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    Structured Review

    ATCC human crc cell lines
    Knockdown of NPEPL1 rescues the carcinogenesis of LINC00342 on <t>CRC</t> progression. The expression of NPEPL1 and LINC00342 in NPEPL1 and LINC00342-overexpressed <t>SW480</t> cells ( a ); ** P
    Human Crc Cell Lines, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "LncRNA LINC00342 contributes to the growth and metastasis of colorectal cancer via targeting miR-19a-3p/NPEPL1 axis"

    Article Title: LncRNA LINC00342 contributes to the growth and metastasis of colorectal cancer via targeting miR-19a-3p/NPEPL1 axis

    Journal: Cancer Cell International

    doi: 10.1186/s12935-020-01705-x

    Knockdown of NPEPL1 rescues the carcinogenesis of LINC00342 on CRC progression. The expression of NPEPL1 and LINC00342 in NPEPL1 and LINC00342-overexpressed SW480 cells ( a ); ** P
    Figure Legend Snippet: Knockdown of NPEPL1 rescues the carcinogenesis of LINC00342 on CRC progression. The expression of NPEPL1 and LINC00342 in NPEPL1 and LINC00342-overexpressed SW480 cells ( a ); ** P

    Techniques Used: Expressing

    LINC00342 is significantly upregulated in CRC tissues and cells. a Expression of LINC00342 in non-tumor tissues and CRC tumor tissues determined by real-time PCR; b expression of LINC00342 in colonic epithelial NCM460 cells and four CRC cells (HCT-8, SW480, HT-29 and DLD-1); c subcellular fractionation assay of LINC00342 in SW480 and HT-29 cells, analyzed by RNA-FISH. ** P
    Figure Legend Snippet: LINC00342 is significantly upregulated in CRC tissues and cells. a Expression of LINC00342 in non-tumor tissues and CRC tumor tissues determined by real-time PCR; b expression of LINC00342 in colonic epithelial NCM460 cells and four CRC cells (HCT-8, SW480, HT-29 and DLD-1); c subcellular fractionation assay of LINC00342 in SW480 and HT-29 cells, analyzed by RNA-FISH. ** P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Fractionation, Fluorescence In Situ Hybridization

    LINC00342 requires for the proliferation, migration and invasion of CRC cells. SW480 and HT-29 cells were infected with lentivirus containing sh-NC or sh-LINC00342. The transfection efficiency was confirmed by real-time PCR ( a ) and green fluorescence microscopy ( b ); cell proliferation was measured with CCK-8 ( c ) and colony formation ( d ) assays; cell migration and invasion was determined by wound healing assay ( e ) and transwell migration and invasion assays ( f ); the protein levels of E-cadherin and Vimentin were measured by western blotting ( g ). * P
    Figure Legend Snippet: LINC00342 requires for the proliferation, migration and invasion of CRC cells. SW480 and HT-29 cells were infected with lentivirus containing sh-NC or sh-LINC00342. The transfection efficiency was confirmed by real-time PCR ( a ) and green fluorescence microscopy ( b ); cell proliferation was measured with CCK-8 ( c ) and colony formation ( d ) assays; cell migration and invasion was determined by wound healing assay ( e ) and transwell migration and invasion assays ( f ); the protein levels of E-cadherin and Vimentin were measured by western blotting ( g ). * P

    Techniques Used: Migration, Infection, Transfection, Real-time Polymerase Chain Reaction, Fluorescence, Microscopy, CCK-8 Assay, Wound Healing Assay, Western Blot

    MiR-19a-3p inhibits CRC cell proliferation, migration and invasion. The proliferation ( a , b ), migration and invasion abilities ( c , d ) and the protein levels of E-cadherin and Vimentin (E) in SW480 and HT-29 cells transfected with mimic NC or miR-19a-3p mimic. ** P
    Figure Legend Snippet: MiR-19a-3p inhibits CRC cell proliferation, migration and invasion. The proliferation ( a , b ), migration and invasion abilities ( c , d ) and the protein levels of E-cadherin and Vimentin (E) in SW480 and HT-29 cells transfected with mimic NC or miR-19a-3p mimic. ** P

    Techniques Used: Migration, Transfection

    2) Product Images from "miR-150-5p suppresses tumor progression by targeting VEGFA in colorectal cancer"

    Article Title: miR-150-5p suppresses tumor progression by targeting VEGFA in colorectal cancer

    Journal: Aging (Albany NY)

    doi: 10.18632/aging.101656

    miR-150-5p inhibited CRC progression by targeting VEGFA. ( A ) VEGFA protein expression was determined in HCT116 and HCT8 cells transfected with agomiR-150-5p with VEGFA expression plasmid or empty vector using western blot; GAPDH was used as the internal control. ( B - D ) Cell proliferation ( B , C ), migration ( D )and invasion ( E ) were evaluated in HCT116 and HCT8 cells transfected with agomiR-150-5p with VEGFA expression plasmid or empty vector. ( F ) HUVECs were cultured in TCM derived from HCT116 and HCT8 cells transfected with agomiR-150-5p plus VEGFA expression plasmid or empty vector. Data are shown as the mean±SD. * p
    Figure Legend Snippet: miR-150-5p inhibited CRC progression by targeting VEGFA. ( A ) VEGFA protein expression was determined in HCT116 and HCT8 cells transfected with agomiR-150-5p with VEGFA expression plasmid or empty vector using western blot; GAPDH was used as the internal control. ( B - D ) Cell proliferation ( B , C ), migration ( D )and invasion ( E ) were evaluated in HCT116 and HCT8 cells transfected with agomiR-150-5p with VEGFA expression plasmid or empty vector. ( F ) HUVECs were cultured in TCM derived from HCT116 and HCT8 cells transfected with agomiR-150-5p plus VEGFA expression plasmid or empty vector. Data are shown as the mean±SD. * p

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Western Blot, Migration, Cell Culture, Derivative Assay

    A. VEGFA was a direct target of miR-150-5p in CRC. ( A ) The direct target genes of miR-150-5p were predicted using the PicTarSites, miRandaSites and Tarbase databases. ( B ) Wild-type and mutant VEGFA-3’UTR sequences were cloned into luciferase reporter. Luciferase activity was determined in HCT116 and 293T cells cotransfected with agomiR-150-5p or agomiR-NC and pmirGLO-VEGFA-3’UTR-WT or pmirGLO-VEGFA-3’UTR-Mut. Luciferase activities were normalized to that of renilla luciferase. C, D. qRT-PCR ( C ) and western blot ( D ) analyses showed that both VEGFA mRNA and protein expression levels were dramatically suppressed by agomiR-150-5p in HCT116 and HCT8 cells, GAPDH was used as the internal control. ** p
    Figure Legend Snippet: A. VEGFA was a direct target of miR-150-5p in CRC. ( A ) The direct target genes of miR-150-5p were predicted using the PicTarSites, miRandaSites and Tarbase databases. ( B ) Wild-type and mutant VEGFA-3’UTR sequences were cloned into luciferase reporter. Luciferase activity was determined in HCT116 and 293T cells cotransfected with agomiR-150-5p or agomiR-NC and pmirGLO-VEGFA-3’UTR-WT or pmirGLO-VEGFA-3’UTR-Mut. Luciferase activities were normalized to that of renilla luciferase. C, D. qRT-PCR ( C ) and western blot ( D ) analyses showed that both VEGFA mRNA and protein expression levels were dramatically suppressed by agomiR-150-5p in HCT116 and HCT8 cells, GAPDH was used as the internal control. ** p

    Techniques Used: Mutagenesis, Clone Assay, Luciferase, Activity Assay, Quantitative RT-PCR, Western Blot, Expressing

    VEGFA knockdown significantly inhibited CRC progression. ( A ) VEGFA expression was downregulated in HCT116 and HCT8 cells transfected with siVEGFA-1 or siVEGFA-2. ( B ) VEGFA knockdown inhibited CRC cell proliferation ( B ), migration ( C ), invasion ( D ) and HUVECs tube formation ( E ). Data are shown as the mean±SD of three independent experiments. * p
    Figure Legend Snippet: VEGFA knockdown significantly inhibited CRC progression. ( A ) VEGFA expression was downregulated in HCT116 and HCT8 cells transfected with siVEGFA-1 or siVEGFA-2. ( B ) VEGFA knockdown inhibited CRC cell proliferation ( B ), migration ( C ), invasion ( D ) and HUVECs tube formation ( E ). Data are shown as the mean±SD of three independent experiments. * p

    Techniques Used: Expressing, Transfection, Migration

    3) Product Images from "An Integrated Network, RNA Sequencing, and Experiment Pharmacology Approach Reveals the Active Component, Potential Target, and Mechanism of Gelsemium elegans in the Treatment of Colorectal Cancer"

    Article Title: An Integrated Network, RNA Sequencing, and Experiment Pharmacology Approach Reveals the Active Component, Potential Target, and Mechanism of Gelsemium elegans in the Treatment of Colorectal Cancer

    Journal: Frontiers in Oncology

    doi: 10.3389/fonc.2020.616628

    KM promoted CRC cells apoptosis in a dose-dependent manner. (A) Flow cytometric analysis of HCT116 and HT29 cell apoptosis after incubation with KM for 24 h. (B) Western blot and quantification of proteins associated with apoptosis (Bcl-2, Bax, Cyt-C and caspase 3) in HCT116 and HT29 cells after incubation with KM for 24 h. Data were presented as mean ± SD (n = 3); significance: * P
    Figure Legend Snippet: KM promoted CRC cells apoptosis in a dose-dependent manner. (A) Flow cytometric analysis of HCT116 and HT29 cell apoptosis after incubation with KM for 24 h. (B) Western blot and quantification of proteins associated with apoptosis (Bcl-2, Bax, Cyt-C and caspase 3) in HCT116 and HT29 cells after incubation with KM for 24 h. Data were presented as mean ± SD (n = 3); significance: * P

    Techniques Used: Incubation, Western Blot

    4) Product Images from "CREPT facilitates colorectal cancer growth through inducing Wnt/β-catenin pathway by enhancing p300-mediated β-catenin acetylation"

    Article Title: CREPT facilitates colorectal cancer growth through inducing Wnt/β-catenin pathway by enhancing p300-mediated β-catenin acetylation

    Journal: Oncogene

    doi: 10.1038/s41388-018-0161-z

    CREPT regulated Wnt/β-catenin signaling pathway in CRC cells. a KEGG analysis based on RNA-sequencing data of HCT116 cells stably overexpressing CREPT or control cells. b Ectopic expression of CREPT in DLD-1 cell line enhanced TopFlash luciferase reporter activity but not FopFlash reporter ( b1 ), while depletion of CREPT reduced dual-luciferase reporter assays in SW480 cells ( b2 ). TopFlash, a classic Wnt-response luciferase reporter containing 3xTCF4 binding site. FopFlash, a negative reporter of which TCF4 binding site is mutant. c The mRNA expression of Wnt downstream targets CCND1 , c-MYC , and AXIN2 were enhanced in the presence of CREPT in DLD-1 cells and HCT116 cells. d The mRNA expression of CCND1 , c-MYC , and AXIN2 was diminished under CREPT knockdown in HT29 and SW480 cells. e The protein expression of β-catenin, c-MYC, CYCLIN D1, and AXIN2 was increased upon ectopic expression of CREPT in DLD1, HCT116 ( e1 ), HCEC 1CT, and 2CT cells ( e2 ), while decreased in CREPT knockdown HT29 and SW480 cells ( e3 ).
    Figure Legend Snippet: CREPT regulated Wnt/β-catenin signaling pathway in CRC cells. a KEGG analysis based on RNA-sequencing data of HCT116 cells stably overexpressing CREPT or control cells. b Ectopic expression of CREPT in DLD-1 cell line enhanced TopFlash luciferase reporter activity but not FopFlash reporter ( b1 ), while depletion of CREPT reduced dual-luciferase reporter assays in SW480 cells ( b2 ). TopFlash, a classic Wnt-response luciferase reporter containing 3xTCF4 binding site. FopFlash, a negative reporter of which TCF4 binding site is mutant. c The mRNA expression of Wnt downstream targets CCND1 , c-MYC , and AXIN2 were enhanced in the presence of CREPT in DLD-1 cells and HCT116 cells. d The mRNA expression of CCND1 , c-MYC , and AXIN2 was diminished under CREPT knockdown in HT29 and SW480 cells. e The protein expression of β-catenin, c-MYC, CYCLIN D1, and AXIN2 was increased upon ectopic expression of CREPT in DLD1, HCT116 ( e1 ), HCEC 1CT, and 2CT cells ( e2 ), while decreased in CREPT knockdown HT29 and SW480 cells ( e3 ).

    Techniques Used: RNA Sequencing Assay, Stable Transfection, Expressing, Luciferase, Activity Assay, Binding Assay, Mutagenesis

    CREPT enhanced CRC metastasis in vitro and in vivo. Ectopic expression of CREPT-promoted cell invasion a and migration b in HCT116 cells. Knockdown of CREPT suppressed cell invasion c and migration d in SW480 cells. e Ectopic expression of CREPT accelerated lung metastasis of HCT116 cells injected via tail vein. Pictures on the left showed representative lung morphology. HE staining showed representative lungs section. Histograph showed statistic result of metastasis nodules in nude mice lung tissues. n = 5, * P
    Figure Legend Snippet: CREPT enhanced CRC metastasis in vitro and in vivo. Ectopic expression of CREPT-promoted cell invasion a and migration b in HCT116 cells. Knockdown of CREPT suppressed cell invasion c and migration d in SW480 cells. e Ectopic expression of CREPT accelerated lung metastasis of HCT116 cells injected via tail vein. Pictures on the left showed representative lung morphology. HE staining showed representative lungs section. Histograph showed statistic result of metastasis nodules in nude mice lung tissues. n = 5, * P

    Techniques Used: In Vitro, In Vivo, Expressing, Migration, Injection, Staining, Mouse Assay

    5) Product Images from "TLE4 promotes colorectal cancer progression through activation of JNK/c-Jun signaling pathway"

    Article Title: TLE4 promotes colorectal cancer progression through activation of JNK/c-Jun signaling pathway

    Journal: Oncotarget

    doi: 10.18632/oncotarget.6694

    TLE4 activates JNK-c-Jun pathway in CRC cells ( A ) TLE4 increases the JNK-c-Jun pathway activity and expression of cyclin D1 and P27Kip1 in CRC cells. ( B ) SW480/TLE4 and HT29/TLE4 cells were treated with JNK inhibitor SP600125 (10 μM) for 24 h. Inhibition of the JNK signaling inhibits the promoting effect of TLE4-overexpression on JNK-c-Jun activity in CRC cells. ( C , D , E and F ) Inhibition of the JNK signaling blocks the promoting effect of TLE4-overexpression on cell proliferation and invasion of CRC cells as determined by MTT assay (C), colony formation assay (D), soft agar assay (E) and migration assay (F) after treatment with SP600125 (10 μM). Error bars represent mean ± SD from 3 independent experiments; * P
    Figure Legend Snippet: TLE4 activates JNK-c-Jun pathway in CRC cells ( A ) TLE4 increases the JNK-c-Jun pathway activity and expression of cyclin D1 and P27Kip1 in CRC cells. ( B ) SW480/TLE4 and HT29/TLE4 cells were treated with JNK inhibitor SP600125 (10 μM) for 24 h. Inhibition of the JNK signaling inhibits the promoting effect of TLE4-overexpression on JNK-c-Jun activity in CRC cells. ( C , D , E and F ) Inhibition of the JNK signaling blocks the promoting effect of TLE4-overexpression on cell proliferation and invasion of CRC cells as determined by MTT assay (C), colony formation assay (D), soft agar assay (E) and migration assay (F) after treatment with SP600125 (10 μM). Error bars represent mean ± SD from 3 independent experiments; * P

    Techniques Used: Activity Assay, Expressing, Inhibition, Over Expression, MTT Assay, Colony Assay, Soft Agar Assay, Migration

    Up-regulation of TLE4 promotes cell proliferation and invasion activity of CRC cells ( A ) Overexpression of TLE4 in SW480 and HT29 cells analyzed by Western blotting. α-Tubulin was used as a loading control. ( B and C ) Overexpression of TLE4 promotes SW480 and HT29 cell proliferation in MTT assays (B) and colony formation assays (C). ( D ) Ectopic expression of TLE4 promotes anchorage independent growth ability of SW480 and HT29 cells as determined by Soft agar assays. Colonies containing more than 50 cells were scored. Each bar represents the mean ± SD of 3 independent experiments. ( E ) The invasive abilities of CRC cells evaluated using the Matrigel-coated Boyden chamber invasion assay. Each bar represents the mean ± SD of three independent experiments ( F ) Xenograft model was built by injected SW480/Vector and SW480/TLE4 cells in nude mice ( n = 5/group). Tumor volumes were measured on the indicated days. Data points are the mean tumor volumes ± SD. ( G ) The sections of tumor were under H E staining or subjected to IHC staining using an antibody against Ki-67. ( H ) The expression of TLE4 in xenograft tumor was analyzed with Real-time PCR. Error bar represents the mean ± SD.
    Figure Legend Snippet: Up-regulation of TLE4 promotes cell proliferation and invasion activity of CRC cells ( A ) Overexpression of TLE4 in SW480 and HT29 cells analyzed by Western blotting. α-Tubulin was used as a loading control. ( B and C ) Overexpression of TLE4 promotes SW480 and HT29 cell proliferation in MTT assays (B) and colony formation assays (C). ( D ) Ectopic expression of TLE4 promotes anchorage independent growth ability of SW480 and HT29 cells as determined by Soft agar assays. Colonies containing more than 50 cells were scored. Each bar represents the mean ± SD of 3 independent experiments. ( E ) The invasive abilities of CRC cells evaluated using the Matrigel-coated Boyden chamber invasion assay. Each bar represents the mean ± SD of three independent experiments ( F ) Xenograft model was built by injected SW480/Vector and SW480/TLE4 cells in nude mice ( n = 5/group). Tumor volumes were measured on the indicated days. Data points are the mean tumor volumes ± SD. ( G ) The sections of tumor were under H E staining or subjected to IHC staining using an antibody against Ki-67. ( H ) The expression of TLE4 in xenograft tumor was analyzed with Real-time PCR. Error bar represents the mean ± SD.

    Techniques Used: Activity Assay, Over Expression, Western Blot, MTT Assay, Expressing, Invasion Assay, Injection, Plasmid Preparation, Mouse Assay, Staining, Immunohistochemistry, Real-time Polymerase Chain Reaction

    6) Product Images from "HDAC8 Promotes Liver Metastasis of Colorectal Cancer via Inhibition of IRF1 and Upregulation of SUCNR1"

    Article Title: HDAC8 Promotes Liver Metastasis of Colorectal Cancer via Inhibition of IRF1 and Upregulation of SUCNR1

    Journal: Oxidative Medicine and Cellular Longevity

    doi: 10.1155/2022/2815187

    SUCNR1 promotes the migration and invasion of CRC cells by blunting tumor cell autophagy. (a) SUCNR1 mRNA expression in HCT-116 cells treated with sh-SUCNR1 determined by RT-qPCR. (b) Viability of HCT-116 cells following SUCNR1 knockdown or combined with 3-MA measured by CCK-8 assay. (c) Migration of HCT-116 cells following SUCNR1 knockdown or combined with 3-MA measured by Transwell assay. (d) Invasion of HCT-116 cells following SUCNR1 knockdown or combined with 3-MA measured by Transwell assay. (e) Flow cytometric analysis of the HCT-116 cell apoptosis following SUCNR1 knockdown or combined with 3-MA. (f) Western blot analysis of LC3-II/LC3-I ratio in HCT-116 cells following SUCNR1 knockdown or combined with 3-MA. (g) Immunofluorescence detection of the number of GFP-LC3 spots in HCT-116 cells following SUCNR1 knockdown or combined with 3-MA (scale bar = 50 μ m). (h) Number of autophagic vacuoles in HCT-116 cells following SUCNR1 knockdown or combined with 3-MA under a TEM. ∗ p
    Figure Legend Snippet: SUCNR1 promotes the migration and invasion of CRC cells by blunting tumor cell autophagy. (a) SUCNR1 mRNA expression in HCT-116 cells treated with sh-SUCNR1 determined by RT-qPCR. (b) Viability of HCT-116 cells following SUCNR1 knockdown or combined with 3-MA measured by CCK-8 assay. (c) Migration of HCT-116 cells following SUCNR1 knockdown or combined with 3-MA measured by Transwell assay. (d) Invasion of HCT-116 cells following SUCNR1 knockdown or combined with 3-MA measured by Transwell assay. (e) Flow cytometric analysis of the HCT-116 cell apoptosis following SUCNR1 knockdown or combined with 3-MA. (f) Western blot analysis of LC3-II/LC3-I ratio in HCT-116 cells following SUCNR1 knockdown or combined with 3-MA. (g) Immunofluorescence detection of the number of GFP-LC3 spots in HCT-116 cells following SUCNR1 knockdown or combined with 3-MA (scale bar = 50 μ m). (h) Number of autophagic vacuoles in HCT-116 cells following SUCNR1 knockdown or combined with 3-MA under a TEM. ∗ p

    Techniques Used: Migration, Expressing, Quantitative RT-PCR, CCK-8 Assay, Transwell Assay, Western Blot, Immunofluorescence, Transmission Electron Microscopy

    HDAC8 suppresses cell autophagy to boost the migration and invasion of CRC cells by regulating the IRF1/SUCNR1 axis. HCT-116 cells were transfected with sh-HDAC8 or combined with oe-SUCNR1. (a) SUCNR1 mRNA expression in HCT-116 cells determined by RT-qPCR. (b) Viability of HCT-116 cells measured by CCK-8 assay. (c) Migration of HCT-116 cells measured by Transwell assay. (d) Invasion of HCT-116 cells measured by Transwell assay. (e) Western blot analysis of LC3-II/LC3-I ratio in HCT-116 cells. (f) Immunofluorescence detection of the number of GFP-LC3 spots in HCT-116 cells (scale bar = 50 μ m). (g) Number of autophagic vacuoles in HCT-116 cells under a TEM. ∗ p
    Figure Legend Snippet: HDAC8 suppresses cell autophagy to boost the migration and invasion of CRC cells by regulating the IRF1/SUCNR1 axis. HCT-116 cells were transfected with sh-HDAC8 or combined with oe-SUCNR1. (a) SUCNR1 mRNA expression in HCT-116 cells determined by RT-qPCR. (b) Viability of HCT-116 cells measured by CCK-8 assay. (c) Migration of HCT-116 cells measured by Transwell assay. (d) Invasion of HCT-116 cells measured by Transwell assay. (e) Western blot analysis of LC3-II/LC3-I ratio in HCT-116 cells. (f) Immunofluorescence detection of the number of GFP-LC3 spots in HCT-116 cells (scale bar = 50 μ m). (g) Number of autophagic vacuoles in HCT-116 cells under a TEM. ∗ p

    Techniques Used: Migration, Transfection, Expressing, Quantitative RT-PCR, CCK-8 Assay, Transwell Assay, Western Blot, Immunofluorescence, Transmission Electron Microscopy

    HDAC8 suppresses the expression of IRF1 and thus facilitates the growth and metastasis of CRC cells. (a) HDAC8 and IRF1 mRNA expression in CRC and adjacent normal tissues determined by RT-qPCR ( n = 58). (b) HDAC8 and IRF1 mRNA expression in SW480, SW620, HT29, HCT-116, and FHC cell lines determined by RT-qPCR. (c) Histone acetylation levels in the IRF1 promoter region in CRC and adjacent normal tissues determined by ChIP. (d) mRNA expression of HDAC8 and IRF1 in HCT-116 cells treated with sh-HDAC8 or oe-HDAC8 determined by RT-qPCR. (e) Western blot analysis of HDAC8 and IRF1 proteins in HCT-116 cells treated with sh-HDAC8 or oe-HDAC8. (f) mRNA expression of HDAC8 and IRF1 in HCT-116 cells treated with PCI-34051 determined by RT-qPCR. (g) H3K9Ac levels in the IRF1 promoter region in HCT-116 cells treated with PCI-34051 determined by ChIP. (h) HDAC8 and IRF1 mRNA expression in HCT-116 cells treated with sh-HDAC8 or combined with sh-IRF1 determined by RT-qPCR. (i) Viability of HCT-116 cells treated with sh-HDAC8 or combined with sh-IRF1 measured by CCK-8 assay. (j) Migration of HCT-116 cells treated with sh-HDAC8 or combined with sh-IRF1 measured by Transwell assay. (k) Invasion of HCT-116 cells treated with sh-HDAC8 or combined with sh-IRF1 measured by Transwell assay. ∗ p
    Figure Legend Snippet: HDAC8 suppresses the expression of IRF1 and thus facilitates the growth and metastasis of CRC cells. (a) HDAC8 and IRF1 mRNA expression in CRC and adjacent normal tissues determined by RT-qPCR ( n = 58). (b) HDAC8 and IRF1 mRNA expression in SW480, SW620, HT29, HCT-116, and FHC cell lines determined by RT-qPCR. (c) Histone acetylation levels in the IRF1 promoter region in CRC and adjacent normal tissues determined by ChIP. (d) mRNA expression of HDAC8 and IRF1 in HCT-116 cells treated with sh-HDAC8 or oe-HDAC8 determined by RT-qPCR. (e) Western blot analysis of HDAC8 and IRF1 proteins in HCT-116 cells treated with sh-HDAC8 or oe-HDAC8. (f) mRNA expression of HDAC8 and IRF1 in HCT-116 cells treated with PCI-34051 determined by RT-qPCR. (g) H3K9Ac levels in the IRF1 promoter region in HCT-116 cells treated with PCI-34051 determined by ChIP. (h) HDAC8 and IRF1 mRNA expression in HCT-116 cells treated with sh-HDAC8 or combined with sh-IRF1 determined by RT-qPCR. (i) Viability of HCT-116 cells treated with sh-HDAC8 or combined with sh-IRF1 measured by CCK-8 assay. (j) Migration of HCT-116 cells treated with sh-HDAC8 or combined with sh-IRF1 measured by Transwell assay. (k) Invasion of HCT-116 cells treated with sh-HDAC8 or combined with sh-IRF1 measured by Transwell assay. ∗ p

    Techniques Used: Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Western Blot, CCK-8 Assay, Migration, Transwell Assay

    Molecular mechanism by which HDAC8 regulates the progression of CRC. Histone deacetylase HDAC8 upregulates SUCNR1 by downregulating IRF1 and consequently inhibits CRC cell autophagy, ultimately contributing to the CRC growth and liver metastasis.
    Figure Legend Snippet: Molecular mechanism by which HDAC8 regulates the progression of CRC. Histone deacetylase HDAC8 upregulates SUCNR1 by downregulating IRF1 and consequently inhibits CRC cell autophagy, ultimately contributing to the CRC growth and liver metastasis.

