hct116  (Thermo Fisher)


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    Name:
    CellSensor Myc bla HCT116 Cell Line
    Description:
    The CellSensor Myc bla HCT116 cell line contains a beta lactamase reporter gene under the control of Myc binding sequences The construct was transduced into HCT116 cells using a lentiviral system HCT116 is a colon cancer cell line which expresses a mutated form of beta catenin This form of beta catenin leads to the accumulation of beta catenin and constitutive activation of downstream genes such as Myc This cell line is a clonal population isolated by flow cytometry It has been validated for cell plating density and DMSO tolerance The signaling pathway has been validated using RNAi against c Myc and ICG 001 an inhibitor of the wnt beta catenin pathway The expression of the mutant beta catenin in HCT116 cells results in constitutive activation of beta lactamase in this CellSensor line which can be knocked down by ICG 001 Figure 1 or Myc RNAi Figure 2
    Catalog Number:
    K1467
    Price:
    None
    Category:
    Kits and Assays
    Applications:
    Cell Analysis|Cell-Based β-Lactamase Assays|Cell-Based Reporter Assays|CellSensor™ Cellular Pathway Assays|Cellular Imaging|Cellular Pathway Analysis Assays|Industrial & Applied Science|Pathway Biology|Pharma & Biopharma|Target & Lead Identification & Validation
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    Structured Review

    Thermo Fisher hct116
    Pol η knockdown increases resistance of cancer cells to IR. Clonogenic survival in (A) <t>HCT116</t> and (C) SQ20B cells after pol η knockdown. Cells were either transfected with non-targeting siRNA or siRNA against pol η for 48h before
    The CellSensor Myc bla HCT116 cell line contains a beta lactamase reporter gene under the control of Myc binding sequences The construct was transduced into HCT116 cells using a lentiviral system HCT116 is a colon cancer cell line which expresses a mutated form of beta catenin This form of beta catenin leads to the accumulation of beta catenin and constitutive activation of downstream genes such as Myc This cell line is a clonal population isolated by flow cytometry It has been validated for cell plating density and DMSO tolerance The signaling pathway has been validated using RNAi against c Myc and ICG 001 an inhibitor of the wnt beta catenin pathway The expression of the mutant beta catenin in HCT116 cells results in constitutive activation of beta lactamase in this CellSensor line which can be knocked down by ICG 001 Figure 1 or Myc RNAi Figure 2
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    Images

    1) Product Images from "Homologous recombination mediates S-phase-dependent radioresistance in cells deficient in DNA polymerase eta"

    Article Title: Homologous recombination mediates S-phase-dependent radioresistance in cells deficient in DNA polymerase eta

    Journal: Carcinogenesis

    doi: 10.1093/carcin/bgs239

    Pol η knockdown increases resistance of cancer cells to IR. Clonogenic survival in (A) HCT116 and (C) SQ20B cells after pol η knockdown. Cells were either transfected with non-targeting siRNA or siRNA against pol η for 48h before
    Figure Legend Snippet: Pol η knockdown increases resistance of cancer cells to IR. Clonogenic survival in (A) HCT116 and (C) SQ20B cells after pol η knockdown. Cells were either transfected with non-targeting siRNA or siRNA against pol η for 48h before

    Techniques Used: Transfection

    2) Product Images from "Downregulation of rho-associated protein kinase 1 by miR-124 in colorectal cancer"

    Article Title: Downregulation of rho-associated protein kinase 1 by miR-124 in colorectal cancer

    Journal: World Journal of Gastroenterology : WJG

    doi: 10.3748/wjg.v21.i18.5454

    miR-124 knockdown promotes cell proliferation. A: New generation of cells were detected by 5-ethynyl-2’ deoxyuridine (EDU; red), nuclei are stained blue (DAPI); B: Proliferative activity of HCT116 and HT29 cells transfected with negative control
    Figure Legend Snippet: miR-124 knockdown promotes cell proliferation. A: New generation of cells were detected by 5-ethynyl-2’ deoxyuridine (EDU; red), nuclei are stained blue (DAPI); B: Proliferative activity of HCT116 and HT29 cells transfected with negative control

    Techniques Used: Staining, Activity Assay, Transfection, Negative Control

    In vitro effects of miR-124 regulation. A: Representative Western blot of rho-associated protein kinase 1 (ROCK1) expression in HCT116 and HT29 cells transfected with negative control (NC), miR-124 mimic and miR-124 inhibitor; B: Relative densitometry
    Figure Legend Snippet: In vitro effects of miR-124 regulation. A: Representative Western blot of rho-associated protein kinase 1 (ROCK1) expression in HCT116 and HT29 cells transfected with negative control (NC), miR-124 mimic and miR-124 inhibitor; B: Relative densitometry

    Techniques Used: In Vitro, Western Blot, Expressing, Transfection, Negative Control

    miR-124 inhibits growth and invasion of HCT116 and HT29 cells. A: Colony-formation assay for HCT116 and HT29 cells transfected with negative control (NC) and miR-124 inhibitor was performed on day 14; B: Quantification of colony formation with miR-124
    Figure Legend Snippet: miR-124 inhibits growth and invasion of HCT116 and HT29 cells. A: Colony-formation assay for HCT116 and HT29 cells transfected with negative control (NC) and miR-124 inhibitor was performed on day 14; B: Quantification of colony formation with miR-124

    Techniques Used: Colony Assay, Transfection, Negative Control

    3) Product Images from "A small molecule, (E)-2-methoxy-4-(3-(4-methoxyphenyl) prop-1-en-1-yl) phenol suppresses tumor growth via inhibition of IkappaB kinase β in colorectal cancer in vivo and in vitro"

    Article Title: A small molecule, (E)-2-methoxy-4-(3-(4-methoxyphenyl) prop-1-en-1-yl) phenol suppresses tumor growth via inhibition of IkappaB kinase β in colorectal cancer in vivo and in vitro

    Journal: Oncotarget

    doi: 10.18632/oncotarget.20440

    Anti-tumor activity of MMPP in colon cancer xenograft mice model (A) , (B) (C) Growth inhibition of subcutaneously transplanted HCT116 xenograft mice treated with MMPP (2.5 mg/kg and 5 mg/kg twice a week) for 3 weeks. Xenograft mice (n=10) were administrated intraperitoneally with 0.01% DMSO or MMPP (2.5 mg/kg and 5 mg/kg). Tumor burden was measured once per week using a caliper, and calculated volume length (mm) × width (mm) × height (mm)/2. Tumor weight and volume are presented as means ± S.D. (D) Immunohistochemistry was used to determine expression levels of PCNA, DR6, DR5, active caspase-3, p-IKKβ in nude mice xenograft tissues by the different treatments as described in the materials and method. DAPI TUNEL assay was carried out to assess the apoptosis rate in the nude mice xenograft tissue. Total number of cells in a given area was determined by using DAPI nuclear staining (fluorescent microscope). A green color in the fixed cells marks TUNEL-labeled cells.
    Figure Legend Snippet: Anti-tumor activity of MMPP in colon cancer xenograft mice model (A) , (B) (C) Growth inhibition of subcutaneously transplanted HCT116 xenograft mice treated with MMPP (2.5 mg/kg and 5 mg/kg twice a week) for 3 weeks. Xenograft mice (n=10) were administrated intraperitoneally with 0.01% DMSO or MMPP (2.5 mg/kg and 5 mg/kg). Tumor burden was measured once per week using a caliper, and calculated volume length (mm) × width (mm) × height (mm)/2. Tumor weight and volume are presented as means ± S.D. (D) Immunohistochemistry was used to determine expression levels of PCNA, DR6, DR5, active caspase-3, p-IKKβ in nude mice xenograft tissues by the different treatments as described in the materials and method. DAPI TUNEL assay was carried out to assess the apoptosis rate in the nude mice xenograft tissue. Total number of cells in a given area was determined by using DAPI nuclear staining (fluorescent microscope). A green color in the fixed cells marks TUNEL-labeled cells.

    Techniques Used: Activity Assay, Mouse Assay, Inhibition, Immunohistochemistry, Expressing, TUNEL Assay, Staining, Microscopy, Labeling

    Effect of MMPP on the growth colon cancer cells and colon epithelial normal cells Concentration-dependent inhibitory effect of MMPP on cancer cell growth was found in HCT116 and SW480 colon cancer cells but not in CCD-18Co colon epithelial normal cells. (A) , (B) (C) HCT116, SW480 and CCD-18Co cells were treated with MMPP (0-20 μg/mL) for 24 h, and then relative cell survival rate was determined by MTT assay. Data was expressed as the mean ± S.D. of three experiments. * p
    Figure Legend Snippet: Effect of MMPP on the growth colon cancer cells and colon epithelial normal cells Concentration-dependent inhibitory effect of MMPP on cancer cell growth was found in HCT116 and SW480 colon cancer cells but not in CCD-18Co colon epithelial normal cells. (A) , (B) (C) HCT116, SW480 and CCD-18Co cells were treated with MMPP (0-20 μg/mL) for 24 h, and then relative cell survival rate was determined by MTT assay. Data was expressed as the mean ± S.D. of three experiments. * p

    Techniques Used: Concentration Assay, MTT Assay

    4) Product Images from "SET7 interacts with HDAC6 and suppresses the development of colon cancer through inactivation of HDAC6"

    Article Title: SET7 interacts with HDAC6 and suppresses the development of colon cancer through inactivation of HDAC6

    Journal: American Journal of Translational Research

    doi:

    SET7 suppressed deacetylating activity of HDAC6 and increased levels of acetylated-α-tubulin partly through ERK signaling pathway. (A, B) Effects of SET7 and HDAC6 up-regulation or down-regulation on levels of acetylated-α-tubulin in colon cancer cell were analyzed by western blotting for 48 hours before harvesting. (C, D) The expression of p-ERK and ERK were detected in HCT116 and SW480 cells treated with Flag-SET7 and HA-HDAC6 plasmid for 48 hours before harvesting. α-tubulin served as the loading control. NOTE: we do the silencing effect on five groups: None, si-NC, si-SET7, si-HDAC6, si-SET7+si-SET7. But we only used the later four groups (si-NC, si-SET7, si-HDAC6, si-SET7+si-SET7) to detected the expression of AC-α-tubulin/α-tubulin/GAPDH. So, when we try to make a whole membrane, we cut the first band (group None: only cell without any treatment). NOTE: like (B). We also do the over-expression effect on five groups: None, over-expression-Veh, over-expression-SET7, over-expression-HDAC6, over-expression-SET7+over-expression-SET7. But we only used the later four groups to detected the expression of P-ERK/ERK/GAPDH. So, when we try to make a whole membrane, we also cut the first band (group None: only cell without any treatment).
    Figure Legend Snippet: SET7 suppressed deacetylating activity of HDAC6 and increased levels of acetylated-α-tubulin partly through ERK signaling pathway. (A, B) Effects of SET7 and HDAC6 up-regulation or down-regulation on levels of acetylated-α-tubulin in colon cancer cell were analyzed by western blotting for 48 hours before harvesting. (C, D) The expression of p-ERK and ERK were detected in HCT116 and SW480 cells treated with Flag-SET7 and HA-HDAC6 plasmid for 48 hours before harvesting. α-tubulin served as the loading control. NOTE: we do the silencing effect on five groups: None, si-NC, si-SET7, si-HDAC6, si-SET7+si-SET7. But we only used the later four groups (si-NC, si-SET7, si-HDAC6, si-SET7+si-SET7) to detected the expression of AC-α-tubulin/α-tubulin/GAPDH. So, when we try to make a whole membrane, we cut the first band (group None: only cell without any treatment). NOTE: like (B). We also do the over-expression effect on five groups: None, over-expression-Veh, over-expression-SET7, over-expression-HDAC6, over-expression-SET7+over-expression-SET7. But we only used the later four groups to detected the expression of P-ERK/ERK/GAPDH. So, when we try to make a whole membrane, we also cut the first band (group None: only cell without any treatment).

    Techniques Used: Activity Assay, Western Blot, Expressing, Plasmid Preparation, Over Expression

    Interaction between SET7 and HDAC6. A. HCT116 cell was transfected with pc-DNA2.0, Flag-SET7 and HA-HDAC6 plasmid, and the over-expression effect was detected by western blotting for 24 and 48 hours before harvesting. B. HCT116 transfected with siRNA targeting si-NC, si-SET7, si-HDAC6, and the siRNA-depletion efficiency was detected by western blotting for 48 hours before harvesting. C. Western blotting analysis of whole cell lysates (WCLs) and immunoprecipitates (IP) derived from HCT116 cells transfected with Flag-SET7 and HA-HDAC6 constructs for 24 hours before harvesting. D. The effect of over-expression Flag-SET7 and HA-HDAC6 on each endogenous expression were detected by western blotting for 48 hours before harvesting. NOTE: there is 5 samples in this member. (si-NC, si-GAPDH, si-SET7-1, si-SET7-2, si-SET7-3). As si-SET7-3 did not played a silencing effect, so we cut it.
    Figure Legend Snippet: Interaction between SET7 and HDAC6. A. HCT116 cell was transfected with pc-DNA2.0, Flag-SET7 and HA-HDAC6 plasmid, and the over-expression effect was detected by western blotting for 24 and 48 hours before harvesting. B. HCT116 transfected with siRNA targeting si-NC, si-SET7, si-HDAC6, and the siRNA-depletion efficiency was detected by western blotting for 48 hours before harvesting. C. Western blotting analysis of whole cell lysates (WCLs) and immunoprecipitates (IP) derived from HCT116 cells transfected with Flag-SET7 and HA-HDAC6 constructs for 24 hours before harvesting. D. The effect of over-expression Flag-SET7 and HA-HDAC6 on each endogenous expression were detected by western blotting for 48 hours before harvesting. NOTE: there is 5 samples in this member. (si-NC, si-GAPDH, si-SET7-1, si-SET7-2, si-SET7-3). As si-SET7-3 did not played a silencing effect, so we cut it.

    Techniques Used: Transfection, Plasmid Preparation, Over Expression, Western Blot, Derivative Assay, Construct, Expressing

    SET7 inhibited cell proliferation and migration in colon cancer cells treated with HDAC6. A. The proliferation in SW480 and HCT116 cells transfected with pc-DNA2.0, Flag-SET7 and HA-HDAC6 plasmid were detected by CCK-8 assays respectively. B, C. The potential migration ability in SW480 and HCT116 cells after transfection were respectively detected by wound healing and transwell assays. D, E. The proliferation and migration ability targeting si-HDAC6 and si-SET7 were detected by CCK-8 assays and wound healing assays respectively in HCT116 cell. Veh and NC served as control.
    Figure Legend Snippet: SET7 inhibited cell proliferation and migration in colon cancer cells treated with HDAC6. A. The proliferation in SW480 and HCT116 cells transfected with pc-DNA2.0, Flag-SET7 and HA-HDAC6 plasmid were detected by CCK-8 assays respectively. B, C. The potential migration ability in SW480 and HCT116 cells after transfection were respectively detected by wound healing and transwell assays. D, E. The proliferation and migration ability targeting si-HDAC6 and si-SET7 were detected by CCK-8 assays and wound healing assays respectively in HCT116 cell. Veh and NC served as control.

