutating the PDP1 promoter and corresponding dual-luciferase promoter activity in HEK293T cells. These data were derived from three independent repetitions. Values are mean ± SD. n.s. (not significant), p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001, determined by two-tailed Student’s t-test (C, I, M, N) and one-way ANOVA (E, G, J, K).

Article Snippet: The human KRAS mutant CRC cell lines (HCT116, SW480, DLD1, HCT8, SW620), human KRAS wild type CRC cell lines (SW48, Caco2, RKO, HT29), human normal colon epithelial cell line (NCM460), mouse KRAS mutant CRC cell line (CT26), and human embryonic kidney epithelial cell line 293T (HEK293T) were obtained from American Type Culture Collection (ATCC).

Techniques: Mutagenesis, Expressing, Transfection, Control, Over Expression, Western Blot, Knockdown, Activity Assay, Luciferase, Binding Assay, ChIP-qPCR, Derivative Assay, Two Tailed Test

Journal: Cancer letters

Article Title: PDP1 promotes KRAS mutant colorectal cancer progression by serving as a scaffold for BRAF and MEK1.

doi: 10.1016/j.canlet.2024.217007

Figure Lengend Snippet: Fig. 5. Mechanism of PDP1-mediated promotion of MAPK signaling. (A) GSEA results from TCGA-COAD database showing enrichment of KRAS signaling hallmark and MAPK pathway in CRC cases with high PDP1 expression. (B-C) Western blot analysis of KRAS mutant CRC cells, transfected with either (B) PDP1 knockdown or (C) overexpression constructs, probed with specific antibodies targeting MAPK signaling components. (D) Representative IHC images showing MAPK signaling in subcutaneous tumors formed by KRAS mutant CRC cells with indicated transfections. Scale bar = 200 μm. (E) Identification of proteins interacting with PDP1 in KRAS mutant CRC cells, as detected by IP-MS. (F) Detection of the interaction between BRAF and PDP1 via LC-MS/MS. The mass spectrometry image of BRAF is displayed. (G) Western blot analysis of co-immunoprecipitation assays in KRAS mutant CRC cells using antibodies against BRAF, MEK1, and PDP1. (H) Observation of reduced endogenous BRAF and MEK1 interaction in KRAS mutant CRC cells with PDP1 knockdown. (I) HEK293T cells with the indicated plasmids transfection, were treated with MG132 (25 μm), followed by immunoprecipitation and immunoblotting. (J) Schematic of truncated PDP1 expression plasmids. (K) HEK293T cells transfected with various truncated PDP1 plasmids, were treated with MG132 (25 μm) and processed to immunoprecipitation and immunoblotting.

Article Snippet: The human KRAS mutant CRC cell lines (HCT116, SW480, DLD1, HCT8, SW620), human KRAS wild type CRC cell lines (SW48, Caco2, RKO, HT29), human normal colon epithelial cell line (NCM460), mouse KRAS mutant CRC cell line (CT26), and human embryonic kidney epithelial cell line 293T (HEK293T) were obtained from American Type Culture Collection (ATCC).

Techniques: Expressing, Western Blot, Mutagenesis, Transfection, Knockdown, Over Expression, Construct, Protein-Protein interactions, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Immunoprecipitation

Journal: Cancer letters

Article Title: PDP1 promotes KRAS mutant colorectal cancer progression by serving as a scaffold for BRAF and MEK1.

doi: 10.1016/j.canlet.2024.217007

Figure Lengend Snippet: Fig. 6. PDP1 knockdown enhances therapeutic effect of KRAS inhibitors. (A) Western blot analysis of SW837 cells with control or PDP1 knockdown, treated with sotorasib (100 nM) for varying durations (0, 8, 24, 48, 72 h), probed with specific antibodies. (B-C) Colony formation assays and (D) cell viability assays of SW837 cells with control or PDP1 knockdown, treated with different concentrations of sotorasib. (E) Representative images of SW837 xenograft tumors in mice treated with either vehicle or sotorasib, and with or without PDP1 knockdown (n = 5 per group). (F) Tumor weights and (G) growth curves of SW837 xenograft tumors under the indicated treatments. (H) Representative IHC images showing p-ERK1/2 and Ki-67 protein levels in SW837 xenograft tumors with various treatments. Scale bar = 200 μm. (I) Schematic model illustrating the role of PDP1 in KRAS mutant CRC. Mutant KRAS upregulates KLF5, leading to increased PDP1 expression. Elevated PDP1 then enhances the BRAF-MEK1 interaction, amplifying MAPK signaling and facilitating CRC progression. Values are mean ± SD. **p < 0.01, ***p < 0.001, determined by one-way ANOVA (C, F, G) and two-tailed Student’s t-test (D).

