Journal: Heliyon

Article Title: Potential actions of capsaicin for preventing vascular calcification of vascular smooth muscle cells in vitro and in vivo

doi: 10.1016/j.heliyon.2024.e28021

Figure Lengend Snippet: Cap inhibits Pi-induced calcification of VSMCs by inhibiting the Wnt/β-catenin signaling pathway by upregulation of TRPV1 receptor expression. A) RT-qPCR showing the mRNA levels of Wnt3a and β-catenin; B) Western blot illustrating the protein expression levels of Wnt3a and β-catenin; C) Alizarin Red staining assay; D) Calcium content in VSMCs assessed using a calcium colorimetric assay kit. E) RT-qPCR showing the expression of osteogenesis-specific and phenotypic markers in VSMC calcification. F) Western blot depicting the protein expression levels of osteogenesis-specific and phenotypic markers in VSMC calcification; G) Western blot showing the protein expression of β-catenin after treatment with DKK1. VSMCs cultured in the complete and pro-calcifying medium were defined as the control and Pi group, respectively; Pi + Cap: VSMCs cultured in the pro-calcifying medium with 20 μM Cap; Pi + Cap + CPZ: VSMCs cultured in the pro-calcifying medium with 20 μM Cap and 10 μM CPZ; Pi + Cap + DKK1: VSMCs cultured in the pro-calcifying medium with 20 μM Cap and 20 ng/mL DKK1; Data are mean ± SD (n = 3); #: p < 0.05, ##: p < 0.01, ###: p < 0.001 vs Control; *: p < 0.05, **: p < 0.01, ***: p < 0.001 vs Pi; ※: p < 0.05, ※※: p < 0.01, ※※※: p < 0.001 vs Pi + Cap; ns: p > 0.05 vs Control; p > 0.05 vs Pi; p > 0.05 vs Pi + Cap. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Antibodies against BMP-2 (PRP100126) and Runx2 (ABP53087) were purchased from Abbkine (Wuhan, Hubei, China); SM22α (AF9266) and Wnt3a (DF6113), Affinity Biosciences LTD; β-actin (81115-1-RR), proteintech (Hubei, China); β-catenin (ab32572), Abcom (USA); and DKK1 ( HY-P73305 ), MCE China (Beijing, China).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Staining, Calcium Colorimetric Assay, Cell Culture, Control

Journal: Scientific Reports

Article Title: HER2 G776S mutation promotes oncogenic potential in colorectal cancer cells when accompanied by loss of APC function

doi: 10.1038/s41598-022-13189-y

Figure Lengend Snippet: Effects of HER2 G776S mutation on the HER2 signaling pathway and anchorage-independent growth in several cell lines. ( A ) Detection of APC proteins in each cell by Western blotting. Full-length APC was detected with APC antibody (#2504, Cell Signaling Technology), and truncated APC was detected with APC antibody (sc-9998, Santa Cruz Biotechnology) raised against the N-terminus of APC. ( B ) The activity of the Wnt/β-catenin signaling pathway measured using the TCF/LEF luciferase reporter assay. The assay was performed in triplicate and the results were standardized to the mean of the activity of HeLa cells. ( C ) Phosphorylation and expression of HER2 and the downstream signaling in WT HER2 - or HER2 G776S-transfected HeLa and colon cells (FHC, CACO-2 and COLO-320). ( D ) Soft agar colony-forming assays showing the effects of stable transfection with HER2 WT or HER2 G776S in FHC (WT APC ) and COLO-320 (mutant APC ) cells. The cells were seeded into six-well plates in triplicate, and the number of colonies per well was counted. ns, not significant. ** P < 0.01 HER2 WT vs HER2 G776S.

Article Snippet: Wnt/β-catenin signaling activity was monitored using the TCF/LEF Reporter Kit (BPS Bioscience, San Diego, CA) following the manufacturer’s instructions.