    Techniques Used: Migration, Transfection, Plasmid Preparation, CCK-8 Assay

    5) Product Images from "Structure-Function Analysis of the Mcl-1 Protein Identifies a Novel Senescence-regulating Domain *"

    Article Title: Structure-Function Analysis of the Mcl-1 Protein Identifies a Novel Senescence-regulating Domain *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.663898

    Non-mitochondrial Mcl-1 is associated with CIS inhibition. A , top panel , schematic of the wild-type Mcl-1 protein, highlighting the location of functional domains of Mcl-1. Center and bottom panels , schematics of the Δ1–79 and Δ328–350 mutant constructs, respectively. P , PEST domain (proline, glutamic acid, serine, and threonine); MP , minor PEST domain; TM , transmembrane domain. B , Western blot analysis of HCT116 p53 −/− shMcl-1 cells transiently transfected with vector control, WT Mcl-1, Δ1–79, or Δ328–350 mutant constructs. C , confocal microscopy of HCT116 p53 −/− shMcl-1 cells expressing the indicated constructs. Cells were stained for Mcl-1 and Tom-20, a mitochondrion-resident protein, 48 h after transfection. D , the images in C were quantified for the percentage of cells displaying significant Mcl-1/Tom20 colocalization. E–H , senescence assays of WT Mcl-1-, Δ1–79-, and Δ328–350 mutant-expressing cells treated or not treated with doxorubicin ( Dox ) compared with the treated empty vector control, as indicated by SA β-galactosidase activity ( E ), PML nuclear body staining ( F ), γH2AX nuclear body staining ( G ), and BrdU staining ( H ). Data are representative of three independent experiments. *, p
    Figure Legend Snippet: Non-mitochondrial Mcl-1 is associated with CIS inhibition. A , top panel , schematic of the wild-type Mcl-1 protein, highlighting the location of functional domains of Mcl-1. Center and bottom panels , schematics of the Δ1–79 and Δ328–350 mutant constructs, respectively. P , PEST domain (proline, glutamic acid, serine, and threonine); MP , minor PEST domain; TM , transmembrane domain. B , Western blot analysis of HCT116 p53 −/− shMcl-1 cells transiently transfected with vector control, WT Mcl-1, Δ1–79, or Δ328–350 mutant constructs. C , confocal microscopy of HCT116 p53 −/− shMcl-1 cells expressing the indicated constructs. Cells were stained for Mcl-1 and Tom-20, a mitochondrion-resident protein, 48 h after transfection. D , the images in C were quantified for the percentage of cells displaying significant Mcl-1/Tom20 colocalization. E–H , senescence assays of WT Mcl-1-, Δ1–79-, and Δ328–350 mutant-expressing cells treated or not treated with doxorubicin ( Dox ) compared with the treated empty vector control, as indicated by SA β-galactosidase activity ( E ), PML nuclear body staining ( F ), γH2AX nuclear body staining ( G ), and BrdU staining ( H ). Data are representative of three independent experiments. *, p

    Techniques Used: Inhibition, Functional Assay, Mutagenesis, Construct, Western Blot, Transfection, Plasmid Preparation, Confocal Microscopy, Expressing, Staining, Activity Assay, BrdU Staining

    The loop region of Mcl-1 is key to tumor CIS resistance and growth potential in vivo . A , tumor growth curves of xenografted HCT116 cells. Mice were injected with HCT116 cells stably overexpressing empty vector or WT-Mcl-1 ( Ai ), the Δ1–157 mutant (Aii), the 208–250 mutant ( Aiii ), or the Δ198–207 mutant ( Aiv ). *, p ≤ 0.05 for doxorubicin ( Dox )-treated vector control tumors compared with growth of tumors expressing the indicated constructs. Error bars represent mean ± S.D. Ten days later, mice received 1.2 mg/kg doxorubicin every other day intraperitoneally or were left untreated. B , tumor growth curves of HCT116 shMcl-1 cells stably expressing the vector control, WT Mcl-1, or the Mcl-1 deletion mutants Δ1–157, Δ208–250, or Δ198–207. *, p ≤ 0.05 for the vector control and Δ198–207 tumors compared with growth of tumors expressing the indicated constructs. Error bars represent mean ± S.D. C , representative xenograft tumor tissue sections from A were analyzed by immunohistochemistry after the final dose of doxorubicin ( Dox ) treatment for Mcl-1 expression, β-gal, PML nuclear body formation ( red circles ), and Ki67 or caspase-3 staining. D , immunohistochemical detection of Mcl-1 protein expression by different anti-Mcl-1 antibodies. Shown are representative micrographs of doxorubicin-treated HCT116 tumors expressing the indicated constructs. Tissue sections were stained with S-19 or K-20 anti-Mcl-1 antibodies.
    Figure Legend Snippet: The loop region of Mcl-1 is key to tumor CIS resistance and growth potential in vivo . A , tumor growth curves of xenografted HCT116 cells. Mice were injected with HCT116 cells stably overexpressing empty vector or WT-Mcl-1 ( Ai ), the Δ1–157 mutant (Aii), the 208–250 mutant ( Aiii ), or the Δ198–207 mutant ( Aiv ). *, p ≤ 0.05 for doxorubicin ( Dox )-treated vector control tumors compared with growth of tumors expressing the indicated constructs. Error bars represent mean ± S.D. Ten days later, mice received 1.2 mg/kg doxorubicin every other day intraperitoneally or were left untreated. B , tumor growth curves of HCT116 shMcl-1 cells stably expressing the vector control, WT Mcl-1, or the Mcl-1 deletion mutants Δ1–157, Δ208–250, or Δ198–207. *, p ≤ 0.05 for the vector control and Δ198–207 tumors compared with growth of tumors expressing the indicated constructs. Error bars represent mean ± S.D. C , representative xenograft tumor tissue sections from A were analyzed by immunohistochemistry after the final dose of doxorubicin ( Dox ) treatment for Mcl-1 expression, β-gal, PML nuclear body formation ( red circles ), and Ki67 or caspase-3 staining. D , immunohistochemical detection of Mcl-1 protein expression by different anti-Mcl-1 antibodies. Shown are representative micrographs of doxorubicin-treated HCT116 tumors expressing the indicated constructs. Tissue sections were stained with S-19 or K-20 anti-Mcl-1 antibodies.

    Techniques Used: In Vivo, Mouse Assay, Injection, Stable Transfection, Plasmid Preparation, Mutagenesis, Expressing, Construct, Immunohistochemistry, Staining

    Posttranslational modification sites and BH domains are dispensable for the ability of Mcl-1 to inhibit CIS. Ai , schematic of the wild-type Mcl-1 protein. Arrows indicate alanine substitutions of important phosphorylation sites. P, pest domain (proline, glutamic acid, serine, and threonine). MP, minor PEST domain. TM, transmembrane domain. Aii , Western blot of Mcl-1 protein levels after transient transfection of the indicated constructs in HCT116 p53 −/− shMcl-1 cells. HCT116 p53 −/− shMcl-1 cells were transiently transfected with vector control, WT Mcl-1, or the indicated constructs and then either treated with doxorubicin or left untreated. Aiii–Avi , data showing a decrease in β-gal ( Aiii ), PML nuclear body ( Aiv ), and γH2AX nuclear body formation ( Av ) and an increase in ki67 staining ( Avi ) in cells expressing the phosphomimetic mutants of Mcl-1. Dox , doxorubicin. Bi , schematic of N-terminal deletions of Mcl-1 equivalent to post-caspase cleavage. HCT116 p53 −/− shMcl-1 cells were transiently transfected with vector control, WT Mcl-1, or Δ1–127 or Δ1–157 constructs. Bii , Western blot of Mcl-1 protein levels after transfection of the indicated constructs. Biii–Bvi , quantitative analysis of CIS in N-terminal deletion mutants as assessed by β-gal activity ( Biii ), staining with antibodies for PML ( Biv ) and γH2AX ( Bv ) nuclear bodies, and BrdU staining ( Bvi ). Ci and Cii , schematic of the Δ208–350 construct. HCT116p53 −/− shMcl-1 cells were transiently transfected with empty pcDNA3.1/myc-His vector, WT-Mcl-1 (non-tagged), or pcDNA3.1/myc-His;Δ208–350 constructs and verified by Western blot ( Cii ). Ciii–Cvi , quantitative analysis of CIS as assessed by β-gal staining ( Ciii ), PML nuclear body formation ( Civ ), γH2AX nuclear body formation ( Cv ), and BrdU staining ( Cvi ). All data are representative of three independent experiments. NS , no statistical differences between the indicated constructs and WT Mcl-1 in doxorubicin-treated cells. *, p
    Figure Legend Snippet: Posttranslational modification sites and BH domains are dispensable for the ability of Mcl-1 to inhibit CIS. Ai , schematic of the wild-type Mcl-1 protein. Arrows indicate alanine substitutions of important phosphorylation sites. P, pest domain (proline, glutamic acid, serine, and threonine). MP, minor PEST domain. TM, transmembrane domain. Aii , Western blot of Mcl-1 protein levels after transient transfection of the indicated constructs in HCT116 p53 −/− shMcl-1 cells. HCT116 p53 −/− shMcl-1 cells were transiently transfected with vector control, WT Mcl-1, or the indicated constructs and then either treated with doxorubicin or left untreated. Aiii–Avi , data showing a decrease in β-gal ( Aiii ), PML nuclear body ( Aiv ), and γH2AX nuclear body formation ( Av ) and an increase in ki67 staining ( Avi ) in cells expressing the phosphomimetic mutants of Mcl-1. Dox , doxorubicin. Bi , schematic of N-terminal deletions of Mcl-1 equivalent to post-caspase cleavage. HCT116 p53 −/− shMcl-1 cells were transiently transfected with vector control, WT Mcl-1, or Δ1–127 or Δ1–157 constructs. Bii , Western blot of Mcl-1 protein levels after transfection of the indicated constructs. Biii–Bvi , quantitative analysis of CIS in N-terminal deletion mutants as assessed by β-gal activity ( Biii ), staining with antibodies for PML ( Biv ) and γH2AX ( Bv ) nuclear bodies, and BrdU staining ( Bvi ). Ci and Cii , schematic of the Δ208–350 construct. HCT116p53 −/− shMcl-1 cells were transiently transfected with empty pcDNA3.1/myc-His vector, WT-Mcl-1 (non-tagged), or pcDNA3.1/myc-His;Δ208–350 constructs and verified by Western blot ( Cii ). Ciii–Cvi , quantitative analysis of CIS as assessed by β-gal staining ( Ciii ), PML nuclear body formation ( Civ ), γH2AX nuclear body formation ( Cv ), and BrdU staining ( Cvi ). All data are representative of three independent experiments. NS , no statistical differences between the indicated constructs and WT Mcl-1 in doxorubicin-treated cells. *, p

    Techniques Used: Modification, Western Blot, Transfection, Construct, Plasmid Preparation, Staining, Expressing, Activity Assay, BrdU Staining

    The Mcl-1 loop domain can be functionally blocked through dominant negative inhibition. A , Western blot for construct expression in endogenous Mcl-1-expressing HCT116 p53 −/− cells transiently transfected with the vector control, WT Mcl-1, or various Mcl-1 constructs. Data are representative of two independent experiments. B–D , quantitative analysis of CIS in cells transiently transfected with the vector control, WT Mcl-1, or various Mcl-1 constructs and analyzed for β-gal activity ( B ), PML nuclear body formation ( C ), or γH2AX nuclear body formation ( D ). *, p
    Figure Legend Snippet: The Mcl-1 loop domain can be functionally blocked through dominant negative inhibition. A , Western blot for construct expression in endogenous Mcl-1-expressing HCT116 p53 −/− cells transiently transfected with the vector control, WT Mcl-1, or various Mcl-1 constructs. Data are representative of two independent experiments. B–D , quantitative analysis of CIS in cells transiently transfected with the vector control, WT Mcl-1, or various Mcl-1 constructs and analyzed for β-gal activity ( B ), PML nuclear body formation ( C ), or γH2AX nuclear body formation ( D ). *, p

    Techniques Used: Dominant Negative Mutation, Inhibition, Western Blot, Construct, Expressing, Transfection, Plasmid Preparation, Activity Assay

    6) Product Images from "MicroRNA-7 inhibits colorectal cancer cell proliferation, migration and invasion via TYRO3 and phosphoinositide 3-kinase/protein B kinase/mammalian target of rapamycin pathway suppression"

    Article Title: MicroRNA-7 inhibits colorectal cancer cell proliferation, migration and invasion via TYRO3 and phosphoinositide 3-kinase/protein B kinase/mammalian target of rapamycin pathway suppression

    Journal: International Journal of Molecular Medicine

    doi: 10.3892/ijmm.2018.3864

    miR-7 was downregulated in CRC tissues and cell lines. The expression of miR-7 in (A) CRC and adjacent normal tissues, and in (B) CRC cells (LoVo, SW480, SW620, HCT116 and HT29) and normal colonic mucosa epithelial NCM460 cells was detected by reverse transcription-quantitative polymerase chain reaction. Data are expressed as the mean ± standard deviation. ### P
    Figure Legend Snippet: miR-7 was downregulated in CRC tissues and cell lines. The expression of miR-7 in (A) CRC and adjacent normal tissues, and in (B) CRC cells (LoVo, SW480, SW620, HCT116 and HT29) and normal colonic mucosa epithelial NCM460 cells was detected by reverse transcription-quantitative polymerase chain reaction. Data are expressed as the mean ± standard deviation. ### P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Standard Deviation

    TYRO3 was upregulated in colorectal cancer cells and is a direct target gene of miR-7. (A) Western blot analysis and (B) reverse transcription-quantitative polymerase chain reaction were used to determine the expression of TYRO3 in LoVo, SW480, SW620, HCT116, HT29 and normal colonic mucosa epithelial cells. (C) Luciferase activity in cells co-transfected with TYRO3 3′UTR WT/MUT and miR-7 mimic/mimic control. (D) Protein expression of TYRO3 in LoVo, SW480 and SW620 cells transfected with miR-7 mimic or miR-NC was detected by western blot analysis. Data are expressed as the mean ± standard deviation. * P
    Figure Legend Snippet: TYRO3 was upregulated in colorectal cancer cells and is a direct target gene of miR-7. (A) Western blot analysis and (B) reverse transcription-quantitative polymerase chain reaction were used to determine the expression of TYRO3 in LoVo, SW480, SW620, HCT116, HT29 and normal colonic mucosa epithelial cells. (C) Luciferase activity in cells co-transfected with TYRO3 3′UTR WT/MUT and miR-7 mimic/mimic control. (D) Protein expression of TYRO3 in LoVo, SW480 and SW620 cells transfected with miR-7 mimic or miR-NC was detected by western blot analysis. Data are expressed as the mean ± standard deviation. * P

    Techniques Used: Western Blot, Real-time Polymerase Chain Reaction, Expressing, Luciferase, Activity Assay, Transfection, Standard Deviation

    7) Product Images from "TAp73-induced phosphofructokinase-1 transcription promotes the Warburg effect and enhances cell proliferation"

    Article Title: TAp73-induced phosphofructokinase-1 transcription promotes the Warburg effect and enhances cell proliferation

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07127-8

    PFKL and G6PD mediate the tumorigenic effect of TAp73. a HCT116 cells stably expressing vector control, PFKL, G6PD, or both PFKL and G6PD were treated with control or TAp73 siRNA for 24 h, and 1000 cells for each condition were plated in soft agar for colony formation. Numbers of colonies with a diameter greater than 10 μm were quantified 10 d later (means ± S.D., n = 6). b , c HCT116 cells stably expressing vector, PFKL, or G6PD were treated with a control or TAp73 siRNA. Cells were xenografted into immunodeficient mice. Average weights ( b , means ± S.D. indicated) and images ( c ) of xenograft tumors at 3 weeks are shown
    Figure Legend Snippet: PFKL and G6PD mediate the tumorigenic effect of TAp73. a HCT116 cells stably expressing vector control, PFKL, G6PD, or both PFKL and G6PD were treated with control or TAp73 siRNA for 24 h, and 1000 cells for each condition were plated in soft agar for colony formation. Numbers of colonies with a diameter greater than 10 μm were quantified 10 d later (means ± S.D., n = 6). b , c HCT116 cells stably expressing vector, PFKL, or G6PD were treated with a control or TAp73 siRNA. Cells were xenografted into immunodeficient mice. Average weights ( b , means ± S.D. indicated) and images ( c ) of xenograft tumors at 3 weeks are shown

    Techniques Used: Stable Transfection, Expressing, Plasmid Preparation, Mouse Assay