    Techniques Used: Histone Deacetylase Assay

    IRF1 downregulates the expression of SUCNR1 by binding to its promoter in CRC cells. (a) SUCNR1 mRNA expression in CRC and adjacent normal tissues determined by RT-qPCR ( n = 58). (b) SUCNR1 mRNA expression in SW480, SW620, HT29, HCT-116, and FHC cell lines determined by RT-qPCR. (c) Prediction of IRF1 binding sites in the SUCNR1 promoter and mutation sequence generated by site mutation determined by ChIP. (d) Binding between IRF1 and SUCNR1 confirmed by dual-luciferase reporter assay. (e) Enrichment of IRF1 in the promoter of SUCNR1 determined by ChIP. (f) IRF1 and SUCNR1 mRNA expression in HCT-116 cells treated with sh-IRF1 or oe-IRF1 determined by RT-qPCR. (g) Western blot analysis of IRF1 and SUCNR1 proteins in HCT-116 cells treated with sh-IRF1 or oe-IRF1. ∗∗ p
    Figure Legend Snippet: IRF1 downregulates the expression of SUCNR1 by binding to its promoter in CRC cells. (a) SUCNR1 mRNA expression in CRC and adjacent normal tissues determined by RT-qPCR ( n = 58). (b) SUCNR1 mRNA expression in SW480, SW620, HT29, HCT-116, and FHC cell lines determined by RT-qPCR. (c) Prediction of IRF1 binding sites in the SUCNR1 promoter and mutation sequence generated by site mutation determined by ChIP. (d) Binding between IRF1 and SUCNR1 confirmed by dual-luciferase reporter assay. (e) Enrichment of IRF1 in the promoter of SUCNR1 determined by ChIP. (f) IRF1 and SUCNR1 mRNA expression in HCT-116 cells treated with sh-IRF1 or oe-IRF1 determined by RT-qPCR. (g) Western blot analysis of IRF1 and SUCNR1 proteins in HCT-116 cells treated with sh-IRF1 or oe-IRF1. ∗∗ p

    Techniques Used: Expressing, Binding Assay, Quantitative RT-PCR, Mutagenesis, Sequencing, Generated, Chromatin Immunoprecipitation, Luciferase, Reporter Assay, Western Blot

    Significance of the HDAC8/IRF1/SUCNR1 axis in CRC. (a) A box plot of the differential expression of HDAC8 in the colon adenocarcinoma (COAD) and rectum adenocarcinoma (READ) samples included in TCGA and GTEx (red box plots represent tumor samples, and gray box plots represent normal samples; in COAD, there are 275 tumor samples and 349 normal samples; in READ, there are 92 tumor samples and 318 normal samples). (b) Venn diagram of HDAC8 downstream regulatory genes and transcription factors (the left is the downstream genes of HDAC8 predicted by the starBase database, the right is the transcription factor annotation, and the center represents the intersection of the two sets of data). (c) Interaction analysis of the candidate transcription factors; each circle in the figure represents a gene, and the line between circles indicates interaction between two genes; the darker color of the circle where the gene is located reflects more interaction genes, higher core degree in the interaction network, and higher degree value. (d) Statistics of degree value of core genes in the gene interaction network (the abscissa represents the degree value and the ordinate represents the gene name). (e) KEGG enrichment analysis of the candidate transcription factors (the abscissa represents the gene ratio, the ordinate represents the KEGG entry identifier, and the histogram on the right is the color scale). (f) A box plot of the differential expression of SUCNR1 in the CRC included in TCGA and GTEx (red box plots represent tumor samples, and gray box plots represent normal samples; in COAD, there are 275 tumor samples and 349 normal samples; in READ, there are 92 tumor samples and 318 normal samples). ∗ p
    Figure Legend Snippet: Significance of the HDAC8/IRF1/SUCNR1 axis in CRC. (a) A box plot of the differential expression of HDAC8 in the colon adenocarcinoma (COAD) and rectum adenocarcinoma (READ) samples included in TCGA and GTEx (red box plots represent tumor samples, and gray box plots represent normal samples; in COAD, there are 275 tumor samples and 349 normal samples; in READ, there are 92 tumor samples and 318 normal samples). (b) Venn diagram of HDAC8 downstream regulatory genes and transcription factors (the left is the downstream genes of HDAC8 predicted by the starBase database, the right is the transcription factor annotation, and the center represents the intersection of the two sets of data). (c) Interaction analysis of the candidate transcription factors; each circle in the figure represents a gene, and the line between circles indicates interaction between two genes; the darker color of the circle where the gene is located reflects more interaction genes, higher core degree in the interaction network, and higher degree value. (d) Statistics of degree value of core genes in the gene interaction network (the abscissa represents the degree value and the ordinate represents the gene name). (e) KEGG enrichment analysis of the candidate transcription factors (the abscissa represents the gene ratio, the ordinate represents the KEGG entry identifier, and the histogram on the right is the color scale). (f) A box plot of the differential expression of SUCNR1 in the CRC included in TCGA and GTEx (red box plots represent tumor samples, and gray box plots represent normal samples; in COAD, there are 275 tumor samples and 349 normal samples; in READ, there are 92 tumor samples and 318 normal samples). ∗ p

    Techniques Used: Expressing

    HDAC8 promotes tumorigenesis and liver metastasis of CRC cells by regulating the IRF1/SUCNR1 axis in nude mice. (a) Tumor growth of mice treated with sh-HDAC8 or combined with oe-SUCNR1. (b) Ki67 immunohistochemical staining images of tumor tissues of nude mice treated with sh-HDAC8 or combined with oe-SUCNR1 as well as the semiquantitative analysis. (c) mRNA expression of HDAC8, IRF1, and SUCNR1 in tumor tissues of mice treated with sh-HDAC8 or combined with oe-SUCNR1 determined by RT-qPCR. (d) HE staining analysis of number of liver metastases in the liver tissues of nude mice treated with sh-HDAC8 or combined with oe-SUCNR1 (scale bar = 50 μ m). n = 10 for mice upon each treatment. ∗ p
    Figure Legend Snippet: HDAC8 promotes tumorigenesis and liver metastasis of CRC cells by regulating the IRF1/SUCNR1 axis in nude mice. (a) Tumor growth of mice treated with sh-HDAC8 or combined with oe-SUCNR1. (b) Ki67 immunohistochemical staining images of tumor tissues of nude mice treated with sh-HDAC8 or combined with oe-SUCNR1 as well as the semiquantitative analysis. (c) mRNA expression of HDAC8, IRF1, and SUCNR1 in tumor tissues of mice treated with sh-HDAC8 or combined with oe-SUCNR1 determined by RT-qPCR. (d) HE staining analysis of number of liver metastases in the liver tissues of nude mice treated with sh-HDAC8 or combined with oe-SUCNR1 (scale bar = 50 μ m). n = 10 for mice upon each treatment. ∗ p

    Techniques Used: Mouse Assay, Immunohistochemistry, Staining, Expressing, Quantitative RT-PCR

    7) Product Images from "Significance of the E3 ubiquitin protein UBR5 as an oncogene and a prognostic biomarker in colorectal cancer"

    Article Title: Significance of the E3 ubiquitin protein UBR5 as an oncogene and a prognostic biomarker in colorectal cancer

    Journal: Oncotarget

    doi: 10.18632/oncotarget.22531

    Knockdown of UBR5 reduces the growth of CRC xenografts in BALB/c nude mice The effect of UBR5 knockdown in SW480 and RKO cells on the xenograft model was assessed by evaluating the tumor volume (A, B) and weight (C) in a xenograft model. (D) The UBR5 expression levels were examined by qPCR and western blotting in the xenograft tumors with or without doxycycline-inducible UBR5 shRNA expression. The ctrl group was treated in the absence of doxycycline (Dox-), and the Dox group was supplied doxycycline (Dox+) in the drinking water. As lanes show, the Control group included the ctrl1, ctrl2, and ctrl3 xenograft tumor specimens, and the Dox group included the Dox1, Dox2, and Dox3 specimens. Ctrl, Control. All statistical tests were two-sided. * P
    Figure Legend Snippet: Knockdown of UBR5 reduces the growth of CRC xenografts in BALB/c nude mice The effect of UBR5 knockdown in SW480 and RKO cells on the xenograft model was assessed by evaluating the tumor volume (A, B) and weight (C) in a xenograft model. (D) The UBR5 expression levels were examined by qPCR and western blotting in the xenograft tumors with or without doxycycline-inducible UBR5 shRNA expression. The ctrl group was treated in the absence of doxycycline (Dox-), and the Dox group was supplied doxycycline (Dox+) in the drinking water. As lanes show, the Control group included the ctrl1, ctrl2, and ctrl3 xenograft tumor specimens, and the Dox group included the Dox1, Dox2, and Dox3 specimens. Ctrl, Control. All statistical tests were two-sided. * P

    Techniques Used: Mouse Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot, shRNA

    8) Product Images from "Bone marrow-derived mesenchymal stem cell-secreted IL-8 promotes the angiogenesis and growth of colorectal cancer"

    Article Title: Bone marrow-derived mesenchymal stem cell-secreted IL-8 promotes the angiogenesis and growth of colorectal cancer

    Journal: Oncotarget

    doi:

    MSCs promote tumor angiogenesis and growth in vivo A. A total of 2 × 10 6 CRC cells (SW480, LS174T and HT29) alone or together with 2 × 10 6 MSCs or shIL-8-MSCs was injected into the flanks of nude athymic mice ( n = 5). The tumors were harvested after 4 weeks and sectioned. The slides were stained with an anti-CD31 antibody for evaluating vessel morphology. Scale bar, 100 μm. B. MVD was determined by measuring the percentage of each field occupied by a CD31-positive signal. The vessel numbers per field were also counted. For each group, four randomly chosen high power fields per tumor ( n = 5) were analyzed (*, p
    Figure Legend Snippet: MSCs promote tumor angiogenesis and growth in vivo A. A total of 2 × 10 6 CRC cells (SW480, LS174T and HT29) alone or together with 2 × 10 6 MSCs or shIL-8-MSCs was injected into the flanks of nude athymic mice ( n = 5). The tumors were harvested after 4 weeks and sectioned. The slides were stained with an anti-CD31 antibody for evaluating vessel morphology. Scale bar, 100 μm. B. MVD was determined by measuring the percentage of each field occupied by a CD31-positive signal. The vessel numbers per field were also counted. For each group, four randomly chosen high power fields per tumor ( n = 5) were analyzed (*, p

    Techniques Used: In Vivo, Injection, Mouse Assay, Staining

    MSC-secreted IL-8 stimulates CRC cell proliferation A. The number of CRC cells (SW480, LS174T and HT29) following culture with fresh medium or conditioned medium from CRC cell/MSC co-cultures or CRC cell/shIL-8-MSC co-cultures was determined. The cell counts are presented as the mean values from three independent experiments using a hemocytometer (*, p
    Figure Legend Snippet: MSC-secreted IL-8 stimulates CRC cell proliferation A. The number of CRC cells (SW480, LS174T and HT29) following culture with fresh medium or conditioned medium from CRC cell/MSC co-cultures or CRC cell/shIL-8-MSC co-cultures was determined. The cell counts are presented as the mean values from three independent experiments using a hemocytometer (*, p

    Techniques Used:

    9) Product Images from "LncRNA-LINC00152 down-regulated by miR-376c-3p restricts viability and promotes apoptosis of colorectal cancer cells"

    Article Title: LncRNA-LINC00152 down-regulated by miR-376c-3p restricts viability and promotes apoptosis of colorectal cancer cells

    Journal: American Journal of Translational Research

    doi:

    miR-367c-3p enhances the growth of CRC cells by down-regulating LINC00152. CCK-8 (A, B) and apoptosis (C, D) assays were performed to analyze the viability and apoptosis of HT-29 and SW480 cells with or without co-overexpression of LINC00152 and/or miR-367c-3p. Data are expressed as means ± SEM. * P
    Figure Legend Snippet: miR-367c-3p enhances the growth of CRC cells by down-regulating LINC00152. CCK-8 (A, B) and apoptosis (C, D) assays were performed to analyze the viability and apoptosis of HT-29 and SW480 cells with or without co-overexpression of LINC00152 and/or miR-367c-3p. Data are expressed as means ± SEM. * P

    Techniques Used: CCK-8 Assay, Over Expression

    The expression of LINC00152 on CRC tissue and cell lines is aberrantly decreased. A-C. qRT-PCR analysis for LINC00152 expression in CRC tissues (n=49) and paired adjacent normal tissues. Ctrl: adjacent normal tissues; tumor: CRC tissues; I-II: patient with the early stage (TNM: I-II); III-IV: patient with advanced stage (TNM: III-IV). D. qRT-PCR analysis for LINC00152 expression in CRC cell lines (CACO2, HCT-116, HT-29, SW480 and SW620) and non-tumorigenic cell lines (CCC-HIE-2 and HER293). Data are expressed as means ± SEM. * P
    Figure Legend Snippet: The expression of LINC00152 on CRC tissue and cell lines is aberrantly decreased. A-C. qRT-PCR analysis for LINC00152 expression in CRC tissues (n=49) and paired adjacent normal tissues. Ctrl: adjacent normal tissues; tumor: CRC tissues; I-II: patient with the early stage (TNM: I-II); III-IV: patient with advanced stage (TNM: III-IV). D. qRT-PCR analysis for LINC00152 expression in CRC cell lines (CACO2, HCT-116, HT-29, SW480 and SW620) and non-tumorigenic cell lines (CCC-HIE-2 and HER293). Data are expressed as means ± SEM. * P

    Techniques Used: Expressing, Quantitative RT-PCR, Countercurrent Chromatography

    miR-367c-3p enhances viability and restricts apoptosis of CRC cells. CCK-8 (A, B) and apoptosis (C, D) assays were performed to analyze the viability and apoptosis of HT-29 and SW480 cells with or without miR-367c-3p overexpression. Data are expressed as means ± SEM. * P
    Figure Legend Snippet: miR-367c-3p enhances viability and restricts apoptosis of CRC cells. CCK-8 (A, B) and apoptosis (C, D) assays were performed to analyze the viability and apoptosis of HT-29 and SW480 cells with or without miR-367c-3p overexpression. Data are expressed as means ± SEM. * P

    Techniques Used: CCK-8 Assay, Over Expression

    miR-367c-3p negatively correlates with LINC00152 in CRC tissue and cells. A. qRT-PCR analysis for miR-367c-3p expression in CRC tissues (n=49) and adjacent normal tissues. B. Relationship analysis between miR-376c-3p and LINC00152 in CRC tissues (R 2 =0.511). C. Construction of miR-367c-3p overexpression in HT-29 and SW480 cells by transfection. Negative Ctrl: HT-29 or SW480 cells transfected with the control miRNA lentivirus (C-miRNA); miR-376c-3p-mimics: HT-29 or SW480 cells transfected with the miR-376c-3p mimic lentivirus. D. qRT-PCR analysis for miR-367c-3p expression in HT-29 and SW480 cells with or without miR-367c-3p overexpression. Data are expressed as means ± SEM. ** P
    Figure Legend Snippet: miR-367c-3p negatively correlates with LINC00152 in CRC tissue and cells. A. qRT-PCR analysis for miR-367c-3p expression in CRC tissues (n=49) and adjacent normal tissues. B. Relationship analysis between miR-376c-3p and LINC00152 in CRC tissues (R 2 =0.511). C. Construction of miR-367c-3p overexpression in HT-29 and SW480 cells by transfection. Negative Ctrl: HT-29 or SW480 cells transfected with the control miRNA lentivirus (C-miRNA); miR-376c-3p-mimics: HT-29 or SW480 cells transfected with the miR-376c-3p mimic lentivirus. D. qRT-PCR analysis for miR-367c-3p expression in HT-29 and SW480 cells with or without miR-367c-3p overexpression. Data are expressed as means ± SEM. ** P

    Techniques Used: Quantitative RT-PCR, Expressing, Over Expression, Transfection

    LINC00152 down-regulates Ki-67 and Bcl-2, and up-regulates Fas expression in CRC cells. The expression of Ki-67, Bcl-2, Bax and Fas in HT-29 (A, B) and SW480 (C, D) cells after transfection with pWPXL or pWPXL-LINC00152 was detected by FCM. MFI: Median Fluorescence Intensity. Data are expressed as means ± SEM. ** P
    Figure Legend Snippet: LINC00152 down-regulates Ki-67 and Bcl-2, and up-regulates Fas expression in CRC cells. The expression of Ki-67, Bcl-2, Bax and Fas in HT-29 (A, B) and SW480 (C, D) cells after transfection with pWPXL or pWPXL-LINC00152 was detected by FCM. MFI: Median Fluorescence Intensity. Data are expressed as means ± SEM. ** P

    Techniques Used: Expressing, Transfection, Fluorescence

    LINC00152 suppresses viability and promotes apoptosis of CRC cells. (A) The construction of LINC00152 overexpression in HT-29 and SW480 cells by transfection. CCK-8 (B) and apoptosis (C, D) assays were performed to analyze viability and apoptosis of HT-29 and SW480 cells with or without LINC00152 overexpression. pWPXL: HT-29 or SW480 cells transfected with the negative control lentivirus (Vector: pWPXL); pWPXL-LINC00152: HT-29 or SW480 cells transfected with lentiviruses expressing the LINC00152 sequence. Data are expressed as means ± SEM. * P
    Figure Legend Snippet: LINC00152 suppresses viability and promotes apoptosis of CRC cells. (A) The construction of LINC00152 overexpression in HT-29 and SW480 cells by transfection. CCK-8 (B) and apoptosis (C, D) assays were performed to analyze viability and apoptosis of HT-29 and SW480 cells with or without LINC00152 overexpression. pWPXL: HT-29 or SW480 cells transfected with the negative control lentivirus (Vector: pWPXL); pWPXL-LINC00152: HT-29 or SW480 cells transfected with lentiviruses expressing the LINC00152 sequence. Data are expressed as means ± SEM. * P

    Techniques Used: Over Expression, Transfection, CCK-8 Assay, Negative Control, Plasmid Preparation, Expressing, Sequencing

    10) Product Images from "Overexpression of the long non-coding RNA BLACAT1 promotes cell proliferation and invasion in colorectal cancer"

    Article Title: Overexpression of the long non-coding RNA BLACAT1 promotes cell proliferation and invasion in colorectal cancer

    Journal: Translational Cancer Research

    doi: 10.21037/tcr.2018.12.26

    BLACAT1 knockdown inhibits proliferation of CRC cells. The interference efficiency of si- BLACAT1 was verified in HCT116 cells. HCT116 cells were transfected with either si-NC or si-BLACAT1 (1#, 2#, 1+2#) for 48 h, following which BLACAT1 expression was analyzed using qRT-PCR (A) and CCK-8 assay (B). (C) The clonogenic assay was used to detect the proliferative ability after transfecting HCT116 cells with si-NC or si- BLACAT1 for 48 h. Data show mean ± SEM. *, P
    Figure Legend Snippet: BLACAT1 knockdown inhibits proliferation of CRC cells. The interference efficiency of si- BLACAT1 was verified in HCT116 cells. HCT116 cells were transfected with either si-NC or si-BLACAT1 (1#, 2#, 1+2#) for 48 h, following which BLACAT1 expression was analyzed using qRT-PCR (A) and CCK-8 assay (B). (C) The clonogenic assay was used to detect the proliferative ability after transfecting HCT116 cells with si-NC or si- BLACAT1 for 48 h. Data show mean ± SEM. *, P

    Techniques Used: Transfection, Expressing, Quantitative RT-PCR, CCK-8 Assay, Clonogenic Assay

    BLACAT1 silencing suppresses proliferation and expression of EMT markers in CRC. HCT116 cells were transfected with si- NC or si-BLACAT1-1 + si-BLACAT1-2 for 48 h, following which proliferation and expression of EMT markers were determined using western blotting and qRT-PCR. (A) Protein levels of cyclin D1 and CDK6 were determined using western blotting and (B) mRNA levels were quantitated using qRT-PCR. (C) Protein levels of E-cadherin and vimentin were analyzed using western blotting and (D) mRNA levels were determined using qRT-PCR. Data show mean ± SEM. *, P
    Figure Legend Snippet: BLACAT1 silencing suppresses proliferation and expression of EMT markers in CRC. HCT116 cells were transfected with si- NC or si-BLACAT1-1 + si-BLACAT1-2 for 48 h, following which proliferation and expression of EMT markers were determined using western blotting and qRT-PCR. (A) Protein levels of cyclin D1 and CDK6 were determined using western blotting and (B) mRNA levels were quantitated using qRT-PCR. (C) Protein levels of E-cadherin and vimentin were analyzed using western blotting and (D) mRNA levels were determined using qRT-PCR. Data show mean ± SEM. *, P

    Techniques Used: Expressing, Transfection, Western Blot, Quantitative RT-PCR

    BLACAT1 knockdown inhibits invasiveness of CRC cells. The cell invasion ability was determined using the transwell Matrigel assay after transfecting HCT116 cells with si-NC or si- BLACAT1 for 48 h. Cells that had invaded through the filter pores were stained with 5% crystal violet. The images were taken at ×200. Data show mean ± SEM. **, P
    Figure Legend Snippet: BLACAT1 knockdown inhibits invasiveness of CRC cells. The cell invasion ability was determined using the transwell Matrigel assay after transfecting HCT116 cells with si-NC or si- BLACAT1 for 48 h. Cells that had invaded through the filter pores were stained with 5% crystal violet. The images were taken at ×200. Data show mean ± SEM. **, P

    Techniques Used: Matrigel Assay, Staining

    BLACAT1 is highly expressed in CRC tissues and cell lines. BLACAT1 expression, as measured using Affymetrix microarray, was higher in CRC tissues than in normal colon mucosal tissues in (A) GSE22598 (consisting of 17 pairs of CRC tissues and corresponding normal colorectal tissues) samples from the GEO database, (B) GSE23878 (consisting of 24 normal colorectal tissues and 35 CRC tissue biopsies), and (C) GSE9348 (consisting of 12 normal colorectal tissues and 70 CRC tissue biopsies). (D) BLACAT1 expression increased significantly in CRC cell lines (SW480, SW620, HCT116, and CaCO 2 ) compared to in NCM460, a normal colon mucosal cell line. Data show mean ± SEM. *, P
    Figure Legend Snippet: BLACAT1 is highly expressed in CRC tissues and cell lines. BLACAT1 expression, as measured using Affymetrix microarray, was higher in CRC tissues than in normal colon mucosal tissues in (A) GSE22598 (consisting of 17 pairs of CRC tissues and corresponding normal colorectal tissues) samples from the GEO database, (B) GSE23878 (consisting of 24 normal colorectal tissues and 35 CRC tissue biopsies), and (C) GSE9348 (consisting of 12 normal colorectal tissues and 70 CRC tissue biopsies). (D) BLACAT1 expression increased significantly in CRC cell lines (SW480, SW620, HCT116, and CaCO 2 ) compared to in NCM460, a normal colon mucosal cell line. Data show mean ± SEM. *, P

    Techniques Used: Expressing, Microarray

    11) Product Images from "LncRNA LINC00963 promotes colorectal cancer cell proliferation and metastasis by regulating miR-1281 and TRIM65"

    Article Title: LncRNA LINC00963 promotes colorectal cancer cell proliferation and metastasis by regulating miR-1281 and TRIM65

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2021.12421

    LINC00963 expression is upregulated in CRC tissues and cell lines. (A) LINC00963 expression in 53 pairs of CRC tissues and adjacent tissues was detected by RT-qPCR. (B) LINC00963 expression in normal colonic epithelial cell line (NCM460 cells) and 4 CRC cell lines (HCT116, HT29, SW480 and LoVo cells) was detected by RT-qPCR. All experiments were performed in triplicate. **P
    Figure Legend Snippet: LINC00963 expression is upregulated in CRC tissues and cell lines. (A) LINC00963 expression in 53 pairs of CRC tissues and adjacent tissues was detected by RT-qPCR. (B) LINC00963 expression in normal colonic epithelial cell line (NCM460 cells) and 4 CRC cell lines (HCT116, HT29, SW480 and LoVo cells) was detected by RT-qPCR. All experiments were performed in triplicate. **P

    Techniques Used: Expressing, Quantitative RT-PCR

    miR-1281 is a target of LINC00963 in CRC. (A) LINC00963-WT and LINC00963-MUT luciferase reporter plasmids containing the binding site for miR-1281 were constructed. (B) Targeting relationship between miR-1281 and LINC00963 was validated by a dual-luciferase reporter assay. (C) RNA pull-down and (D) RNA immunoprecipitation assays were performed to confirm that LINC00963 interacted with miR-1281 directly. (E) Expression level of miR-1281 in LoVo cells with LINC00963 overexpression and HCT116 cells with LINC00963 knockdown was detected by RT-qPCR. (F) miR-1281 expression in 53 pairs of CRC and adjacent tissues was detected by RT-qPCR. (G) The correlation between miR-1281 and LINC00963 expressions in CRC tissues was analyzed by Pearson's analysis. All experiments were performed in triplicate. ***P
    Figure Legend Snippet: miR-1281 is a target of LINC00963 in CRC. (A) LINC00963-WT and LINC00963-MUT luciferase reporter plasmids containing the binding site for miR-1281 were constructed. (B) Targeting relationship between miR-1281 and LINC00963 was validated by a dual-luciferase reporter assay. (C) RNA pull-down and (D) RNA immunoprecipitation assays were performed to confirm that LINC00963 interacted with miR-1281 directly. (E) Expression level of miR-1281 in LoVo cells with LINC00963 overexpression and HCT116 cells with LINC00963 knockdown was detected by RT-qPCR. (F) miR-1281 expression in 53 pairs of CRC and adjacent tissues was detected by RT-qPCR. (G) The correlation between miR-1281 and LINC00963 expressions in CRC tissues was analyzed by Pearson's analysis. All experiments were performed in triplicate. ***P

    Techniques Used: Luciferase, Binding Assay, Construct, Reporter Assay, Immunoprecipitation, Expressing, Over Expression, Quantitative RT-PCR

    12) Product Images from "Metformin selectively inhibits metastatic colorectal cancer with the KRAS mutation by intracellular accumulation through silencing MATE1"

    Article Title: Metformin selectively inhibits metastatic colorectal cancer with the KRAS mutation by intracellular accumulation through silencing MATE1

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1918845117

    Transcriptional silencing of MATE1 by hypermethylation in KRAS -mutation CRC cells is associated with the up-regulation of DNMT1. ( A ) Representative images of DNMT1 immunohistochemistry on cross-sections from mCRC patients with T2DM were shown ( L eft ); cells with high positive DNMT1 expression were counted by ImageJ software, and the proportion was presented by two-way ANOVA ( M iddle ); the association between the IOD of MATE1 (shown in Fig. 3 D and E ) and cell proportion with high positive DNMT1 expression was determined by Pearson’s correlation analysis ( R ight ). ( B and C ) Expression levels of DNMT1 in 374469 KRAS WT colon adenocarcinoma and 386650 KRAS G12D colon mucinous adenocarcinoma ( B ) and in KRAS G13D SW48 ( C ) were analyzed by immunoblot. ( D and E ) Immunoblot analysis of MATE1 level ( Left ) and 48-h cell viability ( Right ) in LoVo ( D ) or KRAS G13D SW48 ( E ) cultured in 10 μM azacitidine for more than three generations ( n = 3). Data are shown as mean ± SEM. * P
    Figure Legend Snippet: Transcriptional silencing of MATE1 by hypermethylation in KRAS -mutation CRC cells is associated with the up-regulation of DNMT1. ( A ) Representative images of DNMT1 immunohistochemistry on cross-sections from mCRC patients with T2DM were shown ( L eft ); cells with high positive DNMT1 expression were counted by ImageJ software, and the proportion was presented by two-way ANOVA ( M iddle ); the association between the IOD of MATE1 (shown in Fig. 3 D and E ) and cell proportion with high positive DNMT1 expression was determined by Pearson’s correlation analysis ( R ight ). ( B and C ) Expression levels of DNMT1 in 374469 KRAS WT colon adenocarcinoma and 386650 KRAS G12D colon mucinous adenocarcinoma ( B ) and in KRAS G13D SW48 ( C ) were analyzed by immunoblot. ( D and E ) Immunoblot analysis of MATE1 level ( Left ) and 48-h cell viability ( Right ) in LoVo ( D ) or KRAS G13D SW48 ( E ) cultured in 10 μM azacitidine for more than three generations ( n = 3). Data are shown as mean ± SEM. * P

    Techniques Used: Mutagenesis, Immunohistochemistry, Expressing, Software, Cell Culture

    KRAS mutation down-regulates MATE1 through mediating the hypermethylation status on the CpG island of the MATE1 promoter. ( A ) The correlation between MATE1 transcriptional levels and methylation levels of CpG sites on the promoter of MATE1. Data from TCGA-COAD RNA-seq-HTseq-FPKM-521 (workflow type HTSeq-FPKM, normalized from RNA-seq of 521 samples) and methyArray 450k were conducted by Pearson’s correlation analysis. ( B ) Bisulfite sequencing PCR analysis showing the methylation status of CpG sites on the MATE1 promoter in CRC cell lines. ( C ) Immunoblot analysis of MATE1 levels and BSP analysis of the MATE1 promoter are shown between KRAS G13D SW48 and its counterpart SW48. ( D ) Immunoblot analysis monitoring MATE1 levels and CpG-site methylation on the MATE1 promoter in KRAS shRNA-LoVo cells treated with dimethyl sulfoxide (DMSO) as a negative control or azacitidine as a positive control. ( E ) The MATE1 expression in 374469 KRAS WT colon adenocarcinoma and 386650 KRAS G12D colon mucinous adenocarcinoma was analyzed by immunoblot, and the methylation status of CpG sites on the MATE1 promoter was determined by BSP. Data are shown as mean ± SEM. All P values were determined by two-way ANOVA. * P
    Figure Legend Snippet: KRAS mutation down-regulates MATE1 through mediating the hypermethylation status on the CpG island of the MATE1 promoter. ( A ) The correlation between MATE1 transcriptional levels and methylation levels of CpG sites on the promoter of MATE1. Data from TCGA-COAD RNA-seq-HTseq-FPKM-521 (workflow type HTSeq-FPKM, normalized from RNA-seq of 521 samples) and methyArray 450k were conducted by Pearson’s correlation analysis. ( B ) Bisulfite sequencing PCR analysis showing the methylation status of CpG sites on the MATE1 promoter in CRC cell lines. ( C ) Immunoblot analysis of MATE1 levels and BSP analysis of the MATE1 promoter are shown between KRAS G13D SW48 and its counterpart SW48. ( D ) Immunoblot analysis monitoring MATE1 levels and CpG-site methylation on the MATE1 promoter in KRAS shRNA-LoVo cells treated with dimethyl sulfoxide (DMSO) as a negative control or azacitidine as a positive control. ( E ) The MATE1 expression in 374469 KRAS WT colon adenocarcinoma and 386650 KRAS G12D colon mucinous adenocarcinoma was analyzed by immunoblot, and the methylation status of CpG sites on the MATE1 promoter was determined by BSP. Data are shown as mean ± SEM. All P values were determined by two-way ANOVA. * P

    Techniques Used: Mutagenesis, Methylation, RNA Sequencing Assay, Methylation Sequencing, Polymerase Chain Reaction, shRNA, Negative Control, Positive Control, Expressing

    KRAS mutation enhances the antitumor activity of metformin in the CRC PDX animal model and CRC cells. ( A and B ) The representative morphology ( A , Left ), tumor weight ( A , Middle ), mean tumor growth rate ( A , Right ), and representative images of Ki67 immunohistochemistry and their corresponding H E staining ( B , Left ) and statistical graph ( B , Right ) are shown as a result of 30-d treatment with metformin in 374469 KRAS WT colon adenocarcinoma and 386650 KRAS G12D colon mucinous adenocarcinoma patient-derived xenograft models. Data are shown as mean ± SEM, and differences between metformin and vehicle were analyzed by two-way ANOVA. ( C – E ) The distribution of G1, S, and G2 phases in KRAS WT CRC cell lines SW48 and CaCO2, KRAS G13D CRC cell lines LoVo and HCT-116 ( C ), KRAS G13D SW48 established by the CRISPR-Cas9 system ( D ), and LoVo infected by shRNA lentivirus ( E ) were detected after treatment with 0, 2.5, 5, and 10 mM metformin for 24 h ( n = 3). Data are shown as mean ± SEM. * P
    Figure Legend Snippet: KRAS mutation enhances the antitumor activity of metformin in the CRC PDX animal model and CRC cells. ( A and B ) The representative morphology ( A , Left ), tumor weight ( A , Middle ), mean tumor growth rate ( A , Right ), and representative images of Ki67 immunohistochemistry and their corresponding H E staining ( B , Left ) and statistical graph ( B , Right ) are shown as a result of 30-d treatment with metformin in 374469 KRAS WT colon adenocarcinoma and 386650 KRAS G12D colon mucinous adenocarcinoma patient-derived xenograft models. Data are shown as mean ± SEM, and differences between metformin and vehicle were analyzed by two-way ANOVA. ( C – E ) The distribution of G1, S, and G2 phases in KRAS WT CRC cell lines SW48 and CaCO2, KRAS G13D CRC cell lines LoVo and HCT-116 ( C ), KRAS G13D SW48 established by the CRISPR-Cas9 system ( D ), and LoVo infected by shRNA lentivirus ( E ) were detected after treatment with 0, 2.5, 5, and 10 mM metformin for 24 h ( n = 3). Data are shown as mean ± SEM. * P