Techniques: Mutagenesis, Western Blot, Activity Assay, Luciferase, Reporter Assay, Expressing, Transfection, Stable Transfection

Journal: Scientific Reports

Article Title: HER2 G776S mutation promotes oncogenic potential in colorectal cancer cells when accompanied by loss of APC function

doi: 10.1038/s41598-022-13189-y

Figure Lengend Snippet: Effects of APC KO on HER signaling in HeLa cells (with WT APC ). ( A ) Confirmation of APC KO and its effect on the HER2–ERK pathway in APC -KO HeLa cells using Western blotting. β-Actin served as a loading control. ( B ) Activity of the Wnt/β-catenin signaling pathway measured using the TCF/LEF luciferase reporter assay. Nontargeting control cells (NTC) incubated with Wnt3a (100 ng/ml) for 24 h were used as a positive control (rightmost bar). The data were standardized to the mean of the activity of NTC cells. One-way ANOVA: P < 0.01, * P < 0.05, ** P < 0.01 vs NTC cells ( n = 3). ( C ) Measurement of activated RAS (RAS–GTP) using the G-LISA assay. NTC plus Wnt3a (100 ng/ml) was used as a control (rightmost bar). The data were standardized to the mean of the activity of NTC cells. One-way ANOVA: P < 0.05, * P < 0.05 vs NTC cells ( n = 3). ( D ) Western blot results showing the effects of HER2 WT or HER2 G776S transfection on the HER2 signaling pathway in APC -KO cells. APC -KO cells or NTC cells were transfected with mock or HER2 expression vectors, and the experiments were performed 48 h after transfection. β-Actin served as a loading control. ( E ) Measurement of RAS-GTP using the G-LISA assay performed 48 h after HER2 expression vector transfection. The data were standardized to the mean of the activity of NTC cells transfected with mock vectors. Two-way ANOVA: interaction P < 0.01, * P < 0.05, ** P < 0.01 ( n = 3). ( F ) Colony-forming assay results showing the effects of stable transfection with HER2 WT or HER2 G776S in APC -KO cells. The number of colonies was counted in six random low-power fields. Scale bar, 200 μm. Two-way ANOVA: interaction P < 0.01, ** P < 0.01.

Article Snippet: Wnt/β-catenin signaling activity was monitored using the TCF/LEF Reporter Kit (BPS Bioscience, San Diego, CA) following the manufacturer’s instructions.

Techniques: Western Blot, Activity Assay, Luciferase, Reporter Assay, Incubation, Positive Control, Transfection, Expressing, Plasmid Preparation, Stable Transfection

Journal: Scientific Reports

Article Title: HER2 G776S mutation promotes oncogenic potential in colorectal cancer cells when accompanied by loss of APC function

doi: 10.1038/s41598-022-13189-y

Figure Lengend Snippet: Effects of WT APC overexpression on the HER2 signaling pathway in COLO-320 cells (with mutant APC ). ( A ) Confirmation of APC overexpression in cells transfected with pCMV_APC vector using Western blotting. ( B ) Activity of the Wnt/β-catenin pathway in COLO-320 cells transfected with pCMV_APC measured using the TCF/LEF luciferase reporter assay. The reporter vectors were cotransfected into COLO-320 cells along with the pCMV empty vector (mock) or pCMV-APC. The data were standardized to the mean of the activity of mock cells. ** P < 0.01 vs mock ( n = 3). ( C ) Measurement of activated RAS using the G-LISA assay performed 48 h after the transfection with pCMV. The data were standardized to the mean of the activity of cells transfected with mock vectors. * P < 0 .05 vs mock ( n = 3) ( D ) Western blot results showing the effects of APC overexpression on the HER2 signaling pathway in COLO-320 cells. The cells were cotransfected with pcDNA_HER2-WT/G776S vectors and/or pCMV_APC vectors. Western blotting was performed 48 h after transfection. β-Actin served as a loading control. ( E ) Measurement of activated RAS using the G-LISA assay performed 48 h after transfection. The data were standardized to the mean of the activity of cells transfected with mock vectors. Two-way ANOVA: interaction P < 0.01, ** P < 0.01 ( n = 3). ( F ) Activity of the Wnt/β-catenin pathway in COLO-320 cells measured 24 h after addition of ICG-001 at different concentrations. The assay was performed in triplicate. ( G ) Measurement of activated RAS using the G-LISA assay in COLO-320 cells 24 h after addition of ICG-001 at different concentrations. The assay was performed in triplicate.