    TAp73 regulates the expression of PFKL. a Relative mRNA levels of glycolytic enzymes in TAp73 +/+ and TAp73 −/− MEFs (clone #1), as analyzed by qRT-PCR. Data are means ± S.D. ( n = 3). b , c TAp73 +/+ and TAp73 −/− MEFs (clone #1 and #2) were analyzed by qRT-PCR for expressions of PFKL, PFKM, and PFKP, and G6PD. Data are means ± S.D. (n = 3). d TAp73 +/+ and TAp73 −/− MEFs (clone #2) were analyzed by semi-quantitative RT-PCR (top), and Western blot (WB, bottom). e mRNA levels of PFKL, PFKM, and G6PD in ΔNp73 +/+ and ΔNp73 −/− MEF cells. Results are representative of three independent experiments. f U2OS cells were transfected with control or p73 siRNA as indicated. mRNA expression was detected by qRT-PCR (means ± S.D., n = 3). g , h U2OS cells were transfected with control or TAp73 siRNA were analyzed by qRT-PCR ( g , means ± S.D., n = 3), semi-quantitative RT-PCR ( h , top), and Western blot (WB) ( h , bottom). i U2OS, HCT116, and H1299 cells transfected with control or TAp73 siRNA were analyzed by Western blot. Results are representative of three independent experiments. j , k HeLa cells transfected with control or TAp73 siRNA were analyzed for mRNA and protein expression. Data are means ± S.D. ( n = 3). l U2OS cells stably expressing control plasmid or an siRNA-resistant TAp73 plasmid were transfected with a control or TAp73 siRNA. PFKL expression was analyzed by RT-PCR and Western blotting
    Figure Legend Snippet: TAp73 regulates the expression of PFKL. a Relative mRNA levels of glycolytic enzymes in TAp73 +/+ and TAp73 −/− MEFs (clone #1), as analyzed by qRT-PCR. Data are means ± S.D. ( n = 3). b , c TAp73 +/+ and TAp73 −/− MEFs (clone #1 and #2) were analyzed by qRT-PCR for expressions of PFKL, PFKM, and PFKP, and G6PD. Data are means ± S.D. (n = 3). d TAp73 +/+ and TAp73 −/− MEFs (clone #2) were analyzed by semi-quantitative RT-PCR (top), and Western blot (WB, bottom). e mRNA levels of PFKL, PFKM, and G6PD in ΔNp73 +/+ and ΔNp73 −/− MEF cells. Results are representative of three independent experiments. f U2OS cells were transfected with control or p73 siRNA as indicated. mRNA expression was detected by qRT-PCR (means ± S.D., n = 3). g , h U2OS cells were transfected with control or TAp73 siRNA were analyzed by qRT-PCR ( g , means ± S.D., n = 3), semi-quantitative RT-PCR ( h , top), and Western blot (WB) ( h , bottom). i U2OS, HCT116, and H1299 cells transfected with control or TAp73 siRNA were analyzed by Western blot. Results are representative of three independent experiments. j , k HeLa cells transfected with control or TAp73 siRNA were analyzed for mRNA and protein expression. Data are means ± S.D. ( n = 3). l U2OS cells stably expressing control plasmid or an siRNA-resistant TAp73 plasmid were transfected with a control or TAp73 siRNA. PFKL expression was analyzed by RT-PCR and Western blotting

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Transfection, Stable Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction

    PFKL is a physiologically relevant target of TAp73. a Schematic representation of human PFKL genomic structure. The sequences of potential p73 response elements RE1–3 and the corresponding mutant RE3 are shown. b , c Luciferase constructs containing RE1, RE2, and RE3 ( b ), or RE3 and mutant RE3 ( c ) were transfected into 293T cells together with Flag-TAp73α or vector control. Renilla vector pRL-CMV was used as a transfection internal control. The relative luciferase activity was normalized to the co-transfected Renila activity. Data are means ± S.D. ( n = 3). d Luciferase reporter constructs containing RE1, RE2, RE3, or RE3mut were transfected into 293T cells together vector control, p53, or TAp73. Renilla vector pRL-CMV was used as a transfection internal control. Relative levels of luciferase are shown. Data are means ± S.D. ( n = 3). Insert shows protein expression. e , f U2OS cells ( e ), or 293T cells transfected with control vector or Flag-TAp73 ( f ), were analyzed by ChIP assay using normal mouse IgG and anti-p73 antibody ( e ), or anti-Flag antibody ( f ). Bound DNA was amplified by PCR and quantified. Results are representative of three independent experiments. g , h U2OS ( g ) and HCT116 ( h ) cells transfected with the indicated siRNAs were analyzed for protein and mRNA expression. Results are representative of three independent experiments. i , j U2OS cells ( i ), or 293T cells transfected with Flag-p53 or vector control ( j ), were analyzed by ChIP assay using normal mouse IgG and anti-p53 antibody ( i ), or anti-Flag antibody ( j ). Bound DNA was amplified by PCR and quantified. Results are representative of three independent experiments. k p53 −/− HCT116 cells stable expressing Tet-inducible p53 were cultured in medium containing [1,2– 13 C 2 ]glucose and treated with doxycycline to induce p53 expression (Tet-on). p53, p73 and PFKL expressions were determined by Western blot analysis. TIGAR expression was analyzed by qRT-PCR. (means ± S.D., n = 3). Relative glycolytic flux is shown in Supplementary Fig. 4b
    Figure Legend Snippet: PFKL is a physiologically relevant target of TAp73. a Schematic representation of human PFKL genomic structure. The sequences of potential p73 response elements RE1–3 and the corresponding mutant RE3 are shown. b , c Luciferase constructs containing RE1, RE2, and RE3 ( b ), or RE3 and mutant RE3 ( c ) were transfected into 293T cells together with Flag-TAp73α or vector control. Renilla vector pRL-CMV was used as a transfection internal control. The relative luciferase activity was normalized to the co-transfected Renila activity. Data are means ± S.D. ( n = 3). d Luciferase reporter constructs containing RE1, RE2, RE3, or RE3mut were transfected into 293T cells together vector control, p53, or TAp73. Renilla vector pRL-CMV was used as a transfection internal control. Relative levels of luciferase are shown. Data are means ± S.D. ( n = 3). Insert shows protein expression. e , f U2OS cells ( e ), or 293T cells transfected with control vector or Flag-TAp73 ( f ), were analyzed by ChIP assay using normal mouse IgG and anti-p73 antibody ( e ), or anti-Flag antibody ( f ). Bound DNA was amplified by PCR and quantified. Results are representative of three independent experiments. g , h U2OS ( g ) and HCT116 ( h ) cells transfected with the indicated siRNAs were analyzed for protein and mRNA expression. Results are representative of three independent experiments. i , j U2OS cells ( i ), or 293T cells transfected with Flag-p53 or vector control ( j ), were analyzed by ChIP assay using normal mouse IgG and anti-p53 antibody ( i ), or anti-Flag antibody ( j ). Bound DNA was amplified by PCR and quantified. Results are representative of three independent experiments. k p53 −/− HCT116 cells stable expressing Tet-inducible p53 were cultured in medium containing [1,2– 13 C 2 ]glucose and treated with doxycycline to induce p53 expression (Tet-on). p53, p73 and PFKL expressions were determined by Western blot analysis. TIGAR expression was analyzed by qRT-PCR. (means ± S.D., n = 3). Relative glycolytic flux is shown in Supplementary Fig. 4b

    Techniques Used: Mutagenesis, Luciferase, Construct, Transfection, Plasmid Preparation, Activity Assay, Expressing, Chromatin Immunoprecipitation, Amplification, Polymerase Chain Reaction, Cell Culture, Western Blot, Quantitative RT-PCR

    TAp73 regulates PFKL under stressed conditions. a , b U2OS cells transfected with control or TAp73 siRNA were treated with increasing amounts of etoposide (ETP) for 24 h, and analyzed by qRT-PCR ( a , means ± S.D., n = 3) and Western blot ( b ). Data are representative of three independent experiments. c – e HCT116 ( c , d ) and U2OS ( e ) cells transfected with control siRNA or TAp73 siRNA were cultured in complete medium for 24 h and then in serum-free medium for different times. Cells were analyzed by qRT-PCR ( c , means ± S.D., n = 3) and Western blot ( d , e ). f HCT116 cells were cultured in medium containing serum or no serum for 3 h, and then subjected to cycloheximide (CHX) chase in the presence or absence of serum. Whole cell extracts were collected using a pellet buffer as described previously 32 . Data are representative of three independent experiments. g U2OS cells transfected with control siRNA or TAp73 siRNA were subjected to CHX chase. Data are representative of three independent experiments
    Figure Legend Snippet: TAp73 regulates PFKL under stressed conditions. a , b U2OS cells transfected with control or TAp73 siRNA were treated with increasing amounts of etoposide (ETP) for 24 h, and analyzed by qRT-PCR ( a , means ± S.D., n = 3) and Western blot ( b ). Data are representative of three independent experiments. c – e HCT116 ( c , d ) and U2OS ( e ) cells transfected with control siRNA or TAp73 siRNA were cultured in complete medium for 24 h and then in serum-free medium for different times. Cells were analyzed by qRT-PCR ( c , means ± S.D., n = 3) and Western blot ( d , e ). f HCT116 cells were cultured in medium containing serum or no serum for 3 h, and then subjected to cycloheximide (CHX) chase in the presence or absence of serum. Whole cell extracts were collected using a pellet buffer as described previously 32 . Data are representative of three independent experiments. g U2OS cells transfected with control siRNA or TAp73 siRNA were subjected to CHX chase. Data are representative of three independent experiments

    Techniques Used: Transfection, Quantitative RT-PCR, Western Blot, Cell Culture

    8) Product Images from "The RNA-binding Protein RNPC1 Stabilizes the mRNA Encoding the RNA-binding Protein HuR and Cooperates with HuR to Suppress Cell Proliferation *"

    Article Title: The RNA-binding Protein RNPC1 Stabilizes the mRNA Encoding the RNA-binding Protein HuR and Cooperates with HuR to Suppress Cell Proliferation *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.326827

    HuR-mediated c-Myc repression is critical for the growth suppression by RNPC1. A , the level of c-Myc is repressed by RNPC1. MCF7 and HCT116 cells were un-induced or induced to express RNPC1 for 48 h, and the level of RNPC1a, HuR, c-Myc, and actin was measured by Western blot analysis. The basal level of c-Myc or HuR was arbitrarily set at 1.0, and relative -fold change of each protein level is shown below each lane . The data are representative of three independent experiments. B and C , RNPC1 repression of c-Myc is HuR-dependent. HCT116 ( B ) and H1299 ( C ) cells were transiently transfected with scrambled or RNPC1a siRNA for 24 h, and then un-induced or induced to express RNPC1 for 48 h. The level of RNPC1, HuR, c-Myc, and actin was measured by Western blot analysis. The basal level of HuR or c-Myc was arbitrarily set at 1.0, and relative -fold change of each protein level is shown below each lane . The data are representative of three independent experiments. D , H1299 cells were transduced with a lentivirus expressing control luciferase shRNA or shRNAs against HuR and/or c-Myc, selected by puromycin for 3 days and then un-induced or induced to express RNPC1a for 48 h. Cell lysates were collected, and the levels of RNPC1, HuR, c-Myc, and actin were measured by Western blot analysis. The basal level of HuR or c-Myc was arbitrarily set at 1.0, and -fold change is shown below each lane . The data are representative of three independent experiments. E , HuR and/or c-Myc knockdown attenuates RNPC1-induced growth suppression. Upper panel , colony formation assay was performed with H1299 cells treated as in D. Lower panel , colony numbers from the wells in the upper panel were quantified and the rate of colony formation at the control condition was set as 100%. The -fold change is the ratio of colonies formed in the absence of RNPC1 versus that in the presence of RNPC1. *, p
    Figure Legend Snippet: HuR-mediated c-Myc repression is critical for the growth suppression by RNPC1. A , the level of c-Myc is repressed by RNPC1. MCF7 and HCT116 cells were un-induced or induced to express RNPC1 for 48 h, and the level of RNPC1a, HuR, c-Myc, and actin was measured by Western blot analysis. The basal level of c-Myc or HuR was arbitrarily set at 1.0, and relative -fold change of each protein level is shown below each lane . The data are representative of three independent experiments. B and C , RNPC1 repression of c-Myc is HuR-dependent. HCT116 ( B ) and H1299 ( C ) cells were transiently transfected with scrambled or RNPC1a siRNA for 24 h, and then un-induced or induced to express RNPC1 for 48 h. The level of RNPC1, HuR, c-Myc, and actin was measured by Western blot analysis. The basal level of HuR or c-Myc was arbitrarily set at 1.0, and relative -fold change of each protein level is shown below each lane . The data are representative of three independent experiments. D , H1299 cells were transduced with a lentivirus expressing control luciferase shRNA or shRNAs against HuR and/or c-Myc, selected by puromycin for 3 days and then un-induced or induced to express RNPC1a for 48 h. Cell lysates were collected, and the levels of RNPC1, HuR, c-Myc, and actin were measured by Western blot analysis. The basal level of HuR or c-Myc was arbitrarily set at 1.0, and -fold change is shown below each lane . The data are representative of three independent experiments. E , HuR and/or c-Myc knockdown attenuates RNPC1-induced growth suppression. Upper panel , colony formation assay was performed with H1299 cells treated as in D. Lower panel , colony numbers from the wells in the upper panel were quantified and the rate of colony formation at the control condition was set as 100%. The -fold change is the ratio of colonies formed in the absence of RNPC1 versus that in the presence of RNPC1. *, p

    Techniques Used: Western Blot, Transfection, Transduction, Expressing, Luciferase, shRNA, Colony Assay

    9) Product Images from "Diffusion kurtosis imaging evaluating epithelial–mesenchymal transition in colorectal carcinoma xenografts model: a preliminary study"

    Article Title: Diffusion kurtosis imaging evaluating epithelial–mesenchymal transition in colorectal carcinoma xenografts model: a preliminary study

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-11808-7

    Migration and invasion results of HCT116/Control and HCT116/Snail1 cells. HCT116/Snail1 cells increased number of migrating cells (A, P
    Figure Legend Snippet: Migration and invasion results of HCT116/Control and HCT116/Snail1 cells. HCT116/Snail1 cells increased number of migrating cells (A, P

    Techniques Used: Migration

    Morphologic anylysis of HCT116/Control and HCT116/Snail1 cells. The HCT116/Snail1 cells overexpressing Snail1 (B, 200× ) showed morphologic changes consistent with EMT including spindle shape with loss of cell polarity, increased intercellular separation, and increased formation of pseudopodia but not in HCT116/Control cells (A, 200× ).
    Figure Legend Snippet: Morphologic anylysis of HCT116/Control and HCT116/Snail1 cells. The HCT116/Snail1 cells overexpressing Snail1 (B, 200× ) showed morphologic changes consistent with EMT including spindle shape with loss of cell polarity, increased intercellular separation, and increased formation of pseudopodia but not in HCT116/Control cells (A, 200× ).