    Techniques Used: Mutagenesis, Activity Assay, Animal Model, Immunohistochemistry, Staining, Derivative Assay, CRISPR, Infection, shRNA

    Down-regulation of MATE1 under KRAS mutation is associated with the sensitivity of CRC cells to metformin. ( A – C ) Transcriptional levels of SLC29A4 (PMAT), SLC22A1 (OCT1), SLC22A2 (OCT2), SLC22A3 (OCT3), SLC47A1 (MATE1), and SLC47A2 (MATE2k) in KRAS WT CRC cell lines SW48 and CaCO2, KRAS G13D CRC cell lines HCT-116 and LoVo ( A ), KRAS G13D SW48 and its counterpart SW48 ( B ), and sh-KRAS LoVo and sh-ctrl LoVo ( C ) were determined by qRT-PCR ( n = 3). Data are shown as mean ± SEM. P values were determined with the black bar as a control in A – C as * P
    Figure Legend Snippet: Down-regulation of MATE1 under KRAS mutation is associated with the sensitivity of CRC cells to metformin. ( A – C ) Transcriptional levels of SLC29A4 (PMAT), SLC22A1 (OCT1), SLC22A2 (OCT2), SLC22A3 (OCT3), SLC47A1 (MATE1), and SLC47A2 (MATE2k) in KRAS WT CRC cell lines SW48 and CaCO2, KRAS G13D CRC cell lines HCT-116 and LoVo ( A ), KRAS G13D SW48 and its counterpart SW48 ( B ), and sh-KRAS LoVo and sh-ctrl LoVo ( C ) were determined by qRT-PCR ( n = 3). Data are shown as mean ± SEM. P values were determined with the black bar as a control in A – C as * P

    Techniques Used: Mutagenesis, Quantitative RT-PCR

    KRAS mutation up-regulates the intracellular accumulation of metformin in vitro and in vivo. ( A and B ) Forty-eight-hour growth of KRAS G13D CRC cell lines HCT-116 ( A ) and LoVo ( B ) cultured with or without lansoprazole was determined after treatment with metformin ( Top ; n = 5), and the IC 50 and 95% CI were calculated by SPSS 21.0 software ( Bottom ). ( C and D ) Levels of metformin in KRAS WT SW48, KRAS G13D SW48 ( C ), sh-KRAS LoVo, and its control cell strain sh-ctrl LoVo ( D ) were detected by the LC-MS method after treatment with 2.5, 5, and 10 mM metformin for 24 h ( n = 3 at each time point). Data are shown as mean ± SEM. The significance of KRAS G13D SW48 vs. KRAS WT SW48 ( C ) and sh-KRAS LoVo vs. sh-ctrl LoVo ( D ) was determined as * P
    Figure Legend Snippet: KRAS mutation up-regulates the intracellular accumulation of metformin in vitro and in vivo. ( A and B ) Forty-eight-hour growth of KRAS G13D CRC cell lines HCT-116 ( A ) and LoVo ( B ) cultured with or without lansoprazole was determined after treatment with metformin ( Top ; n = 5), and the IC 50 and 95% CI were calculated by SPSS 21.0 software ( Bottom ). ( C and D ) Levels of metformin in KRAS WT SW48, KRAS G13D SW48 ( C ), sh-KRAS LoVo, and its control cell strain sh-ctrl LoVo ( D ) were detected by the LC-MS method after treatment with 2.5, 5, and 10 mM metformin for 24 h ( n = 3 at each time point). Data are shown as mean ± SEM. The significance of KRAS G13D SW48 vs. KRAS WT SW48 ( C ) and sh-KRAS LoVo vs. sh-ctrl LoVo ( D ) was determined as * P

    Techniques Used: Mutagenesis, In Vitro, In Vivo, Cell Culture, Software, Liquid Chromatography with Mass Spectroscopy

    13) Product Images from "xCT contributes to colorectal cancer tumorigenesis through upregulation of the MELK oncogene and activation of the AKT/mTOR cascade"

    Article Title: xCT contributes to colorectal cancer tumorigenesis through upregulation of the MELK oncogene and activation of the AKT/mTOR cascade

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-022-04827-4

    Silencing of xCT suppresses CRC growth and blocks spontaneous lung metastasis of CRC cells. A xCT knockdown inhibited the growth of tumors in CRC xenograft mouse models. Differences in tumor weight ( B ) and volume ( C ) in nude mouse CRC models subcutaneously injected with HCT116 cells with or without xCT inhibition. D IHC staining showed the changes in the expression of Ki67, N-cadherin, E-cadherin, CD133 and P-AKT in CRC tissues after xCT knockdown. E The process to establish a CRC lung metastasis mouse model. F qRT-PCR and western blotting assays confirmed the knockdown efficiency with xCT shRNA administration in CT26 cells. Macroscopic changes in the lungs ( G ) and pathological changes in the lung tissues ( H ) of mice after caudal vein injection of CT26 cells with or without xCT inhibition. I xCT knockdown significantly inhibited the metastatic capability of CRC. * P
    Figure Legend Snippet: Silencing of xCT suppresses CRC growth and blocks spontaneous lung metastasis of CRC cells. A xCT knockdown inhibited the growth of tumors in CRC xenograft mouse models. Differences in tumor weight ( B ) and volume ( C ) in nude mouse CRC models subcutaneously injected with HCT116 cells with or without xCT inhibition. D IHC staining showed the changes in the expression of Ki67, N-cadherin, E-cadherin, CD133 and P-AKT in CRC tissues after xCT knockdown. E The process to establish a CRC lung metastasis mouse model. F qRT-PCR and western blotting assays confirmed the knockdown efficiency with xCT shRNA administration in CT26 cells. Macroscopic changes in the lungs ( G ) and pathological changes in the lung tissues ( H ) of mice after caudal vein injection of CT26 cells with or without xCT inhibition. I xCT knockdown significantly inhibited the metastatic capability of CRC. * P

    Techniques Used: Injection, Inhibition, Immunohistochemistry, Staining, Expressing, Quantitative RT-PCR, Western Blot, shRNA, Mouse Assay

    MELK is required for xCT-mediated CRC tumorigenesis and AKT/mTOR signaling. A , B The colony formation assay showed that the suppressive effect of xCT inhibition on the proliferation of CRC cells was reversed by MELK upregulation. C , D Transwell assays indicated that MELK overexpression effectively recovered the migration capability inhibited by xCT knockdown in CRC cells. E , F Cell cycle assay confirmed that MELK rescued the suppression of xCT inhibition on G1/S transition. G , H The sphere formation assay indicated that upregulated MELK rescued the suppressive effect of xCT inhibition on the sphere-forming capability of CRC cells. I The interaction of MELK with different factors expressed in various organelles. J , K GSEA indicated that upregulated expression of MELK was closely related to the activation of signaling pathways, including hallmark_mTORC1_signaling and hallmark_MYC_targets_V1. L , M Western blot analysis confirmed that upregulated MELK rescued the suppression of the expression of tumorigenesis- and stemness-related factors in CRC cells with xCT knockdown. N , O The suppressive effect of xCT knockdown on the AKT/mTOR signaling pathway was rescued by MELK overexpression. * P
    Figure Legend Snippet: MELK is required for xCT-mediated CRC tumorigenesis and AKT/mTOR signaling. A , B The colony formation assay showed that the suppressive effect of xCT inhibition on the proliferation of CRC cells was reversed by MELK upregulation. C , D Transwell assays indicated that MELK overexpression effectively recovered the migration capability inhibited by xCT knockdown in CRC cells. E , F Cell cycle assay confirmed that MELK rescued the suppression of xCT inhibition on G1/S transition. G , H The sphere formation assay indicated that upregulated MELK rescued the suppressive effect of xCT inhibition on the sphere-forming capability of CRC cells. I The interaction of MELK with different factors expressed in various organelles. J , K GSEA indicated that upregulated expression of MELK was closely related to the activation of signaling pathways, including hallmark_mTORC1_signaling and hallmark_MYC_targets_V1. L , M Western blot analysis confirmed that upregulated MELK rescued the suppression of the expression of tumorigenesis- and stemness-related factors in CRC cells with xCT knockdown. N , O The suppressive effect of xCT knockdown on the AKT/mTOR signaling pathway was rescued by MELK overexpression. * P

    Techniques Used: Colony Assay, Inhibition, Over Expression, Migration, Cell Cycle Assay, Tube Formation Assay, Expressing, Activation Assay, Western Blot

    E2F1 was identified and confirmed as an upstream transcription factor of SLC7A11. A Prediction of potential upstream transcription factors of SLC7A11 in the oPOSSUM and GeneCards databases. B qRT-PCR showed that knockdown of E2F1 significantly suppressed the expression of SLC7A11. C Western blot analysis confirmed that E2F1 inhibition led to the downregulation of xCT expression in 293T cells. D , E The correlation between the expression of E2F1 and SLC7A11 in GSE17538 and GSE87211. F The potential binding sites of E2F1 to the SLC7A11 promoter region and binding site mutation strategy. G , H A luciferase assay was used to identify the binding sites of E2F1 to the SLC7A11 promoter region in 293T cells and HCT116 cells. I , J The expression of E2F1 was higher in CRC tissues than in normal samples from the TCGA-COAD cohort and TCGA-READ cohort. K Knockdown efficiency of E2F1 via lentiviral shRNA transfection was confirmed through western blot assay. L CCK-8 assay showed that E2F1 knockdown significantly inhibited the proliferation of HCT15 cells. M Transwell assays indicated that E2F1 inhibition effectively weakened the migration capability of HCT15 cells. N Sphere formation assays confirmed that E2F1 inhibition significantly suppressed the stemness of HCT15 cells. (O) Cell cycle assay showed E2F1 regulated cell cycle by inducing the G1/S transition. * P
    Figure Legend Snippet: E2F1 was identified and confirmed as an upstream transcription factor of SLC7A11. A Prediction of potential upstream transcription factors of SLC7A11 in the oPOSSUM and GeneCards databases. B qRT-PCR showed that knockdown of E2F1 significantly suppressed the expression of SLC7A11. C Western blot analysis confirmed that E2F1 inhibition led to the downregulation of xCT expression in 293T cells. D , E The correlation between the expression of E2F1 and SLC7A11 in GSE17538 and GSE87211. F The potential binding sites of E2F1 to the SLC7A11 promoter region and binding site mutation strategy. G , H A luciferase assay was used to identify the binding sites of E2F1 to the SLC7A11 promoter region in 293T cells and HCT116 cells. I , J The expression of E2F1 was higher in CRC tissues than in normal samples from the TCGA-COAD cohort and TCGA-READ cohort. K Knockdown efficiency of E2F1 via lentiviral shRNA transfection was confirmed through western blot assay. L CCK-8 assay showed that E2F1 knockdown significantly inhibited the proliferation of HCT15 cells. M Transwell assays indicated that E2F1 inhibition effectively weakened the migration capability of HCT15 cells. N Sphere formation assays confirmed that E2F1 inhibition significantly suppressed the stemness of HCT15 cells. (O) Cell cycle assay showed E2F1 regulated cell cycle by inducing the G1/S transition. * P

    Techniques Used: Quantitative RT-PCR, Expressing, Western Blot, Inhibition, Binding Assay, Mutagenesis, Luciferase, shRNA, Transfection, CCK-8 Assay, Migration, Cell Cycle Assay

    xCT activates the AKT/mTOR signaling pathway and enhances MELK expression in CRC cells. A DEGs in HCT15 cells with or without xCT shRNA administration. B KEGG analysis was performed on the DEGs. C GSEA showed that xCT was upregulated in samples with the activation of the hallmark_mTORC1_signaling pathway. D Identification of potential factors whose expression might correlate with that of xCT in CRC. E , F The correlation between the expression of MELK and xCT in GSE39582 and GSE87211. G , H IF assays confirmed that MELK and xCT were strongly colocalized in HCT116 and HCT15 cells. I , J Western blot analysis showed that xCT knockdown inhibited the expression level of MELK and restrained the activation of the AKT/mTOR signaling pathway in HCT116 and HCT15 cells.
    Figure Legend Snippet: xCT activates the AKT/mTOR signaling pathway and enhances MELK expression in CRC cells. A DEGs in HCT15 cells with or without xCT shRNA administration. B KEGG analysis was performed on the DEGs. C GSEA showed that xCT was upregulated in samples with the activation of the hallmark_mTORC1_signaling pathway. D Identification of potential factors whose expression might correlate with that of xCT in CRC. E , F The correlation between the expression of MELK and xCT in GSE39582 and GSE87211. G , H IF assays confirmed that MELK and xCT were strongly colocalized in HCT116 and HCT15 cells. I , J Western blot analysis showed that xCT knockdown inhibited the expression level of MELK and restrained the activation of the AKT/mTOR signaling pathway in HCT116 and HCT15 cells.

    Techniques Used: Expressing, shRNA, Activation Assay, Western Blot

    xCT is highly expressed in patients with primary CRC and predicts a poor prognosis. A Differences in the expression of xCT between tumors and normal tissues of various organs were observed in the TCGA database. B An overall exploration of the expression characteristics of xCT in the TCGA pancancer dataset. C , D Difference in the expression of xCT between patients with COAD or READ and matched normal samples in the TCGA database. E Difference in the expression of xCT between 203 CRC samples and 160 mucosa control samples in the GSE87211 dataset. F Difference in the expression of xCT between CRC samples and matched normal samples from our collected 49 CRC subjects was assessed using IHC staining. G IHC staining showed that xCT was more positively expressed in CRC tissues than in normal tissues from our collected CRC samples. H Western blot analysis showed that the expression of xCT was higher in CRC tissues than in normal tissues. I Relationship between xCT expression and clinicopathological characteristics in CRC patients from our collected CRC samples. J The prognosis of CRC patients with high expression of xCT was worse than that of CRC patients with low expression of xCT in GSE87211. K The ROC curve shows that xCT levels could predict the prognosis of CRC patients with great accuracy. L CRC patients with high expression of xCT had a poorer prognosis than those with low expression of xCT in our validation cohort. M The ROC curve confirmed the prognostic predictive accuracy in our validation cohort. ** P
    Figure Legend Snippet: xCT is highly expressed in patients with primary CRC and predicts a poor prognosis. A Differences in the expression of xCT between tumors and normal tissues of various organs were observed in the TCGA database. B An overall exploration of the expression characteristics of xCT in the TCGA pancancer dataset. C , D Difference in the expression of xCT between patients with COAD or READ and matched normal samples in the TCGA database. E Difference in the expression of xCT between 203 CRC samples and 160 mucosa control samples in the GSE87211 dataset. F Difference in the expression of xCT between CRC samples and matched normal samples from our collected 49 CRC subjects was assessed using IHC staining. G IHC staining showed that xCT was more positively expressed in CRC tissues than in normal tissues from our collected CRC samples. H Western blot analysis showed that the expression of xCT was higher in CRC tissues than in normal tissues. I Relationship between xCT expression and clinicopathological characteristics in CRC patients from our collected CRC samples. J The prognosis of CRC patients with high expression of xCT was worse than that of CRC patients with low expression of xCT in GSE87211. K The ROC curve shows that xCT levels could predict the prognosis of CRC patients with great accuracy. L CRC patients with high expression of xCT had a poorer prognosis than those with low expression of xCT in our validation cohort. M The ROC curve confirmed the prognostic predictive accuracy in our validation cohort. ** P

    Techniques Used: Expressing, Immunohistochemistry, Staining, Western Blot

    xCT contributes to proliferation and colony formation in CRC cells. A – D Western blot assay confirming the knockdown efficiency of xCT siRNA and xCT shRNA in CRC cells. E – H CCK-8 assay showed that xCT knockdown by both xCT siRNA and xCT shRNA significantly suppressed the proliferation of HCT116 and HCT15 cells. I , J Colony formation assays indicated that xCT inhibition markedly reduced HCT116 and HCT15 cell colony formation ability. K , L EdU assay confirmed that xCT inhibition attenuated HCT116 and HCT15 cell proliferation. M , N Cell cycle assay indicated that xCT inhibition hampered G1/S transition of HCT116 and HCT15 cells. O , P IF staining showed that xCT knockdown diminished the expression of PCNA in HCT116 and HCT15 cells. Q , R Western blot analysis revealed that xCT inhibition downregulated the expression of proliferation-related factors, including PCNA and C-myc, in HCT116 and HCT15 cells. S Western blot analysis indicated that xCT was overexpressed in HCT116 cells upon transfection. T CCK-8 assay showed that overexpression of xCT significantly promoted the proliferation of HCT116 cells. U EdU assay confirmed the promoting effect of xCT overexpression on the proliferation of HCT116 cells. * P
    Figure Legend Snippet: xCT contributes to proliferation and colony formation in CRC cells. A – D Western blot assay confirming the knockdown efficiency of xCT siRNA and xCT shRNA in CRC cells. E – H CCK-8 assay showed that xCT knockdown by both xCT siRNA and xCT shRNA significantly suppressed the proliferation of HCT116 and HCT15 cells. I , J Colony formation assays indicated that xCT inhibition markedly reduced HCT116 and HCT15 cell colony formation ability. K , L EdU assay confirmed that xCT inhibition attenuated HCT116 and HCT15 cell proliferation. M , N Cell cycle assay indicated that xCT inhibition hampered G1/S transition of HCT116 and HCT15 cells. O , P IF staining showed that xCT knockdown diminished the expression of PCNA in HCT116 and HCT15 cells. Q , R Western blot analysis revealed that xCT inhibition downregulated the expression of proliferation-related factors, including PCNA and C-myc, in HCT116 and HCT15 cells. S Western blot analysis indicated that xCT was overexpressed in HCT116 cells upon transfection. T CCK-8 assay showed that overexpression of xCT significantly promoted the proliferation of HCT116 cells. U EdU assay confirmed the promoting effect of xCT overexpression on the proliferation of HCT116 cells. * P

    Techniques Used: Western Blot, shRNA, CCK-8 Assay, Inhibition, EdU Assay, Cell Cycle Assay, Staining, Expressing, Transfection, Over Expression

    Effects of xCT knockdown on the stemness of HCT116 and HCT15 CRC cells. A An overview of the correlations between xCT expression, mRNAsi, and known clinical characteristics (TNM stage, sex, and survival status) in the TCGA-COAD cohort. B The association between the expression of xCT and mRNAsi in the TCGA-COAD cohort. C , D Western blot assay was applied to detect the expression of xCT in CRC cells and CRC stem cells. E , F Sphere formation assays showed that xCT inhibition suppressed the sphere-forming capability of CRC stem cells. G , H Western blot analysis showed that xCT knockdown reduced the expression level of stemness-related molecules in HCT116 cells and HCT15 cells. I , J Flow cytometry indicated that xCT inhibition significantly diminished the expression of EPCAM and CD133 in CRC cells. K , L IF staining showed that xCT knockdown suppressed the expression of CD133 in CRC cells. ** P
    Figure Legend Snippet: Effects of xCT knockdown on the stemness of HCT116 and HCT15 CRC cells. A An overview of the correlations between xCT expression, mRNAsi, and known clinical characteristics (TNM stage, sex, and survival status) in the TCGA-COAD cohort. B The association between the expression of xCT and mRNAsi in the TCGA-COAD cohort. C , D Western blot assay was applied to detect the expression of xCT in CRC cells and CRC stem cells. E , F Sphere formation assays showed that xCT inhibition suppressed the sphere-forming capability of CRC stem cells. G , H Western blot analysis showed that xCT knockdown reduced the expression level of stemness-related molecules in HCT116 cells and HCT15 cells. I , J Flow cytometry indicated that xCT inhibition significantly diminished the expression of EPCAM and CD133 in CRC cells. K , L IF staining showed that xCT knockdown suppressed the expression of CD133 in CRC cells. ** P

    Techniques Used: Expressing, Western Blot, Inhibition, Flow Cytometry, Staining

    Upregulation of xCT in CRC cells promotes their migration and reverses the EMT process. A – D Transwell assays indicated that xCT knockdown with xCT siRNA and xCT shRNA significantly suppressed the migration of CRC cells. E – G The wound scratch assay and quantitative analysis showed that xCT knockdown inhibited the migration of CRC cells. H – K IF staining indicated that xCT inhibition reduced the expression of vimentin ( H – I ) and N-cadherin ( J – K ) in CRC cells. L , M Western blot analysis confirmed that xCT knockdown decreased the expression of MMP1 and vimentin in CRC cells. N , O Transwell assays showed that overexpression of xCT promoted the migration of CRC cells. ** P
    Figure Legend Snippet: Upregulation of xCT in CRC cells promotes their migration and reverses the EMT process. A – D Transwell assays indicated that xCT knockdown with xCT siRNA and xCT shRNA significantly suppressed the migration of CRC cells. E – G The wound scratch assay and quantitative analysis showed that xCT knockdown inhibited the migration of CRC cells. H – K IF staining indicated that xCT inhibition reduced the expression of vimentin ( H – I ) and N-cadherin ( J – K ) in CRC cells. L , M Western blot analysis confirmed that xCT knockdown decreased the expression of MMP1 and vimentin in CRC cells. N , O Transwell assays showed that overexpression of xCT promoted the migration of CRC cells. ** P

    Techniques Used: Migration, shRNA, Wound Healing Assay, Staining, Inhibition, Expressing, Western Blot, Over Expression

    14) Product Images from "Expression of Epidermal Growth Factor Receptor Detected by Cetuximab Indicates Its Efficacy to Inhibit In Vitro and In Vivo Proliferation of Colorectal Cancer Cells"

    Article Title: Expression of Epidermal Growth Factor Receptor Detected by Cetuximab Indicates Its Efficacy to Inhibit In Vitro and In Vivo Proliferation of Colorectal Cancer Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0066302

    EGFR expression in subcloned CRC cell lines. (A) Histogram of WiDR subcloned cell lines showing differences in EGFR expression. Various EGFR expressions were seen in subcloned cell lines. (B) High and low EGFR subclones with high or low EGFR expression were selected on the basis of FCM results. Subclones derived from four CRC cell lines were analyzed, and the subclones exhibiting highest (blue) and lowest (green) levels of EGFR expression were selected for further studies. Controls are described in red line.
    Figure Legend Snippet: EGFR expression in subcloned CRC cell lines. (A) Histogram of WiDR subcloned cell lines showing differences in EGFR expression. Various EGFR expressions were seen in subcloned cell lines. (B) High and low EGFR subclones with high or low EGFR expression were selected on the basis of FCM results. Subclones derived from four CRC cell lines were analyzed, and the subclones exhibiting highest (blue) and lowest (green) levels of EGFR expression were selected for further studies. Controls are described in red line.

    Techniques Used: Expressing, Derivative Assay

    EGFR expression levels in CRC cell lines. (A) Each cell line was evaluated by FCM with biotinylated cetuximab. All four CRC cell lines differed in EGFR expression levels. (B) Histogram of each CRC cell lines. Expression level of EGFR was measured by the intensity of FITC fluorescence.
    Figure Legend Snippet: EGFR expression levels in CRC cell lines. (A) Each cell line was evaluated by FCM with biotinylated cetuximab. All four CRC cell lines differed in EGFR expression levels. (B) Histogram of each CRC cell lines. Expression level of EGFR was measured by the intensity of FITC fluorescence.

    Techniques Used: Expressing, Fluorescence

    Proliferation assay of subcloned CRC cell lines. When the cetuximab was administered to low CRC subclones with low EGFR expression, weak inhibition of cell growth was observed. However, growth of subclones with high EGFR was strongly inhibited in comparison.
    Figure Legend Snippet: Proliferation assay of subcloned CRC cell lines. When the cetuximab was administered to low CRC subclones with low EGFR expression, weak inhibition of cell growth was observed. However, growth of subclones with high EGFR was strongly inhibited in comparison.

    Techniques Used: Proliferation Assay, Expressing, Inhibition

    15) Product Images from "miR-133b suppresses metastasis by targeting HOXA9 in human colorectal cancer"

    Article Title: miR-133b suppresses metastasis by targeting HOXA9 in human colorectal cancer

    Journal: Oncotarget

    doi: 10.18632/oncotarget.19212

    Knockdown of HOXA9 suppresses CRC tumorigenesis and metastasis HCT8 cells were infected with a lentivirus expressing specific shRNA of HOXA9 genes (HOXA9-KD) or shRNA scramble controls (Lv-scramble). (A) qPCR analysis of the knockdown effect in HCT8 and HCT116 cells infected with HOXA9-KD or Lv-scramble. (B) in vitro cell proliferation was detected with CCK8 assay. (C) in vitro cell proliferation was detected with colony formation assay. (D, E) In vitro cell migration was analyzed with a gel invasion assay (D) and a wound-healing assay (E) . (F) Knock down of HOXA9 decreased ZEB1 while increased E-cadherin expression. (G) Overexpression of miR133-b decreased HOXA9 and ZEB1, while increased E-cadherin expression. *P
    Figure Legend Snippet: Knockdown of HOXA9 suppresses CRC tumorigenesis and metastasis HCT8 cells were infected with a lentivirus expressing specific shRNA of HOXA9 genes (HOXA9-KD) or shRNA scramble controls (Lv-scramble). (A) qPCR analysis of the knockdown effect in HCT8 and HCT116 cells infected with HOXA9-KD or Lv-scramble. (B) in vitro cell proliferation was detected with CCK8 assay. (C) in vitro cell proliferation was detected with colony formation assay. (D, E) In vitro cell migration was analyzed with a gel invasion assay (D) and a wound-healing assay (E) . (F) Knock down of HOXA9 decreased ZEB1 while increased E-cadherin expression. (G) Overexpression of miR133-b decreased HOXA9 and ZEB1, while increased E-cadherin expression. *P

    Techniques Used: Infection, Expressing, shRNA, Real-time Polymerase Chain Reaction, In Vitro, CCK-8 Assay, Colony Assay, Migration, Invasion Assay, Wound Healing Assay, Over Expression

    MiR-133b suppresses CRC tumorigenesis and metastasis in vivo HCT8 and HCT116 cells were transfected with miR-133b-mimic or scramble and then subcutaneously injected into nude mice. (A) in vivo subcutaneous tumor cell transplantation, the gross morphology of tumors are represented. (B, C) The statistical data for tumor volume (B) and tumor weight (C) are shown. (D) Representative image of hepatic metastatic sites. (E) Haptic metastatic tumor volume are shown. *P
    Figure Legend Snippet: MiR-133b suppresses CRC tumorigenesis and metastasis in vivo HCT8 and HCT116 cells were transfected with miR-133b-mimic or scramble and then subcutaneously injected into nude mice. (A) in vivo subcutaneous tumor cell transplantation, the gross morphology of tumors are represented. (B, C) The statistical data for tumor volume (B) and tumor weight (C) are shown. (D) Representative image of hepatic metastatic sites. (E) Haptic metastatic tumor volume are shown. *P

    Techniques Used: In Vivo, Transfection, Injection, Mouse Assay, Transplantation Assay

    HOXA9 was identified as miR-133b targets in CRC (A) miR-133b targeting genes and the corresponding mutations. (B) examination of luciferase activity. Co-transfection of a wildtype or a mutant HOXA9 3′UTR with miR-133b mimics into HEK 293 cells. Firefly luciferase activity was measured and standardized by Renilla luciferase activity. (C) Left, HCT8 cells were infected with miR-133b mimics or scramble control, and protein levels of HOXA9 was analyzed with western blot. Right, the expression of HOXA9 in normal control (N), CRC without metastasis (T) and CRC with metastasis(M). 3 pair samples were used and a representative sample is shown. (D) Relative expression of HOXA9 in TCGA of CRC, which contained 40 normal samples, 45 stage I, 111 stage II, 83 stage III and 39 stage IV samples. (E) Screenshot of the profiles of the human gene HOXA9 within the BioGPS online portal. All data used for this study are available through the BioGPS database ( biogps.org ). (F) the expression correlation analysis for miR-133b and HOXA9 in 66 CRC tumor samples. *P
    Figure Legend Snippet: HOXA9 was identified as miR-133b targets in CRC (A) miR-133b targeting genes and the corresponding mutations. (B) examination of luciferase activity. Co-transfection of a wildtype or a mutant HOXA9 3′UTR with miR-133b mimics into HEK 293 cells. Firefly luciferase activity was measured and standardized by Renilla luciferase activity. (C) Left, HCT8 cells were infected with miR-133b mimics or scramble control, and protein levels of HOXA9 was analyzed with western blot. Right, the expression of HOXA9 in normal control (N), CRC without metastasis (T) and CRC with metastasis(M). 3 pair samples were used and a representative sample is shown. (D) Relative expression of HOXA9 in TCGA of CRC, which contained 40 normal samples, 45 stage I, 111 stage II, 83 stage III and 39 stage IV samples. (E) Screenshot of the profiles of the human gene HOXA9 within the BioGPS online portal. All data used for this study are available through the BioGPS database ( biogps.org ). (F) the expression correlation analysis for miR-133b and HOXA9 in 66 CRC tumor samples. *P

    Techniques Used: Luciferase, Activity Assay, Cotransfection, Mutagenesis, Infection, Western Blot, Expressing

    16) Product Images from "CircPTK2 (hsa_circ_0005273) as a novel therapeutic target for metastatic colorectal cancer"

    Article Title: CircPTK2 (hsa_circ_0005273) as a novel therapeutic target for metastatic colorectal cancer