Article Snippet: Wnt/β-catenin signaling activity was monitored using the TCF/LEF Reporter Kit (BPS Bioscience, San Diego, CA) following the manufacturer’s instructions.

Techniques: Over Expression, Mutagenesis, Transfection, Plasmid Preparation, Western Blot, Activity Assay, Luciferase, Reporter Assay

Journal: Scientific Reports

Article Title: HER2 G776S mutation promotes oncogenic potential in colorectal cancer cells when accompanied by loss of APC function

doi: 10.1038/s41598-022-13189-y

Figure Lengend Snippet: Schema of the relationship between HER2 mutations and APC mutations in colorectal cancer cells. HER2 G776S is a mutation that increases HER2 kinase activity, although the activity is weak. APC loss-of-function increases Wnt/β-catenin pathway activation but also increases the amount of RAS-GTP, which helps the HER2 G776S mutation to activate the HER2-ERK pathway.

Article Snippet: Wnt/β-catenin signaling activity was monitored using the TCF/LEF Reporter Kit (BPS Bioscience, San Diego, CA) following the manufacturer’s instructions.

Techniques: Mutagenesis, Activity Assay, Activation Assay

Journal: Frontiers in Oncology

Article Title: AXL Overexpression in Tumor-Derived Endothelial Cells Promotes Vessel Metastasis in Patients With Hepatocellular Carcinoma

doi: 10.3389/fonc.2021.650963

Figure Lengend Snippet: AXL/SOX2/DKK-1 axis in HUVECs promotes HCC metastasis. (A) DKK-1 and CCL14 secretion was significantly downregulated in CM from HUVEC-AXL-KD compared with that from HUVEC-AXL-NC, as detected with a human cytokine antibody array. (B) DKK-1 and CCL14 expression was markedly upregulated in the CM of HUVECs overexpressing AXL (CCL14: p < 0.001; DKK-1: p = 0.003) compared with the CM of HUVEC-AXL-NC, as detected by ELISA assay. (C) AXL siRNA downregulated DKK-1 and CCL14 secretion in the CM of HUVEC-AXL-NC and HUVEC-AXL-OE cells (CCL14: p < 0.001 and p < 0.001; DKK-1: p < 0.001 and p < 0.001). (D) DKK1 siRNA (MHCC-97L: p < 0.001 and p < 0.001; HCC-LM3: p <0.001 and p < 0.001), but not CCL14 siRNA (MHCC-97L: p = 0.126 and p = 0.711; HCC-LM3: p = 0.901 and p = 0.694) could attenuate the effect of the CM from HUVEC-AXL-NC and HUVEC-AXL-OE cells on the migration of HCC-LM3 cells and MHCC-97L cells. (E) SOX2 mRNA expression was significantly increased in HUVEC-AXL-OE cells and decreased in HUVEC-AXL-KD cells compared with HUVEC-AXL-NC cells (HUVEC-AXL-KD: p < 0.001, HUVEC-AXL-OE: p < 0.001). (F) AXL overexpression could significantly increase SOX2 and DKK-1 protein expression in HUVEC-AXL-OE cells compared with HUVEC-AXL-NC cells, and SOX2 siRNA inhibited SOX2 and DKK-1 protein expression in HUVEC-AXL-OE and HUVEC-AXL-NC cells.

Article Snippet: The protein concentrations of CCL14 and DKK-1 in the supernatants were also measured using an enzyme-linked immunosorbent assay (ELISA) kit (CCL14: EK1123 Boster, Wuhan, China; DKK-1: EK0867 Boster, Wuhan, China) according to the manufacturer’s instructions.