    Techniques Used:

    RT-PCR and Western blot analysis of HCT116/Control and HCT116/Snail1 cells. RT-PCR analysis showed that Snail1-mRNA expression ( A ) was significantly higher ( P = 0.001), E-cadherin-mRNA expression ( B ) was significantly lower ( P = 0.007), and vimentin-mRNA expression ( C ) was significantly higher ( P
    Figure Legend Snippet: RT-PCR and Western blot analysis of HCT116/Control and HCT116/Snail1 cells. RT-PCR analysis showed that Snail1-mRNA expression ( A ) was significantly higher ( P = 0.001), E-cadherin-mRNA expression ( B ) was significantly lower ( P = 0.007), and vimentin-mRNA expression ( C ) was significantly higher ( P

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing

    10) Product Images from "Targeting nonsense-mediated mRNA decay in colorectal cancers with microsatellite instability"

    Article Title: Targeting nonsense-mediated mRNA decay in colorectal cancers with microsatellite instability

    Journal: Oncogenesis

    doi: 10.1038/s41389-018-0079-x

    Inhibition of NMD system and differential RNA decay. a Upper panel: schematic representation of the HSP110DE9-specific NMD reporter system used in this work. The NMD.reporter gene consisted of an in-frame HSP110 construction and contains the cDNA sequence from exon 1 to exon 8, intron 9, exon 10, intron 16, and exon 18. As in the case of the T 17 mutation of HSP110 located near the splice acceptor site of intron 8, a nonsense mutation appears in exon 10 due to the frameshift mutation caused by skipping of exon 9, making the exogenous mRNA a target of NMD. This construction is placed in an EBV stable vector. Lower panel: relative expression of HSP110DE9-PTC mRNA from NMD reporter stably transfected in the SW480 CRC cell line treated with several NMD inhibitors (siUPF1, cycloheximide (CHX), amlexanox, and NMDI-1). b Relative expression levels of TGFBR2 , MSH3 , or HSP110DE9 mRNAs determined by quantitative RT-PCR in CRC cell lines. After cycloheximide [CHX] treatment (4 h, 400 μg/ml). RNA expression levels compared to untreated cells [UT] in cell lines were analyzed with the TGFBR2 probe, [No Del] (SW480 and FET), Heterozygote [Htz] (HCT8 and RKO), Homozygote [Hmz] (HCT116 and LS174T); with the MSH3 probe, [No Del] (SW480 and HCT8), Heterozygote [Htz] = (LS174T), Homozygote [Hmz] (HCT116); with HSP110DE9 probes, [No Del] (SW480 and FET); [Del S ] (HCT116 and HCT8); [Del L ] (RKO and LS174T). Dashed line refers to the ratio calculated between treated and untreated cells with CHX. c Relative mRNA expression levels of MSI target genes (containing coding DNA repeats) as determined by quantitative RT-PCR in HCT116 (MSI) and SW480 (MSS) CRC cell lines transfected with siUPF1 (24 h post-transfection; top panel) or treated for 24 h with 5 µM amlexanox (lower panel). IGF2R is used as an internal control (not mutated in HCT116 and SW480 CRC cell line) for experimental conditions. All data are means ± SEM. Unpaired t -test was performed to determine significance. * p
    Figure Legend Snippet: Inhibition of NMD system and differential RNA decay. a Upper panel: schematic representation of the HSP110DE9-specific NMD reporter system used in this work. The NMD.reporter gene consisted of an in-frame HSP110 construction and contains the cDNA sequence from exon 1 to exon 8, intron 9, exon 10, intron 16, and exon 18. As in the case of the T 17 mutation of HSP110 located near the splice acceptor site of intron 8, a nonsense mutation appears in exon 10 due to the frameshift mutation caused by skipping of exon 9, making the exogenous mRNA a target of NMD. This construction is placed in an EBV stable vector. Lower panel: relative expression of HSP110DE9-PTC mRNA from NMD reporter stably transfected in the SW480 CRC cell line treated with several NMD inhibitors (siUPF1, cycloheximide (CHX), amlexanox, and NMDI-1). b Relative expression levels of TGFBR2 , MSH3 , or HSP110DE9 mRNAs determined by quantitative RT-PCR in CRC cell lines. After cycloheximide [CHX] treatment (4 h, 400 μg/ml). RNA expression levels compared to untreated cells [UT] in cell lines were analyzed with the TGFBR2 probe, [No Del] (SW480 and FET), Heterozygote [Htz] (HCT8 and RKO), Homozygote [Hmz] (HCT116 and LS174T); with the MSH3 probe, [No Del] (SW480 and HCT8), Heterozygote [Htz] = (LS174T), Homozygote [Hmz] (HCT116); with HSP110DE9 probes, [No Del] (SW480 and FET); [Del S ] (HCT116 and HCT8); [Del L ] (RKO and LS174T). Dashed line refers to the ratio calculated between treated and untreated cells with CHX. c Relative mRNA expression levels of MSI target genes (containing coding DNA repeats) as determined by quantitative RT-PCR in HCT116 (MSI) and SW480 (MSS) CRC cell lines transfected with siUPF1 (24 h post-transfection; top panel) or treated for 24 h with 5 µM amlexanox (lower panel). IGF2R is used as an internal control (not mutated in HCT116 and SW480 CRC cell line) for experimental conditions. All data are means ± SEM. Unpaired t -test was performed to determine significance. * p

    Techniques Used: Inhibition, Sequencing, Mutagenesis, Plasmid Preparation, Expressing, Stable Transfection, Transfection, Quantitative RT-PCR, RNA Expression

    Impact of NMD inhibition in cell proliferation and tumor growth. a Cell proliferation of HCT116 (MSI), RKO (MSI), SW480 (MSS), and LS513 (MSS) colon cancer cells were analyzed with Xcelligence technology (see Materials and methods). Twenty-four hours post-transfection with a control (siCTL) or siRNA against UPF1 (siUPF1), cells were seeded in E-plate 96 to allow measurement of proliferation rates during 48 h. b HCT116 (MSI) and SW480 (MSS) CRC cancer cells were treated once a day during 4 days with or without 5 μM amlexanox. OD was measured every day after drug treatment using the WST-1 assay. c Upper panel: schematic representation of the protocol for treating mice with the NMD inhibitor amlexanox. The osmotic pump contained either a mock buffer made with 50% DMSO and 50% PEG400, or amlexanox diluted in the mock buffer in order to deliver 0.15 mg of amlexanox per day to each mouse during 28 days. Lower panel: comparative analysis of tumor growth (mean tumor volumes) in mice treated with or without amlexanox. Eight mice per group. Experiments were performed with MSI (HCT116) or MSS (SW480) CRC cells (left and right panels, respectively). All data are means ± SEM. Unpaired t -test was performed to determine significance. * p
    Figure Legend Snippet: Impact of NMD inhibition in cell proliferation and tumor growth. a Cell proliferation of HCT116 (MSI), RKO (MSI), SW480 (MSS), and LS513 (MSS) colon cancer cells were analyzed with Xcelligence technology (see Materials and methods). Twenty-four hours post-transfection with a control (siCTL) or siRNA against UPF1 (siUPF1), cells were seeded in E-plate 96 to allow measurement of proliferation rates during 48 h. b HCT116 (MSI) and SW480 (MSS) CRC cancer cells were treated once a day during 4 days with or without 5 μM amlexanox. OD was measured every day after drug treatment using the WST-1 assay. c Upper panel: schematic representation of the protocol for treating mice with the NMD inhibitor amlexanox. The osmotic pump contained either a mock buffer made with 50% DMSO and 50% PEG400, or amlexanox diluted in the mock buffer in order to deliver 0.15 mg of amlexanox per day to each mouse during 28 days. Lower panel: comparative analysis of tumor growth (mean tumor volumes) in mice treated with or without amlexanox. Eight mice per group. Experiments were performed with MSI (HCT116) or MSS (SW480) CRC cells (left and right panels, respectively). All data are means ± SEM. Unpaired t -test was performed to determine significance. * p

    Techniques Used: Inhibition, Transfection, WST-1 Assay, Mouse Assay

    The overexpression of NMD factor in MSI primary CRC. a Microarray analysis of NMD-related factors in MSI/MSS tumor tissues ([MSS primary CRC], n = 48; [MSI primary CRC], n = 40). b Microarray analysis associated with Exome sequencing data of 30 MSI tumors to compare the expression of genes with mutation in microsatellite located in the last exon (LE; n = 98) or before the last exon (NLE; n = 569). c Schematic structure of mutant proteins (15 mutated tumor suppressor genes in green or 16 mutated oncogenes in red) with NLE mutations and significant down-regulation in MSI tumors. d Left panel: relative expression levels of HSP110wt and HSP110DE9 mRNAs by quantitative RT-PCR in MSS and MSI CRC primary tumors. No Del (wild type status; MSS CRCs) n = 36; DelS (small deletion: ≤4 pb; MSI CRCs), n = 28; DelL (large deletion: > 4 pb; MSI CRCs), n = 7. Right panel: relative mRNA expression levels of HSP110wt and HSP110DE9 determined by quantitative RT-PCR in MSS and MSI CRC cell lines. No T17 deletion [No Del], n = 6 CRC cell lines (ISI, SW1116, V9P, ALA, FET, SW480); small T17 Deletion [DelS], n = 4 CRC cell lines (HCT8, HCT116, TC71, LIM1215); large T17 Deletion [DelL], n = 6 CRC cell lines (TC7, Lovo, KM12, LS411, Ls174T, Co115). Data are means ± SEM. e Quantification of all HSP110 mRNAs. Densities and bar plots of HSP110 log2 intensities in normal colonic mucosa (Muc), adenomas (Ade), MSS tumors (MSS), and MSI tumors (MSI). All Data are means ± SEM. Unpaired t -test was performed to determine significance. ** p
    Figure Legend Snippet: The overexpression of NMD factor in MSI primary CRC. a Microarray analysis of NMD-related factors in MSI/MSS tumor tissues ([MSS primary CRC], n = 48; [MSI primary CRC], n = 40). b Microarray analysis associated with Exome sequencing data of 30 MSI tumors to compare the expression of genes with mutation in microsatellite located in the last exon (LE; n = 98) or before the last exon (NLE; n = 569). c Schematic structure of mutant proteins (15 mutated tumor suppressor genes in green or 16 mutated oncogenes in red) with NLE mutations and significant down-regulation in MSI tumors. d Left panel: relative expression levels of HSP110wt and HSP110DE9 mRNAs by quantitative RT-PCR in MSS and MSI CRC primary tumors. No Del (wild type status; MSS CRCs) n = 36; DelS (small deletion: ≤4 pb; MSI CRCs), n = 28; DelL (large deletion: > 4 pb; MSI CRCs), n = 7. Right panel: relative mRNA expression levels of HSP110wt and HSP110DE9 determined by quantitative RT-PCR in MSS and MSI CRC cell lines. No T17 deletion [No Del], n = 6 CRC cell lines (ISI, SW1116, V9P, ALA, FET, SW480); small T17 Deletion [DelS], n = 4 CRC cell lines (HCT8, HCT116, TC71, LIM1215); large T17 Deletion [DelL], n = 6 CRC cell lines (TC7, Lovo, KM12, LS411, Ls174T, Co115). Data are means ± SEM. e Quantification of all HSP110 mRNAs. Densities and bar plots of HSP110 log2 intensities in normal colonic mucosa (Muc), adenomas (Ade), MSS tumors (MSS), and MSI tumors (MSI). All Data are means ± SEM. Unpaired t -test was performed to determine significance. ** p

    Techniques Used: Over Expression, Microarray, Sequencing, Expressing, Mutagenesis, Quantitative RT-PCR

    11) Product Images from "HECTD3 Mediates an HSP90-Dependent Degradation Pathway for Protein Kinase Clients"

    Article Title: HECTD3 Mediates an HSP90-Dependent Degradation Pathway for Protein Kinase Clients

    Journal: Cell Reports

    doi: 10.1016/j.celrep.2017.05.078

    HECTD3 Is Downregulated in Cancer Cell Lines with Activated MAPK Signaling (A) Western blot of HECTD3 in lysates from HEK293, COS7, and four human cancer cells lines: U2OS, HT29, HCT116, and A549. Tumor cell lines either lack immunoreactive protein or express a truncated isoform (also visible in HEK293) that is recognized by the C-terminal epitope of the α-HECTD3 antiserum. The molecular weight of the truncated product corresponds to that predicted for an experimentally documented, alternatively spliced isoform of HECTD3. (B) Western blot of HECTD3 from lysates of HEK293 cells expressing eYFP-CRAF. While both 97-kDa and 65-kDa HECTD3 isoforms are present in the input, only the 97-kDa species corresponding to the full-length protein is co-immunprecipitated with eYFP-CRAF. (C) The truncated splice isoform of HECTD3 in HCT116 cells is effectively knocked down by siRNA, but unlike knockdown of the full-length protein in HEK293 cells, this does not stabilize endogenous CRAF protein to degradation triggered by AUY922. This shows that the 65-kDa isoform is not an active participant in CRAF ubiquitylation and degradation.
    Figure Legend Snippet: HECTD3 Is Downregulated in Cancer Cell Lines with Activated MAPK Signaling (A) Western blot of HECTD3 in lysates from HEK293, COS7, and four human cancer cells lines: U2OS, HT29, HCT116, and A549. Tumor cell lines either lack immunoreactive protein or express a truncated isoform (also visible in HEK293) that is recognized by the C-terminal epitope of the α-HECTD3 antiserum. The molecular weight of the truncated product corresponds to that predicted for an experimentally documented, alternatively spliced isoform of HECTD3. (B) Western blot of HECTD3 from lysates of HEK293 cells expressing eYFP-CRAF. While both 97-kDa and 65-kDa HECTD3 isoforms are present in the input, only the 97-kDa species corresponding to the full-length protein is co-immunprecipitated with eYFP-CRAF. (C) The truncated splice isoform of HECTD3 in HCT116 cells is effectively knocked down by siRNA, but unlike knockdown of the full-length protein in HEK293 cells, this does not stabilize endogenous CRAF protein to degradation triggered by AUY922. This shows that the 65-kDa isoform is not an active participant in CRAF ubiquitylation and degradation.

    Techniques Used: Western Blot, Molecular Weight, Expressing

    12) Product Images from "RNF183 promotes proliferation and metastasis of colorectal cancer cells via activation of NF-κB-IL-8 axis"

    Article Title: RNF183 promotes proliferation and metastasis of colorectal cancer cells via activation of NF-κB-IL-8 axis

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2017.400

    RNF183 promotes metastasis of CRC cells in vivo . ( a ) Lungs were removed from mice injected with control or DLD-1 stable cell line with RNF183 overexpression. Pulmonary histopathology was examined by H E staining. Mean±S.D. ( n =7). ( b ) Livers were removed from mice injected with control or DLD-1 stable cell line with RNF183 overexpression. Hepatic histopathology was examined by H E staining. Mean±S.D. ( n =7). ( c ) Lungs were dissected from mice injected with control or RNF183 stable silenced HCT116 cells. Pulmonary histopathology was examined by H E staining mean±S.D. ( n =8). ( d ) Livers were dissected from mice injected with control or RNF183 stable silenced HCT116 cells. Hepatic histopathology was examined by H E staining. Mean±S.D. ( n =7). Scale bar: 100 μ m (× 100 and × 200) or 500 μ m (× 40). ** P
    Figure Legend Snippet: RNF183 promotes metastasis of CRC cells in vivo . ( a ) Lungs were removed from mice injected with control or DLD-1 stable cell line with RNF183 overexpression. Pulmonary histopathology was examined by H E staining. Mean±S.D. ( n =7). ( b ) Livers were removed from mice injected with control or DLD-1 stable cell line with RNF183 overexpression. Hepatic histopathology was examined by H E staining. Mean±S.D. ( n =7). ( c ) Lungs were dissected from mice injected with control or RNF183 stable silenced HCT116 cells. Pulmonary histopathology was examined by H E staining mean±S.D. ( n =8). ( d ) Livers were dissected from mice injected with control or RNF183 stable silenced HCT116 cells. Hepatic histopathology was examined by H E staining. Mean±S.D. ( n =7). Scale bar: 100 μ m (× 100 and × 200) or 500 μ m (× 40). ** P

    Techniques Used: In Vivo, Mouse Assay, Injection, Stable Transfection, Over Expression, Histopathology, Staining

    RNF183 promotes IL-8 transcription through NF- κ B. ( a ) The effects of RNF183 knockdown on the mRNA abundance of several NF- κ B downstream genes in HCT116 cells. ( b ) Knockdown RNF183 expression significantly reduced IL-8 secretion in HCT116 (left) and DLD-1 (right) cells. ( c ) Enforced RNF183 expression augments IL-8 transcription (left) and IL-8 secretion (right) in DLD-1 cells. ( d ) Stable RNF183 overexpression increased IL-8 transcription in xenograft tumors as shown in Figure 2g. ( e ) Effects of RNF183 on the activity of luciferase reporter with wild-type or NF- κ B binding site deleted (△NF- κ B) IL-8 promoter in HCT116 cells. ( f ) Expression of several proteins in NF- κ B pathway was examined by western blots with enforced RNF183 expression in HCT116 cells or with RNF183 knockdown in DLD-1 cells. ( g ) Chromatin immunoprecipitation (ChIP) assays were carried out to determine the binding of P65 on IL-8 promoter with or without RNF183 enforced expression. ( h ) The expression of P65 and RNF183 were evaluated in forty CRC tissues. The correlation of these two proteins and the significance were also calculated. Mean±S.D. ( n =3). Scale bar: 100 μ m. *** P
    Figure Legend Snippet: RNF183 promotes IL-8 transcription through NF- κ B. ( a ) The effects of RNF183 knockdown on the mRNA abundance of several NF- κ B downstream genes in HCT116 cells. ( b ) Knockdown RNF183 expression significantly reduced IL-8 secretion in HCT116 (left) and DLD-1 (right) cells. ( c ) Enforced RNF183 expression augments IL-8 transcription (left) and IL-8 secretion (right) in DLD-1 cells. ( d ) Stable RNF183 overexpression increased IL-8 transcription in xenograft tumors as shown in Figure 2g. ( e ) Effects of RNF183 on the activity of luciferase reporter with wild-type or NF- κ B binding site deleted (△NF- κ B) IL-8 promoter in HCT116 cells. ( f ) Expression of several proteins in NF- κ B pathway was examined by western blots with enforced RNF183 expression in HCT116 cells or with RNF183 knockdown in DLD-1 cells. ( g ) Chromatin immunoprecipitation (ChIP) assays were carried out to determine the binding of P65 on IL-8 promoter with or without RNF183 enforced expression. ( h ) The expression of P65 and RNF183 were evaluated in forty CRC tissues. The correlation of these two proteins and the significance were also calculated. Mean±S.D. ( n =3). Scale bar: 100 μ m. *** P