    Journal: Molecular Cancer

    doi: 10.1186/s12943-020-1139-3

    CircPTK2 targeted vimentin protein to regulate growth and metastasis of CRC. a The correlation between the expression level of circPTK2 and vimentin. b Western blot assay showing the protein vimentin pulled down by biotin-labeled circular RNA probes from the lysates of SW620 and LOVO cells. c Mass spectrometry assays revealing the protein vimentin pulled down by biotin-labeled circPTK2 from the lysates of SW620 and LOVO cells. d Western blot assay showing circPTK2 overexpression up-regulated vimentin, MMP2/9 and CXCR4, and down-regulated E-cadherin. e Dual RNAscope and IHC assay indicating the co-localization of circPTK2 (red) and vimentin (brown) in CRC tissues. f RNA pulldown and western blotting assay showing the target sites for circPTK2 on vimentin. g - h SW480 cells were transfected with circPTK2-overexpressing plasmid as well as vimentin siRNA for 48 h. Cells were used for staining with crystal violet or injected into subcutaneous tissue of nude mice. Colony formation and tumor growth were detected. Colony formation and tumor growth of SW480 cells were shown. i - j SW620 cells were transfected with circPTK2 siRNA plasmid as well as vimentin-overexpressing plasmid for 48 h. Cells were used for staining with crystal violet or injected into subcutaneous tissue of nude mice. Colony formation and tumor growth of SW620 cells were shown. P values were calculated by t- test
    Figure Legend Snippet: CircPTK2 targeted vimentin protein to regulate growth and metastasis of CRC. a The correlation between the expression level of circPTK2 and vimentin. b Western blot assay showing the protein vimentin pulled down by biotin-labeled circular RNA probes from the lysates of SW620 and LOVO cells. c Mass spectrometry assays revealing the protein vimentin pulled down by biotin-labeled circPTK2 from the lysates of SW620 and LOVO cells. d Western blot assay showing circPTK2 overexpression up-regulated vimentin, MMP2/9 and CXCR4, and down-regulated E-cadherin. e Dual RNAscope and IHC assay indicating the co-localization of circPTK2 (red) and vimentin (brown) in CRC tissues. f RNA pulldown and western blotting assay showing the target sites for circPTK2 on vimentin. g - h SW480 cells were transfected with circPTK2-overexpressing plasmid as well as vimentin siRNA for 48 h. Cells were used for staining with crystal violet or injected into subcutaneous tissue of nude mice. Colony formation and tumor growth were detected. Colony formation and tumor growth of SW480 cells were shown. i - j SW620 cells were transfected with circPTK2 siRNA plasmid as well as vimentin-overexpressing plasmid for 48 h. Cells were used for staining with crystal violet or injected into subcutaneous tissue of nude mice. Colony formation and tumor growth of SW620 cells were shown. P values were calculated by t- test

    Techniques Used: Expressing, Western Blot, Labeling, Mass Spectrometry, Over Expression, Immunohistochemistry, Transfection, Plasmid Preparation, Staining, Injection, Mouse Assay

    Expression profiles of circRNAs in CRC and characterization of circPTK2. a Heatmap of the differentially expressed circRNAs in six pairs of human CRC tissues and adjacent non-tumor tissues. Red: upregulated circRNAs in CRC; green: downregulated circRNAs in CRC. b circPTK2 is back-spliced by exons 27, 28, and 29 of PTK2. c , d Cells were treated with hypoxia. circPTK2 was detected by RNAscope. Location and expression of circPTK2 in SW620 and LOVO cells. e , f Location and expression of circPTK2 in SW480 and HCT15 cells. g Location and expression of circPTK2 in adjacent and tumor of patient tissue. P values were calculated by one-way ANOVA
    Figure Legend Snippet: Expression profiles of circRNAs in CRC and characterization of circPTK2. a Heatmap of the differentially expressed circRNAs in six pairs of human CRC tissues and adjacent non-tumor tissues. Red: upregulated circRNAs in CRC; green: downregulated circRNAs in CRC. b circPTK2 is back-spliced by exons 27, 28, and 29 of PTK2. c , d Cells were treated with hypoxia. circPTK2 was detected by RNAscope. Location and expression of circPTK2 in SW620 and LOVO cells. e , f Location and expression of circPTK2 in SW480 and HCT15 cells. g Location and expression of circPTK2 in adjacent and tumor of patient tissue. P values were calculated by one-way ANOVA

    Techniques Used: Expressing

    CircPTK2 promoted aggressive phenotypes of CRC cells in vitro. Cells were transfected with circPTK2 siRNA or circPTK2- overexpressing plasmid for 48 h. Then cells were harvested for annexin-V and PI staining. Cell apoptosis was detected with flow cytometry analysis. The cells were stained by crystal violet to evaluate cell proliferation, migration, and invasion capability. a , b The effect of circPTK2 knockdown on SW620 apoptosis. c , d The effect of circPTK2 overexpression on SW480 apoptosis. e - h Representative images and quantification results of the colony formation of cells. i - l Representative images and quantification results of the migration and invasion of cells. The data are the mean ± SEM. * P
    Figure Legend Snippet: CircPTK2 promoted aggressive phenotypes of CRC cells in vitro. Cells were transfected with circPTK2 siRNA or circPTK2- overexpressing plasmid for 48 h. Then cells were harvested for annexin-V and PI staining. Cell apoptosis was detected with flow cytometry analysis. The cells were stained by crystal violet to evaluate cell proliferation, migration, and invasion capability. a , b The effect of circPTK2 knockdown on SW620 apoptosis. c , d The effect of circPTK2 overexpression on SW480 apoptosis. e - h Representative images and quantification results of the colony formation of cells. i - l Representative images and quantification results of the migration and invasion of cells. The data are the mean ± SEM. * P

    Techniques Used: In Vitro, Transfection, Plasmid Preparation, Staining, Flow Cytometry, Migration, Over Expression

    17) Product Images from "Loss of ZNF677 expression is a predictive biomarker for lymph node metastasis in Middle Eastern Colorectal Cancer"

    Article Title: Loss of ZNF677 expression is a predictive biomarker for lymph node metastasis in Middle Eastern Colorectal Cancer

    Journal: Scientific Reports

    doi: 10.1038/s41598-021-01869-0

    Loss of ZNF677 protein expression is associated with ZNF677 promoter methylation. ( A ) Methylation status of CRC cell lines were assessed by methylation-specific PCR for the ZNF677 gene. MSP analyses of both methylated (M) and unmethylated (U) reactions were amplified from bisulfite-treated DNA and run in a 2% agarose gel. ( B ) Demethylation of the ZNF677 gene restored ZNF677 expression in COLO-320 and HT29 cells. COLO-320 and HT29 cell lines were treated with different doses (0.5, 1, and 2 µM) of 5-aza-2′deoxycytidine for 72 h before lysis. Equal amounts of proteins were immunoblotted with antibodies against ZNF677 and β-actin.
    Figure Legend Snippet: Loss of ZNF677 protein expression is associated with ZNF677 promoter methylation. ( A ) Methylation status of CRC cell lines were assessed by methylation-specific PCR for the ZNF677 gene. MSP analyses of both methylated (M) and unmethylated (U) reactions were amplified from bisulfite-treated DNA and run in a 2% agarose gel. ( B ) Demethylation of the ZNF677 gene restored ZNF677 expression in COLO-320 and HT29 cells. COLO-320 and HT29 cell lines were treated with different doses (0.5, 1, and 2 µM) of 5-aza-2′deoxycytidine for 72 h before lysis. Equal amounts of proteins were immunoblotted with antibodies against ZNF677 and β-actin.

    Techniques Used: Expressing, Methylation, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Lysis

    18) Product Images from "miR-150-5p suppresses tumor progression by targeting VEGFA in colorectal cancer"

    Article Title: miR-150-5p suppresses tumor progression by targeting VEGFA in colorectal cancer

    Journal: Aging (Albany NY)

    doi: 10.18632/aging.101656

    miR-150-5p inhibited VEGFA/VEGFR2/Akt/mTOR signaling pathway in CRC. Western blot was used to measure the expression of VEGFA, VEGFR2, p-VEGFR2, Akt, p-Akt, mTOR, p-mTOR in transfected HCT116 and HCT8 cells. GAPDH was used as a loading control. Data are shown as the mean±SD of three independent experiments. * p
    Figure Legend Snippet: miR-150-5p inhibited VEGFA/VEGFR2/Akt/mTOR signaling pathway in CRC. Western blot was used to measure the expression of VEGFA, VEGFR2, p-VEGFR2, Akt, p-Akt, mTOR, p-mTOR in transfected HCT116 and HCT8 cells. GAPDH was used as a loading control. Data are shown as the mean±SD of three independent experiments. * p

    Techniques Used: Western Blot, Expressing, Transfection

    miR-150-5p inhibited CRC progression by targeting VEGFA. ( A ) VEGFA protein expression was determined in HCT116 and HCT8 cells transfected with agomiR-150-5p with VEGFA expression plasmid or empty vector using western blot; GAPDH was used as the internal control. ( B - D ) Cell proliferation ( B , C ), migration ( D )and invasion ( E ) were evaluated in HCT116 and HCT8 cells transfected with agomiR-150-5p with VEGFA expression plasmid or empty vector. ( F ) HUVECs were cultured in TCM derived from HCT116 and HCT8 cells transfected with agomiR-150-5p plus VEGFA expression plasmid or empty vector. Data are shown as the mean±SD. * p
    Figure Legend Snippet: miR-150-5p inhibited CRC progression by targeting VEGFA. ( A ) VEGFA protein expression was determined in HCT116 and HCT8 cells transfected with agomiR-150-5p with VEGFA expression plasmid or empty vector using western blot; GAPDH was used as the internal control. ( B - D ) Cell proliferation ( B , C ), migration ( D )and invasion ( E ) were evaluated in HCT116 and HCT8 cells transfected with agomiR-150-5p with VEGFA expression plasmid or empty vector. ( F ) HUVECs were cultured in TCM derived from HCT116 and HCT8 cells transfected with agomiR-150-5p plus VEGFA expression plasmid or empty vector. Data are shown as the mean±SD. * p

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Western Blot, Migration, Cell Culture, Derivative Assay

    A. VEGFA was a direct target of miR-150-5p in CRC. ( A ) The direct target genes of miR-150-5p were predicted using the PicTarSites, miRandaSites and Tarbase databases. ( B ) Wild-type and mutant VEGFA-3’UTR sequences were cloned into luciferase reporter. Luciferase activity was determined in HCT116 and 293T cells cotransfected with agomiR-150-5p or agomiR-NC and pmirGLO-VEGFA-3’UTR-WT or pmirGLO-VEGFA-3’UTR-Mut. Luciferase activities were normalized to that of renilla luciferase. C, D. qRT-PCR ( C ) and western blot ( D ) analyses showed that both VEGFA mRNA and protein expression levels were dramatically suppressed by agomiR-150-5p in HCT116 and HCT8 cells, GAPDH was used as the internal control. ** p
    Figure Legend Snippet: A. VEGFA was a direct target of miR-150-5p in CRC. ( A ) The direct target genes of miR-150-5p were predicted using the PicTarSites, miRandaSites and Tarbase databases. ( B ) Wild-type and mutant VEGFA-3’UTR sequences were cloned into luciferase reporter. Luciferase activity was determined in HCT116 and 293T cells cotransfected with agomiR-150-5p or agomiR-NC and pmirGLO-VEGFA-3’UTR-WT or pmirGLO-VEGFA-3’UTR-Mut. Luciferase activities were normalized to that of renilla luciferase. C, D. qRT-PCR ( C ) and western blot ( D ) analyses showed that both VEGFA mRNA and protein expression levels were dramatically suppressed by agomiR-150-5p in HCT116 and HCT8 cells, GAPDH was used as the internal control. ** p

    Techniques Used: Mutagenesis, Clone Assay, Luciferase, Activity Assay, Quantitative RT-PCR, Western Blot, Expressing

    VEGFA knockdown significantly inhibited CRC progression. ( A ) VEGFA expression was downregulated in HCT116 and HCT8 cells transfected with siVEGFA-1 or siVEGFA-2. ( B ) VEGFA knockdown inhibited CRC cell proliferation ( B ), migration ( C ), invasion ( D ) and HUVECs tube formation ( E ). Data are shown as the mean±SD of three independent experiments. * p
    Figure Legend Snippet: VEGFA knockdown significantly inhibited CRC progression. ( A ) VEGFA expression was downregulated in HCT116 and HCT8 cells transfected with siVEGFA-1 or siVEGFA-2. ( B ) VEGFA knockdown inhibited CRC cell proliferation ( B ), migration ( C ), invasion ( D ) and HUVECs tube formation ( E ). Data are shown as the mean±SD of three independent experiments. * p

    Techniques Used: Expressing, Transfection, Migration

    19) Product Images from "Reactive oxygen species modulator-1 (Romo1) predicts unfavorable prognosis in colorectal cancer patients"

    Article Title: Reactive oxygen species modulator-1 (Romo1) predicts unfavorable prognosis in colorectal cancer patients

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0176834

    (A) Western blotting was performed to confirm upregulation of Romo1. (B) The cell viability of the colorectal cancer (CRC) cells was determined by MTT assay after transfection with pFlag-c1 or pFlag-c1 Romo1. Romo1 had no effect on cell viability and proliferation in CRC cells. (C) The effects of Romo1 on cell motility in CRC cells were determined by wound healing assay. Cell monolayers were scratched with a pipette tip and incubated with 5% FBS medium. Cell migration to the wound area was then monitored for 0hr, 24hr, and 48h post-wound, and the percentage of total area covered by the cells was then assessed using the NIH Image program. (D) Matrigel invasion assay was performed to determine the effects of Romo1 on the invasive ability of CRC cells. The invaded cells on the bottom chamber were stained with crystal violet and counted. Images of invasive HCT116 and DLD-1 cells are shown.
    Figure Legend Snippet: (A) Western blotting was performed to confirm upregulation of Romo1. (B) The cell viability of the colorectal cancer (CRC) cells was determined by MTT assay after transfection with pFlag-c1 or pFlag-c1 Romo1. Romo1 had no effect on cell viability and proliferation in CRC cells. (C) The effects of Romo1 on cell motility in CRC cells were determined by wound healing assay. Cell monolayers were scratched with a pipette tip and incubated with 5% FBS medium. Cell migration to the wound area was then monitored for 0hr, 24hr, and 48h post-wound, and the percentage of total area covered by the cells was then assessed using the NIH Image program. (D) Matrigel invasion assay was performed to determine the effects of Romo1 on the invasive ability of CRC cells. The invaded cells on the bottom chamber were stained with crystal violet and counted. Images of invasive HCT116 and DLD-1 cells are shown.

    Techniques Used: Western Blot, MTT Assay, Transfection, Wound Healing Assay, Transferring, Incubation, Migration, Invasion Assay, Staining

    (A) Western blotting was performed to confirm knockdown of Romo1. (B) The cell viability of the colorectal cancer (CRC) cells was determined by MTT assay after transfection with Romo1 siRNA, Romo1 shRNA, control siRNA, or control shRNA. Romo1 had no effect on cell viability and proliferation in CRC cells. (C) The effects of Romo1 on cell motility in CRC cells were determined by wound healing assay. Cell monolayers were scratched with a pipette tip and incubated with 5% FBS medium. Cell migration to the wound area was then monitored for 0hr, 24hr, and 48h post-wound, and the percentage of total area covered by the cells was then assessed using the NIH Image program. (D) Matrigel invasion assay was performed to determine the effects of Romo1 on the invasive ability of CRC cells. The invaded cells on the bottom chamber were stained with crystal violet and counted. Images of invasive HCT116 and DLD-1 cells are shown.
    Figure Legend Snippet: (A) Western blotting was performed to confirm knockdown of Romo1. (B) The cell viability of the colorectal cancer (CRC) cells was determined by MTT assay after transfection with Romo1 siRNA, Romo1 shRNA, control siRNA, or control shRNA. Romo1 had no effect on cell viability and proliferation in CRC cells. (C) The effects of Romo1 on cell motility in CRC cells were determined by wound healing assay. Cell monolayers were scratched with a pipette tip and incubated with 5% FBS medium. Cell migration to the wound area was then monitored for 0hr, 24hr, and 48h post-wound, and the percentage of total area covered by the cells was then assessed using the NIH Image program. (D) Matrigel invasion assay was performed to determine the effects of Romo1 on the invasive ability of CRC cells. The invaded cells on the bottom chamber were stained with crystal violet and counted. Images of invasive HCT116 and DLD-1 cells are shown.

    Techniques Used: Western Blot, MTT Assay, Transfection, shRNA, Wound Healing Assay, Transferring, Incubation, Migration, Invasion Assay, Staining

    20) Product Images from "Empty Spiracles Homeobox 2 (EMX2) Inhibits the Invasion and Tumorigenesis in Colorectal Cancer Cells"

    Article Title: Empty Spiracles Homeobox 2 (EMX2) Inhibits the Invasion and Tumorigenesis in Colorectal Cancer Cells

    Journal: Oncology Research

    doi: 10.3727/096504016X14756640150695

    EMX2 inhibits the tumorigenicity of CRC cells in vivo. HCT116 cells (1 × 10 6 cells/0.1 ml) transfected with pcDNA3.1-EMX2 or empty vector were subcutaneously injected into the flank of BALB/c nude mice. (A) Tumor volume was monitored every week. (B) Four weeks after injection, mice were euthanized, and tumors were dissected and weighed. * p
    Figure Legend Snippet: EMX2 inhibits the tumorigenicity of CRC cells in vivo. HCT116 cells (1 × 10 6 cells/0.1 ml) transfected with pcDNA3.1-EMX2 or empty vector were subcutaneously injected into the flank of BALB/c nude mice. (A) Tumor volume was monitored every week. (B) Four weeks after injection, mice were euthanized, and tumors were dissected and weighed. * p

    Techniques Used: In Vivo, Transfection, Plasmid Preparation, Injection, Mouse Assay

    EMX2 inhibits the migration and invasion of CRC cells. HCT116 cells were transfected with pcDNA3.1-EMX2 or empty vector for 24 h. (A) Cell migration assay was detected using the Transwell chamber assay. (B) Cell invasion assay was detected using the Transwell Matrigel invasion assay. (C) The protein levels of E-cadherin, N-cadherin, and vimentin were determined by Western blotting. * p
    Figure Legend Snippet: EMX2 inhibits the migration and invasion of CRC cells. HCT116 cells were transfected with pcDNA3.1-EMX2 or empty vector for 24 h. (A) Cell migration assay was detected using the Transwell chamber assay. (B) Cell invasion assay was detected using the Transwell Matrigel invasion assay. (C) The protein levels of E-cadherin, N-cadherin, and vimentin were determined by Western blotting. * p

    Techniques Used: Migration, Transfection, Plasmid Preparation, Cell Migration Assay, Transwell Chamber Assay, Invasion Assay, Western Blot

    EMX2 inhibits the activation of the Wnt/β-catenin pathway in CRC cells. HCT116 cells were transfected with pcDNA3.1-EMX2 or empty vector for 24 h. (A) β-Catenin, cyclin D1, and c-Myc expression levels were detected by Western blot. (B) The relative protein expression levels of β-catenin, cyclin D1, and c-Myc were quantified using Image-Pro Plus 6.0 software and normalized to GAPDH. * p
    Figure Legend Snippet: EMX2 inhibits the activation of the Wnt/β-catenin pathway in CRC cells. HCT116 cells were transfected with pcDNA3.1-EMX2 or empty vector for 24 h. (A) β-Catenin, cyclin D1, and c-Myc expression levels were detected by Western blot. (B) The relative protein expression levels of β-catenin, cyclin D1, and c-Myc were quantified using Image-Pro Plus 6.0 software and normalized to GAPDH. * p

    Techniques Used: Activation Assay, Transfection, Plasmid Preparation, Expressing, Western Blot, Software

    EMX2 inhibits proliferation in CRC cells. HCT116 cells were transfected with pcDNA3.1-EMX2 or empty vector for 24 h. (A) EMX2 mRNA expression was determined using RT-qPCR analysis. (B) EMX2 protein expression was determined using Western blot analysis. (C) Cell proliferation was evaluated by the CCK-8 assay. * p
    Figure Legend Snippet: EMX2 inhibits proliferation in CRC cells. HCT116 cells were transfected with pcDNA3.1-EMX2 or empty vector for 24 h. (A) EMX2 mRNA expression was determined using RT-qPCR analysis. (B) EMX2 protein expression was determined using Western blot analysis. (C) Cell proliferation was evaluated by the CCK-8 assay. * p

    Techniques Used: Transfection, Plasmid Preparation, Expressing, Quantitative RT-PCR, Western Blot, CCK-8 Assay

    21) Product Images from "Overexpressed ACP5 has prognostic value in colorectal cancer and promotes cell proliferation and tumorigenesis via FAK/PI3K/AKT signaling pathway"

    Article Title: Overexpressed ACP5 has prognostic value in colorectal cancer and promotes cell proliferation and tumorigenesis via FAK/PI3K/AKT signaling pathway

    Journal: American Journal of Cancer Research

    doi:

    The Akt inhibitors could abolish the oncogenic role of ACP5 in CRC cells. A, B. Proliferation of HT-29 and SW480 cells treated with pcDNA3.1-ACP5 and (or) 1 μmol/L MK2206 by CCK-8 assay. C. Invasion of HT-29 and SW480 cells treated with pcDNA3.1-ACP5 and (or) 1 μmol/L MK2206 by transwell assay. Results shown are the mean ± SD (*P
    Figure Legend Snippet: The Akt inhibitors could abolish the oncogenic role of ACP5 in CRC cells. A, B. Proliferation of HT-29 and SW480 cells treated with pcDNA3.1-ACP5 and (or) 1 μmol/L MK2206 by CCK-8 assay. C. Invasion of HT-29 and SW480 cells treated with pcDNA3.1-ACP5 and (or) 1 μmol/L MK2206 by transwell assay. Results shown are the mean ± SD (*P

    Techniques Used: CCK-8 Assay, Transwell Assay

    ACP5 regulated FAK/PI3K/AKT signaling pathway in CRC cells. A. Expression of total-FAK, phospho-FAK, total-PI3K, phospho-PI3K, total-AKT, phospho-AKT proteins were determined in HT-29 and SW480 cells transfected with pcDNA3.1-ACP5. B. Expression of total-FAK, phospho-FAK, total-PI3K, phospho-PI3K, total-AKT, phospho-AKT proteins were determined in HCT116 and SW1116 cells transfected with siRNAs-ACP5. Results shown are the mean ± SD (**P
    Figure Legend Snippet: ACP5 regulated FAK/PI3K/AKT signaling pathway in CRC cells. A. Expression of total-FAK, phospho-FAK, total-PI3K, phospho-PI3K, total-AKT, phospho-AKT proteins were determined in HT-29 and SW480 cells transfected with pcDNA3.1-ACP5. B. Expression of total-FAK, phospho-FAK, total-PI3K, phospho-PI3K, total-AKT, phospho-AKT proteins were determined in HCT116 and SW1116 cells transfected with siRNAs-ACP5. Results shown are the mean ± SD (**P

    Techniques Used: Expressing, Transfection

    22) Product Images from "Long noncoding RNA CRNDE promotes colorectal cancer cell proliferation via epigenetically silencing DUSP5/CDKN1A expression"

    Article Title: Long noncoding RNA CRNDE promotes colorectal cancer cell proliferation via epigenetically silencing DUSP5/CDKN1A expression

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2017.328

    Knockdown of CRNDE inhibits CRC tumorigenesis in vivo. ( a ) The total numbers of tumors after removal from the mice. ( b ) qRT-PCR analyses indicated that the CRNDE expression was significantly increased in vivo . ( c ) The tumor volumes were calculated every 3 days after inoculation. ( d ) The tumor weights after the tumors were harvested. The data represent the mean±S.D. ( e ) Representative images of H E and immunohistochemical staining of the tumor. IHC revealed a downregulation of the proliferation index Ki-67. * P
    Figure Legend Snippet: Knockdown of CRNDE inhibits CRC tumorigenesis in vivo. ( a ) The total numbers of tumors after removal from the mice. ( b ) qRT-PCR analyses indicated that the CRNDE expression was significantly increased in vivo . ( c ) The tumor volumes were calculated every 3 days after inoculation. ( d ) The tumor weights after the tumors were harvested. The data represent the mean±S.D. ( e ) Representative images of H E and immunohistochemical staining of the tumor. IHC revealed a downregulation of the proliferation index Ki-67. * P

    Techniques Used: In Vivo, Mouse Assay, Quantitative RT-PCR, Expressing, Immunohistochemistry, Staining

    CRNDE knockdown increases the expression of genes involved in cell proliferation. ( a ) Mean-centered, hierarchical clustering of transcripts altered in scrambled siRNA-treated cells and si-CRNDE-treated cells, with three repeats. ( b ) Gene Ontology analysis for all genes with altered expressions between si-NC- and si-CRNDE-treated DLD1 cells in vitro . ( c ) GSEA analysis indicated that cell proliferation process gene sets were significantly altered in genes upregulated by CRNDE knockdown. qRT-PCR was used to validate the changes of several mRNAs involved in cell proliferation. ( d ) qRT-PCR was used to validate the changes of DUSP5 and CDKN1A mRNAs after si-CRNDE 1 or si-NC transfection in DLD1 and HCT116 cells. ( e ) Western blot analysis of DUSP5 and CDKN1A after si-CRNDE 1 or si-NC transfection in DLD1 and HCT116 cells. GAPDH protein was used as an internal control. * P
    Figure Legend Snippet: CRNDE knockdown increases the expression of genes involved in cell proliferation. ( a ) Mean-centered, hierarchical clustering of transcripts altered in scrambled siRNA-treated cells and si-CRNDE-treated cells, with three repeats. ( b ) Gene Ontology analysis for all genes with altered expressions between si-NC- and si-CRNDE-treated DLD1 cells in vitro . ( c ) GSEA analysis indicated that cell proliferation process gene sets were significantly altered in genes upregulated by CRNDE knockdown. qRT-PCR was used to validate the changes of several mRNAs involved in cell proliferation. ( d ) qRT-PCR was used to validate the changes of DUSP5 and CDKN1A mRNAs after si-CRNDE 1 or si-NC transfection in DLD1 and HCT116 cells. ( e ) Western blot analysis of DUSP5 and CDKN1A after si-CRNDE 1 or si-NC transfection in DLD1 and HCT116 cells. GAPDH protein was used as an internal control. * P

    Techniques Used: Expressing, In Vitro, Quantitative RT-PCR, Transfection, Western Blot

    CRNDE promotes CRC cell proliferation in vitro. ( a ) A MTT assay was performed to determine the proliferation of DLD1 and HCT116 cells following treatment with si-CRNDE 1, 2, pcDNA-CRNDE, or the negative control. The data represent the mean±S.D. from three independent experiments. ( b ) Colony formation assays were performed to determine the proliferation of sh-CRNDE-transfected DLD1 and HCT116 cells or overexpression plasmid-transfected DLD1 and HCT116 cells. Experiments were performed in triplicate. ( c ) The colonies were counted and captured. Error bars indicate mean±S.D. * P
    Figure Legend Snippet: CRNDE promotes CRC cell proliferation in vitro. ( a ) A MTT assay was performed to determine the proliferation of DLD1 and HCT116 cells following treatment with si-CRNDE 1, 2, pcDNA-CRNDE, or the negative control. The data represent the mean±S.D. from three independent experiments. ( b ) Colony formation assays were performed to determine the proliferation of sh-CRNDE-transfected DLD1 and HCT116 cells or overexpression plasmid-transfected DLD1 and HCT116 cells. Experiments were performed in triplicate. ( c ) The colonies were counted and captured. Error bars indicate mean±S.D. * P

    Techniques Used: In Vitro, MTT Assay, Negative Control, Transfection, Over Expression, Plasmid Preparation

    DUSP5 function as the tumor suppressor in CRC. ( a ) qRT-PCR analysis of DUSP5expression levels following the treatment of DLD1 and HCT116 cells with pcDNA- DUSP5 or empty vector. ( b ) A MTT assay was performed to determine DLD1 and HCT116 cell proliferation following treatment with pcDNA- DUSP5 or empty vector. The data represent the mean±S.D. from three independent experiments. ( c ) Colony-forming growth assays were performed to determine CRC cell proliferation. The colonies were counted and captured. ( d ) The percentage of apoptotic cells was determined by flow cytometric analysis. The data represent the mean±S.D. from three independent experiments. * P
    Figure Legend Snippet: DUSP5 function as the tumor suppressor in CRC. ( a ) qRT-PCR analysis of DUSP5expression levels following the treatment of DLD1 and HCT116 cells with pcDNA- DUSP5 or empty vector. ( b ) A MTT assay was performed to determine DLD1 and HCT116 cell proliferation following treatment with pcDNA- DUSP5 or empty vector. The data represent the mean±S.D. from three independent experiments. ( c ) Colony-forming growth assays were performed to determine CRC cell proliferation. The colonies were counted and captured. ( d ) The percentage of apoptotic cells was determined by flow cytometric analysis. The data represent the mean±S.D. from three independent experiments. * P

    Techniques Used: Quantitative RT-PCR, Plasmid Preparation, MTT Assay

    Silencing DUSP5 potentially involves the oncogenic function of CRNDE. ( a ) MTT assays were used to determine the cell viability for pcDNA-CRNDE and pcDNA-DUSP5 co-transfected DLD1 and HCT116 cells. ( b ) Colony-forming growth assays were performed to determine CRC cell proliferation. ( c ) The colonies were counted and captured. Experiments were performed in triplicate. Error bars indicate mean±S.D. * P
    Figure Legend Snippet: Silencing DUSP5 potentially involves the oncogenic function of CRNDE. ( a ) MTT assays were used to determine the cell viability for pcDNA-CRNDE and pcDNA-DUSP5 co-transfected DLD1 and HCT116 cells. ( b ) Colony-forming growth assays were performed to determine CRC cell proliferation. ( c ) The colonies were counted and captured. Experiments were performed in triplicate. Error bars indicate mean±S.D. * P

    Techniques Used: MTT Assay, Transfection

    Downregulation of CRNDE promotes G1 arrest and causes apoptosis in CRC cells in vitro. ( a and b ) The bar chart represents the percentage of cells in G0/G1, S, or G2/M phase, as indicated. ( c and d ) Western blot analysis of CDK2 and CDK 6 after si-CRNDE 1 or si-NC transfection in DLD1 and HCT116 cells. GAPDH protein was used as an internal control. ( e and f ) The percentage of apoptotic cells was determined by flow cytometric analysis. The data represent the mean±S.D. from three independent experiments. * P
    Figure Legend Snippet: Downregulation of CRNDE promotes G1 arrest and causes apoptosis in CRC cells in vitro. ( a and b ) The bar chart represents the percentage of cells in G0/G1, S, or G2/M phase, as indicated. ( c and d ) Western blot analysis of CDK2 and CDK 6 after si-CRNDE 1 or si-NC transfection in DLD1 and HCT116 cells. GAPDH protein was used as an internal control. ( e and f ) The percentage of apoptotic cells was determined by flow cytometric analysis. The data represent the mean±S.D. from three independent experiments. * P