Techniques: Ab Array, Expressing, Enzyme-linked Immunosorbent Assay, Migration, Over Expression

Journal: BMC Bioinformatics

Article Title: PAGED: a pathway and gene-set enrichment database to enable molecular phenotype discoveries

doi: 10.1186/1471-2105-13-S15-S2

Figure Lengend Snippet: Top search results of colorectal cancer advanced search

Article Snippet: Protein Lounge suggests "Molecular Mechanisms of Cancer," "Akt Signaling," and other important pathways in colorectal cancer; BioCarta suggests "wnt signaling pathway"; and NCI Nature curated suggests "Canonical Wnt signaling pathway."

Techniques: Clinical Proteomics, Membrane

Journal: International Journal of Stem Cells

Article Title: Upregulation of C-Reactive Protein by Placenta-Derived Mesenchymal Stem Cells Promotes Angiogenesis in A Rat Model with Cirrhotic Liver

doi: 10.15283/ijsc20052

Figure Lengend Snippet: PD-MSC transplantation induces Wnt signaling and angiogene-sis in CCl 4 -injured rats. Angiogenic (A) and Wnt signaling (B) factors were detected by Western blotting. Localization and expression of VEGF and β -catenin visualized using immunofluorescence (C). ×630, Scale bars; 20 μ m. Positive correlations were found between CRP and β -catenin (D) also, between β -catenin and VEGF (E). NTX and TTX refer to the non-transplanted group and the PD-MSC-transplanted group, respec-tively.

Article Snippet: The suppression of the angiocrine Wnt signaling pathway exacerbated metabolic zonation in the liver through the loss of β -catenin-dependent genes such as Axin2, glutamine synthase, and cytochrome P450 2E1.

Techniques: Transplantation Assay, Western Blot, Expressing, Immunofluorescence

Journal: International Journal of Stem Cells

Article Title: Upregulation of C-Reactive Protein by Placenta-Derived Mesenchymal Stem Cells Promotes Angiogenesis in A Rat Model with Cirrhotic Liver

doi: 10.15283/ijsc20052

Figure Lengend Snippet: CRP regulates angiogenesis and Wnt signaling in rat hepatocytes (WB-F344s). Protein levels of CRP (A), VEGF (B), β -catenin (C), ALB (D), HNF1 α (E), and CyclinD1 (F) were evaluated in siRNA-CRP-transfected rat hepatocytes by western blot. Data are represented as the triplicated mean±SD of. *, p<0.05. Mock and si-CRP refer to non-transfected and siRNA-CRP-transfected rat hepatocytes, respectively.

Article Snippet: The suppression of the angiocrine Wnt signaling pathway exacerbated metabolic zonation in the liver through the loss of β -catenin-dependent genes such as Axin2, glutamine synthase, and cytochrome P450 2E1.

Techniques: Transfection, Western Blot

Journal: International Journal of Stem Cells

Article Title: Upregulation of C-Reactive Protein by Placenta-Derived Mesenchymal Stem Cells Promotes Angiogenesis in A Rat Model with Cirrhotic Liver

doi: 10.15283/ijsc20052

Figure Lengend Snippet: PD-MSCs promote the expression of CRP and proliferation of hepatocytes through Wnt signaling. Expression of CRP was analyzed by qRT-PCR (A) and ELISA (B) after CCl 4 treatment and co-culture with PD-MSCs. Immunofluorescence staining shows the localization and expression of BrdU and β -catenin in rat hepatocytes treated with CCl 4 and co-cultured with PD-MSCs (C). ×400, Scale bars; 20 μ m. BrdU- or β -ca-tenin-(D) positive rat hepatocytes were quantified after immunofluore-scence staining. Protein levels of HNF1 α , and CyclinD1 were detected by western blotting in rat hepatocytes treated with CCl 4 and co-cultured with PD-MSCs (E). Intensity of HNF1 α (F) and Cyclin D1 (G) protein bands was calculated. Tripli-cated data are represented as the mean±SD. *, p<0.05.

Article Snippet: The suppression of the angiocrine Wnt signaling pathway exacerbated metabolic zonation in the liver through the loss of β -catenin-dependent genes such as Axin2, glutamine synthase, and cytochrome P450 2E1.

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Immunofluorescence, Staining, Cell Culture, Western Blot