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

    RNF183 promotes proliferation of CRC cells in vitro and in vivo . ( a ) Western blots detect endogenous RNF183 protein expression in human normal colorectal epithelial cell line and CRC cells. ( b ) RNF183 knockdown efficiency in two CRC cell lines was levels were examined by western blots. ( c and d ) Effects of RNF183 silencing ( c ) or overexpression ( d ) on proliferation of HCT116 and DLD-1 cells was monitored by MTT assays. Mean±S.D. ( n =6). ( e and f ) Effects of RNF183 silencing ( e ) or enforced expression ( f ) on the colony formation of HCT116 and DLD-1 cells. ( g – j ) RNF183 overexpression accelerates tumor growth in vivo . ( g ) Tumor pictures from mice inoculated with stable RNF183 overexpression cell line DLD-1 or control. ( h ) Growth curve of tumor volume measured on indicated days (left) and tumor weight at the end of experiment (right). Mean±S.D. ( n =6). ( i ) Photographs exhibited the H E staining (left), IHC staining for RNF183 (middle) and ki-67 (right) in tumors. Scale bar: 100 μ m. ( j ) Number of Ki-67 positive cells in control and RNF183 stable expression tumors. * P
    Figure Legend Snippet: RNF183 promotes proliferation of CRC cells in vitro and in vivo . ( a ) Western blots detect endogenous RNF183 protein expression in human normal colorectal epithelial cell line and CRC cells. ( b ) RNF183 knockdown efficiency in two CRC cell lines was levels were examined by western blots. ( c and d ) Effects of RNF183 silencing ( c ) or overexpression ( d ) on proliferation of HCT116 and DLD-1 cells was monitored by MTT assays. Mean±S.D. ( n =6). ( e and f ) Effects of RNF183 silencing ( e ) or enforced expression ( f ) on the colony formation of HCT116 and DLD-1 cells. ( g – j ) RNF183 overexpression accelerates tumor growth in vivo . ( g ) Tumor pictures from mice inoculated with stable RNF183 overexpression cell line DLD-1 or control. ( h ) Growth curve of tumor volume measured on indicated days (left) and tumor weight at the end of experiment (right). Mean±S.D. ( n =6). ( i ) Photographs exhibited the H E staining (left), IHC staining for RNF183 (middle) and ki-67 (right) in tumors. Scale bar: 100 μ m. ( j ) Number of Ki-67 positive cells in control and RNF183 stable expression tumors. * P

    Techniques Used: In Vitro, In Vivo, Western Blot, Expressing, Over Expression, MTT Assay, Mouse Assay, Staining, Immunohistochemistry

    RNF183 prmotes migration, invasion and epithelial–mesenchymal transition (EMT) of CRC cells. ( a and b ) Effects of RNF183 silencing on migration and invasion of HCT116 ( a ) and DLD-1 ( b ) cells evaluated by transwell assays. Mean±S.D. ( n =3). ( c and d ) Effects of RNF183 overexpression on migration and invasion of HCT116 ( c ) and DLD-1 ( d ) cells detected by transwell assays. Mean±S.D. ( n =3). ( e ) Effects of RNF183 overexpression (upper) and silencing (bottom) on the mRNA abundance of selected EMT markers in HCT116 cells. ( f ) Effects of RNF183 overexpression (left) and silencing (right) on the protein expression of selected EMT markers in HCT116 cells. Scale bar: 100 μ m. * P
    Figure Legend Snippet: RNF183 prmotes migration, invasion and epithelial–mesenchymal transition (EMT) of CRC cells. ( a and b ) Effects of RNF183 silencing on migration and invasion of HCT116 ( a ) and DLD-1 ( b ) cells evaluated by transwell assays. Mean±S.D. ( n =3). ( c and d ) Effects of RNF183 overexpression on migration and invasion of HCT116 ( c ) and DLD-1 ( d ) cells detected by transwell assays. Mean±S.D. ( n =3). ( e ) Effects of RNF183 overexpression (upper) and silencing (bottom) on the mRNA abundance of selected EMT markers in HCT116 cells. ( f ) Effects of RNF183 overexpression (left) and silencing (right) on the protein expression of selected EMT markers in HCT116 cells. Scale bar: 100 μ m. * P

    Techniques Used: Migration, Over Expression, Expressing

    NF- κ B-IL-8 axis is indispensible for the oncogenic function of RNF183. ( a and b ) NF- κ B inhibitor BAY11-7085 attenuates RNF183 overexpression induced migration ( a ) and invasion ( b ) of DLD-1 cells. ( c ) HCT116 cells were co-transfected with RNF183 plasmid and siRNA-targeting IL-8 as indicated. Recombinant IL-8 was also supplemented in the knockdown group to rescue the phenotype. The pictures of migrated cells (left) and quantification (right) were showed. ( d ) Luciferase activity of IL-8 promoter was evaluated in HCT116 cells with overexpression of control plasmid, wild-type RNF183 and truncated RNF183 without E3 ubiquitin ligase activity. ( e–g ) Effects of wild-type and truncated RNF183 on proliferation ( e ), colony formation ( f ) and migration ( g ) of HCT116 cells. Mean±S.D. ( n =3). Scale bar: 50 μ m. ** P
    Figure Legend Snippet: NF- κ B-IL-8 axis is indispensible for the oncogenic function of RNF183. ( a and b ) NF- κ B inhibitor BAY11-7085 attenuates RNF183 overexpression induced migration ( a ) and invasion ( b ) of DLD-1 cells. ( c ) HCT116 cells were co-transfected with RNF183 plasmid and siRNA-targeting IL-8 as indicated. Recombinant IL-8 was also supplemented in the knockdown group to rescue the phenotype. The pictures of migrated cells (left) and quantification (right) were showed. ( d ) Luciferase activity of IL-8 promoter was evaluated in HCT116 cells with overexpression of control plasmid, wild-type RNF183 and truncated RNF183 without E3 ubiquitin ligase activity. ( e–g ) Effects of wild-type and truncated RNF183 on proliferation ( e ), colony formation ( f ) and migration ( g ) of HCT116 cells. Mean±S.D. ( n =3). Scale bar: 50 μ m. ** P

    Techniques Used: Over Expression, Migration, Transfection, Plasmid Preparation, Recombinant, Luciferase, Activity Assay

    13) Product Images from "ZBTB7A functioned as an oncogene in colorectal cancer"

    Article Title: ZBTB7A functioned as an oncogene in colorectal cancer

    Journal: BMC Gastroenterology

    doi: 10.1186/s12876-020-01456-z

    ZBTB7A promotes cell proliferation of CRC cells in vitro. a, b Cell growth capacity was evaluated by colony formation for about 2 weeks. Silencing ZBTB7A significantly inhibited the growth of DLD1 cells, while upregulation of ZBTB7A evidently promoted the growth of HCT116 cells. c, d Cell viability was analyzed using CCK8 assays in CRC cells. Cell proliferation of knock-down cells and overexpression cells were attenuated and enhanced, respectively
    Figure Legend Snippet: ZBTB7A promotes cell proliferation of CRC cells in vitro. a, b Cell growth capacity was evaluated by colony formation for about 2 weeks. Silencing ZBTB7A significantly inhibited the growth of DLD1 cells, while upregulation of ZBTB7A evidently promoted the growth of HCT116 cells. c, d Cell viability was analyzed using CCK8 assays in CRC cells. Cell proliferation of knock-down cells and overexpression cells were attenuated and enhanced, respectively

    Techniques Used: In Vitro, Over Expression

    14) Product Images from "MACC1 promotes carcinogenesis of colorectal cancer via β-catenin signaling pathway"

    Article Title: MACC1 promotes carcinogenesis of colorectal cancer via β-catenin signaling pathway

    Journal: Oncotarget

    doi:

    MACC1 over-expression significantly promoted tumor growth of HCT116 cells implanted subcutaneously in BALB/c-nu mice compared with the control group (p
    Figure Legend Snippet: MACC1 over-expression significantly promoted tumor growth of HCT116 cells implanted subcutaneously in BALB/c-nu mice compared with the control group (p

    Techniques Used: Over Expression, Mouse Assay

    Nuclear β-catenin expression was suppressed in SW620 cells with shMACC1 compared with the control groups by western blot analysis (A); β-catenin knockdown had no remarkable effect on MACC1 protein expression in SW620 cells transfected with β-catenin-siRNA compared with the control group by western blot analysis (B). GAPDH level was considered loading control, Histone3 was considered as nuclear loading control. MACC1 expression significantly increased β-catenin transcriptional activity in SW620 cells transfected with MACC1 expression plasmid at 48 hours compared with the empty vector control group by dual-luciferase reporter assay (p=0.014; C). MACC1 expression increased Met, c-Myc, cyclin D1, MMP9, phos-GSK-3β (Ser9), and vimentin expression, but suppressed cleaved caspase-3 and E-cadherin expression in HCT116 cells transfected with MACC1 over-expression plasmid compared with the control group by western blot analysis. However, MACC1 knockdown reversed all these changes in SW620 cells by western blot analysis (D).
    Figure Legend Snippet: Nuclear β-catenin expression was suppressed in SW620 cells with shMACC1 compared with the control groups by western blot analysis (A); β-catenin knockdown had no remarkable effect on MACC1 protein expression in SW620 cells transfected with β-catenin-siRNA compared with the control group by western blot analysis (B). GAPDH level was considered loading control, Histone3 was considered as nuclear loading control. MACC1 expression significantly increased β-catenin transcriptional activity in SW620 cells transfected with MACC1 expression plasmid at 48 hours compared with the empty vector control group by dual-luciferase reporter assay (p=0.014; C). MACC1 expression increased Met, c-Myc, cyclin D1, MMP9, phos-GSK-3β (Ser9), and vimentin expression, but suppressed cleaved caspase-3 and E-cadherin expression in HCT116 cells transfected with MACC1 over-expression plasmid compared with the control group by western blot analysis. However, MACC1 knockdown reversed all these changes in SW620 cells by western blot analysis (D).

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

    MACC1 protein expression reduced in SW620 cells transfected with MACC1-siRNA at 48, 72, and 96 hours. GAPDH was used as loading control (A); MACC1 knockdown dramatically suppressed cell proliferation of SW620 at 48, 72, and 96 hours compared with the scramble siRNA(NC-siRNA) and untreated SW620 group by MTT analysis, respectively (B); MACC1 mRNA and protein expression was suppressed in SW620 cells (left) stably transfected shMACC1 compared with the scramble and untreated control groups by real-time PCR and western blot analysis, respectively. MACC1 mRNA and protein expression was elevated in HCT116 cells (right) stably transfected with MACC1 expression plasmid compared with empty vector and untreated control groups, respectively. **P
    Figure Legend Snippet: MACC1 protein expression reduced in SW620 cells transfected with MACC1-siRNA at 48, 72, and 96 hours. GAPDH was used as loading control (A); MACC1 knockdown dramatically suppressed cell proliferation of SW620 at 48, 72, and 96 hours compared with the scramble siRNA(NC-siRNA) and untreated SW620 group by MTT analysis, respectively (B); MACC1 mRNA and protein expression was suppressed in SW620 cells (left) stably transfected shMACC1 compared with the scramble and untreated control groups by real-time PCR and western blot analysis, respectively. MACC1 mRNA and protein expression was elevated in HCT116 cells (right) stably transfected with MACC1 expression plasmid compared with empty vector and untreated control groups, respectively. **P

    Techniques Used: Expressing, Transfection, MTT Assay, Stable Transfection, Real-time Polymerase Chain Reaction, Western Blot, Plasmid Preparation

    MACC1 inhibited or induced apoptosis in HCT116 cells (A) stably with MACC1 over-expression or SW620 cells (B) stably with shMACC1 compared with the control groups by flow cytometry analysis; MACC1 knockdown inhibited migration (C) and invasion (F) of SW620 cells compared with control groups by scratch wound assay and transwell invasion assay, respectively. MACC1 overexpression induced migration (D) and invasion (E) of HCT116 cells compared with control groups by transwell migration assay and transwell invasion assay, respectively.
    Figure Legend Snippet: MACC1 inhibited or induced apoptosis in HCT116 cells (A) stably with MACC1 over-expression or SW620 cells (B) stably with shMACC1 compared with the control groups by flow cytometry analysis; MACC1 knockdown inhibited migration (C) and invasion (F) of SW620 cells compared with control groups by scratch wound assay and transwell invasion assay, respectively. MACC1 overexpression induced migration (D) and invasion (E) of HCT116 cells compared with control groups by transwell migration assay and transwell invasion assay, respectively.

    Techniques Used: Stable Transfection, Over Expression, Flow Cytometry, Cytometry, Migration, Scratch Wound Assay Assay, Transwell Invasion Assay, Transwell Migration Assay

    MACC1 and β-catenin mRNA (A-B) and protein (C-D) expression were much increased or decreased in enucleated tumors derived from HCT116 cells stably transfected with MACC1 over-expression plasmid or SW620 stably transfected with shMACC1 compared with the control group by real-time PCR and immunohistochemistry staining (×400), respectively; β-catenin mRNA expression was dramatically increased or decreased in HCT116 with MACC1 over-expression or SW620 cells with shMACC1 compared with the control groups by real-time PCR analysis (E); MACC1 and nuclear β-catenin protein expression was dramatically decreased in SW620 cells with shMACC1 compared with the control group by immunohistochemistry staining, ×400. **p=0.004 (F).
    Figure Legend Snippet: MACC1 and β-catenin mRNA (A-B) and protein (C-D) expression were much increased or decreased in enucleated tumors derived from HCT116 cells stably transfected with MACC1 over-expression plasmid or SW620 stably transfected with shMACC1 compared with the control group by real-time PCR and immunohistochemistry staining (×400), respectively; β-catenin mRNA expression was dramatically increased or decreased in HCT116 with MACC1 over-expression or SW620 cells with shMACC1 compared with the control groups by real-time PCR analysis (E); MACC1 and nuclear β-catenin protein expression was dramatically decreased in SW620 cells with shMACC1 compared with the control group by immunohistochemistry staining, ×400. **p=0.004 (F).

    Techniques Used: Expressing, Derivative Assay, Stable Transfection, Transfection, Over Expression, Plasmid Preparation, Real-time Polymerase Chain Reaction, Immunohistochemistry, Staining

    MACC1 protein expression in CRC cell lines (LOVO, SW1116, SW480, HCT116, SW620, and HT29) and normal colonic mucosa epithelial cell (NCM460) by western blot analysis (A); MACC1 and β-catenin mRNA expression in 12 pairs of fresh CRC and adjacent non-tumour colorectal mucosa (ANM) tissues by real-time PCR analysis (B); Significant positive correlation between MACC1 and β-catenin mRNA expression in such 12 pairs of fresh CRC and ANM tissues (C); MACC1 and β-catenin protein expression in 8 pairs of fresh CRC and ANM tissues by western blot analysis (D).
    Figure Legend Snippet: MACC1 protein expression in CRC cell lines (LOVO, SW1116, SW480, HCT116, SW620, and HT29) and normal colonic mucosa epithelial cell (NCM460) by western blot analysis (A); MACC1 and β-catenin mRNA expression in 12 pairs of fresh CRC and adjacent non-tumour colorectal mucosa (ANM) tissues by real-time PCR analysis (B); Significant positive correlation between MACC1 and β-catenin mRNA expression in such 12 pairs of fresh CRC and ANM tissues (C); MACC1 and β-catenin protein expression in 8 pairs of fresh CRC and ANM tissues by western blot analysis (D).