    Techniques Used: In Vitro, Western Blot, Transfection

    Relative expression of CRNDE in colorectal cancer tissues and cells compared with adjacent normal tissues and normal colonic epithelial cells. ( a ) Relative expression of CRNDE in human colorectal cancerous tissues compared with noncancerous tissue via GSE21510 data analysis. ( b ) The relative expression of CRNDE in colorectal cancer tissues ( n =80) compared with corresponding nontumor tissues ( n =80). CRNDE expression was examined by qRT-PCR and normalized to GAPDH expression. The results are presented as the fold change in tumor tissues relative to normal tissues (presented as −ΔΔCT). ΔCt value was determined by subtracting the GAPDH Ct value from the CRNDE Ct value. The −ΔΔCt value was calculated from the ΔCt value of the normal tissue control minus the ΔCt value of the CRC tissue. ( c ) CRNDE expression was assessed by qRT-PCR in colorectal cancer cell lines (DLD1, HCT116, SW480, and LOVO) and compared with the normal human colonic epithelial cell line (HCoEpiC). ( d and e ) The data are presented as the relative expression levels in tumor tissues. CRNDE expression was significantly increased in patients with a higher pathological stage and larger tumors. ( f and g ) qRT-PCR analysis of CRNDE expression levels following the treatment of DLD1 and HCT116 cells with si-CRNDE 1, 2, pcDNA-CRNDE, or the negative control. * P
    Figure Legend Snippet: Relative expression of CRNDE in colorectal cancer tissues and cells compared with adjacent normal tissues and normal colonic epithelial cells. ( a ) Relative expression of CRNDE in human colorectal cancerous tissues compared with noncancerous tissue via GSE21510 data analysis. ( b ) The relative expression of CRNDE in colorectal cancer tissues ( n =80) compared with corresponding nontumor tissues ( n =80). CRNDE expression was examined by qRT-PCR and normalized to GAPDH expression. The results are presented as the fold change in tumor tissues relative to normal tissues (presented as −ΔΔCT). ΔCt value was determined by subtracting the GAPDH Ct value from the CRNDE Ct value. The −ΔΔCt value was calculated from the ΔCt value of the normal tissue control minus the ΔCt value of the CRC tissue. ( c ) CRNDE expression was assessed by qRT-PCR in colorectal cancer cell lines (DLD1, HCT116, SW480, and LOVO) and compared with the normal human colonic epithelial cell line (HCoEpiC). ( d and e ) The data are presented as the relative expression levels in tumor tissues. CRNDE expression was significantly increased in patients with a higher pathological stage and larger tumors. ( f and g ) qRT-PCR analysis of CRNDE expression levels following the treatment of DLD1 and HCT116 cells with si-CRNDE 1, 2, pcDNA-CRNDE, or the negative control. * P

    Techniques Used: Expressing, Quantitative RT-PCR, Negative Control

    CRNDE epigenetically silences DUSP5 and CDKN1A transcription by binding to EZH2. ( a ) qRT-PCR analysis of CRNDE nuclear and cytoplasmic expression levels in DLD1 and HCT116 cells. U6 was used as a nucleus marker, and GAPDH was used as a cytosol marker. ( b ) RIP experiments were performed in DLD1 and HCT116 cells, and the coprecipitated RNA was subjected to qRT-PCR for CRNDE. The fold enrichment of CRNDE in EZH2/SUZ12 RIP is relative to its matched IgG control. ( c ) The RNA pull-down experiments also confirmed CRNDE could interact with EZH2 in DLD1 cell line. Protein levels in immunoprecipitates were determined by western blot assay. The expression levels of EZH2 protein were presented. ( d ) qRT-PCR was used to validate the changes of DUSP5 and CDKN1A mRNAs after si-EZH2 or si-NC transfection in DLD1 and HCT116 cells. ( e ) ChIP-qRT-PCR of EZH2 occupancy and H3K27me3 binding in the DUSP5 and CDKN1A promoters in DLD1 and HCT116 cells treated with si-CRNDE 1 (48 h) or si-NC; IgG as a negative control. Error bars indicate mean±S.D. * P
    Figure Legend Snippet: CRNDE epigenetically silences DUSP5 and CDKN1A transcription by binding to EZH2. ( a ) qRT-PCR analysis of CRNDE nuclear and cytoplasmic expression levels in DLD1 and HCT116 cells. U6 was used as a nucleus marker, and GAPDH was used as a cytosol marker. ( b ) RIP experiments were performed in DLD1 and HCT116 cells, and the coprecipitated RNA was subjected to qRT-PCR for CRNDE. The fold enrichment of CRNDE in EZH2/SUZ12 RIP is relative to its matched IgG control. ( c ) The RNA pull-down experiments also confirmed CRNDE could interact with EZH2 in DLD1 cell line. Protein levels in immunoprecipitates were determined by western blot assay. The expression levels of EZH2 protein were presented. ( d ) qRT-PCR was used to validate the changes of DUSP5 and CDKN1A mRNAs after si-EZH2 or si-NC transfection in DLD1 and HCT116 cells. ( e ) ChIP-qRT-PCR of EZH2 occupancy and H3K27me3 binding in the DUSP5 and CDKN1A promoters in DLD1 and HCT116 cells treated with si-CRNDE 1 (48 h) or si-NC; IgG as a negative control. Error bars indicate mean±S.D. * P

    Techniques Used: Binding Assay, Quantitative RT-PCR, Expressing, Marker, Western Blot, Transfection, Chromatin Immunoprecipitation, Negative Control

    23) Product Images from "Engineered sTRAIL-armed MSCs overcome STING deficiency to enhance the therapeutic efficacy of radiotherapy for immune checkpoint blockade"

    Article Title: Engineered sTRAIL-armed MSCs overcome STING deficiency to enhance the therapeutic efficacy of radiotherapy for immune checkpoint blockade

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-022-05069-0

    CRC-specific TRAIL-armed MSC significantly enhanced the therapeutic efficacy of radiotherapy and immunotherapy. A Schematic depiction of soluble TRAIL (sTRAIL) and IFNβ1 expression cassette. The sTRAIL construct consists of a signal peptide, a Furin cleavage site (Furin CS), an Isoleucine Zipper (ILZ), sTRAIL (aa 114–281) a P2A cleavage site and IFNβ1 sequence. This construct is under the control of the CER promoter within the AAV plasmid. After AAV transduction in MSC, the level of TRAIL mRNA was evaluated by qRT-PCR ( n = 3). *** p
    Figure Legend Snippet: CRC-specific TRAIL-armed MSC significantly enhanced the therapeutic efficacy of radiotherapy and immunotherapy. A Schematic depiction of soluble TRAIL (sTRAIL) and IFNβ1 expression cassette. The sTRAIL construct consists of a signal peptide, a Furin cleavage site (Furin CS), an Isoleucine Zipper (ILZ), sTRAIL (aa 114–281) a P2A cleavage site and IFNβ1 sequence. This construct is under the control of the CER promoter within the AAV plasmid. After AAV transduction in MSC, the level of TRAIL mRNA was evaluated by qRT-PCR ( n = 3). *** p

    Techniques Used: Expressing, Construct, Sequencing, Plasmid Preparation, Transduction, Quantitative RT-PCR

    Deficiency of cGAS/STING led to less type I IFN production as well as TRAIL signaling after radiotherapy. A The expression of cGAS and STING in colorectal cancer patients ( n = 259). B The deficiency of cGAS and STING was frequently in CRC patients. C Low STING1 on tumor cells was associated with poor CSS in colorectal cancer patients who received postoperative chemotherapy and radiotherapy ( n = 108, Log-rank p = 0.0479). CT chemotherapy, RT radiotherapy. D SW480, HCT116 (cGAS-deficient), and HT29 (cGAS/STING-proficient) cells were treated with RT (5 Gy) for 12 and 24 h. The mRNA level of IFNα , IFNβ , TRAIL receptor DR4 and DR5 was examined by qRT-PCR (mean ± SEM, n = 3). ** p
    Figure Legend Snippet: Deficiency of cGAS/STING led to less type I IFN production as well as TRAIL signaling after radiotherapy. A The expression of cGAS and STING in colorectal cancer patients ( n = 259). B The deficiency of cGAS and STING was frequently in CRC patients. C Low STING1 on tumor cells was associated with poor CSS in colorectal cancer patients who received postoperative chemotherapy and radiotherapy ( n = 108, Log-rank p = 0.0479). CT chemotherapy, RT radiotherapy. D SW480, HCT116 (cGAS-deficient), and HT29 (cGAS/STING-proficient) cells were treated with RT (5 Gy) for 12 and 24 h. The mRNA level of IFNα , IFNβ , TRAIL receptor DR4 and DR5 was examined by qRT-PCR (mean ± SEM, n = 3). ** p

    Techniques Used: Expressing, Quantitative RT-PCR

    Type I IFN significantly enhanced radiotherapy-induced cell death by TRAIL signaling. A Three colorectal cancer cells were treated with IFNβ (10 ng/mL) and RT (5 Gy) for 24 h. The level of caspase-3 and PARP cleavage was evaluated by western blot. B Three colorectal cancer cells were treated with IFNAR1 (10 μM) and RT (5 Gy) for 24 h. The level of caspase-3 and PARP cleavage was evaluated by western blot. C HT29 (cGAS/STING-proficient) cells were treated with IFNAR1 (10 μM) and RT (5 Gy) for 24 h. The mRNA level of DR4 , DR5 , and TRAIL was evaluated by qRT-PCR (mean ± SEM, n = 3). * p
    Figure Legend Snippet: Type I IFN significantly enhanced radiotherapy-induced cell death by TRAIL signaling. A Three colorectal cancer cells were treated with IFNβ (10 ng/mL) and RT (5 Gy) for 24 h. The level of caspase-3 and PARP cleavage was evaluated by western blot. B Three colorectal cancer cells were treated with IFNAR1 (10 μM) and RT (5 Gy) for 24 h. The level of caspase-3 and PARP cleavage was evaluated by western blot. C HT29 (cGAS/STING-proficient) cells were treated with IFNAR1 (10 μM) and RT (5 Gy) for 24 h. The mRNA level of DR4 , DR5 , and TRAIL was evaluated by qRT-PCR (mean ± SEM, n = 3). * p

    Techniques Used: Western Blot, Quantitative RT-PCR

    Type I IFN significantly enhanced radiotherapy-induced cell death by TRAIL signaling in cGAS-deficient CRC cells. A SW480 and HT29 cells were treated with conditioned medium (CM) from MSCs and RT (5 Gy). After 7 days, the survival fraction was examined by clonogenic assay. B SW480 (cGAS-deficient) and HT29 (cGAS/STING-proficient) cells were treated with MSC-CM and RT (5 Gy) for 24 h. The mRNA level of DR4 and DR5 was evaluated by qRT-PCR (mean ± SEM, n = 3). ** p
    Figure Legend Snippet: Type I IFN significantly enhanced radiotherapy-induced cell death by TRAIL signaling in cGAS-deficient CRC cells. A SW480 and HT29 cells were treated with conditioned medium (CM) from MSCs and RT (5 Gy). After 7 days, the survival fraction was examined by clonogenic assay. B SW480 (cGAS-deficient) and HT29 (cGAS/STING-proficient) cells were treated with MSC-CM and RT (5 Gy) for 24 h. The mRNA level of DR4 and DR5 was evaluated by qRT-PCR (mean ± SEM, n = 3). ** p

    Techniques Used: Clonogenic Assay, Quantitative RT-PCR

    24) Product Images from "Antibody targeting of claudin-1 as a potential colorectal cancer therapy"

    Article Title: Antibody targeting of claudin-1 as a potential colorectal cancer therapy

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-017-0558-5

    Specificity and affinity of the anti-CLDN1 mAb 6F6. a Reactivity of 10 μg/ml of purified 6F6 mAb towards different CRC cell lines that express or not CLDN1, determined by FACS analysis (Additional file 3 ). Left : FACS histograms of cells incubated with ( gray ) or not ( dotted line ) the 6F6 mAb; Right : quantification of CLDN1 expression by western blotting using the anti-CLDN1 polyclonal antibody JAY8. b Determination of the half saturation binding considered as the apparent Kd. SW620 cells were incubated with increasing concentrations of the 6F6 mAb and binding was assessed by FACS c Biodistribution of 125 I-labeled 6F6. Images were acquired three days after intravenous injection of 500 μCi of 125 I-6F6 mAb in the tail vein of mice bearing SW480 or SW480-CLDN1 cell xenografts
    Figure Legend Snippet: Specificity and affinity of the anti-CLDN1 mAb 6F6. a Reactivity of 10 μg/ml of purified 6F6 mAb towards different CRC cell lines that express or not CLDN1, determined by FACS analysis (Additional file 3 ). Left : FACS histograms of cells incubated with ( gray ) or not ( dotted line ) the 6F6 mAb; Right : quantification of CLDN1 expression by western blotting using the anti-CLDN1 polyclonal antibody JAY8. b Determination of the half saturation binding considered as the apparent Kd. SW620 cells were incubated with increasing concentrations of the 6F6 mAb and binding was assessed by FACS c Biodistribution of 125 I-labeled 6F6. Images were acquired three days after intravenous injection of 500 μCi of 125 I-6F6 mAb in the tail vein of mice bearing SW480 or SW480-CLDN1 cell xenografts

    Techniques Used: Purification, FACS, Incubation, Expressing, Western Blot, Binding Assay, Labeling, Injection, Mouse Assay

    25) Product Images from "Deguelin induces apoptosis in colorectal cancer cells by activating the p38 MAPK pathway"

    Article Title: Deguelin induces apoptosis in colorectal cancer cells by activating the p38 MAPK pathway

    Journal: Cancer Management and Research

    doi: 10.2147/CMAR.S169476

    Deguelin induced apoptosis in colorectal cancer cells via activation of p38 MAPK pathway. Notes: ( A ) RKO and ( B ) SW620 cells were treated with indicated concentrations of deguelin for 24 hours. The total lysates were subjected to Western blot analysis, and β-actin was used as an internal reference. ( C ) Analysis of MAPK and NF-κB pathways after deguelin treatment. As a result, phosphorylated form of p38 was upregulated, while no obvious changes were detected in p-JNK, p-ERK1/2, or p65. Administration of SB203580 at a dose of 10 µM, a specific inhibitor of p38 MAPK, could reverse deguelin-induced ( E ) cell proliferation inhibition and ( D and F ) apoptosis in RKO cells. Briefly, SB203580 was dissolved in DMSO at 10 mM as stocking solution, which was further diluted into 10 µM as working solution. ** P
    Figure Legend Snippet: Deguelin induced apoptosis in colorectal cancer cells via activation of p38 MAPK pathway. Notes: ( A ) RKO and ( B ) SW620 cells were treated with indicated concentrations of deguelin for 24 hours. The total lysates were subjected to Western blot analysis, and β-actin was used as an internal reference. ( C ) Analysis of MAPK and NF-κB pathways after deguelin treatment. As a result, phosphorylated form of p38 was upregulated, while no obvious changes were detected in p-JNK, p-ERK1/2, or p65. Administration of SB203580 at a dose of 10 µM, a specific inhibitor of p38 MAPK, could reverse deguelin-induced ( E ) cell proliferation inhibition and ( D and F ) apoptosis in RKO cells. Briefly, SB203580 was dissolved in DMSO at 10 mM as stocking solution, which was further diluted into 10 µM as working solution. ** P

    Techniques Used: Activation Assay, Western Blot, Inhibition

    Deguelin inhibited proliferation of human colorectal cancer cell lines in a dose- and time-dependent manner. Notes: Cell viability was determined using the CCK-8 assay. ( A ) SW480, ( B ) SW620, and ( C ) RKO cells were treated without or with different concentrations of deguelin for 24 and 48 hours. The IC 50 of deguelin for 24 hours in SW480 and SW620 were 122.02 nM and 36.57 nM, respectively. And the IC 50 of deguelin for 48 hours in SW480, SW620, and RKO cells were 40.86 nM, 17.73 nM, and 13.25 µM, respectively. ( D ) SW480 and ( E ) SW620 cells were cultured with deguelin (100 nM) for different time points. ( F ) RKO cells were cultured with deguelin (10 µM) for different time points. The data are presented as mean ± SEM for three different experiments performed in triplicate. * P
    Figure Legend Snippet: Deguelin inhibited proliferation of human colorectal cancer cell lines in a dose- and time-dependent manner. Notes: Cell viability was determined using the CCK-8 assay. ( A ) SW480, ( B ) SW620, and ( C ) RKO cells were treated without or with different concentrations of deguelin for 24 and 48 hours. The IC 50 of deguelin for 24 hours in SW480 and SW620 were 122.02 nM and 36.57 nM, respectively. And the IC 50 of deguelin for 48 hours in SW480, SW620, and RKO cells were 40.86 nM, 17.73 nM, and 13.25 µM, respectively. ( D ) SW480 and ( E ) SW620 cells were cultured with deguelin (100 nM) for different time points. ( F ) RKO cells were cultured with deguelin (10 µM) for different time points. The data are presented as mean ± SEM for three different experiments performed in triplicate. * P

    Techniques Used: CCK-8 Assay, Cell Culture

    Deguelin induced apoptosis in human colorectal cancer cell lines. Notes: Cell apoptosis was evaluated by flow cytometry after annexin V and PI staining as described in the “Materials and methods” section. SW620 cells were treated without or with different concentrations of deguelin for ( A ) 24 hours or ( B ) 48 hours. RKO cells were treated without or with different concentrations of deguelin for ( C ) 24 hours or ( D ) 48 hours. The data are presented as the mean ± SEM of three independent experiments. * P
    Figure Legend Snippet: Deguelin induced apoptosis in human colorectal cancer cell lines. Notes: Cell apoptosis was evaluated by flow cytometry after annexin V and PI staining as described in the “Materials and methods” section. SW620 cells were treated without or with different concentrations of deguelin for ( A ) 24 hours or ( B ) 48 hours. RKO cells were treated without or with different concentrations of deguelin for ( C ) 24 hours or ( D ) 48 hours. The data are presented as the mean ± SEM of three independent experiments. * P

    Techniques Used: Flow Cytometry, Staining

    26) Product Images from "Downregulation of Siah1 promotes colorectal cancer cell proliferation and migration by regulating AKT and YAP ubiquitylation and proteasome degradation"

    Article Title: Downregulation of Siah1 promotes colorectal cancer cell proliferation and migration by regulating AKT and YAP ubiquitylation and proteasome degradation

    Journal: Cancer Cell International

    doi: 10.1186/s12935-020-1124-3

    Silencing of Siah1 promotes migration and invasion of CRC cells. a Depletion of Siah1 in SW480 and HCT116 accelerates the migratory speed of CRC cells, as detected by wound-healing assays. Scale bar: 50 μm. b Silencing of Siah1 in SW480 and HCT116 increases the migratory number of CRC cells, as determined by Transwell chamber invasion assays. Each bar represents the mean ± SD of three independent experiments. *P
    Figure Legend Snippet: Silencing of Siah1 promotes migration and invasion of CRC cells. a Depletion of Siah1 in SW480 and HCT116 accelerates the migratory speed of CRC cells, as detected by wound-healing assays. Scale bar: 50 μm. b Silencing of Siah1 in SW480 and HCT116 increases the migratory number of CRC cells, as determined by Transwell chamber invasion assays. Each bar represents the mean ± SD of three independent experiments. *P

    Techniques Used: Migration

    Siah1 regulates the activity of MAPK, PI3K-AKT and Hippo pathways by promoting the ubiquitylation of AKT and YAP in CRC cells. a The bioinformatics analysis revealed that the MAPK, Hippo, Wnt, and VEGF signaling pathways were significantly enriched. b Western blot analysis of the expression of the indicated proteins in CRC cells with Siah1 stable overexpression. c Western blot analysis of the expression of the indicated proteins in the CRC cells with Siah1 knockdown. d Ascertain the interaction between Siah1 and AKT/YAP through Co-IP. e Ubiquitylation detection assays analysis of the K48-linked polyubiquitin levels of AKT and YAP in CRC cells treated with MG132
    Figure Legend Snippet: Siah1 regulates the activity of MAPK, PI3K-AKT and Hippo pathways by promoting the ubiquitylation of AKT and YAP in CRC cells. a The bioinformatics analysis revealed that the MAPK, Hippo, Wnt, and VEGF signaling pathways were significantly enriched. b Western blot analysis of the expression of the indicated proteins in CRC cells with Siah1 stable overexpression. c Western blot analysis of the expression of the indicated proteins in the CRC cells with Siah1 knockdown. d Ascertain the interaction between Siah1 and AKT/YAP through Co-IP. e Ubiquitylation detection assays analysis of the K48-linked polyubiquitin levels of AKT and YAP in CRC cells treated with MG132

    Techniques Used: Activity Assay, Western Blot, Expressing, Over Expression, Co-Immunoprecipitation Assay

    Model: Siah1, downstream of miR450b-5p, promoting AKT and YAP ubiquitylation and proteasome degradation to regulate the activity of the PI3K-AKT and Hippo signaling pathways, ultimately leading to an aggressive CRC phenotype
    Figure Legend Snippet: Model: Siah1, downstream of miR450b-5p, promoting AKT and YAP ubiquitylation and proteasome degradation to regulate the activity of the PI3K-AKT and Hippo signaling pathways, ultimately leading to an aggressive CRC phenotype

    Techniques Used: Activity Assay

    Decreased expression of Siah1 indicates poor clinical prognosis in colorectal cancer. a Expression of Siah1 protein in ten primary CRC (T) and adjacent noncancerous tissues (N) paired from the same patient, assessed by Western blot (left). The protein expression levels were quantified by comparing the gray level of each band using Quantity one Software (right). b Average N/T ratio of Siah1 mRNA expression by RT-QPCR(n = 50). The expression of mRNA levels was normalized with GAPDH. Error bars represent mean ± SD calculated from 3 parallel experiments. c Representative images of Siah1 expression in normal intestinal epithelium and CRC specimens with different differentiation examined by IHC. Siah1 was positively detected in normal intestinal epithelial cells (left), whereas it was only weakly (middle) or negatively (right) detected in CRC cells. Scale bar: 50 μm. d Influence of Siah1 expression level on overall survival (upper left) and disease-free survival (upper right) of CRC patients by Kaplan–Meier analyses. Green, patients with low Siah1 expression (n = 115); blue, patients with high expression of Siah1 (n = 55). GSE38832 showed the effect of Siah1 expression level on overall survival of CRC patients(lower)
    Figure Legend Snippet: Decreased expression of Siah1 indicates poor clinical prognosis in colorectal cancer. a Expression of Siah1 protein in ten primary CRC (T) and adjacent noncancerous tissues (N) paired from the same patient, assessed by Western blot (left). The protein expression levels were quantified by comparing the gray level of each band using Quantity one Software (right). b Average N/T ratio of Siah1 mRNA expression by RT-QPCR(n = 50). The expression of mRNA levels was normalized with GAPDH. Error bars represent mean ± SD calculated from 3 parallel experiments. c Representative images of Siah1 expression in normal intestinal epithelium and CRC specimens with different differentiation examined by IHC. Siah1 was positively detected in normal intestinal epithelial cells (left), whereas it was only weakly (middle) or negatively (right) detected in CRC cells. Scale bar: 50 μm. d Influence of Siah1 expression level on overall survival (upper left) and disease-free survival (upper right) of CRC patients by Kaplan–Meier analyses. Green, patients with low Siah1 expression (n = 115); blue, patients with high expression of Siah1 (n = 55). GSE38832 showed the effect of Siah1 expression level on overall survival of CRC patients(lower)

    Techniques Used: Expressing, Western Blot, Software, Quantitative RT-PCR, Immunohistochemistry

    Overexpression of Siah1 inhibits proliferation in CRC cells. a Ectopic expression of Siah1 in SW480 and HCT116 cells, analyzed by Western blot. α-Tubulin was used as a loading control. b , c Ectopic expression of Siah1 inhibits cell proliferation, as determined by MTT assays ( b ) and colony formation assays ( c ). d Overexpression of Siah1 inhibits SW480 and HCT116 cell growth in soft-agar assays. Only colonies containing > 50 cells were counted. Each error bar represents the mean ± SD from 3 independent experiments. *P
    Figure Legend Snippet: Overexpression of Siah1 inhibits proliferation in CRC cells. a Ectopic expression of Siah1 in SW480 and HCT116 cells, analyzed by Western blot. α-Tubulin was used as a loading control. b , c Ectopic expression of Siah1 inhibits cell proliferation, as determined by MTT assays ( b ) and colony formation assays ( c ). d Overexpression of Siah1 inhibits SW480 and HCT116 cell growth in soft-agar assays. Only colonies containing > 50 cells were counted. Each error bar represents the mean ± SD from 3 independent experiments. *P

    Techniques Used: Over Expression, Expressing, Western Blot, MTT Assay

    Silencing of Siah1 promotes proliferation the of CRC cells. a RNAi-silencing of Siah1 in specific shRNA-transduced stable SW480 and HCT116 cells by Western blot. α-Tubulin was used as a loading control. b , c Knockdown of endogenous Siah1 promoted cell growth, as assessed by MTT assays ( b ) and colony formation assays ( c ). d Silencing of Siah1 promotes the growth ability of SW480 and HCT116, as determined by Soft agar assays. Colonies containing > 50 cells were scored. Each error bar represents the mean ± SD from3 independent experiments. Scale bar: 50 μm. e , f SW480/GV248 and SW480/Siah1-shRNA cells (2 × 10 6 ) were injected in the hindlimbs of nude mice (n = 6). The volumes of tumor were measured on the indicated days. Panel upper shows tumors after inoculation. Data points are displayed as the mean tumor volumes ± SD (lower panel). f The tumor histological sections were viewed H E staining and IHC staining using an antibody against Siah1 and Ki-67 (left), right panel shows average percentage of staining cells among the total cell as the Ki-67 index. Scale bar: 50 μm
    Figure Legend Snippet: Silencing of Siah1 promotes proliferation the of CRC cells. a RNAi-silencing of Siah1 in specific shRNA-transduced stable SW480 and HCT116 cells by Western blot. α-Tubulin was used as a loading control. b , c Knockdown of endogenous Siah1 promoted cell growth, as assessed by MTT assays ( b ) and colony formation assays ( c ). d Silencing of Siah1 promotes the growth ability of SW480 and HCT116, as determined by Soft agar assays. Colonies containing > 50 cells were scored. Each error bar represents the mean ± SD from3 independent experiments. Scale bar: 50 μm. e , f SW480/GV248 and SW480/Siah1-shRNA cells (2 × 10 6 ) were injected in the hindlimbs of nude mice (n = 6). The volumes of tumor were measured on the indicated days. Panel upper shows tumors after inoculation. Data points are displayed as the mean tumor volumes ± SD (lower panel). f The tumor histological sections were viewed H E staining and IHC staining using an antibody against Siah1 and Ki-67 (left), right panel shows average percentage of staining cells among the total cell as the Ki-67 index. Scale bar: 50 μm

    Techniques Used: shRNA, Western Blot, MTT Assay, Injection, Mouse Assay, Staining, Immunohistochemistry

    Overexpression of Siah1 inhibits the migration and invasion of CRC cells. a Overexpression of Siah1 in SW480 and HCT116 inhibits the migratory speed of CRC cells, as detected by wound-healing assays. Scale bar: 50 μm. b Overexpression of Siah1 in SW480 and HCT116 reduces the migratory number of CRC cells, as determined by Transwell chamber invasion assays. Each bar represents the mean ± SD of three independent experiments. *P
    Figure Legend Snippet: Overexpression of Siah1 inhibits the migration and invasion of CRC cells. a Overexpression of Siah1 in SW480 and HCT116 inhibits the migratory speed of CRC cells, as detected by wound-healing assays. Scale bar: 50 μm. b Overexpression of Siah1 in SW480 and HCT116 reduces the migratory number of CRC cells, as determined by Transwell chamber invasion assays. Each bar represents the mean ± SD of three independent experiments. *P

    Techniques Used: Over Expression, Migration

    27) Product Images from "FBW7 mutations mediate resistance of colorectal cancer to targeted therapies by blocking Mcl-1 degradation"

    Article Title: FBW7 mutations mediate resistance of colorectal cancer to targeted therapies by blocking Mcl-1 degradation

    Journal: Oncogene

    doi: 10.1038/onc.2016.247

    CRC cells with FBW7 mutations are defective in apoptosis and Mcl-1 degradation (A) Indicated FBW7 -WT (black) and -mutant (red) CRC cell lines were treated with 40 μM regorafenib for 48 hr. Apoptosis was analyzed by counting condensed and fragmented nuclei after nuclear staining. (B) Western blotting of Mcl-1 in HCT116 cells treated with regorafenib at indicated concentrations and time points. (C) Western blotting of Mcl-1 in FBW7 -WT (black) and -mutant CRC (red) cell lines treated with 40 μM regorafenib at indicated time points. (D) Western blotting of Mcl-1 in FBW7 -WT (black) and -mutant (red) CRC cell lines treated with 20 μM sorafenib at indicated time points.
    Figure Legend Snippet: CRC cells with FBW7 mutations are defective in apoptosis and Mcl-1 degradation (A) Indicated FBW7 -WT (black) and -mutant (red) CRC cell lines were treated with 40 μM regorafenib for 48 hr. Apoptosis was analyzed by counting condensed and fragmented nuclei after nuclear staining. (B) Western blotting of Mcl-1 in HCT116 cells treated with regorafenib at indicated concentrations and time points. (C) Western blotting of Mcl-1 in FBW7 -WT (black) and -mutant CRC (red) cell lines treated with 40 μM regorafenib at indicated time points. (D) Western blotting of Mcl-1 in FBW7 -WT (black) and -mutant (red) CRC cell lines treated with 20 μM sorafenib at indicated time points.