Article Snippet: The human KRAS mutant CRC cell lines (HCT116, SW480, DLD1, HCT8, SW620), human KRAS wild type CRC cell lines (SW48, Caco2, RKO, HT29), human normal colon epithelial cell line (NCM460), mouse KRAS mutant CRC cell line (CT26), and human embryonic kidney epithelial cell line 293T (HEK293T) were obtained from American Type Culture Collection (ATCC).

Techniques: Knockdown, Western Blot, Control, Mutagenesis, Expressing, Two Tailed Test

Journal: Nature biomedical engineering

Article Title: A complex human gut microbiome cultured in an anaerobic intestine-on-a-chip

doi: 10.1038/s41551-019-0397-0

Figure Lengend Snippet: a, Schematic showing the position of the human intestinal epithelium overlaid with its own mucus layer and complex gut microbiota on top, with vascular endothelium on bottom side of the same ECM-coated porous membrane, within a 2-channel microfluidic Organ Chip device in presence of oxygen gradients. Orange and blue colors indicated high and low levels of oxygen concentration, respectively. b, Schematic representation of the Intestine Chip with 6 oxygen quenched fluorescent particles embed in inlet, middle and outlet of top and bottom channels. (T, top channel; B, bottom channel). c, Sensitivity analysis of oxygen spots located in the Intestine Chip in response to defined, standard oxygen concentrations. d , Anaerobic chamber validation at various N 2 inflow pressures; N 2 was introduced into the chamber at 81 mL min −1 , 162 mL min −1 , or 243 mL min −1 for 1 h before gas flow was stopped and the chamber was allowed to recover (n=3, shaded regions indicate standard deviation; data are presented as mean ± s.d.). e, Microscopic views showing the villus morphology of the human Caco-2 intestinal epithelium (top left; scale bar, 100 μm) and vascular endothelium (top right; scale bar, 100 μm) cultured for 6 days in the Intestine Chip under anaerobic conditions, when viewed from above by DIC and phase contrast imaging, respectively, or by immunofluorescence staining for the tight junction protein, ZO-1 (red, bottom left; scale bar, 100 μm) and endothelial cell junction-associated protein, VE-cadherin (red, bottom right; scale bar, 20 μm). Gray indicates DAPI-stained nuclei; white dashed lines indicate the border of the oxygen sensor spot). f, Oxygen concentration profiles within aerobically- and anaerobically-cultured Intestine Chips. Representative pseudocolor insets indicate average oxygen concentration in aerobic chip (1), and inlet (2), middle (3) and outlet (4) of the anaerobically-cultured epithelium channel, at day 7 of culture. Scale bar, 200 μm. (n=3 individual chips; data are presented as mean ± s.d.; significance was calculated by one-way analysis of variance; *P = 0.046).

Article Snippet: To fabricate the Caco2 Intestine Chip, human intestinal microvascular endothelial cells (HIMECs; ScienCell) were first seeded (1.5×10 5 cells cm −2 ) in the bottom channel of the chips, on opposite side of the porous membrane.

Techniques: Membrane, Concentration Assay, Biomarker Discovery, Standard Deviation, Cell Culture, Imaging, Immunofluorescence, Staining

Journal: Nature biomedical engineering

Article Title: A complex human gut microbiome cultured in an anaerobic intestine-on-a-chip