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

    15) Product Images from "Effects of Microchannel Shape and Ultrasonic Mixing on Microfluidic Padlock Probe Rolling Circle Amplification (RCA) Reactions"

    Article Title: Effects of Microchannel Shape and Ultrasonic Mixing on Microfluidic Padlock Probe Rolling Circle Amplification (RCA) Reactions

    Journal: Micromachines

    doi: 10.3390/mi9060272

    Imaging of mRNAs by rolling circle amplification (RCA) in the circular-shaped microchamber. ( a ) Detection of KRAS codon 12 (wild type) mRNA in the choriocarcinoma cell line BeWo. ( b ) Detection of β-actin mRNA in the colorectal carcinoma cell line HCT116.
    Figure Legend Snippet: Imaging of mRNAs by rolling circle amplification (RCA) in the circular-shaped microchamber. ( a ) Detection of KRAS codon 12 (wild type) mRNA in the choriocarcinoma cell line BeWo. ( b ) Detection of β-actin mRNA in the colorectal carcinoma cell line HCT116.

    Techniques Used: Imaging, Amplification

    16) Product Images from "Long non-coding RNA ZFAS1 interacts with CDK1 and is involved in p53-dependent cell cycle control and apoptosis in colorectal cancer"

    Article Title: Long non-coding RNA ZFAS1 interacts with CDK1 and is involved in p53-dependent cell cycle control and apoptosis in colorectal cancer

    Journal: Oncotarget

    doi:

    Effects of ZFAS1 knockdown on colorectal cancer cell proliferation in vitro A. Trypan blue exclusion method was performed to determine the proliferation of HCT116+/+, DLD-1, and HCT116−/− cells. Data represent the mean ± S.D. from three independent experiments. B. The effect of ZFAS1 silencing on cell cycle. The bar chart represents the percentage of cells in G0/G1, S, or G2/M phase, as indicated. C. Colony-forming growth assays were performed to determine the proliferation of HCT116+/+, DLD-1, and HCT116−/− cells after silencing of ZFAS1. The colonies were counted and captured. * P
    Figure Legend Snippet: Effects of ZFAS1 knockdown on colorectal cancer cell proliferation in vitro A. Trypan blue exclusion method was performed to determine the proliferation of HCT116+/+, DLD-1, and HCT116−/− cells. Data represent the mean ± S.D. from three independent experiments. B. The effect of ZFAS1 silencing on cell cycle. The bar chart represents the percentage of cells in G0/G1, S, or G2/M phase, as indicated. C. Colony-forming growth assays were performed to determine the proliferation of HCT116+/+, DLD-1, and HCT116−/− cells after silencing of ZFAS1. The colonies were counted and captured. * P

    Techniques Used: In Vitro

    Western blot analysis of p53, Cyclin B1 and PARP cleavage after ZFAS1 siRNA transfection of HCT116+/+ and DLD-1 cells
    Figure Legend Snippet: Western blot analysis of p53, Cyclin B1 and PARP cleavage after ZFAS1 siRNA transfection of HCT116+/+ and DLD-1 cells

    Techniques Used: Western Blot, Transfection

    17) Product Images from "Estrogen Activation by Steroid Sulfatase Increases Colorectal Cancer Proliferation via GPER"

    Article Title: Estrogen Activation by Steroid Sulfatase Increases Colorectal Cancer Proliferation via GPER

    Journal: The Journal of Clinical Endocrinology and Metabolism

    doi: 10.1210/jc.2016-3716

    Estrogens increase proliferation in CRC cell lines. (A) Expression profile of HSD17B1, HSD17B2, HSD17B7, and HSD17B12 in HCT116, HT29, Caco-2, and Colo205 cells. β -Actin was used as a loading control. One representative blot from three independent experiments. (B and C) E 1 and E 2 increased proliferation rates in a dose-dependent manner in HCT116 and HT-29 cells. Caco-2 cells did not respond to E 1 or E 2 treatment (n = 4 independent experiments). (D and E) HCT116 cells did not readily metabolize E 1 , E 2 , and E 1 S. HT-29 cells metabolized E 1 and E 2 to an unknown metabolite. Caco-2 cells rapidly metabolized E 2 to E 1 (n = 3 independent experiments). All data presented as mean ± standard deviation.
    Figure Legend Snippet: Estrogens increase proliferation in CRC cell lines. (A) Expression profile of HSD17B1, HSD17B2, HSD17B7, and HSD17B12 in HCT116, HT29, Caco-2, and Colo205 cells. β -Actin was used as a loading control. One representative blot from three independent experiments. (B and C) E 1 and E 2 increased proliferation rates in a dose-dependent manner in HCT116 and HT-29 cells. Caco-2 cells did not respond to E 1 or E 2 treatment (n = 4 independent experiments). (D and E) HCT116 cells did not readily metabolize E 1 , E 2 , and E 1 S. HT-29 cells metabolized E 1 and E 2 to an unknown metabolite. Caco-2 cells rapidly metabolized E 2 to E 1 (n = 3 independent experiments). All data presented as mean ± standard deviation.

    Techniques Used: Expressing, Standard Deviation

    Overexpression of STS in HCT116 cells increased estrogen-dependent proliferation in vitro and in vivo . (A) HCT116[sts] cells proliferated at a greater rate compared with HCT116[vo] cells. This proliferation was significantly inhibited by STX64 (1 mM). ** P
    Figure Legend Snippet: Overexpression of STS in HCT116 cells increased estrogen-dependent proliferation in vitro and in vivo . (A) HCT116[sts] cells proliferated at a greater rate compared with HCT116[vo] cells. This proliferation was significantly inhibited by STX64 (1 mM). ** P

    Techniques Used: Over Expression, In Vitro, In Vivo

    E 2 acts through GPER signaling to increase CRC proliferation. (A) ER α and ER β were not expressed in HCT116 or HT-29 but were present in Caco2 and Colo205 cells. GPER was expressed in all cell lines tested. β -Actin was used as a loading control. One representative blot from three independent experiments. (B) Schematic of the downstream molecular signaling factors stimulated by GPER action as defined in breast cancer. (C) The GPER agonist G1 increased the proliferation rates in a dose-dependent manner compared with cells grown only in media with sFBS (two-tailed Student t test used; n = 4 independent experiments). (C) The GPER antagonist G15 (1 mM) inhibits the increased proliferation induced by E 2 (100 nM for 72 hours) and G1 (100 nM for 72 hours) in HCT116 and HT-29 cells. ** P
    Figure Legend Snippet: E 2 acts through GPER signaling to increase CRC proliferation. (A) ER α and ER β were not expressed in HCT116 or HT-29 but were present in Caco2 and Colo205 cells. GPER was expressed in all cell lines tested. β -Actin was used as a loading control. One representative blot from three independent experiments. (B) Schematic of the downstream molecular signaling factors stimulated by GPER action as defined in breast cancer. (C) The GPER agonist G1 increased the proliferation rates in a dose-dependent manner compared with cells grown only in media with sFBS (two-tailed Student t test used; n = 4 independent experiments). (C) The GPER antagonist G15 (1 mM) inhibits the increased proliferation induced by E 2 (100 nM for 72 hours) and G1 (100 nM for 72 hours) in HCT116 and HT-29 cells. ** P

    Techniques Used: Two Tailed Test

    18) Product Images from "Topoisomerase 3B (TOP3B) DNA and RNA Cleavage Complexes and Pathway to Repair TOP3B-linked RNA and DNA Breaks"

    Article Title: Topoisomerase 3B (TOP3B) DNA and RNA Cleavage Complexes and Pathway to Repair TOP3B-linked RNA and DNA Breaks

    Journal: bioRxiv

    doi: 10.1101/2020.03.22.002691

    TDP2 Excises Cellular TOP3Bccs both from DNA and RNA (A-D) DNA RNA TOP3Bccs detection by RADAR assays. (A) HEK293 cells were transfected with the indicated siRNA: siTDP1, siTDP2 or both siTDP1 and siTDP2 and co-transfected with R338W TOP3B. After 72 h, nucleic acids and protein-nucleic acid adducts were isolated by RADAR assay, slot-blotted and detected with anti-FLAG antibody. The figure is representative of two independent experiments. (B) Quantitation of TOP3Bcc of RADAR assays as shown in panel A. TOP3Bccs were measured by densitometric analyses of slot-blot signals and plotted as a function of total nucleic acid (DNA and RNA) concentration. Two independent experiments are plotted. (C) Wild-type or TDP2KO HCT116 cells were transfected with FLAG-tagged R338W TOP3B. After 72 h, nucleic acids and protein-nucleic acid adducts were isolated by RADAR assay and slot-blotted. TOP3Bccs were detected using anti-FLAG antibody. The figure is representative of three independent experiments. (D) Quantitation of TOP3Bcc formation in RADAR assays as shown in panel C. Three independent experiments are plotted. (E-H) DNA RNA TOP3Bccs detection by ICE assays. (E) HEK293 cells were transfected with R338W TOP3B alone or co-transfected with siTDP2. After 72 h, ICE bioassay was performed to separate DNA and RNA from free proteins. DNA and RNA fractions were slot-blotted and TOP3Bccs were detected using anti-FLAG antibody. The figure is representative of three independent experiments. (F) Quantitation of TOP3Bcc in the DNA and RNA fractions as shown in panel E. Three independent experiments are plotted. (G) HCT116 WT and TDP2KO cells were transfected either with wild-type TOP3B or R338W TOP3B or a combination of R338W TOP3B and siTDP2 constructs as indicated. After 72 h, ICE bioassays were performed to separate DNA and RNA from free proteins. DNA and RNA fractions were slot-blotted and TOP3Bccs were detected using anti-FLAG antibody. The figure is representative of three independent experiments. (H) Quantitation of TOP3Bcc formation in ICE assays as shown in panel G. Three independent experiments are plotted. (I-L) Detection of RNA TOP3Bccs. (I) HEK293 cells were transfected with R338W TOP3B alone or co-transfected with siTDP2. After 72 h, covalent protein-RNA adducts were isolated using TRIzol ® , slot-blotted and TOP3Bccs were detected using anti-FLAG Antibody. The figure is representative of three independent experiments. (J) Quantitation of TOP3Bcc in RNA as shown in panel I. Three independent experiments are plotted. (K) Wild-type and TDP2KO HCT116 cells were transfected with FLAG-tagged R338W TOP3B. After 72 h, covalent protein-RNA adducts were isolated using TRIzol ® , slot-blotted and TOP3Bccs were detected using anti-FLAG Antibody. The figure is representative of three independent experiments. (L) Quantitation of RNA TOP3Bcc in RNA as shown in panel K. Three independent experiments are plotted. (M-N) Ectopic expression of TDP2 reduces TOP3Bccs. (M) Wild-type (WT) and TDP2KO HCT116 cells were transfected with FLAG-tagged R338W TOP3B alone or co-transfected with HA-tagged TDP2. After 72 h, nucleic acids and protein-nucleic acid adducts were isolated by RADAR assay, slot-blotted and TOP3Bccs were detected with anti-FLAG antibody. The figure is representative of three independent experiments. (N) Quantitation of TOP3Bcc formation using the RADAR assays as shown in panel M. Three independent experiments are plotted.
    Figure Legend Snippet: TDP2 Excises Cellular TOP3Bccs both from DNA and RNA (A-D) DNA RNA TOP3Bccs detection by RADAR assays. (A) HEK293 cells were transfected with the indicated siRNA: siTDP1, siTDP2 or both siTDP1 and siTDP2 and co-transfected with R338W TOP3B. After 72 h, nucleic acids and protein-nucleic acid adducts were isolated by RADAR assay, slot-blotted and detected with anti-FLAG antibody. The figure is representative of two independent experiments. (B) Quantitation of TOP3Bcc of RADAR assays as shown in panel A. TOP3Bccs were measured by densitometric analyses of slot-blot signals and plotted as a function of total nucleic acid (DNA and RNA) concentration. Two independent experiments are plotted. (C) Wild-type or TDP2KO HCT116 cells were transfected with FLAG-tagged R338W TOP3B. After 72 h, nucleic acids and protein-nucleic acid adducts were isolated by RADAR assay and slot-blotted. TOP3Bccs were detected using anti-FLAG antibody. The figure is representative of three independent experiments. (D) Quantitation of TOP3Bcc formation in RADAR assays as shown in panel C. Three independent experiments are plotted. (E-H) DNA RNA TOP3Bccs detection by ICE assays. (E) HEK293 cells were transfected with R338W TOP3B alone or co-transfected with siTDP2. After 72 h, ICE bioassay was performed to separate DNA and RNA from free proteins. DNA and RNA fractions were slot-blotted and TOP3Bccs were detected using anti-FLAG antibody. The figure is representative of three independent experiments. (F) Quantitation of TOP3Bcc in the DNA and RNA fractions as shown in panel E. Three independent experiments are plotted. (G) HCT116 WT and TDP2KO cells were transfected either with wild-type TOP3B or R338W TOP3B or a combination of R338W TOP3B and siTDP2 constructs as indicated. After 72 h, ICE bioassays were performed to separate DNA and RNA from free proteins. DNA and RNA fractions were slot-blotted and TOP3Bccs were detected using anti-FLAG antibody. The figure is representative of three independent experiments. (H) Quantitation of TOP3Bcc formation in ICE assays as shown in panel G. Three independent experiments are plotted. (I-L) Detection of RNA TOP3Bccs. (I) HEK293 cells were transfected with R338W TOP3B alone or co-transfected with siTDP2. After 72 h, covalent protein-RNA adducts were isolated using TRIzol ® , slot-blotted and TOP3Bccs were detected using anti-FLAG Antibody. The figure is representative of three independent experiments. (J) Quantitation of TOP3Bcc in RNA as shown in panel I. Three independent experiments are plotted. (K) Wild-type and TDP2KO HCT116 cells were transfected with FLAG-tagged R338W TOP3B. After 72 h, covalent protein-RNA adducts were isolated using TRIzol ® , slot-blotted and TOP3Bccs were detected using anti-FLAG Antibody. The figure is representative of three independent experiments. (L) Quantitation of RNA TOP3Bcc in RNA as shown in panel K. Three independent experiments are plotted. (M-N) Ectopic expression of TDP2 reduces TOP3Bccs. (M) Wild-type (WT) and TDP2KO HCT116 cells were transfected with FLAG-tagged R338W TOP3B alone or co-transfected with HA-tagged TDP2. After 72 h, nucleic acids and protein-nucleic acid adducts were isolated by RADAR assay, slot-blotted and TOP3Bccs were detected with anti-FLAG antibody. The figure is representative of three independent experiments. (N) Quantitation of TOP3Bcc formation using the RADAR assays as shown in panel M. Three independent experiments are plotted.