    Techniques Used: Mutagenesis, Staining, Western Blot

    FBW7 is essential for regorafenib sensitivity and Mcl-1 degradation in CRC cells (A) Regorafenib sensitivity of WT and FBW7 -KO HCT116 cells with or without HA-tagged FBW7 transfection or Mcl-1 knockdown, which was analyzed by western blotting (left panel). (B) Left , western blotting of Mcl-1 and FBW7 in WT and FBW7 -KO HCT116 cells treated with 5 μM regorafenib at indicated time points; right , plotting of FBW7 and Mcl-1 expression quantified by the Image J program and normalized to β-actin. (C) Regorafenib sensitivity of FBW7 -KO HCT116 cells transfected with HA-tagged WT FBW7 or indicated mutants (R465C, R479Q or R505C). Transfected FBW7 and endogenous Mcl-1 were analyzed by western blotting (left panel). (D) Regorafenib sensitivity of FBW7 -mutant SW837 cells with or without HA-tagged FBW7 transfection or Mcl-1 knockdown, which was analyzed by western blotting (left panel). In (A), (C) and (D), regorafenib sensitivity was analyzed by MTS assay on cells treated with regorafenib at indicated concentrations for 72 hr. Western blotting was performed on untreated cells at 24 hr after transfection. Results were expressed as means ± s.d. of three independent experiments.
    Figure Legend Snippet: FBW7 is essential for regorafenib sensitivity and Mcl-1 degradation in CRC cells (A) Regorafenib sensitivity of WT and FBW7 -KO HCT116 cells with or without HA-tagged FBW7 transfection or Mcl-1 knockdown, which was analyzed by western blotting (left panel). (B) Left , western blotting of Mcl-1 and FBW7 in WT and FBW7 -KO HCT116 cells treated with 5 μM regorafenib at indicated time points; right , plotting of FBW7 and Mcl-1 expression quantified by the Image J program and normalized to β-actin. (C) Regorafenib sensitivity of FBW7 -KO HCT116 cells transfected with HA-tagged WT FBW7 or indicated mutants (R465C, R479Q or R505C). Transfected FBW7 and endogenous Mcl-1 were analyzed by western blotting (left panel). (D) Regorafenib sensitivity of FBW7 -mutant SW837 cells with or without HA-tagged FBW7 transfection or Mcl-1 knockdown, which was analyzed by western blotting (left panel). In (A), (C) and (D), regorafenib sensitivity was analyzed by MTS assay on cells treated with regorafenib at indicated concentrations for 72 hr. Western blotting was performed on untreated cells at 24 hr after transfection. Results were expressed as means ± s.d. of three independent experiments.

    Techniques Used: Transfection, Western Blot, Expressing, Mutagenesis, MTS Assay

    FBW7 mutations are enriched in in CRC cells with acquired regorafenib resistance (A) Crystal violet staining of indicated parental and regorafenib-resistant (-R) CRC cells plated at equal density in triplicate after regorafenib (40 μM) treatment for 72 hr. (B) MTS analysis of cell viability of indicated parental (black) and regorafenib-resistant (-R) (red) CRC cells treated with increasing concentrations of regorafenib for 72 hr. Results were expressed as means ± s.d. of three independent experiments. (C) Sequencing of FBW7 genomic region from indicated regorafenib-resistant CRC cell lines highlighting the identified mutations. (D) FBW7 c.1513C > T and c.1393C > T mutations were analyzed by allele-specific PCR in HCT116, Lim1215, and Lim2405 cells after 1-4 rounds (R1-R4) of regorafenib selection. Genomic DNA from LoVo and CCK-81 cells spiked into FBW7 -WT HEK293 cells at indicated ratios were used as controls for c.1513C > T and c.1393C > T mutations, respectively.
    Figure Legend Snippet: FBW7 mutations are enriched in in CRC cells with acquired regorafenib resistance (A) Crystal violet staining of indicated parental and regorafenib-resistant (-R) CRC cells plated at equal density in triplicate after regorafenib (40 μM) treatment for 72 hr. (B) MTS analysis of cell viability of indicated parental (black) and regorafenib-resistant (-R) (red) CRC cells treated with increasing concentrations of regorafenib for 72 hr. Results were expressed as means ± s.d. of three independent experiments. (C) Sequencing of FBW7 genomic region from indicated regorafenib-resistant CRC cell lines highlighting the identified mutations. (D) FBW7 c.1513C > T and c.1393C > T mutations were analyzed by allele-specific PCR in HCT116, Lim1215, and Lim2405 cells after 1-4 rounds (R1-R4) of regorafenib selection. Genomic DNA from LoVo and CCK-81 cells spiked into FBW7 -WT HEK293 cells at indicated ratios were used as controls for c.1513C > T and c.1393C > T mutations, respectively.

    Techniques Used: Staining, Sequencing, Polymerase Chain Reaction, Selection

    28) Product Images from "MicroRNA-204 modulates colorectal cancer cell sensitivity in response to 5-fluorouracil-based treatment by targeting high mobility group protein A2"

    Article Title: MicroRNA-204 modulates colorectal cancer cell sensitivity in response to 5-fluorouracil-based treatment by targeting high mobility group protein A2

    Journal: Biology Open

    doi: 10.1242/bio.015008

    Knockdown HMGA2 inhibits CRC cell growth in response to 5-Fu. (A,B) HMGA2 inhibition was achieved by si-HMGA2 and the inhibitory efficiency was verified by real-time PCR and western blot. (C,D) MTT and BrdU assays were performed and results showed that the viabilities of HCT116 and SW480 cell lines were significantly abrogated when HMGA2 inhibited without 5-Fu treatment. (E,F) Under a dose-dependent 5-Fu treatment at concentrations of 2, 4, 16, 32 and 64 μg/ml, proliferation levels of HMGA2-inhibited CRC cell lines were examined. Cell inhibition rate of HCT116 and SW480 cell lines increased with 5-Fu dose, and the cell inhibition rate of HMGA2 inhibited CRC cell lines was higher compared with null- transfected cell lines. Data represented as means±s.d.; * P
    Figure Legend Snippet: Knockdown HMGA2 inhibits CRC cell growth in response to 5-Fu. (A,B) HMGA2 inhibition was achieved by si-HMGA2 and the inhibitory efficiency was verified by real-time PCR and western blot. (C,D) MTT and BrdU assays were performed and results showed that the viabilities of HCT116 and SW480 cell lines were significantly abrogated when HMGA2 inhibited without 5-Fu treatment. (E,F) Under a dose-dependent 5-Fu treatment at concentrations of 2, 4, 16, 32 and 64 μg/ml, proliferation levels of HMGA2-inhibited CRC cell lines were examined. Cell inhibition rate of HCT116 and SW480 cell lines increased with 5-Fu dose, and the cell inhibition rate of HMGA2 inhibited CRC cell lines was higher compared with null- transfected cell lines. Data represented as means±s.d.; * P

    Techniques Used: Inhibition, Real-time Polymerase Chain Reaction, Western Blot, MTT Assay, Transfection

    Forced expression of HMGA2 restores the effects of miR-204 in CRC cells when exposed to 5-Fu treatment. (A) Time axis of the experiment. The mimics NC, miR-204 mimics, pcDNA3.1 and HMGA2 ORF clone were given 4 h before 5-Fu (32 μg/ml) treatment. At 48 h after 5-Fu treatment, the cells were harvested for further analysis. (B-E) MTT and BrdU assays were used to determine cell proliferation. (F) PI3K, p-PI3K, AKT and p-AKT protein level were determined by western blot assay. Cell viability and PI3K/AKT pathway activation were significantly decreased in cells treated with miR-204 mimics compared to mimics NC group. Forced expression of HMGA2 restored significant inhibition of cell viability and PI3K/AKT pathway activation by miR-204 in HCT116 and SW480 cell lines when exposed to 5-Fu treatment. Figure is representative of three experiments with similar results. Data represented as means±s.d.; * P
    Figure Legend Snippet: Forced expression of HMGA2 restores the effects of miR-204 in CRC cells when exposed to 5-Fu treatment. (A) Time axis of the experiment. The mimics NC, miR-204 mimics, pcDNA3.1 and HMGA2 ORF clone were given 4 h before 5-Fu (32 μg/ml) treatment. At 48 h after 5-Fu treatment, the cells were harvested for further analysis. (B-E) MTT and BrdU assays were used to determine cell proliferation. (F) PI3K, p-PI3K, AKT and p-AKT protein level were determined by western blot assay. Cell viability and PI3K/AKT pathway activation were significantly decreased in cells treated with miR-204 mimics compared to mimics NC group. Forced expression of HMGA2 restored significant inhibition of cell viability and PI3K/AKT pathway activation by miR-204 in HCT116 and SW480 cell lines when exposed to 5-Fu treatment. Figure is representative of three experiments with similar results. Data represented as means±s.d.; * P

    Techniques Used: Expressing, MTT Assay, Western Blot, Activation Assay, Inhibition

    Enhanced miR-204 CRC cell lines repressed HMGA2 protein expression and miR-204 directly targets HMGA2 by binding to its 3′ UTR. (A-C) The results showed that, at 48 h after transfection, enhanced miR-204 in HCT116 and SW480 cell lines significantly repressed HMGA2 protein expression as compared with mimics NC. (D) We created a wt-HMGA2 3′ UTR luciferase reporter vector (wt-HMGA2), as well as a mut-HMGA2 3′ UTR luciferase reporter vector (mut-HMGA2) by sequentially mutating the predicted 8 bp miR-204 binding site in the HMGA2 3′ UTR. (E) The luciferase activity of the HMGA2 3′ UTR luciferase reporter vector was significantly reduced in miR-204 mimics transfected cells compared to scrambled control cells, while miR-204-mediated repression of HMGA2 3′ UTR luciferase reporter activity was abolished by mutation of the putative miR-204 binding site in the HMGA2 3′ UTR. Figure is representative of three experiments with similar results. Data represented as means±s.d.; ** P
    Figure Legend Snippet: Enhanced miR-204 CRC cell lines repressed HMGA2 protein expression and miR-204 directly targets HMGA2 by binding to its 3′ UTR. (A-C) The results showed that, at 48 h after transfection, enhanced miR-204 in HCT116 and SW480 cell lines significantly repressed HMGA2 protein expression as compared with mimics NC. (D) We created a wt-HMGA2 3′ UTR luciferase reporter vector (wt-HMGA2), as well as a mut-HMGA2 3′ UTR luciferase reporter vector (mut-HMGA2) by sequentially mutating the predicted 8 bp miR-204 binding site in the HMGA2 3′ UTR. (E) The luciferase activity of the HMGA2 3′ UTR luciferase reporter vector was significantly reduced in miR-204 mimics transfected cells compared to scrambled control cells, while miR-204-mediated repression of HMGA2 3′ UTR luciferase reporter activity was abolished by mutation of the putative miR-204 binding site in the HMGA2 3′ UTR. Figure is representative of three experiments with similar results. Data represented as means±s.d.; ** P

    Techniques Used: Expressing, Binding Assay, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Mutagenesis

    miR-204 facilitates the inhibitory effect of 5-Fu on CRC cell lines. (A) CRC cell proliferation was examined under conditions of miR-204 overexpression by miR-204 mimic transfection or HMGA2 inhibition by si-HMGA2 without 5-Fu treatment to verify the role of miR-204/HMGA2 in CRC cell growth. Results showed that proliferation of both HCT116 and SW480 cell lines were inhibited by miR-204 overexpression or HMGA2 inhibition compared with null-transfected cell lines. (B) Under a dose-dependent 5-Fu treatment at concentrations of 2, 4, 16, 32 and 64 μg/ml, proliferation levels of miR-204 mimic-transfected CRC cell lines were examined. Results showed that cell inhibition rate of HCT116 and SW480 cell lines increased with 5-Fu dose, and the cell inhibition rate of miR-204 mimic-transfected CRC cell lines was higher compared with null-transfected cell lines. Figure is representative of three experiments with similar results. Data represented as means±s.d.; * P
    Figure Legend Snippet: miR-204 facilitates the inhibitory effect of 5-Fu on CRC cell lines. (A) CRC cell proliferation was examined under conditions of miR-204 overexpression by miR-204 mimic transfection or HMGA2 inhibition by si-HMGA2 without 5-Fu treatment to verify the role of miR-204/HMGA2 in CRC cell growth. Results showed that proliferation of both HCT116 and SW480 cell lines were inhibited by miR-204 overexpression or HMGA2 inhibition compared with null-transfected cell lines. (B) Under a dose-dependent 5-Fu treatment at concentrations of 2, 4, 16, 32 and 64 μg/ml, proliferation levels of miR-204 mimic-transfected CRC cell lines were examined. Results showed that cell inhibition rate of HCT116 and SW480 cell lines increased with 5-Fu dose, and the cell inhibition rate of miR-204 mimic-transfected CRC cell lines was higher compared with null-transfected cell lines. Figure is representative of three experiments with similar results. Data represented as means±s.d.; * P

    Techniques Used: Over Expression, Transfection, Inhibition

    29) Product Images from "Linc00659, a long noncoding RNA, acts as novel oncogene in regulating cancer cell growth in colorectal cancer"

    Article Title: Linc00659, a long noncoding RNA, acts as novel oncogene in regulating cancer cell growth in colorectal cancer

    Journal: Molecular Cancer

    doi: 10.1186/s12943-018-0821-1

    Linc00659 knockdown mediated PI3K-Akt signaling regulation leading to cell growth inhibition and cell death in CRC. a The expression levels of PI3K, Akt, phosphorylation of Akt and GSK3β were examined in HCT116 cells with Linc00659 knockdown. b The downstream effectors of PI3K-Akt pathway were examined by western blotting in HCT116 cells with Linc00659 knockdown. β-Actin was used for an input control. c the expression levels ofβ-catenin, cytoplasmic β-catenin and nuclear β-catenin were analyzed in HCT116 cells after Linc00659 knockdown. GAPDH was used as a cytoplasmic marker and Histone H3 was used as a nucleus marker. d Model for determining the role of Linc00659 in modulating the growth of colon cancer cells
    Figure Legend Snippet: Linc00659 knockdown mediated PI3K-Akt signaling regulation leading to cell growth inhibition and cell death in CRC. a The expression levels of PI3K, Akt, phosphorylation of Akt and GSK3β were examined in HCT116 cells with Linc00659 knockdown. b The downstream effectors of PI3K-Akt pathway were examined by western blotting in HCT116 cells with Linc00659 knockdown. β-Actin was used for an input control. c the expression levels ofβ-catenin, cytoplasmic β-catenin and nuclear β-catenin were analyzed in HCT116 cells after Linc00659 knockdown. GAPDH was used as a cytoplasmic marker and Histone H3 was used as a nucleus marker. d Model for determining the role of Linc00659 in modulating the growth of colon cancer cells

    Techniques Used: Inhibition, Expressing, Western Blot, Marker

    30) Product Images from "eIF4E S209 phosphorylation licenses myc- and stress-driven oncogenesis"

    Article Title: eIF4E S209 phosphorylation licenses myc- and stress-driven oncogenesis

    Journal: eLife

    doi: 10.7554/eLife.60151

    Glutamine deprivation leads to ISR hyperactivation in mutant KRAS/BRAF CRC cells. RKO and HT29 cells were subjected to glutamine deprivation for 24 hr. The indicated proteins were analyzed by western blotting. *, non specific band.
    Figure Legend Snippet: Glutamine deprivation leads to ISR hyperactivation in mutant KRAS/BRAF CRC cells. RKO and HT29 cells were subjected to glutamine deprivation for 24 hr. The indicated proteins were analyzed by western blotting. *, non specific band.

    Techniques Used: Mutagenesis, Western Blot

    31) Product Images from "Low dose of zearalenone elevated colon cancer cell growth through G protein-coupled estrogenic receptor"

    Article Title: Low dose of zearalenone elevated colon cancer cell growth through G protein-coupled estrogenic receptor

    Journal: Scientific Reports

    doi: 10.1038/s41598-021-86788-w

    ZEA increases GPER expression in CRC cell. ( A ) RT-qPCR analysis of GPER gene expression in SW480 and HT29 cells with and without 3 μM ZEA was investigated. ( B ) Western blot analysis of GPER in SW480 and HT29 with and without G15 treatment was investigated. Results shown are mean ± SEM, with n = 3. *p
    Figure Legend Snippet: ZEA increases GPER expression in CRC cell. ( A ) RT-qPCR analysis of GPER gene expression in SW480 and HT29 cells with and without 3 μM ZEA was investigated. ( B ) Western blot analysis of GPER in SW480 and HT29 with and without G15 treatment was investigated. Results shown are mean ± SEM, with n = 3. *p

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot

    Zearalenone promoted the growth of human CRC cells. ( A ) SW480 and HT29 cells were incubated with 3 μM ZEA with or without G15 (GPER antagonist) for 24, 48 and 72 h. CRCs were then trypsinized and fixed with trypan blue solution. Cell counting was carried out using LUNA Automated Cell Counter. *p
    Figure Legend Snippet: Zearalenone promoted the growth of human CRC cells. ( A ) SW480 and HT29 cells were incubated with 3 μM ZEA with or without G15 (GPER antagonist) for 24, 48 and 72 h. CRCs were then trypsinized and fixed with trypan blue solution. Cell counting was carried out using LUNA Automated Cell Counter. *p

    Techniques Used: Incubation, Cell Counting

    ZEA triggered the MAPK-ERK pathway. ( A ) Western blot analysis of the total and phosphorylation of ERK1/2 protein expression after CRC cells treated with 3 μM ZEA (A) in 1 h, and ( B ) at 15 min. (B) RT-qPCR analysis of downstream marker c-jun ( C ) and c-fos ( D ) expression in SW480 and HT29 incubated with and without 3 μM ZEA. Results shown are mean ± SEM, n = 3. *p
    Figure Legend Snippet: ZEA triggered the MAPK-ERK pathway. ( A ) Western blot analysis of the total and phosphorylation of ERK1/2 protein expression after CRC cells treated with 3 μM ZEA (A) in 1 h, and ( B ) at 15 min. (B) RT-qPCR analysis of downstream marker c-jun ( C ) and c-fos ( D ) expression in SW480 and HT29 incubated with and without 3 μM ZEA. Results shown are mean ± SEM, n = 3. *p

    Techniques Used: Western Blot, Expressing, Quantitative RT-PCR, Marker, Incubation

    ZEA promoted YAP nuclear localization and activity through GPER in human CRC cells. ( A )Western blot analysis of the total and phosphorylation of YAP1 and TAZ protein expression after CRC cells treated with ZEA. Western blot analysis of YAP expression in total ( B ), cytoplasmic and nuclear fraction ( C ) of SW480 and HT29 cells after incubated with 3 μM ZEA with or without G15 (GPER antagonist).GADPH a-tubulin and lamin B1 were used as a loading control for total, cytoplasmic and nucleus protein. ( D ) YAP nuclear localisation was determined by immunofluorescence staining for endogenous YAP1 (green). Nuclei was visualized using DAPI for DNA (blue). Results shown are mean ± SEM, n = 3. *p
    Figure Legend Snippet: ZEA promoted YAP nuclear localization and activity through GPER in human CRC cells. ( A )Western blot analysis of the total and phosphorylation of YAP1 and TAZ protein expression after CRC cells treated with ZEA. Western blot analysis of YAP expression in total ( B ), cytoplasmic and nuclear fraction ( C ) of SW480 and HT29 cells after incubated with 3 μM ZEA with or without G15 (GPER antagonist).GADPH a-tubulin and lamin B1 were used as a loading control for total, cytoplasmic and nucleus protein. ( D ) YAP nuclear localisation was determined by immunofluorescence staining for endogenous YAP1 (green). Nuclei was visualized using DAPI for DNA (blue). Results shown are mean ± SEM, n = 3. *p

    Techniques Used: Activity Assay, Western Blot, Expressing, Incubation, Immunofluorescence, Staining

    32) Product Images from "NCOA5 promotes proliferation, migration and invasion of colorectal cancer cells via activation of PI3K/AKT pathway"

    Article Title: NCOA5 promotes proliferation, migration and invasion of colorectal cancer cells via activation of PI3K/AKT pathway

    Journal: Oncotarget

    doi: 10.18632/oncotarget.22429

    Knockdown of NCOA5 suppresses CRC cell migration and invasion, whereas forced expression of NCOA5 enhances these processes (A and B) Wound healing assay. The SW620-shNCOA5 2 # , SW620-shNCOA5 3 # and SW620-shNTC; SW480-LV-NCOA5 and SW480-LV CRC cells were scratched in the monolayer with a pipette tip. Wound closures were photographed at 0, 24 and 48 hours after wounding. The representative figures of wound healing assay were shown (A). The relative migratory ability of control group was presented (B). SW620-shNCOA5 2 # compared with SW620-shNTC group: * , P
    Figure Legend Snippet: Knockdown of NCOA5 suppresses CRC cell migration and invasion, whereas forced expression of NCOA5 enhances these processes (A and B) Wound healing assay. The SW620-shNCOA5 2 # , SW620-shNCOA5 3 # and SW620-shNTC; SW480-LV-NCOA5 and SW480-LV CRC cells were scratched in the monolayer with a pipette tip. Wound closures were photographed at 0, 24 and 48 hours after wounding. The representative figures of wound healing assay were shown (A). The relative migratory ability of control group was presented (B). SW620-shNCOA5 2 # compared with SW620-shNTC group: * , P

    Techniques Used: Migration, Expressing, Wound Healing Assay, Transferring

    Lentivirus-mediated knockdown and overexpression of NCOA5 in CRC cells (A) Western blot analysis of NCOA5 in a panel of CRC cell lines. The lysates of HT29, HCT8, HCE8693, SW620 and SW480 CRC cells were immunoblotted with anti-NCOA5 or anti-GAPDH (a loading control) antibody. ** , P
    Figure Legend Snippet: Lentivirus-mediated knockdown and overexpression of NCOA5 in CRC cells (A) Western blot analysis of NCOA5 in a panel of CRC cell lines. The lysates of HT29, HCT8, HCE8693, SW620 and SW480 CRC cells were immunoblotted with anti-NCOA5 or anti-GAPDH (a loading control) antibody. ** , P

    Techniques Used: Over Expression, Western Blot

    NCOA5 promotes CRC cell proliferation and G1 to S phase transition (A) CCK-8 assay. The SW620-shNCOA5 2 # , SW620-shNCOA5 3 # and SW620-shNTC; SW480-LV-NCOA5 and SW480-LV CRC cells were cultured and the cell proliferation ability was evaluated by CCK-8 assay at the indicated culture time points. SW620-shNCOA5 2 # compared with SW620-shNTC group: * , P
    Figure Legend Snippet: NCOA5 promotes CRC cell proliferation and G1 to S phase transition (A) CCK-8 assay. The SW620-shNCOA5 2 # , SW620-shNCOA5 3 # and SW620-shNTC; SW480-LV-NCOA5 and SW480-LV CRC cells were cultured and the cell proliferation ability was evaluated by CCK-8 assay at the indicated culture time points. SW620-shNCOA5 2 # compared with SW620-shNTC group: * , P

    Techniques Used: Sublimation, CCK-8 Assay, Cell Culture

    Knockdown of NCOA5 inhibits CRC xenografted tumor growth, whereas overexpression of NCOA5 promotes its growth The SW620-shNCOA5 3 # and SW620-shNTC; SW480-LV-NCOA5 and SW480-LV CRC cells s.c. injected into nude mice. The tumor volume (A) was measured after implantation of tumor cells.SW620-shNCOA5 3 # compared with SW620-shNTC group: * , P
    Figure Legend Snippet: Knockdown of NCOA5 inhibits CRC xenografted tumor growth, whereas overexpression of NCOA5 promotes its growth The SW620-shNCOA5 3 # and SW620-shNTC; SW480-LV-NCOA5 and SW480-LV CRC cells s.c. injected into nude mice. The tumor volume (A) was measured after implantation of tumor cells.SW620-shNCOA5 3 # compared with SW620-shNTC group: * , P

    Techniques Used: Over Expression, Injection, Mouse Assay

    NCOA5 upregulates Cyclin D1 and MMP9 as well as downreuglates P27 in CRC cells via the PI3K/AKT pathway (A) Western blot analysis of NCOA5, p-AKT, AKT, p-ERK1/2, ERK1/2, Cyclin D1, P27 and MMP9. The lysates derived from SW620-shNCOA5 2 # , SW620-shNCOA5 3 # and SW620-shNTC; SW480-LV-NCOA5 and SW480-LV CRC cells were immunoblotted with a panel of antibodies specific for NCOA5, p-AKT, AKT, p-ERK1/2, ERK1/2, Cyclin D1, P27, MMP9 and GAPDH (a loading control), respectively. The representative figures of Western blot assay were shown. (B) Western blot analysis after PI3K inhibition. The SW480-LV-NCOA5 CRC cells were pretreated with different concentrations (0, 5, 10 and 20 μM) of PI3K inhibitor LY294002. The lysates of LY294002-treated and -untreated SW480-LV-NCOA5 CRC cells were harvested and then immunoblotted with a panel of antibodies specific for NCOA5, p-AKT, AKT, Cyclin D1, P27, MMP9 and GAPDH (a loading control). The representative figures of Western blot assay were shown. (C) ELISA analysis of MMP9 secretion.SW620-shNCOA5 2 # and SW620-shNCOA5 3 # compared with SW620-shNTC group: ** , P
    Figure Legend Snippet: NCOA5 upregulates Cyclin D1 and MMP9 as well as downreuglates P27 in CRC cells via the PI3K/AKT pathway (A) Western blot analysis of NCOA5, p-AKT, AKT, p-ERK1/2, ERK1/2, Cyclin D1, P27 and MMP9. The lysates derived from SW620-shNCOA5 2 # , SW620-shNCOA5 3 # and SW620-shNTC; SW480-LV-NCOA5 and SW480-LV CRC cells were immunoblotted with a panel of antibodies specific for NCOA5, p-AKT, AKT, p-ERK1/2, ERK1/2, Cyclin D1, P27, MMP9 and GAPDH (a loading control), respectively. The representative figures of Western blot assay were shown. (B) Western blot analysis after PI3K inhibition. The SW480-LV-NCOA5 CRC cells were pretreated with different concentrations (0, 5, 10 and 20 μM) of PI3K inhibitor LY294002. The lysates of LY294002-treated and -untreated SW480-LV-NCOA5 CRC cells were harvested and then immunoblotted with a panel of antibodies specific for NCOA5, p-AKT, AKT, Cyclin D1, P27, MMP9 and GAPDH (a loading control). The representative figures of Western blot assay were shown. (C) ELISA analysis of MMP9 secretion.SW620-shNCOA5 2 # and SW620-shNCOA5 3 # compared with SW620-shNTC group: ** , P

    Techniques Used: Western Blot, Derivative Assay, Inhibition, Enzyme-linked Immunosorbent Assay

    33) Product Images from "LncRNA MCF2L-AS1 aggravates the malignant development of colorectal cancer via targeting miR-105-5p/RAB22A axis"

    Article Title: LncRNA MCF2L-AS1 aggravates the malignant development of colorectal cancer via targeting miR-105-5p/RAB22A axis

    Journal: BMC Cancer

    doi: 10.1186/s12885-021-08668-w

    MCF2L-AS1 is highly expressed in CRC cells and accelerates the progression of CRC. A . The expression of MCF2L-AS1 in COAD tissues and adjacent normal tissues was assessed via GEPIA database. B . MCF2L-AS1 expression was measured in CRC cell lines (HCT15, SW620, SW116 and LOVO) and human normal colon epithelial cells (NCM-460) by RT-qPCR. C . The interference efficiency of MCF2L-AS1 in HCT15 and SW116 cells was tested by RT-qPCR assay. D and E . Cell proliferation in HCT15 and SW116 cells after MCF2L-AS1 silencing was evaluated through IF staining and EdU assay. F and G . The migratory and invasive abilities of CRC cells upon MCF2L-AS1 silencing were evaluated through Transwell assays. H and I . Flow cytometry assay and caspase-3/8/9 activity analysis were utilized to test cell apoptosis upon MCF2L-AS1 silencing. J . The EMT process was assessed through IF assay after MCF2L-AS1 was silenced in HCT15 and SW116 cells. Adjustments of individual color channels were made on ‘Merge’ figures. The statistical analysis of Fig. 1B-I was tested with one-way ANOVA. * P
    Figure Legend Snippet: MCF2L-AS1 is highly expressed in CRC cells and accelerates the progression of CRC. A . The expression of MCF2L-AS1 in COAD tissues and adjacent normal tissues was assessed via GEPIA database. B . MCF2L-AS1 expression was measured in CRC cell lines (HCT15, SW620, SW116 and LOVO) and human normal colon epithelial cells (NCM-460) by RT-qPCR. C . The interference efficiency of MCF2L-AS1 in HCT15 and SW116 cells was tested by RT-qPCR assay. D and E . Cell proliferation in HCT15 and SW116 cells after MCF2L-AS1 silencing was evaluated through IF staining and EdU assay. F and G . The migratory and invasive abilities of CRC cells upon MCF2L-AS1 silencing were evaluated through Transwell assays. H and I . Flow cytometry assay and caspase-3/8/9 activity analysis were utilized to test cell apoptosis upon MCF2L-AS1 silencing. J . The EMT process was assessed through IF assay after MCF2L-AS1 was silenced in HCT15 and SW116 cells. Adjustments of individual color channels were made on ‘Merge’ figures. The statistical analysis of Fig. 1B-I was tested with one-way ANOVA. * P

    Techniques Used: Expressing, Quantitative RT-PCR, Staining, EdU Assay, Flow Cytometry, Activity Assay

    34) Product Images from "Elf3 drives β-catenin transactivation and associates with poor prognosis in colorectal cancer"

    Article Title: Elf3 drives β-catenin transactivation and associates with poor prognosis in colorectal cancer