doi: 10.1038/s41551-019-0397-0

Figure Lengend Snippet: a, Oxygen concentration profiles in aerobic and anaerobic Intestine Chips co-cultured with Bacteroides fragilis (n=4 individual chips; data are presented as mean ± s.d.). b, Representative vertical cross-sectional, confocal micrographic views through the intestinal epithelium-microbiome interface within the Intestine Chip cultured under anaerobic conditions, when immunostained for villin (cyan), ZO-1 (magenta) and nuclei with DAPI (blue). (scale bar, 50 μm; B. fragilis was HADA (yellow) labelled; representative images from 4 intestine chips are shown). c, Changes in apparent paracellular permeability ( P app ) measured by quantitating cascade blue transport across the tissue-tissue interface within the Intestine Chip microdevices co-cultured with Bacteroides fragilis under aerobic and anaerobic conditions (n=4 individual chips; data are presented as mean ± s.d.; significance was calculated by one-way analysis of variance; *P = 0.042 and 0.033 for anaerobic day 2 vs. day 0 and aerobic vs. anaerobic day 3, respectively). d, CFU counts/mL of Bacteroides fragilis co-cultured in the Intestine Chip under aerobic and anaerobic conditions (n=3 individual chips; data are presented as mean ± s.d.; significance was calculated by one-way analysis of variance; *P = 0.031 for day 2 and ***P<0.001 for day 3). e, Cross-sectional fluorescence microscopic view of the Caco2 epithelium (nuclei stained in blue with DAPI), overlying mucus layer stained with Alexa Fluor 488-conjugated WGA (yellow), and B. fragilis bacteria (GalCCP labelled, white) when co-cultured in the Intestine Chip. Scale bar, 10 μm. f, SEM views of the apical surface of the Caco2 epithelium in the Intestine Chip comparing the morphology on day 4 of culture before it accumulates a mucus layer and when the surface microvilli are visible (top) versus when Bacteroides fragilis have been added on day 12 after the mucus layer has accumulated, which can be seen as a dense mat that separates the bacteria from the epithelial cell surface (bottom). Scale bar, 2 μm.

Article Snippet: To fabricate the Caco2 Intestine Chip, human intestinal microvascular endothelial cells (HIMECs; ScienCell) were first seeded (1.5×10 5 cells cm −2 ) in the bottom channel of the chips, on opposite side of the porous membrane.

Techniques: Concentration Assay, Cell Culture, Permeability, Fluorescence, Staining, Bacteria

Journal: Nature biomedical engineering

Article Title: A complex human gut microbiome cultured in an anaerobic intestine-on-a-chip

doi: 10.1038/s41551-019-0397-0

Figure Lengend Snippet: a, Observed alpha diversity (richness) in our complex gut microbiome samples when cultured for 1 to 3 days in direct contact with human Caco2 intestinal epithelium (each data point represents one Intestine Chip). b, Relative abundance of genera measured across all samples highlighting changes in the abundance of the different genera observed over time. Data points represent each of the 3 replicates cultured under aerobic or anaerobic conditions at 0, 1, 2 or 3 days of culture (left to right, respectively); Hmb indicates genera abundance in the complex microbiome stock at time 0. c, Changes in apparent paracellular permeability (P app ) measured by quantifying cascade blue transport across the tissue-tissue interface within the Intestine Chip after co-culture with complex gut microbiome under aerobic and anaerobic conditions (n=4 individual chips; data are presented as mean ± s.d.; significance was calculated by one-way analysis of variance; *P = 0.048, ***P<0.001, ns = not significant). d, Differences in microbial abundance between Intestine Chip samples (dark blue: aerobic; light blue: anaerobic) and human microbiome stool sample from the Human Microbiome Project (red). Data are shown as log10 of the total number of reads; each data point corresponds to a single sample (error bars represent the s.d.; data are presented as box plots with individual data points overlaid, where lower or upper edges of the box represent 25 th or 75 th percentiles and the middle bar is the median).

Article Snippet: To fabricate the Caco2 Intestine Chip, human intestinal microvascular endothelial cells (HIMECs; ScienCell) were first seeded (1.5×10 5 cells cm −2 ) in the bottom channel of the chips, on opposite side of the porous membrane.

Techniques: Cell Culture, Permeability, Co-Culture Assay

Journal: Nature biomedical engineering

Article Title: A complex human gut microbiome cultured in an anaerobic intestine-on-a-chip

doi: 10.1038/s41551-019-0397-0

Figure Lengend Snippet: a, Differential abundance bacterial genera in the Caco2 Intestine Chip measured under anaerobic conditions over 3 days of culture (top), or at day 3 in the anaerobic chip compared to the aerobic chip or anaerobic liquid culture (bottom). Data are presented as log10 of the total read counts for each genus; each data point represents one chip.The total read counts for all genera at the bottom are normalized to their counts in liquid culture (data are presented as box plots with individual data points overlaid, where lower or upper edges of the box represent 25 th or 75 th percentiles and the middle bar is the median). b, Differential abundance in bacterial genera measured over 1 to 3 days of co-culture in the anaerobic versus aerobic Intestine Chip (data are represented as log2 fold change; each data point corresponds to the differential abundance for a given genus at a given day, comparing anaerobic to aerobic cultures; n=4 chips for each group on each day; error bars represent the s.d.).

Article Snippet: To fabricate the Caco2 Intestine Chip, human intestinal microvascular endothelial cells (HIMECs; ScienCell) were first seeded (1.5×10 5 cells cm −2 ) in the bottom channel of the chips, on opposite side of the porous membrane.

Techniques: Co-Culture Assay