    Techniques Used: Transfection, Isolation, Quantitation Assay, Dot Blot, Concentration Assay, Construct, Expressing

    TRIM41 acts as Ubiquitin Ligase for TOP3Bccs and Promotes the Repair of TOP3Bccs (A) Immunoblots showing TRIM41 and R338W TOP3B expression after TRIM41 down-regulation (GAPDH as loading control). HCT116 cells were either transfected with FLAG-tagged R338W TOP3B plasmid construct alone or co-transfected with siTRIM41construct for 72 h. (B) HCT116 cells were transfected with FLAG-tagged wild-type or R338W TOP3Bs or co-transfected with siTRIM41construct for 72 h. Cells were harvested and nucleic acids and protein-nucleic acid adducts isolated by RADAR assay. TOP3Bcc were detected with anti-FLAG antibody. The figure is representative of two independent experiments. (C) Quantitation of TOP3Bccs in 2 independent RADAR assays as shown in panel B. (D) HCT116 cells were transfected with FLAG-tagged R338W TOP3B alone or with siTRIM41construct. After 72 h, TOP3Bcc ubiquitylation was detected by DUST assay. Samples were also subjected to slot-blotting and probed with anti-dsDNA antibody as loading control. (E) Immunoblot showing expression of TRIM41 with GAPDH as loading control. HCT116 cells transfected with FLAG-tagged TRIM41 for 48 h. (F) HCT116 cells were either transfected with HA-tagged R338W TOP3B plasmid construct alone or co-transfected with FLAG-tagged TRIM41 construct. After 48 h cells were harvested and nucleic acids and protein-nucleic acid adducts were isolated by RADAR assay. TOP3Bccs were detected using anti-HA antibody. The figure is representative of three independent experiments. (G) Quantitation of TOP3Bcc formation in 3 independent RADAR assays as shown in panel F. (H) HCT116 cells were either transfected with HA-tagged R338W TOP3B plasmid construct alone or co-transfected with FLAG-tagged TRIM41construct. After 48 h, TOP3Bcc ubiquitylation was detected by DUST assay. Samples were also subjected to slot-blotting and probing with anti-dsDNA antibody as loading control.
    Figure Legend Snippet: TRIM41 acts as Ubiquitin Ligase for TOP3Bccs and Promotes the Repair of TOP3Bccs (A) Immunoblots showing TRIM41 and R338W TOP3B expression after TRIM41 down-regulation (GAPDH as loading control). HCT116 cells were either transfected with FLAG-tagged R338W TOP3B plasmid construct alone or co-transfected with siTRIM41construct for 72 h. (B) HCT116 cells were transfected with FLAG-tagged wild-type or R338W TOP3Bs or co-transfected with siTRIM41construct for 72 h. Cells were harvested and nucleic acids and protein-nucleic acid adducts isolated by RADAR assay. TOP3Bcc were detected with anti-FLAG antibody. The figure is representative of two independent experiments. (C) Quantitation of TOP3Bccs in 2 independent RADAR assays as shown in panel B. (D) HCT116 cells were transfected with FLAG-tagged R338W TOP3B alone or with siTRIM41construct. After 72 h, TOP3Bcc ubiquitylation was detected by DUST assay. Samples were also subjected to slot-blotting and probed with anti-dsDNA antibody as loading control. (E) Immunoblot showing expression of TRIM41 with GAPDH as loading control. HCT116 cells transfected with FLAG-tagged TRIM41 for 48 h. (F) HCT116 cells were either transfected with HA-tagged R338W TOP3B plasmid construct alone or co-transfected with FLAG-tagged TRIM41 construct. After 48 h cells were harvested and nucleic acids and protein-nucleic acid adducts were isolated by RADAR assay. TOP3Bccs were detected using anti-HA antibody. The figure is representative of three independent experiments. (G) Quantitation of TOP3Bcc formation in 3 independent RADAR assays as shown in panel F. (H) HCT116 cells were either transfected with HA-tagged R338W TOP3B plasmid construct alone or co-transfected with FLAG-tagged TRIM41construct. After 48 h, TOP3Bcc ubiquitylation was detected by DUST assay. Samples were also subjected to slot-blotting and probing with anti-dsDNA antibody as loading control.

    Techniques Used: Western Blot, Expressing, Transfection, Plasmid Preparation, Construct, Isolation, Quantitation Assay

    Cellular TOP3Bccs are Ubiquitylated and Degraded by the Proteasomal Pathway (A-D) Proteasome inhibition enhances cellular TOP3Bccs. HEK293 (A) and HCT116 cells (B) were transfected with FLAG-tagged wild-type TOP3B and R338W TOP3B for 72 h. Before harvest, cells were treated with MG132 (10 µM, 2 h). TOP3Bccs were detected by RADAR assays using anti-FLAG antibody. The figure is representative of two independent experiments. (C-D) Quantitation of TOP3Bccs in two independent RADAR assays as shown in panels A and B. Two independent experiments are plotted for each panels. (E-H) Ubiquitylation inhibition enhances cellular TOP3Bccs. HEK293 (E) and HCT116 cells (G) were transfected with FLAG-tagged R338W TOP3B for 72 h. Before harvesting, the cells were treated with the UAE inhibitor TAK-243 (10 µM, 2 h). TOP3Bccs were detected by RADAR assays using anti-FLAG antibody. The figure is representative of three independent experiments. (F H) Quantitation of RADAR assays as shown in panels E and G. Three independent experiments are plotted for each panels. (I) Scheme of the DUST (Detection of Ubiquitylated-SUMOylated TOPccs) assay. Following the isolation of TOP3Bccs by RADAR assay, the covalently attached nucleic acids are digested with micrococcal nuclease (MNase). Ubiquitylated TOP3B can then be detected by immunoblotting following SDS-PAGE. (J) Ubiquitylation of TOP3Bccs in HCT116 cells transfected with FLAG-tagged R338W TOP3B for 72 h, as detected by the DUST Assay. Equal loading was tested by slot-blotting and probing with anti-dsDNA antibody. (K) TOP3Bcc ubiquitylation involves the classical proteasomal-specific linkages to lysines K11, K27, K48 and K63. HCT116 cells were co-transfected with FLAG-tagged R338W TOP3B plasmid construct and HA-tagged wild-type or mutant ubiquitin constructs. DUST assays were performed after 72 h. (L) Inhibition of TOP3Bcc ubiquitylation by the UBE1 inhibitor TAK-243 and enhancement by the proteasome inhibitor MG132. HCT116 cells transfected with FLAG-tagged R338W TOP3B for 72 h were treated with either MG132 (10 µM, 2 h) or TAK-243 (10 µM, 2 h), as detected by the DUST Assay.
    Figure Legend Snippet: Cellular TOP3Bccs are Ubiquitylated and Degraded by the Proteasomal Pathway (A-D) Proteasome inhibition enhances cellular TOP3Bccs. HEK293 (A) and HCT116 cells (B) were transfected with FLAG-tagged wild-type TOP3B and R338W TOP3B for 72 h. Before harvest, cells were treated with MG132 (10 µM, 2 h). TOP3Bccs were detected by RADAR assays using anti-FLAG antibody. The figure is representative of two independent experiments. (C-D) Quantitation of TOP3Bccs in two independent RADAR assays as shown in panels A and B. Two independent experiments are plotted for each panels. (E-H) Ubiquitylation inhibition enhances cellular TOP3Bccs. HEK293 (E) and HCT116 cells (G) were transfected with FLAG-tagged R338W TOP3B for 72 h. Before harvesting, the cells were treated with the UAE inhibitor TAK-243 (10 µM, 2 h). TOP3Bccs were detected by RADAR assays using anti-FLAG antibody. The figure is representative of three independent experiments. (F H) Quantitation of RADAR assays as shown in panels E and G. Three independent experiments are plotted for each panels. (I) Scheme of the DUST (Detection of Ubiquitylated-SUMOylated TOPccs) assay. Following the isolation of TOP3Bccs by RADAR assay, the covalently attached nucleic acids are digested with micrococcal nuclease (MNase). Ubiquitylated TOP3B can then be detected by immunoblotting following SDS-PAGE. (J) Ubiquitylation of TOP3Bccs in HCT116 cells transfected with FLAG-tagged R338W TOP3B for 72 h, as detected by the DUST Assay. Equal loading was tested by slot-blotting and probing with anti-dsDNA antibody. (K) TOP3Bcc ubiquitylation involves the classical proteasomal-specific linkages to lysines K11, K27, K48 and K63. HCT116 cells were co-transfected with FLAG-tagged R338W TOP3B plasmid construct and HA-tagged wild-type or mutant ubiquitin constructs. DUST assays were performed after 72 h. (L) Inhibition of TOP3Bcc ubiquitylation by the UBE1 inhibitor TAK-243 and enhancement by the proteasome inhibitor MG132. HCT116 cells transfected with FLAG-tagged R338W TOP3B for 72 h were treated with either MG132 (10 µM, 2 h) or TAK-243 (10 µM, 2 h), as detected by the DUST Assay.

    Techniques Used: Inhibition, Transfection, Quantitation Assay, Isolation, SDS Page, Plasmid Preparation, Construct, Mutagenesis

    TOP3B Forms TOP3Bccs both with DNA and RNA in cells Transfected with R338W TOP3B (A) Alignment of the active site regions of E. coli Topo I and Y. pestis Topo I with corresponding region of Human TOP3B. (B) Schematic representation of the domain organization of human TOP3B (1-862 aa) (top) and ribbon representation of human TOP3B (residues 1-612 aa) based on X-ray crystal structure by ( Goto-Ito et al., 2017 )(bottom). Positions of the active site residue (Tyrosine 336) and the self-trapping mutation site (Arginine 338) are indicated. (C) Western Blots showing ectopic expression of wild-type, P337V and R338W TOP3B. HEK293 and HCT116 cells were transfected with the indicated FLAG-tagged TOP3B constructs for 72 h and subjected to Western blotting with anti-FLAG antibody. (D)-(E) Detection of TOP3Bccs by RADAR assay in HEK293 and HCT116 cells transfected with FLAG-tagged wild-type, P337V or R338W TOP3B plasmid constructs for 72 h. Cells were lysed with DNAzol, nucleic acids and protein-nucleic acid adducts were isolated, slot-blotted and TOP3Bccs were detected with anti-FLAG antibody. The figure is representative of three independent experiments. (F) Scheme of the modified RADAR assay. Cells transfected with FLAG-tagged TOP3B (blue Circle) form TOP3Bccs in DNA (red) and RNA (green). Nucleic acids containing TOP3Bccs were isolated using the RADAR assay and digested with micrococcal nuclease (MNase) followed by SDS-PAGE and immunoblotting with anti-FLAG antibody. (G) Modified RADAR assay in HEK293 cells transfected with wild-type or R338W TOP3B for 72 h. TOP3B was detected with anti-FLAG antibody. (H) Detection of TOP3Bccs both in DNA and RNA of HEK293 cells transfected for 72 h with wild-type or R338W TOP3B constructs. Equal numbers of cells were lysed in 1% sarkosyl and ICE (In vivo complex of enzymes) bioassay by cesium chloride gradient ultracentrifugation was performed to separate DNA (middle of the gradient) and RNA (bottom of the gradient) from free proteins (top of the gradient). DNA and RNA fractions were then slot-blotted. TOP3Bccs were detected using anti-FLAG antibody. The figure is representative of three independent experiments. (I) Detection of RNA TOP3Bccs. HEK293 cells were transfected with wild-type or R338W TOP3B. 72 h later, covalent protein-RNA adducts were isolated using TRIzol® (Thermo Scientific), slot-blotted and TOP3Bccs were detected with anti-FLAG Antibody. The figure represents three independent experiments.
    Figure Legend Snippet: TOP3B Forms TOP3Bccs both with DNA and RNA in cells Transfected with R338W TOP3B (A) Alignment of the active site regions of E. coli Topo I and Y. pestis Topo I with corresponding region of Human TOP3B. (B) Schematic representation of the domain organization of human TOP3B (1-862 aa) (top) and ribbon representation of human TOP3B (residues 1-612 aa) based on X-ray crystal structure by ( Goto-Ito et al., 2017 )(bottom). Positions of the active site residue (Tyrosine 336) and the self-trapping mutation site (Arginine 338) are indicated. (C) Western Blots showing ectopic expression of wild-type, P337V and R338W TOP3B. HEK293 and HCT116 cells were transfected with the indicated FLAG-tagged TOP3B constructs for 72 h and subjected to Western blotting with anti-FLAG antibody. (D)-(E) Detection of TOP3Bccs by RADAR assay in HEK293 and HCT116 cells transfected with FLAG-tagged wild-type, P337V or R338W TOP3B plasmid constructs for 72 h. Cells were lysed with DNAzol, nucleic acids and protein-nucleic acid adducts were isolated, slot-blotted and TOP3Bccs were detected with anti-FLAG antibody. The figure is representative of three independent experiments. (F) Scheme of the modified RADAR assay. Cells transfected with FLAG-tagged TOP3B (blue Circle) form TOP3Bccs in DNA (red) and RNA (green). Nucleic acids containing TOP3Bccs were isolated using the RADAR assay and digested with micrococcal nuclease (MNase) followed by SDS-PAGE and immunoblotting with anti-FLAG antibody. (G) Modified RADAR assay in HEK293 cells transfected with wild-type or R338W TOP3B for 72 h. TOP3B was detected with anti-FLAG antibody. (H) Detection of TOP3Bccs both in DNA and RNA of HEK293 cells transfected for 72 h with wild-type or R338W TOP3B constructs. Equal numbers of cells were lysed in 1% sarkosyl and ICE (In vivo complex of enzymes) bioassay by cesium chloride gradient ultracentrifugation was performed to separate DNA (middle of the gradient) and RNA (bottom of the gradient) from free proteins (top of the gradient). DNA and RNA fractions were then slot-blotted. TOP3Bccs were detected using anti-FLAG antibody. The figure is representative of three independent experiments. (I) Detection of RNA TOP3Bccs. HEK293 cells were transfected with wild-type or R338W TOP3B. 72 h later, covalent protein-RNA adducts were isolated using TRIzol® (Thermo Scientific), slot-blotted and TOP3Bccs were detected with anti-FLAG Antibody. The figure represents three independent experiments.

    Techniques Used: Transfection, Mutagenesis, Western Blot, Expressing, Construct, Plasmid Preparation, Isolation, Modification, SDS Page, In Vivo

    TDP2-Mediated Repair of TOP3Bccs is Dependent on Ubiquitination and Proteasomal Degradation (A) Wild-type and TDP2KO HCT116 cells were transfected with FLAG-tagged R338W TOP3B alone or co-transfected with HA-tagged TDP2 plasmid construct and incubated for 72 h. Before harvest, cells were treated with MG132 (10 µM, 2 h). Nucleic acids and protein-nucleic acid adducts were recovered by RADAR assay, slot-blotted and TOP3Bccs were detected using anti-FLAG antibody. The figure is representative of two independent experiments. (B) Quantitation of TOP3Bccs in 2 independent RADAR assays as shown in panel A. TOP3Bccs were measured by densitometric analyses of slot-blot signals and plotted individually (x2) as a function of total nucleic acid (DNA and RNA) concentration. (C) Wild-type and TDP2KO HCT116 cells were transfected with FLAG-tagged R338W TOP3B alone or co-transfected with HA-tagged TDP2 plasmid construct and incubated for 72 h. Before harvest, cells were treated with TAK-243 (10 µM, 2 h). Nucleic acids and protein-nucleic acid adducts were recovered by RADAR assay, slot-blotted and TOP3Bccs were detected using anti-FLAG antibody. The figure is representative of two independent experiments. (D) Quantitation of TOP3Bccs in 2 independent RADAR assays as shown in panel C. (E) Wild-type and TDP2KO HCT116 cells were transfected with FLAG-tagged R338W TOP3B alone or co-transfected with siTRIM41 constructs and incubated for 72 h. Cells were harvested, nucleic acids containing protein adducts isolated by RADAR assay and slot blotted. TOP3Bccs were detected with anti-FLAG antibody. The figure is representative of two independent experiments. (F) Quantitation of TOP3Bccs in 2 independent RADAR assays as shown in panel E.
    Figure Legend Snippet: TDP2-Mediated Repair of TOP3Bccs is Dependent on Ubiquitination and Proteasomal Degradation (A) Wild-type and TDP2KO HCT116 cells were transfected with FLAG-tagged R338W TOP3B alone or co-transfected with HA-tagged TDP2 plasmid construct and incubated for 72 h. Before harvest, cells were treated with MG132 (10 µM, 2 h). Nucleic acids and protein-nucleic acid adducts were recovered by RADAR assay, slot-blotted and TOP3Bccs were detected using anti-FLAG antibody. The figure is representative of two independent experiments. (B) Quantitation of TOP3Bccs in 2 independent RADAR assays as shown in panel A. TOP3Bccs were measured by densitometric analyses of slot-blot signals and plotted individually (x2) as a function of total nucleic acid (DNA and RNA) concentration. (C) Wild-type and TDP2KO HCT116 cells were transfected with FLAG-tagged R338W TOP3B alone or co-transfected with HA-tagged TDP2 plasmid construct and incubated for 72 h. Before harvest, cells were treated with TAK-243 (10 µM, 2 h). Nucleic acids and protein-nucleic acid adducts were recovered by RADAR assay, slot-blotted and TOP3Bccs were detected using anti-FLAG antibody. The figure is representative of two independent experiments. (D) Quantitation of TOP3Bccs in 2 independent RADAR assays as shown in panel C. (E) Wild-type and TDP2KO HCT116 cells were transfected with FLAG-tagged R338W TOP3B alone or co-transfected with siTRIM41 constructs and incubated for 72 h. Cells were harvested, nucleic acids containing protein adducts isolated by RADAR assay and slot blotted. TOP3Bccs were detected with anti-FLAG antibody. The figure is representative of two independent experiments. (F) Quantitation of TOP3Bccs in 2 independent RADAR assays as shown in panel E.