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2014.206

    Elf3 enhances the Wnt/ β -catenin signaling pathway in CRC cells. ( a , b ) Representative Western blot and summarized data show that Elf3 siRNA transfection significantly decreases total β -catenin expression, while Elf3 overexpression significantly increased total β -catenin levels in HCT116 cells. Statistical result based on three independent experiments is indicated in ( a ), and representative western blot images are shown in ( b ). ( c , d ) Representative Western blot and summarized data show that Elf3 siRNA transfection significantly decreases nuclear β -catenin expression, while Elf3 overexpression significantly increased nuclear β -catenin levels in HCT116 cells. Statistical result based on three independent experiments is indicated in ( c ), and representative western blot images are shown in ( d ). ( e , f ) TOP/FOP flash assays indicate that Elf3 knockdown significantly reduces the β -catenin/TCF transcriptional activity ( e ), while Elf3 overexpression significantly increases the β -catenin/TCF transcriptional activity in HCT116 cells ( f ). ( g , h ) qRT-PCR assay showing Elf3 could transactivate the Wnt/ β -catenin downstream genes in CRC cells. Knockdown of Elf3 could suppress the expression of Wnt/ β -catenin target (c-Myc, VEGF, CCND1, MMP-7 and c-Jun) downstream ( g ), while ectopic expression of Elf3 transactivated these downstream genes ( h )
    Figure Legend Snippet: Elf3 enhances the Wnt/ β -catenin signaling pathway in CRC cells. ( a , b ) Representative Western blot and summarized data show that Elf3 siRNA transfection significantly decreases total β -catenin expression, while Elf3 overexpression significantly increased total β -catenin levels in HCT116 cells. Statistical result based on three independent experiments is indicated in ( a ), and representative western blot images are shown in ( b ). ( c , d ) Representative Western blot and summarized data show that Elf3 siRNA transfection significantly decreases nuclear β -catenin expression, while Elf3 overexpression significantly increased nuclear β -catenin levels in HCT116 cells. Statistical result based on three independent experiments is indicated in ( c ), and representative western blot images are shown in ( d ). ( e , f ) TOP/FOP flash assays indicate that Elf3 knockdown significantly reduces the β -catenin/TCF transcriptional activity ( e ), while Elf3 overexpression significantly increases the β -catenin/TCF transcriptional activity in HCT116 cells ( f ). ( g , h ) qRT-PCR assay showing Elf3 could transactivate the Wnt/ β -catenin downstream genes in CRC cells. Knockdown of Elf3 could suppress the expression of Wnt/ β -catenin target (c-Myc, VEGF, CCND1, MMP-7 and c-Jun) downstream ( g ), while ectopic expression of Elf3 transactivated these downstream genes ( h )

    Techniques Used: Western Blot, Transfection, Expressing, Over Expression, Activity Assay, Quantitative RT-PCR

    Expression of β -catenin rescued CRC cell malignant phenotypes that were suppressed by knockdown of Elf3. ( a ) The statistical analysis of CRC cell apoptosis in different experimental groups. The HCT116 and SW1116 cells were treated by specific siRNAs for Elf3 expression in the absence or presence of β -catenin expression vector. Statistical result based on three independent experiments ( P -values indicated, t -test). ( b , c ) Representative flow cytometry images in experiments described above. ( d , e ) Statistical result and representative images of Transwell assay of HCT116 and SW116 cells treated by specific siRNAs for Elf3 expression in the absence or presence of β -catenin expression vector. ( f , g ) Proliferation of HCT116 and SW1116 cells treated by specific siRNAs for Elf3 expression in the absence or presence of β -catenin expression vector ( P -values indicated, t -test)
    Figure Legend Snippet: Expression of β -catenin rescued CRC cell malignant phenotypes that were suppressed by knockdown of Elf3. ( a ) The statistical analysis of CRC cell apoptosis in different experimental groups. The HCT116 and SW1116 cells were treated by specific siRNAs for Elf3 expression in the absence or presence of β -catenin expression vector. Statistical result based on three independent experiments ( P -values indicated, t -test). ( b , c ) Representative flow cytometry images in experiments described above. ( d , e ) Statistical result and representative images of Transwell assay of HCT116 and SW116 cells treated by specific siRNAs for Elf3 expression in the absence or presence of β -catenin expression vector. ( f , g ) Proliferation of HCT116 and SW1116 cells treated by specific siRNAs for Elf3 expression in the absence or presence of β -catenin expression vector ( P -values indicated, t -test)

    Techniques Used: Expressing, Plasmid Preparation, Flow Cytometry, Transwell Assay

    Elf3 could serve as a potential therapeutic target in CRC. ( a , b ) Knockdown of Elf3 inhibited growth of xenograft in nude mice. Human CRC HCT116 cells were injected subcutaneously into the right armpit to establish a xenograft model, and specific siRNA for Elf3 or control siRNA was introduced to tumors cells by in vivo -jetPEI nanoparticles. The sizes of in situ and resected tumors treated with/without Elf3 siRNAs in the last time point (12 days after first injection) are shown in panels ( a ) and ( b ), respectively. ( c ) Tumor growth curves in the Elf3 siRNA group and the control group ( P
    Figure Legend Snippet: Elf3 could serve as a potential therapeutic target in CRC. ( a , b ) Knockdown of Elf3 inhibited growth of xenograft in nude mice. Human CRC HCT116 cells were injected subcutaneously into the right armpit to establish a xenograft model, and specific siRNA for Elf3 or control siRNA was introduced to tumors cells by in vivo -jetPEI nanoparticles. The sizes of in situ and resected tumors treated with/without Elf3 siRNAs in the last time point (12 days after first injection) are shown in panels ( a ) and ( b ), respectively. ( c ) Tumor growth curves in the Elf3 siRNA group and the control group ( P

    Techniques Used: Mouse Assay, Injection, In Vivo, In Situ

    35) Product Images from "E2A attenuates tumor-initiating capacity of colorectal cancer cells via the Wnt/beta-catenin pathway"

    Article Title: E2A attenuates tumor-initiating capacity of colorectal cancer cells via the Wnt/beta-catenin pathway

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-019-1261-5

    E2A suppressed the tumor-initiating capacity of CRC cells in vitro. a Effect of shE2A on SW480 cell sphere-formation ability. The sphere-formation ability of SW480 cells was enhanced by shE2A. P
    Figure Legend Snippet: E2A suppressed the tumor-initiating capacity of CRC cells in vitro. a Effect of shE2A on SW480 cell sphere-formation ability. The sphere-formation ability of SW480 cells was enhanced by shE2A. P

    Techniques Used: In Vitro

    Wnt/β-catenin pathway is crucial for E2A in CRC cell tumor-initiating capacity. a Effect of E2A on the expression of β-catenin in cell nuclei. shE2A expression enhanced the β-catenin expression in SW480 cell nuclei, and E12/E47 attenuated the β-catenin expression in Caco-2 cell nuclei. Lower panel: Densitometric analysis of beta-catenin normalized to SP1. Data in the histograms is expressed as means ± SD from three separate experiments. b , c E2A suppressed the Wnt pathway in CRC cells. shE2A increased expression of the Wnt pathway effectors TCF-1 and cyclin D1 in SW480 cells, whereas E12 and E47 had the opposite effect in Caco-2 cells. Right panel: Densitometric analysis of TCF-1 and cyclin D1 normalized to GAPDH. Data in the histograms is expressed as mean ± SD from three separate experiments. d Wnt pathway is crucial for E2A suppressing tumor-initiating ability. SW480/shE2A cells treated with 5 μM CGP049090, which is a small-molecular compound inhibiting Wnt/β-catenin pathway activity, had decreased tumor-initiating ability compared with that of SW480/shE2A without CGP049090 (upper panel). wnt3a, at 100 ng/mL, added Caco-2 restored the tumor-initiating ability inhibited by E12/E47 to a level like that in Caco-2/NC cells (lower panel). e Lgr5 expression in SW480/shE2A cells treated with 5 μM CGP049090 was like that in SW480/shNC, which was lower than that in SW480/shE2A without CGP049090. wnt3a, at 100 ng/mL, added to Caco-2 cells, restored the Lgr5 expression inhibited by E12/E47 to a level like that in Caco-2/NC cells (lower panel). Data are the mean ± SD from at least three separate experiments. *, P
    Figure Legend Snippet: Wnt/β-catenin pathway is crucial for E2A in CRC cell tumor-initiating capacity. a Effect of E2A on the expression of β-catenin in cell nuclei. shE2A expression enhanced the β-catenin expression in SW480 cell nuclei, and E12/E47 attenuated the β-catenin expression in Caco-2 cell nuclei. Lower panel: Densitometric analysis of beta-catenin normalized to SP1. Data in the histograms is expressed as means ± SD from three separate experiments. b , c E2A suppressed the Wnt pathway in CRC cells. shE2A increased expression of the Wnt pathway effectors TCF-1 and cyclin D1 in SW480 cells, whereas E12 and E47 had the opposite effect in Caco-2 cells. Right panel: Densitometric analysis of TCF-1 and cyclin D1 normalized to GAPDH. Data in the histograms is expressed as mean ± SD from three separate experiments. d Wnt pathway is crucial for E2A suppressing tumor-initiating ability. SW480/shE2A cells treated with 5 μM CGP049090, which is a small-molecular compound inhibiting Wnt/β-catenin pathway activity, had decreased tumor-initiating ability compared with that of SW480/shE2A without CGP049090 (upper panel). wnt3a, at 100 ng/mL, added Caco-2 restored the tumor-initiating ability inhibited by E12/E47 to a level like that in Caco-2/NC cells (lower panel). e Lgr5 expression in SW480/shE2A cells treated with 5 μM CGP049090 was like that in SW480/shNC, which was lower than that in SW480/shE2A without CGP049090. wnt3a, at 100 ng/mL, added to Caco-2 cells, restored the Lgr5 expression inhibited by E12/E47 to a level like that in Caco-2/NC cells (lower panel). Data are the mean ± SD from at least three separate experiments. *, P

    Techniques Used: Expressing, Activity Assay

    E2A suppress CRC liver metastasis in vivo. a Representative images of liver metastasis in SW480 cells in vivo. Scale bar: 100 μm. Magnification: 40×. Six mice are included in each group; ( b ) The number of metastatic nodules in each group. Data are presented as mean ± SD. *, P
    Figure Legend Snippet: E2A suppress CRC liver metastasis in vivo. a Representative images of liver metastasis in SW480 cells in vivo. Scale bar: 100 μm. Magnification: 40×. Six mice are included in each group; ( b ) The number of metastatic nodules in each group. Data are presented as mean ± SD. *, P

    Techniques Used: In Vivo, Mouse Assay

    Foxm1 is a downstream target through which E2A suppressed CRC cell tumor-initiating capacity. a , b Heat map and volcano plot displaying the hierarchical clustering of the mRNA expression profiles. c PPI protein network generated by Genemania showing the relationship between E2A, FoxM1 and β-catenin. d Downregulation of E2A by shE2A resulted in increased FoxM1 mRNA expression in SW480 cells, whereas transfection of plasmids encoding E12 or E47 decreased FoxM1 mRNA in Caco-2 cells, as revealed by semi-qRT-PCR. e E2A decreased FoxM1 protein expression, as determined by immunoblot analysis. Lower panel: Densitometric analysis of FoxM1 normalized to GAPDH. Data in the histograms is expressed as the mean ± SD from three separate experiments. f Immunoblot analysis of FoxM1, with GAPDH as loading control. g shFoxM1 decreased tumor-initiating ability of SW480/shE2A cells to a level like that of SW480/shE2A cells. h Lgr5 expression in SW480/shE2A cells treated with shFoxM1 is like that in SW480/shNC, which is lower than in SW480/shE2A. Lower panel: Densitometric analysis of Lgr5 normalized to GAPDH. Data in the histograms is expressed as mean ± SD from three separate experiments. i Schematic diagram illustrates that E2A inhibited FoxM1 transcription to attenuate FoxM1, depending on β-catenin translocation to cell nuclei, which decreased the Wnt pathway in CRC cells
    Figure Legend Snippet: Foxm1 is a downstream target through which E2A suppressed CRC cell tumor-initiating capacity. a , b Heat map and volcano plot displaying the hierarchical clustering of the mRNA expression profiles. c PPI protein network generated by Genemania showing the relationship between E2A, FoxM1 and β-catenin. d Downregulation of E2A by shE2A resulted in increased FoxM1 mRNA expression in SW480 cells, whereas transfection of plasmids encoding E12 or E47 decreased FoxM1 mRNA in Caco-2 cells, as revealed by semi-qRT-PCR. e E2A decreased FoxM1 protein expression, as determined by immunoblot analysis. Lower panel: Densitometric analysis of FoxM1 normalized to GAPDH. Data in the histograms is expressed as the mean ± SD from three separate experiments. f Immunoblot analysis of FoxM1, with GAPDH as loading control. g shFoxM1 decreased tumor-initiating ability of SW480/shE2A cells to a level like that of SW480/shE2A cells. h Lgr5 expression in SW480/shE2A cells treated with shFoxM1 is like that in SW480/shNC, which is lower than in SW480/shE2A. Lower panel: Densitometric analysis of Lgr5 normalized to GAPDH. Data in the histograms is expressed as mean ± SD from three separate experiments. i Schematic diagram illustrates that E2A inhibited FoxM1 transcription to attenuate FoxM1, depending on β-catenin translocation to cell nuclei, which decreased the Wnt pathway in CRC cells

    Techniques Used: Expressing, Generated, Transfection, Quantitative RT-PCR, Translocation Assay

    36) Product Images from "STX2 promotes colorectal cancer metastasis through a positive feedback loop that activates the NF-κB pathway"

    Article Title: STX2 promotes colorectal cancer metastasis through a positive feedback loop that activates the NF-κB pathway

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-0675-x

    NF-κB increased STX2 expression by directly binding to the STX2 promoter. a Western blot analysis of the P65 and STX2 protein levels in SW480 and LOVO cells that had been treated with TNFα (20 ng/ml) for 24 h. b ChIP analysis. Schematic illustration of the STX2 promoter. (The region that binds with NF-κB p50 is indicated with “+”; top). ChIP analysis of NF-κB p50 binding with the STX2 promoter in LOVO cells (bottom). c Luciferase activity analysis of the indicated cells transfected with the indicated plasmids (Error bars represent the mean ± s.d. from three independent experiments. d Model: STX2 increased the activity of the NF-κB pathway by interacting with TRAF6, led to the increased expression of STX2 and formed a positive feedback loop, ultimately promoted CRC metastasis.** p
    Figure Legend Snippet: NF-κB increased STX2 expression by directly binding to the STX2 promoter. a Western blot analysis of the P65 and STX2 protein levels in SW480 and LOVO cells that had been treated with TNFα (20 ng/ml) for 24 h. b ChIP analysis. Schematic illustration of the STX2 promoter. (The region that binds with NF-κB p50 is indicated with “+”; top). ChIP analysis of NF-κB p50 binding with the STX2 promoter in LOVO cells (bottom). c Luciferase activity analysis of the indicated cells transfected with the indicated plasmids (Error bars represent the mean ± s.d. from three independent experiments. d Model: STX2 increased the activity of the NF-κB pathway by interacting with TRAF6, led to the increased expression of STX2 and formed a positive feedback loop, ultimately promoted CRC metastasis.** p

    Techniques Used: Expressing, Binding Assay, Western Blot, Chromatin Immunoprecipitation, Luciferase, Activity Assay, Transfection

    STX2 upregulation was associated with CRC metastasis and a poorer clinical outcome. a qPCR analysis of STX2 expression in the fresh human CRC tissues of 55 CRC cases and the matched adjacent normal tissues; STX2 expression was normalized to GAPDH and expressed relative to the matched adjacent normal tissues (2-ΔΔCт). b qPCR analysis of STX2 mRNA expression in the normal intestinal mucosa and in primary CRC with or without metastasis. Boundaries of boxes represent bounding of the boxes and stand for the lower and upper quartile. Lines within the boxes and whiskers represent median and extremum(maximum and minimum). c IHC analysis of STX2 protein expression in the normal intestinal mucosa and in primary CRC with or without metastasis. d , e Kaplan–Meier analysis of the influence of STX2 expression and LN metastasis on overall survival. ** p
    Figure Legend Snippet: STX2 upregulation was associated with CRC metastasis and a poorer clinical outcome. a qPCR analysis of STX2 expression in the fresh human CRC tissues of 55 CRC cases and the matched adjacent normal tissues; STX2 expression was normalized to GAPDH and expressed relative to the matched adjacent normal tissues (2-ΔΔCт). b qPCR analysis of STX2 mRNA expression in the normal intestinal mucosa and in primary CRC with or without metastasis. Boundaries of boxes represent bounding of the boxes and stand for the lower and upper quartile. Lines within the boxes and whiskers represent median and extremum(maximum and minimum). c IHC analysis of STX2 protein expression in the normal intestinal mucosa and in primary CRC with or without metastasis. d , e Kaplan–Meier analysis of the influence of STX2 expression and LN metastasis on overall survival. ** p

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Immunohistochemistry

    Overexpression of STX2 promoted the metastasis of CRC cells in vitro and in vivo. a qPCR and western blot analyses of endogenous STX2 expression in the CRC cell lines. b qPCR and western blot analyses of STX2 overexpression in SW480 and HCT116 cells. c , d Analysis of the migratory and invasive properties of SW480/Vector, SW480/STX2, and HCT116/Vector, HCT116/STX2 cells using Boyden chambers or Matrigel-coated Boyden chambers, respectively. Error bars represent the mean ± s.d. from three independent experiments. e , f Wound-healing assay. Histograms represent the average migrated distances at the indicated times. Error bars represent the mean ± s.d. from three independent experiments. g Three-dimensional morphology analysis. Histograms represent the average number of filopodia formed by each cell sphere from three independent experiments. Error bars represent mean ± s.d. h Representative gross and microscopic images of the intestines and livers are shown. The sections were stained with H E. The arrows indicate the primary tumors in the intestines, visible metastatic nodules in the liver and the infiltration of the primary tumors into the intestines. i The number of visible metastatic nodules in the liver. j Overall survival time of the mice bearing liver metastases of SW480/Vector and SW480/STX2 tumors. ** p
    Figure Legend Snippet: Overexpression of STX2 promoted the metastasis of CRC cells in vitro and in vivo. a qPCR and western blot analyses of endogenous STX2 expression in the CRC cell lines. b qPCR and western blot analyses of STX2 overexpression in SW480 and HCT116 cells. c , d Analysis of the migratory and invasive properties of SW480/Vector, SW480/STX2, and HCT116/Vector, HCT116/STX2 cells using Boyden chambers or Matrigel-coated Boyden chambers, respectively. Error bars represent the mean ± s.d. from three independent experiments. e , f Wound-healing assay. Histograms represent the average migrated distances at the indicated times. Error bars represent the mean ± s.d. from three independent experiments. g Three-dimensional morphology analysis. Histograms represent the average number of filopodia formed by each cell sphere from three independent experiments. Error bars represent mean ± s.d. h Representative gross and microscopic images of the intestines and livers are shown. The sections were stained with H E. The arrows indicate the primary tumors in the intestines, visible metastatic nodules in the liver and the infiltration of the primary tumors into the intestines. i The number of visible metastatic nodules in the liver. j Overall survival time of the mice bearing liver metastases of SW480/Vector and SW480/STX2 tumors. ** p

    Techniques Used: Over Expression, In Vitro, In Vivo, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Plasmid Preparation, Wound Healing Assay, Staining, Mouse Assay

    Downregulation of STX2 repressed the metastatic potential of CRC cells in vitro and in vivo. a qPCR and western blot analyses of STX2 in specific shRNA-transduced stable cells derived from SW620 and LOVO. b , c The migratory and invasive properties of the stable cell lines were analyzed using Boyden chambers or Matrigel-coated Boyden chambers. Error bars represent the mean ± s.d. from three independent experiments. d , e Wound-healing assay. Histograms represent the average migrated distances at the indicated times. Error bars represent the mean ± s.d. from three independent experiments. f Three-dimensional morphology analysis. Histograms represent the average number of filopodia formed by each cell sphere from three independent experiments. Error bars represent the mean ± s.d. g Representative gross and microscopic images of the intestines and livers are shown. The sections were stained with H E. The arrows indicate the primary tumors in the intestines, visible metastatic nodules in the liver and infiltrative tumors in the intestine. h The number of visible metastatic nodules in the liver. i Overall survival times of the different group of mice bearing liver metastases. ** p
    Figure Legend Snippet: Downregulation of STX2 repressed the metastatic potential of CRC cells in vitro and in vivo. a qPCR and western blot analyses of STX2 in specific shRNA-transduced stable cells derived from SW620 and LOVO. b , c The migratory and invasive properties of the stable cell lines were analyzed using Boyden chambers or Matrigel-coated Boyden chambers. Error bars represent the mean ± s.d. from three independent experiments. d , e Wound-healing assay. Histograms represent the average migrated distances at the indicated times. Error bars represent the mean ± s.d. from three independent experiments. f Three-dimensional morphology analysis. Histograms represent the average number of filopodia formed by each cell sphere from three independent experiments. Error bars represent the mean ± s.d. g Representative gross and microscopic images of the intestines and livers are shown. The sections were stained with H E. The arrows indicate the primary tumors in the intestines, visible metastatic nodules in the liver and infiltrative tumors in the intestine. h The number of visible metastatic nodules in the liver. i Overall survival times of the different group of mice bearing liver metastases. ** p

    Techniques Used: In Vitro, In Vivo, Real-time Polymerase Chain Reaction, Western Blot, shRNA, Derivative Assay, Stable Transfection, Wound Healing Assay, Staining, Mouse Assay

    37) Product Images from "Expression of Zinc Finger and BTB Domain-Containing 4 in Colorectal Cancer and Its Clinical Significance"

    Article Title: Expression of Zinc Finger and BTB Domain-Containing 4 in Colorectal Cancer and Its Clinical Significance

    Journal: Cancer Management and Research

    doi: 10.2147/CMAR.S266529

    ZBTB4 expression was down-regulated in colorectal carcinoma (CRC). ( A ) Relative mRNA expression of ZBTB4 in CRC tissues and their related normal tissues from the TCGA database. P value was calculated by Student’s t -test. The mRNA ( B ) and protein ( C ) expressions of ZBTB4 in CRC cell lines (HT-29, HCT-116, SW480, CaCO2 and SW620) and normal colonic epithelial cell lines (NCM460). ***P
    Figure Legend Snippet: ZBTB4 expression was down-regulated in colorectal carcinoma (CRC). ( A ) Relative mRNA expression of ZBTB4 in CRC tissues and their related normal tissues from the TCGA database. P value was calculated by Student’s t -test. The mRNA ( B ) and protein ( C ) expressions of ZBTB4 in CRC cell lines (HT-29, HCT-116, SW480, CaCO2 and SW620) and normal colonic epithelial cell lines (NCM460). ***P

    Techniques Used: Expressing

    38) Product Images from "Functional assessment of miR-1291 in colon cancer cells"

    Article Title: Functional assessment of miR-1291 in colon cancer cells

    Journal: International Journal of Oncology

    doi: 10.3892/ijo.2022.5303

    Cell cycle analysis. (A) The expression of cell cycle-related proteins was evaluated by western blotting. (B) Two independent experiments using rapid growing cultures evaluated the expression of p21 WAF1/CIP1 and p27 KIP1 in DLD-1, HT29 and HCT116 cells at 48 h. ACTB was used as a loading control. (C) Western blotting for p21 WAF1/CIP1 and p27 KIP1 in DCLK1-OE HCT116 clones. DCLK1 cDNA was subcloned into the pcDNA3.1 plasmid, after which the DCLK1 vector or empty vector was transfected into HCT116 cells by Lipofectamine ® 2000. Cells were collected at 48 h. * P
    Figure Legend Snippet: Cell cycle analysis. (A) The expression of cell cycle-related proteins was evaluated by western blotting. (B) Two independent experiments using rapid growing cultures evaluated the expression of p21 WAF1/CIP1 and p27 KIP1 in DLD-1, HT29 and HCT116 cells at 48 h. ACTB was used as a loading control. (C) Western blotting for p21 WAF1/CIP1 and p27 KIP1 in DCLK1-OE HCT116 clones. DCLK1 cDNA was subcloned into the pcDNA3.1 plasmid, after which the DCLK1 vector or empty vector was transfected into HCT116 cells by Lipofectamine ® 2000. Cells were collected at 48 h. * P

    Techniques Used: Cell Cycle Assay, Expressing, Western Blot, Clone Assay, Plasmid Preparation, Transfection

    Effect of DCLK1-siRNAs on the viability, invasion and gap closure of HCT116 cells. (A) HCT116 cells were treated with miR-1291 or DCLK1-siRNAs at a concentration of 30 nM for 48 and 72 h. Cell viability was assessed by Cell Counting Kit-8 assay. (B) HCT116 cell invasion was assessed following 50 nM DCLK1-siRNAs transfection at 72 h. BD BioCoat Matrigel invasion chambers (BD Biosciences) were used. (C) The gap closure ability of HCT116 cells was observed by transfection of DCLK1-siRNA1 and DCLK1-siRNA2 at concentration of 30 nM. The area of gap (unit, pixel 2 ) was assessed at 0, 24 and 48 h by ImageJ 1.52v software. All experiments were performed in triplicate. All data represent the mean ± SEM. * P
    Figure Legend Snippet: Effect of DCLK1-siRNAs on the viability, invasion and gap closure of HCT116 cells. (A) HCT116 cells were treated with miR-1291 or DCLK1-siRNAs at a concentration of 30 nM for 48 and 72 h. Cell viability was assessed by Cell Counting Kit-8 assay. (B) HCT116 cell invasion was assessed following 50 nM DCLK1-siRNAs transfection at 72 h. BD BioCoat Matrigel invasion chambers (BD Biosciences) were used. (C) The gap closure ability of HCT116 cells was observed by transfection of DCLK1-siRNA1 and DCLK1-siRNA2 at concentration of 30 nM. The area of gap (unit, pixel 2 ) was assessed at 0, 24 and 48 h by ImageJ 1.52v software. All experiments were performed in triplicate. All data represent the mean ± SEM. * P

    Techniques Used: Concentration Assay, Cell Counting, Transfection, Software

    Expression levels of miR-1291. (A) CRC cell lines and non-tumor cell lines. Reverse transcription-quantitative PCR was used to detect the expression of miR-1291 in CRC cell lines (CACO-2, COLO205, DLD-1, HCT116, HT29, KM12SM, LoVo, RKO and SW480) and human non-tumor cell lines (293, CCD-18Co and MRC5). RNU6B was used as a loading control. All experiments were performed in triplicate. All data are presented as the mean ± SEM. (B) Expression levels of miR-1291 were detected in CRC tissues and corresponding normal colon mucosa (n=20). Different colored lines represent the individual patient analyzed. * P
    Figure Legend Snippet: Expression levels of miR-1291. (A) CRC cell lines and non-tumor cell lines. Reverse transcription-quantitative PCR was used to detect the expression of miR-1291 in CRC cell lines (CACO-2, COLO205, DLD-1, HCT116, HT29, KM12SM, LoVo, RKO and SW480) and human non-tumor cell lines (293, CCD-18Co and MRC5). RNU6B was used as a loading control. All experiments were performed in triplicate. All data are presented as the mean ± SEM. (B) Expression levels of miR-1291 were detected in CRC tissues and corresponding normal colon mucosa (n=20). Different colored lines represent the individual patient analyzed. * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Inhibition of colorectal cancer cell gap closure and colony formation by miR-1291. (A) Gap closure assay in DLD-1, HT29 and HCT116 cells treated with miR-NC or miR-1291. The area of gap (unit, pixel 2 ) was assessed 0, 24, and 48 h by ImageJ 1.52v software. (B) The colony formation of DLD-1, HT29 and HCT116 cells was subsequently assessed 10 days after transfection with miR-1291. All data are presented as mean ± SEM. * P
    Figure Legend Snippet: Inhibition of colorectal cancer cell gap closure and colony formation by miR-1291. (A) Gap closure assay in DLD-1, HT29 and HCT116 cells treated with miR-NC or miR-1291. The area of gap (unit, pixel 2 ) was assessed 0, 24, and 48 h by ImageJ 1.52v software. (B) The colony formation of DLD-1, HT29 and HCT116 cells was subsequently assessed 10 days after transfection with miR-1291. All data are presented as mean ± SEM. * P

    Techniques Used: Inhibition, Software, Transfection

    Inhibition of colorectal cancer cell malignancy by miR-1291. (A) miR-1291 levels were recorded at 4 or 24 h after miR-1291 and miR-NC transfection. RNU6B was used as an internal control. (B) The viability of DLD-1, HT29, and HCT116 cells was detected following miR-1291 overexpression. (C) Invasion was assessed using the Matrigel Invasion Chambers. For DLD-1 and HCT116 cells, 1×10 5 cells/chamber were seeded and 2×10 5 cells/chamber were seeded for HT29 cells. Invasive cell numbers were counted at 48, 72 and 96 h after transfection. All experiments were performed in triplicate. All data are presented as the mean ± SEM. * P
    Figure Legend Snippet: Inhibition of colorectal cancer cell malignancy by miR-1291. (A) miR-1291 levels were recorded at 4 or 24 h after miR-1291 and miR-NC transfection. RNU6B was used as an internal control. (B) The viability of DLD-1, HT29, and HCT116 cells was detected following miR-1291 overexpression. (C) Invasion was assessed using the Matrigel Invasion Chambers. For DLD-1 and HCT116 cells, 1×10 5 cells/chamber were seeded and 2×10 5 cells/chamber were seeded for HT29 cells. Invasive cell numbers were counted at 48, 72 and 96 h after transfection. All experiments were performed in triplicate. All data are presented as the mean ± SEM. * P

    Techniques Used: Inhibition, Transfection, Over Expression

    Treatment of miR-1291 inhibits cell proliferation and induces apoptosis. (A) The relative absorbance of BrdU (450/540 nm) was measured in DLD-1, HT29 and HCT116 cell lines at 24 and 48 h after transfection All experiments were performed in triplicate. All data represent the mean ± SEM. (B) The percentage of apoptotic cells (in bold font in the center of the charts) were calculated by combining both the percentage of early apoptosis (Annexin V+, PI-; shown in B-Q2) and late apoptosis (Annexin V+, PI+; shown in B-Q4). * P
    Figure Legend Snippet: Treatment of miR-1291 inhibits cell proliferation and induces apoptosis. (A) The relative absorbance of BrdU (450/540 nm) was measured in DLD-1, HT29 and HCT116 cell lines at 24 and 48 h after transfection All experiments were performed in triplicate. All data represent the mean ± SEM. (B) The percentage of apoptotic cells (in bold font in the center of the charts) were calculated by combining both the percentage of early apoptosis (Annexin V+, PI-; shown in B-Q2) and late apoptosis (Annexin V+, PI+; shown in B-Q4). * P

    Techniques Used: Transfection

    miR-1291 directly targets the 3′UTR of DCLK1 in HCT116 cells. (A) TargetScan ( http://www.targetscan.org/vert_72/ ) was used to identify a binding site at position 2,459-2,465 of the DCLK1 mRNA 3′UTR that was complementary to the seed sequence of miR-1291 (WT). The binding sequence of DCLK1 was mutated by changing two nucleotides (Mut) or deleting three nucleotides (Del). (B) A luciferase reporter assay was performed in HCT116 cells following transfection with miR-1291. (C) DCLK1 expression in HCT116 cells was assessed using an anti-human rabbit polyclonal antibody against DCAMKL1. (D) The effect of miR-1291 on the expression of DCLK1 was assessed by RT-qPCR. (E) A repeat RT-qPCR experiment was performed using antagomir-1291. GAPDH was utilized as an endogenous control. All experiments were performed in triplicate. All data are presented as the mean ± SEM. (F) Western blotting was performed to determine the expression of DCLK1 protein after 48 h of transfection in HCT116 cells. Anti-human rabbit polyclonal antibodies against DCAMKL1 were used. ACTB was used as a loading control and two independent experiments were performed. * P
    Figure Legend Snippet: miR-1291 directly targets the 3′UTR of DCLK1 in HCT116 cells. (A) TargetScan ( http://www.targetscan.org/vert_72/ ) was used to identify a binding site at position 2,459-2,465 of the DCLK1 mRNA 3′UTR that was complementary to the seed sequence of miR-1291 (WT). The binding sequence of DCLK1 was mutated by changing two nucleotides (Mut) or deleting three nucleotides (Del). (B) A luciferase reporter assay was performed in HCT116 cells following transfection with miR-1291. (C) DCLK1 expression in HCT116 cells was assessed using an anti-human rabbit polyclonal antibody against DCAMKL1. (D) The effect of miR-1291 on the expression of DCLK1 was assessed by RT-qPCR. (E) A repeat RT-qPCR experiment was performed using antagomir-1291. GAPDH was utilized as an endogenous control. All experiments were performed in triplicate. All data are presented as the mean ± SEM. (F) Western blotting was performed to determine the expression of DCLK1 protein after 48 h of transfection in HCT116 cells. Anti-human rabbit polyclonal antibodies against DCAMKL1 were used. ACTB was used as a loading control and two independent experiments were performed. * P

    Techniques Used: Binding Assay, Sequencing, Luciferase, Reporter Assay, Transfection, Expressing, Quantitative RT-PCR, Western Blot

    miR-1291 suppresses the stemness of HCT116 cells. (A) Reverse transcription-quantitative PCR was performed to evaluate stemness. GAPDH was utilized as an endogenous control. (B) Flow cytometric analysis was performed to determine the ratio of stem cell surface marker CD133 following miR-1291 and miR-NC transfection. (C) Western blotting was performed to determine the protein expression of CD133 following miR-1291 and miR-NC transfection. (D) Sphere formation ability was assessed in miR-1291-transfected HCT116 cells. The number of spheres > 40 µ m was counted 4 days after seeding. Representative images are presented. (E) The effects of three siRNAs targeting DCLK1 were assessed via western blotting in HCT116 cells. Anti-human rabbit polyclonal antibodies against DCAMKL1 were used with ACTB as a loading control. (F) The number of spheroids ( > 100 µ m) following DCLK1-siRNA transfection was counted at day 7 after seeding. (G) Spheroid formation of shDCLK1 clones. All experiments were performed in triplicate. All data are presented as the mean ± SEM. ShDCLK1 clones of HCT116 were generated in our previous study ( 29 ). * P
    Figure Legend Snippet: miR-1291 suppresses the stemness of HCT116 cells. (A) Reverse transcription-quantitative PCR was performed to evaluate stemness. GAPDH was utilized as an endogenous control. (B) Flow cytometric analysis was performed to determine the ratio of stem cell surface marker CD133 following miR-1291 and miR-NC transfection. (C) Western blotting was performed to determine the protein expression of CD133 following miR-1291 and miR-NC transfection. (D) Sphere formation ability was assessed in miR-1291-transfected HCT116 cells. The number of spheres > 40 µ m was counted 4 days after seeding. Representative images are presented. (E) The effects of three siRNAs targeting DCLK1 were assessed via western blotting in HCT116 cells. Anti-human rabbit polyclonal antibodies against DCAMKL1 were used with ACTB as a loading control. (F) The number of spheroids ( > 100 µ m) following DCLK1-siRNA transfection was counted at day 7 after seeding. (G) Spheroid formation of shDCLK1 clones. All experiments were performed in triplicate. All data are presented as the mean ± SEM. ShDCLK1 clones of HCT116 were generated in our previous study ( 29 ). * P

    Techniques Used: Real-time Polymerase Chain Reaction, Marker, Transfection, Western Blot, Expressing, Clone Assay, Generated

    39) Product Images from "RNA-seq reveals determinants for irinotecan sensitivity/resistance in colorectal cancer cell lines"

    Article Title: RNA-seq reveals determinants for irinotecan sensitivity/resistance in colorectal cancer cell lines

    Journal: International Journal of Clinical and Experimental Pathology

    doi:

    qPCR validation. 7 genes were validated in two CRC cell lines (COLO205 and LS174T) by qPCR. The gene expression was calculated relative to that in COLO205 cells.
    Figure Legend Snippet: qPCR validation. 7 genes were validated in two CRC cell lines (COLO205 and LS174T) by qPCR. The gene expression was calculated relative to that in COLO205 cells.

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing

    40) Product Images from "Integrin-Linked Kinase Expression Characterizes the Immunosuppressive Tumor Microenvironment in Colorectal Cancer and Regulates PD-L1 Expression and Immune Cell Cytotoxicity"

    Article Title: Integrin-Linked Kinase Expression Characterizes the Immunosuppressive Tumor Microenvironment in Colorectal Cancer and Regulates PD-L1 Expression and Immune Cell Cytotoxicity

    Journal: Frontiers in Oncology

    doi: 10.3389/fonc.2022.836005

    ILK KD reduced basal PD-L1 expression in RKO CRC cells via NF-κB p65 regulation. [ (A) , upper] Western blot showing expression of ILK protein. [ (A) , lower] Quantitation of ILK protein, the means were normalized to -Dox (n = 3). Cells were seeded into a 6-well culture plate and duplicated into 2 sub-populations and incubated overnight. One sub-population per cell line was treated with 2 µg/ml doxycycline to induce CRISPR/Cas9 to delete the ILK gene over 3 days. The doxycycline was washed from the cells and the cells sub-cultured for growing. The cells were then harvested for testing ILK protein expression via western blot and the Dox-treated cells are verified as ILK KD cells. (B) The western blot of PD-L1 protein expression in CRC cell lines at the basal level. (C) ILK KD reduced NF-κB p65/PD-L1 protein expression in the RKO CRC cell line. (left) The western blot showed the effect of ILK KD in RKO cells on PD-L1 and NF-κB p65 (total and phosphorylated) protein expression at 4 hr serum stimulation following overnight starvation. (right) The quantitation of PD-L1, p65 phosphorylation (pp65) and p65 total protein expression (t-p65). Quantitation of protein expression normalized to -Dox. Actin was used as an internal control. Error bars are represented as mean ± SEM (n = 3). P -value was analyzed with an unpaired t-test. The significant P -value is
    Figure Legend Snippet: ILK KD reduced basal PD-L1 expression in RKO CRC cells via NF-κB p65 regulation. [ (A) , upper] Western blot showing expression of ILK protein. [ (A) , lower] Quantitation of ILK protein, the means were normalized to -Dox (n = 3). Cells were seeded into a 6-well culture plate and duplicated into 2 sub-populations and incubated overnight. One sub-population per cell line was treated with 2 µg/ml doxycycline to induce CRISPR/Cas9 to delete the ILK gene over 3 days. The doxycycline was washed from the cells and the cells sub-cultured for growing. The cells were then harvested for testing ILK protein expression via western blot and the Dox-treated cells are verified as ILK KD cells. (B) The western blot of PD-L1 protein expression in CRC cell lines at the basal level. (C) ILK KD reduced NF-κB p65/PD-L1 protein expression in the RKO CRC cell line. (left) The western blot showed the effect of ILK KD in RKO cells on PD-L1 and NF-κB p65 (total and phosphorylated) protein expression at 4 hr serum stimulation following overnight starvation. (right) The quantitation of PD-L1, p65 phosphorylation (pp65) and p65 total protein expression (t-p65). Quantitation of protein expression normalized to -Dox. Actin was used as an internal control. Error bars are represented as mean ± SEM (n = 3). P -value was analyzed with an unpaired t-test. The significant P -value is

    Techniques Used: Expressing, Western Blot, Quantitation Assay, Incubation, CRISPR, Cell Culture

    ILK KD reduced IFNγ-induced PD-L1 expression in the CRC cell lines. (A) Western blot showed PD-L1 induction by IFNγ (50 ng) for 24 hr in HCT116, RKO, HT29 and SW480 CRC cells. Actin was used as an internal control. The protein quantitation was determined by relative ratio to actin. The relative PD-L1 expression is normalized to the -Dox unstimulated protein. (B) The quantitation of PD-L1 expression in CRC cell lines. (C) The quantitation of PD-L1 protein expression in the different CRC cell lines combined. The relative PD-L1 expression is normalized to the -Dox protein. Error bars are represented as mean ± SEM (n = 3). P -value was analyzed with an unpaired t-test. The significant P -value is
    Figure Legend Snippet: ILK KD reduced IFNγ-induced PD-L1 expression in the CRC cell lines. (A) Western blot showed PD-L1 induction by IFNγ (50 ng) for 24 hr in HCT116, RKO, HT29 and SW480 CRC cells. Actin was used as an internal control. The protein quantitation was determined by relative ratio to actin. The relative PD-L1 expression is normalized to the -Dox unstimulated protein. (B) The quantitation of PD-L1 expression in CRC cell lines. (C) The quantitation of PD-L1 protein expression in the different CRC cell lines combined. The relative PD-L1 expression is normalized to the -Dox protein. Error bars are represented as mean ± SEM (n = 3). P -value was analyzed with an unpaired t-test. The significant P -value is

    Techniques Used: Expressing, Western Blot, Protein Quantitation, Quantitation Assay

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    ATCC human crc cell lines
    miR-150-5p inhibited <t>CRC</t> progression by targeting VEGFA. ( A ) VEGFA protein expression was determined in <t>HCT116</t> and HCT8 cells transfected with agomiR-150-5p with VEGFA expression plasmid or empty vector using western blot; GAPDH was used as the internal control. ( B - D ) Cell proliferation ( B , C ), migration ( D )and invasion ( E ) were evaluated in HCT116 and HCT8 cells transfected with agomiR-150-5p with VEGFA expression plasmid or empty vector. ( F ) HUVECs were cultured in TCM derived from HCT116 and HCT8 cells transfected with agomiR-150-5p plus VEGFA expression plasmid or empty vector. Data are shown as the mean±SD. * p
    Human Crc Cell Lines, supplied by ATCC, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC human crc cell line ht29
    IL-33/ST2 upregulates COX2 expression through NF-κB signaling. a , b The COX2 mRNA ( a ) and protein ( b ) expression in primary <t>CRC</t> cells or <t>HT29</t> cells responding to the incubation with rhIL33 (100 ng/mL) or/ and ST2 antibody (2 μg/mL) for 24 h. Each experiment was performed three times. Data expressed as mean ± SEM. ** P
    Human Crc Cell Line Ht29, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC human crc cell lines colo320dm
    Downregulation of ERCC1 expression in KRAS-mutant <t>CRC</t> cells might be related to hypermethylation of ERCC1gene, which possibly induced by up-regulation of DNMT3B (DNA methyltransferase 3B). (A) Protein expression of ERCC1 in DLD-1(KRAS G13D mutation) cells is up-regulated after 5′-azacitidine (de-methylating agent) treatment for 96 hours, which implied that the downregulation of ERCC1 in KRAS-mutant CRC cells might be partly through ERCC1 hypermethylation. (B) Downregulation of ERCC1 in COLO320DM (KRAS wild-type) cells transfected by KRAS G13D -mutant-vector for 24 and 96 hours may not only be restored by 5′-azacitidine in 10 µM, but also caused up-regulation of DNMT3B.
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    miR-150-5p inhibited CRC progression by targeting VEGFA. ( A ) VEGFA protein expression was determined in HCT116 and HCT8 cells transfected with agomiR-150-5p with VEGFA expression plasmid or empty vector using western blot; GAPDH was used as the internal control. ( B - D ) Cell proliferation ( B , C ), migration ( D )and invasion ( E ) were evaluated in HCT116 and HCT8 cells transfected with agomiR-150-5p with VEGFA expression plasmid or empty vector. ( F ) HUVECs were cultured in TCM derived from HCT116 and HCT8 cells transfected with agomiR-150-5p plus VEGFA expression plasmid or empty vector. Data are shown as the mean±SD. * p

    Journal: Aging (Albany NY)

    Article Title: miR-150-5p suppresses tumor progression by targeting VEGFA in colorectal cancer

    doi: 10.18632/aging.101656

    Figure Lengend Snippet: miR-150-5p inhibited CRC progression by targeting VEGFA. ( A ) VEGFA protein expression was determined in HCT116 and HCT8 cells transfected with agomiR-150-5p with VEGFA expression plasmid or empty vector using western blot; GAPDH was used as the internal control. ( B - D ) Cell proliferation ( B , C ), migration ( D )and invasion ( E ) were evaluated in HCT116 and HCT8 cells transfected with agomiR-150-5p with VEGFA expression plasmid or empty vector. ( F ) HUVECs were cultured in TCM derived from HCT116 and HCT8 cells transfected with agomiR-150-5p plus VEGFA expression plasmid or empty vector. Data are shown as the mean±SD. * p

    Article Snippet: The human CRC cell lines including HCT116, HCT8, HT29, SW620, SW480 and DLD-1 and normal colonic epithelial cells (FHC) were obtained from ATCC.

    Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Migration, Cell Culture, Derivative Assay

    A. VEGFA was a direct target of miR-150-5p in CRC. ( A ) The direct target genes of miR-150-5p were predicted using the PicTarSites, miRandaSites and Tarbase databases. ( B ) Wild-type and mutant VEGFA-3’UTR sequences were cloned into luciferase reporter. Luciferase activity was determined in HCT116 and 293T cells cotransfected with agomiR-150-5p or agomiR-NC and pmirGLO-VEGFA-3’UTR-WT or pmirGLO-VEGFA-3’UTR-Mut. Luciferase activities were normalized to that of renilla luciferase. C, D. qRT-PCR ( C ) and western blot ( D ) analyses showed that both VEGFA mRNA and protein expression levels were dramatically suppressed by agomiR-150-5p in HCT116 and HCT8 cells, GAPDH was used as the internal control. ** p

    Journal: Aging (Albany NY)

    Article Title: miR-150-5p suppresses tumor progression by targeting VEGFA in colorectal cancer

    doi: 10.18632/aging.101656

    Figure Lengend Snippet: A. VEGFA was a direct target of miR-150-5p in CRC. ( A ) The direct target genes of miR-150-5p were predicted using the PicTarSites, miRandaSites and Tarbase databases. ( B ) Wild-type and mutant VEGFA-3’UTR sequences were cloned into luciferase reporter. Luciferase activity was determined in HCT116 and 293T cells cotransfected with agomiR-150-5p or agomiR-NC and pmirGLO-VEGFA-3’UTR-WT or pmirGLO-VEGFA-3’UTR-Mut. Luciferase activities were normalized to that of renilla luciferase. C, D. qRT-PCR ( C ) and western blot ( D ) analyses showed that both VEGFA mRNA and protein expression levels were dramatically suppressed by agomiR-150-5p in HCT116 and HCT8 cells, GAPDH was used as the internal control. ** p

    Article Snippet: The human CRC cell lines including HCT116, HCT8, HT29, SW620, SW480 and DLD-1 and normal colonic epithelial cells (FHC) were obtained from ATCC.

    Techniques: Mutagenesis, Clone Assay, Luciferase, Activity Assay, Quantitative RT-PCR, Western Blot, Expressing

    VEGFA knockdown significantly inhibited CRC progression. ( A ) VEGFA expression was downregulated in HCT116 and HCT8 cells transfected with siVEGFA-1 or siVEGFA-2. ( B ) VEGFA knockdown inhibited CRC cell proliferation ( B ), migration ( C ), invasion ( D ) and HUVECs tube formation ( E ). Data are shown as the mean±SD of three independent experiments. * p

    Journal: Aging (Albany NY)

    Article Title: miR-150-5p suppresses tumor progression by targeting VEGFA in colorectal cancer

    doi: 10.18632/aging.101656

    Figure Lengend Snippet: VEGFA knockdown significantly inhibited CRC progression. ( A ) VEGFA expression was downregulated in HCT116 and HCT8 cells transfected with siVEGFA-1 or siVEGFA-2. ( B ) VEGFA knockdown inhibited CRC cell proliferation ( B ), migration ( C ), invasion ( D ) and HUVECs tube formation ( E ). Data are shown as the mean±SD of three independent experiments. * p

    Article Snippet: The human CRC cell lines including HCT116, HCT8, HT29, SW620, SW480 and DLD-1 and normal colonic epithelial cells (FHC) were obtained from ATCC.

    Techniques: Expressing, Transfection, Migration

    CREPT regulated Wnt/β-catenin signaling pathway in CRC cells. a KEGG analysis based on RNA-sequencing data of HCT116 cells stably overexpressing CREPT or control cells. b Ectopic expression of CREPT in DLD-1 cell line enhanced TopFlash luciferase reporter activity but not FopFlash reporter ( b1 ), while depletion of CREPT reduced dual-luciferase reporter assays in SW480 cells ( b2 ). TopFlash, a classic Wnt-response luciferase reporter containing 3xTCF4 binding site. FopFlash, a negative reporter of which TCF4 binding site is mutant. c The mRNA expression of Wnt downstream targets CCND1 , c-MYC , and AXIN2 were enhanced in the presence of CREPT in DLD-1 cells and HCT116 cells. d The mRNA expression of CCND1 , c-MYC , and AXIN2 was diminished under CREPT knockdown in HT29 and SW480 cells. e The protein expression of β-catenin, c-MYC, CYCLIN D1, and AXIN2 was increased upon ectopic expression of CREPT in DLD1, HCT116 ( e1 ), HCEC 1CT, and 2CT cells ( e2 ), while decreased in CREPT knockdown HT29 and SW480 cells ( e3 ).

    Journal: Oncogene

    Article Title: CREPT facilitates colorectal cancer growth through inducing Wnt/β-catenin pathway by enhancing p300-mediated β-catenin acetylation

    doi: 10.1038/s41388-018-0161-z

    Figure Lengend Snippet: CREPT regulated Wnt/β-catenin signaling pathway in CRC cells. a KEGG analysis based on RNA-sequencing data of HCT116 cells stably overexpressing CREPT or control cells. b Ectopic expression of CREPT in DLD-1 cell line enhanced TopFlash luciferase reporter activity but not FopFlash reporter ( b1 ), while depletion of CREPT reduced dual-luciferase reporter assays in SW480 cells ( b2 ). TopFlash, a classic Wnt-response luciferase reporter containing 3xTCF4 binding site. FopFlash, a negative reporter of which TCF4 binding site is mutant. c The mRNA expression of Wnt downstream targets CCND1 , c-MYC , and AXIN2 were enhanced in the presence of CREPT in DLD-1 cells and HCT116 cells. d The mRNA expression of CCND1 , c-MYC , and AXIN2 was diminished under CREPT knockdown in HT29 and SW480 cells. e The protein expression of β-catenin, c-MYC, CYCLIN D1, and AXIN2 was increased upon ectopic expression of CREPT in DLD1, HCT116 ( e1 ), HCEC 1CT, and 2CT cells ( e2 ), while decreased in CREPT knockdown HT29 and SW480 cells ( e3 ).

    Article Snippet: Human CRC cell lines (CaCO2, Colo205, DLD-1, HCT116, HT29, LoVo, SW1116, SW480, and SW620) and one immortalized colorectal epithelial cell line (NCM460) were from the American Type Culture Collection (Manassas, VA) and were cultured as described in previous study [ ].

    Techniques: RNA Sequencing Assay, Stable Transfection, Expressing, Luciferase, Activity Assay, Binding Assay, Mutagenesis

    CREPT enhanced CRC metastasis in vitro and in vivo. Ectopic expression of CREPT-promoted cell invasion a and migration b in HCT116 cells. Knockdown of CREPT suppressed cell invasion c and migration d in SW480 cells. e Ectopic expression of CREPT accelerated lung metastasis of HCT116 cells injected via tail vein. Pictures on the left showed representative lung morphology. HE staining showed representative lungs section. Histograph showed statistic result of metastasis nodules in nude mice lung tissues. n = 5, * P

    Journal: Oncogene

    Article Title: CREPT facilitates colorectal cancer growth through inducing Wnt/β-catenin pathway by enhancing p300-mediated β-catenin acetylation

    doi: 10.1038/s41388-018-0161-z

    Figure Lengend Snippet: CREPT enhanced CRC metastasis in vitro and in vivo. Ectopic expression of CREPT-promoted cell invasion a and migration b in HCT116 cells. Knockdown of CREPT suppressed cell invasion c and migration d in SW480 cells. e Ectopic expression of CREPT accelerated lung metastasis of HCT116 cells injected via tail vein. Pictures on the left showed representative lung morphology. HE staining showed representative lungs section. Histograph showed statistic result of metastasis nodules in nude mice lung tissues. n = 5, * P

    Article Snippet: Human CRC cell lines (CaCO2, Colo205, DLD-1, HCT116, HT29, LoVo, SW1116, SW480, and SW620) and one immortalized colorectal epithelial cell line (NCM460) were from the American Type Culture Collection (Manassas, VA) and were cultured as described in previous study [ ].

    Techniques: In Vitro, In Vivo, Expressing, Migration, Injection, Staining, Mouse Assay

    IL-33/ST2 upregulates COX2 expression through NF-κB signaling. a , b The COX2 mRNA ( a ) and protein ( b ) expression in primary CRC cells or HT29 cells responding to the incubation with rhIL33 (100 ng/mL) or/ and ST2 antibody (2 μg/mL) for 24 h. Each experiment was performed three times. Data expressed as mean ± SEM. ** P

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: IL-33 facilitates proliferation of colorectal cancer dependent on COX2/PGE2

    doi: 10.1186/s13046-018-0839-7

    Figure Lengend Snippet: IL-33/ST2 upregulates COX2 expression through NF-κB signaling. a , b The COX2 mRNA ( a ) and protein ( b ) expression in primary CRC cells or HT29 cells responding to the incubation with rhIL33 (100 ng/mL) or/ and ST2 antibody (2 μg/mL) for 24 h. Each experiment was performed three times. Data expressed as mean ± SEM. ** P

    Article Snippet: Human CRC cell line HT29 was bought from American Type Culture Collection (ATCC).

    Techniques: Expressing, Incubation

    Downregulation of ERCC1 expression in KRAS-mutant CRC cells might be related to hypermethylation of ERCC1gene, which possibly induced by up-regulation of DNMT3B (DNA methyltransferase 3B). (A) Protein expression of ERCC1 in DLD-1(KRAS G13D mutation) cells is up-regulated after 5′-azacitidine (de-methylating agent) treatment for 96 hours, which implied that the downregulation of ERCC1 in KRAS-mutant CRC cells might be partly through ERCC1 hypermethylation. (B) Downregulation of ERCC1 in COLO320DM (KRAS wild-type) cells transfected by KRAS G13D -mutant-vector for 24 and 96 hours may not only be restored by 5′-azacitidine in 10 µM, but also caused up-regulation of DNMT3B.

    Journal: PLoS ONE

    Article Title: KRAS Mutation Is a Predictor of Oxaliplatin Sensitivity in Colon Cancer Cells

    doi: 10.1371/journal.pone.0050701

    Figure Lengend Snippet: Downregulation of ERCC1 expression in KRAS-mutant CRC cells might be related to hypermethylation of ERCC1gene, which possibly induced by up-regulation of DNMT3B (DNA methyltransferase 3B). (A) Protein expression of ERCC1 in DLD-1(KRAS G13D mutation) cells is up-regulated after 5′-azacitidine (de-methylating agent) treatment for 96 hours, which implied that the downregulation of ERCC1 in KRAS-mutant CRC cells might be partly through ERCC1 hypermethylation. (B) Downregulation of ERCC1 in COLO320DM (KRAS wild-type) cells transfected by KRAS G13D -mutant-vector for 24 and 96 hours may not only be restored by 5′-azacitidine in 10 µM, but also caused up-regulation of DNMT3B.

    Article Snippet: Cell Culture and Reagents Human CRC cell lines COLO320DM (KRAS-wild-type), DLD-1G13D (KRAS G13D mutation), and SW480G12V (KRAS G12V mutation) were all obtained from American Type Culture Collection.

    Techniques: Expressing, Mutagenesis, Transfection, Plasmid Preparation

    Validating ERCC1 as the predictor of oxaliplatin sensitivity in CRC cells. (A) ERCC1-knocked-down COLO320DM cells were more sensitive to oxaliplatin than parental COLO320DM cells, as demonstrated by MTT assay. (B) Protein and mRNA levels of ERCC1 were downregulated when COLO320DM cells were knocked-down by ERCC1 siRNA. *: p

    Journal: PLoS ONE

    Article Title: KRAS Mutation Is a Predictor of Oxaliplatin Sensitivity in Colon Cancer Cells

    doi: 10.1371/journal.pone.0050701

    Figure Lengend Snippet: Validating ERCC1 as the predictor of oxaliplatin sensitivity in CRC cells. (A) ERCC1-knocked-down COLO320DM cells were more sensitive to oxaliplatin than parental COLO320DM cells, as demonstrated by MTT assay. (B) Protein and mRNA levels of ERCC1 were downregulated when COLO320DM cells were knocked-down by ERCC1 siRNA. *: p

    Article Snippet: Cell Culture and Reagents Human CRC cell lines COLO320DM (KRAS-wild-type), DLD-1G13D (KRAS G13D mutation), and SW480G12V (KRAS G12V mutation) were all obtained from American Type Culture Collection.

    Techniques: MTT Assay

    Overexpressing KRAS by another KRAS overexpression vector (G12V) in KRAS wild-type CRC cells leads to oxaliplatin sensitivity and ERCC1 downregulation. (A) KRAS G12V -DDK-myc-COLO320DM cells were more sensitive to oxaliplatin, but have the same sensitivity to irinotecan, 5FU, and doxorubicin than parental COLO320DM cells, as demonstrated by MTT assay. (B) The protein level of ERCC1, but not those of TOPO1 or TS, was downregulated after COLO320DM cells were transfected by the KRAS G12V mutant vector. (C) The mRNA level of ERCC1, but not those of TOPO1 or TS, was downregulated after COLO320DM cells were transfected by the KRAS G12V mutant vector. **: p

    Journal: PLoS ONE

    Article Title: KRAS Mutation Is a Predictor of Oxaliplatin Sensitivity in Colon Cancer Cells

    doi: 10.1371/journal.pone.0050701

    Figure Lengend Snippet: Overexpressing KRAS by another KRAS overexpression vector (G12V) in KRAS wild-type CRC cells leads to oxaliplatin sensitivity and ERCC1 downregulation. (A) KRAS G12V -DDK-myc-COLO320DM cells were more sensitive to oxaliplatin, but have the same sensitivity to irinotecan, 5FU, and doxorubicin than parental COLO320DM cells, as demonstrated by MTT assay. (B) The protein level of ERCC1, but not those of TOPO1 or TS, was downregulated after COLO320DM cells were transfected by the KRAS G12V mutant vector. (C) The mRNA level of ERCC1, but not those of TOPO1 or TS, was downregulated after COLO320DM cells were transfected by the KRAS G12V mutant vector. **: p

    Article Snippet: Cell Culture and Reagents Human CRC cell lines COLO320DM (KRAS-wild-type), DLD-1G13D (KRAS G13D mutation), and SW480G12V (KRAS G12V mutation) were all obtained from American Type Culture Collection.

    Techniques: Over Expression, Plasmid Preparation, MTT Assay, Transfection, Mutagenesis

    Overexpressing KRAS in KRAS wild-type CRC cells leads to oxaliplatin sensitivity and ERCC1 downregulation. (A) KRAS G13D -DDK-myc-COLO320DM cells were more sensitive to oxaliplatin, but have the same sensitivity to irinotecan, 5FU, and doxorubicin than parental COLO320DM cells, as demonstrated by MTT assay. (B) The protein level of ERCC1, but not those of TOPO1 or TS, was downregulated after COLO320DM cells were transfected by the KRAS G13D mutant vector. (C) The mRNA level of ERCC1, but not those of TOPO1 or TS, was downregulated after COLO320DM cells were transfected by the KRAS G13D mutant vector. **: p

    Journal: PLoS ONE

    Article Title: KRAS Mutation Is a Predictor of Oxaliplatin Sensitivity in Colon Cancer Cells

    doi: 10.1371/journal.pone.0050701

    Figure Lengend Snippet: Overexpressing KRAS in KRAS wild-type CRC cells leads to oxaliplatin sensitivity and ERCC1 downregulation. (A) KRAS G13D -DDK-myc-COLO320DM cells were more sensitive to oxaliplatin, but have the same sensitivity to irinotecan, 5FU, and doxorubicin than parental COLO320DM cells, as demonstrated by MTT assay. (B) The protein level of ERCC1, but not those of TOPO1 or TS, was downregulated after COLO320DM cells were transfected by the KRAS G13D mutant vector. (C) The mRNA level of ERCC1, but not those of TOPO1 or TS, was downregulated after COLO320DM cells were transfected by the KRAS G13D mutant vector. **: p

    Article Snippet: Cell Culture and Reagents Human CRC cell lines COLO320DM (KRAS-wild-type), DLD-1G13D (KRAS G13D mutation), and SW480G12V (KRAS G12V mutation) were all obtained from American Type Culture Collection.

    Techniques: MTT Assay, Transfection, Mutagenesis, Plasmid Preparation