    Techniques Used: Transfection, Plasmid Preparation, Construct, Incubation, Quantitation Assay, Dot Blot, Concentration Assay, Isolation

    19) Product Images from "Discovery of a small molecule targeting autophagy via ATG4B inhibition and cell death of colorectal cancer cells in vitro and in vivo"

    Article Title: Discovery of a small molecule targeting autophagy via ATG4B inhibition and cell death of colorectal cancer cells in vitro and in vivo

    Journal: Autophagy

    doi: 10.1080/15548627.2018.1517073

    Nutrient starvation sensitizes S130-induced cell death. (A-D) HeLa (A), HCT116 (B), ATG4B OE HeLa (C), and ATG4B KO HeLa (D) cells were treated with 0–25 μM of S130 in CM or EBSS for 48 h, cell viability was measured with CCK8.
    Figure Legend Snippet: Nutrient starvation sensitizes S130-induced cell death. (A-D) HeLa (A), HCT116 (B), ATG4B OE HeLa (C), and ATG4B KO HeLa (D) cells were treated with 0–25 μM of S130 in CM or EBSS for 48 h, cell viability was measured with CCK8.

    Techniques Used:

    20) Product Images from "Serine is a natural ligand and allosteric activator of pyruvate kinase M2"

    Article Title: Serine is a natural ligand and allosteric activator of pyruvate kinase M2

    Journal: Nature

    doi: 10.1038/nature11540

    Characterisation of PKM1/2-silenced HCT116 cells
    Figure Legend Snippet: Characterisation of PKM1/2-silenced HCT116 cells

    Techniques Used:

    21) Product Images from "MEK5/ERK5 activation regulates colon cancer stem-like cell properties"

    Article Title: MEK5/ERK5 activation regulates colon cancer stem-like cell properties

    Journal: Cell Death Discovery

    doi: 10.1038/s41420-019-0150-1

    ERK5-mediated signaling regulates IL-8 expression. a , b Gene expression profiling was performed in HCT116 tumorspheres using the Human Cancer Stem Cells RT 2 Profiler PCR Array. a Heatmap representation of differentially expressed genes between vehicle- and XMD8-92-treated tumorspheres (green: downregulation; red: upregulation). b PCR array results are expressed as mean ± SD log 2 -transformed fold change to vehicle control-treated spheres. c The effect of ERK5 inhibition on IL-8 expression was validated by independent qRT-PCR in HCT116, HT29, SW480, and SW620 tumorspheres treated with XMD8-92 versus vehicle control, and d ERK5 small interfering RNA (siRNA) versus control siRNA HCT116 tumorspheres. e IL-8 mRNA levels were further measured in tumorspheres versus adherent cultures, and f CA-MEK5-overexpressing versus empty vector SW480 cells. Results are expressed as mean ± standard error of mean from at least three independent experiments. * p
    Figure Legend Snippet: ERK5-mediated signaling regulates IL-8 expression. a , b Gene expression profiling was performed in HCT116 tumorspheres using the Human Cancer Stem Cells RT 2 Profiler PCR Array. a Heatmap representation of differentially expressed genes between vehicle- and XMD8-92-treated tumorspheres (green: downregulation; red: upregulation). b PCR array results are expressed as mean ± SD log 2 -transformed fold change to vehicle control-treated spheres. c The effect of ERK5 inhibition on IL-8 expression was validated by independent qRT-PCR in HCT116, HT29, SW480, and SW620 tumorspheres treated with XMD8-92 versus vehicle control, and d ERK5 small interfering RNA (siRNA) versus control siRNA HCT116 tumorspheres. e IL-8 mRNA levels were further measured in tumorspheres versus adherent cultures, and f CA-MEK5-overexpressing versus empty vector SW480 cells. Results are expressed as mean ± standard error of mean from at least three independent experiments. * p

    Techniques Used: Expressing, Polymerase Chain Reaction, Transformation Assay, Inhibition, Quantitative RT-PCR, Small Interfering RNA, Plasmid Preparation

    ERK5 pharmacological inhibition blocks NF-kB/IL-8 signaling. a HCT116 cells were cultured for 7 days under sphere-forming conditions in the presence of 4 µM XMD8-92 or dimethyl sulfoxide (DMSO) vehicle control. IκB phosphorylation levels were evaluated by western blot. b Nuclear factor-κB (NF-κB) transcriptional activity was assayed using an inducible luciferase reporter system. c , d HCT116 cells were transfected with NF-κB p65 or DN-IκBα expression constructs, and empty vector, and then treated for 48 h with 4 µM XMD8-92. DMSO was used as vehicle control. c NF-κB and IκBα overexpression was confirmed by immunoblotting. d IL-8 mRNA levels were determined by qRT-PCR. Results are expressed as mean ± standard error of mean from three independent experiments. * p
    Figure Legend Snippet: ERK5 pharmacological inhibition blocks NF-kB/IL-8 signaling. a HCT116 cells were cultured for 7 days under sphere-forming conditions in the presence of 4 µM XMD8-92 or dimethyl sulfoxide (DMSO) vehicle control. IκB phosphorylation levels were evaluated by western blot. b Nuclear factor-κB (NF-κB) transcriptional activity was assayed using an inducible luciferase reporter system. c , d HCT116 cells were transfected with NF-κB p65 or DN-IκBα expression constructs, and empty vector, and then treated for 48 h with 4 µM XMD8-92. DMSO was used as vehicle control. c NF-κB and IκBα overexpression was confirmed by immunoblotting. d IL-8 mRNA levels were determined by qRT-PCR. Results are expressed as mean ± standard error of mean from three independent experiments. * p

    Techniques Used: Inhibition, Cell Culture, Western Blot, Activity Assay, Luciferase, Transfection, Expressing, Construct, Plasmid Preparation, Over Expression, Quantitative RT-PCR

    MEK5/ERK5 signaling activation is increased in colon cancer tumorspheres, and MEK5 constitutive activation correlates with a shift toward a stem-like state. a HCT116, HT29, SW480, and SW620 cells were cultured under sphere-forming or adherent conditions. MEK5 and ERK5 phosphorylation levels were evaluated by western blot. b – d SW480 adherent cultures were transfected with CA-MEK5 expression construct or empty vector. b MEK5 overexpression and ERK5 activation status were confirmed by immunoblotting. c mRNA levels of several stemness- and differentiation-associated markers were determined by qRT-PCR. d Steady-state protein levels of NANOG, OCT4, and SOX2 were evaluated by immunoblot analysis. Blots are representative of three independent experiments with similar results. Results are expressed as mean ± standard error of mean from at least three independent experiments. * p
    Figure Legend Snippet: MEK5/ERK5 signaling activation is increased in colon cancer tumorspheres, and MEK5 constitutive activation correlates with a shift toward a stem-like state. a HCT116, HT29, SW480, and SW620 cells were cultured under sphere-forming or adherent conditions. MEK5 and ERK5 phosphorylation levels were evaluated by western blot. b – d SW480 adherent cultures were transfected with CA-MEK5 expression construct or empty vector. b MEK5 overexpression and ERK5 activation status were confirmed by immunoblotting. c mRNA levels of several stemness- and differentiation-associated markers were determined by qRT-PCR. d Steady-state protein levels of NANOG, OCT4, and SOX2 were evaluated by immunoblot analysis. Blots are representative of three independent experiments with similar results. Results are expressed as mean ± standard error of mean from at least three independent experiments. * p

    Techniques Used: Activation Assay, Cell Culture, Western Blot, Transfection, Expressing, Construct, Plasmid Preparation, Over Expression, Quantitative RT-PCR

    Pharmacological and genetic inhibition of ERK5 impairs sphere formation in colon cancer cells. a – e HCT116, HT29, SW480, and SW620 cells were cultured for two generations under sphere-forming conditions in the presence or absence of 4 µM XMD8-92. a The inhibitory effect of XMD8-92 on ERK5 activation was confirmed by western blot analysis. b Representative images of first-generation tumorspheres at ×100 magnification. Scale bar, 100 μm. c The rate of tumorsphere formation was measured in primary (with XMD8-92 treatment) and secondary cultures (without additional treatment). d Tumorsphere size was determined according to the number of cells per sphere in first-generation cultures. e Single-cell assays were performed using HCT116 and SW620 cells to validate the impact of XMD8-92 on clonal sphere formation. f – i HCT116 cells were transiently transfected with a specific small interfering RNA (siRNA) against ERK5, or control siRNA, and then cultured in non-adherent conditions. f ERK5 silencing was monitored by immunoblot analysis during the period of tumorsphere growth. g Representative images of 7-day tumorspheres at ×100 magnification. Scale bar, 100 μm. h Tumorspheres were scored according to number (left panel) and i size (cells per sphere; right panel). Results are expressed as mean ± standard error of mean from at least four independent experiments. * p
    Figure Legend Snippet: Pharmacological and genetic inhibition of ERK5 impairs sphere formation in colon cancer cells. a – e HCT116, HT29, SW480, and SW620 cells were cultured for two generations under sphere-forming conditions in the presence or absence of 4 µM XMD8-92. a The inhibitory effect of XMD8-92 on ERK5 activation was confirmed by western blot analysis. b Representative images of first-generation tumorspheres at ×100 magnification. Scale bar, 100 μm. c The rate of tumorsphere formation was measured in primary (with XMD8-92 treatment) and secondary cultures (without additional treatment). d Tumorsphere size was determined according to the number of cells per sphere in first-generation cultures. e Single-cell assays were performed using HCT116 and SW620 cells to validate the impact of XMD8-92 on clonal sphere formation. f – i HCT116 cells were transiently transfected with a specific small interfering RNA (siRNA) against ERK5, or control siRNA, and then cultured in non-adherent conditions. f ERK5 silencing was monitored by immunoblot analysis during the period of tumorsphere growth. g Representative images of 7-day tumorspheres at ×100 magnification. Scale bar, 100 μm. h Tumorspheres were scored according to number (left panel) and i size (cells per sphere; right panel). Results are expressed as mean ± standard error of mean from at least four independent experiments. * p

    Techniques Used: Inhibition, Cell Culture, Activation Assay, Western Blot, Transfection, Small Interfering RNA

    ERK5 pharmacological inhibition sensitizes colon cancer stem-like cells to 5-fluorouracil (5-FU)-based chemotherapy. HCT116 cells were grown for 7 days under sphere-forming conditions, and then treated for 4 days with FOLFOX (50 µM 5-FU plus 1.25 µM oxaliplatin) or FOLFIRI (50 µM 5-FU plus 1 µM irinotecan) alone or in combination with 4 µM XMD8-92. a Representative images of chemotherapy-treated tumorspheres at ×100 magnification. Scale bar, 100 μm. b Cell death was evaluated according to adenylate kinase (AK) release using the Toxilight assay. c Caspase-3/7 activity was measured using the Caspase-Glo 3/7 assay. d PARP cleavage and XIAP degradation were evaluated by western blot analysis. Results are expressed as mean ± standard error of mean from at least four independent experiments. ‡ p
    Figure Legend Snippet: ERK5 pharmacological inhibition sensitizes colon cancer stem-like cells to 5-fluorouracil (5-FU)-based chemotherapy. HCT116 cells were grown for 7 days under sphere-forming conditions, and then treated for 4 days with FOLFOX (50 µM 5-FU plus 1.25 µM oxaliplatin) or FOLFIRI (50 µM 5-FU plus 1 µM irinotecan) alone or in combination with 4 µM XMD8-92. a Representative images of chemotherapy-treated tumorspheres at ×100 magnification. Scale bar, 100 μm. b Cell death was evaluated according to adenylate kinase (AK) release using the Toxilight assay. c Caspase-3/7 activity was measured using the Caspase-Glo 3/7 assay. d PARP cleavage and XIAP degradation were evaluated by western blot analysis. Results are expressed as mean ± standard error of mean from at least four independent experiments. ‡ p

    Techniques Used: Inhibition, Activity Assay, Caspase-Glo Assay, Western Blot

    ERK5 pharmacological inhibition suppresses colon cancer stem-like cell molecular features. HCT116, HT29, SW480, and SW620 cells were cultured under sphere-forming conditions in the presence of 4 µM XMD8-92 or dimethyl sulfoxide vehicle control. a The mRNA levels of stemness-associated transcription factors were determined by qRT-PCR. b Steady-state protein levels of NANOG, OCT4, and SOX2 were confirmed by immunoblot analysis in HCT116-derived tumorspheres. Blots are representative of three independent experiments with similar results. c The percentage of cells with high aldehyde dehydrogenase (ALDH) activity was determined by flow cytometry using the Aldefluor assay. Representative side scatter versus green fluorescence intensity plots for HCT116 cells are shown. Results are expressed as mean ± standard error of mean from at least three independent experiments. * p
    Figure Legend Snippet: ERK5 pharmacological inhibition suppresses colon cancer stem-like cell molecular features. HCT116, HT29, SW480, and SW620 cells were cultured under sphere-forming conditions in the presence of 4 µM XMD8-92 or dimethyl sulfoxide vehicle control. a The mRNA levels of stemness-associated transcription factors were determined by qRT-PCR. b Steady-state protein levels of NANOG, OCT4, and SOX2 were confirmed by immunoblot analysis in HCT116-derived tumorspheres. Blots are representative of three independent experiments with similar results. c The percentage of cells with high aldehyde dehydrogenase (ALDH) activity was determined by flow cytometry using the Aldefluor assay. Representative side scatter versus green fluorescence intensity plots for HCT116 cells are shown. Results are expressed as mean ± standard error of mean from at least three independent experiments. * p

    Techniques Used: Inhibition, Cell Culture, Quantitative RT-PCR, Derivative Assay, Activity Assay, Flow Cytometry, Cytometry, Fluorescence

    22) Product Images from "GABARAPs and LC3s have opposite roles in regulating ULK1 for autophagy induction"

    Article Title: GABARAPs and LC3s have opposite roles in regulating ULK1 for autophagy induction

    Journal: Autophagy

    doi: 10.1080/15548627.2019.1632620

    ATG8 binding to ATG13, but not RB1CC1, is important for ULK1 activity. (A) RB1CC1 LIR mutation does not affect ULK1 activity. WT or RB1CC1 LIR mutant HCT116 cells were cultured in full medium or EBSS for 1 h. Two LIR mutant clones (mut1 and mut2) were analyzed. (B) ATG8 binding to ATG13 is important for ULK1 activity. ATG13-depleted HCT116 cells stably reconstituted with ATG13 WT, ATG13 LIR mutant (I447A/D448A) or empty vector were cultured in full medium or EBSS for 1 h. (C) ATG8 binding to ATG13 is important for starvation-induced formation of LC3B puncta. Red bars are mean ± SEM (**** p
    Figure Legend Snippet: ATG8 binding to ATG13, but not RB1CC1, is important for ULK1 activity. (A) RB1CC1 LIR mutation does not affect ULK1 activity. WT or RB1CC1 LIR mutant HCT116 cells were cultured in full medium or EBSS for 1 h. Two LIR mutant clones (mut1 and mut2) were analyzed. (B) ATG8 binding to ATG13 is important for ULK1 activity. ATG13-depleted HCT116 cells stably reconstituted with ATG13 WT, ATG13 LIR mutant (I447A/D448A) or empty vector were cultured in full medium or EBSS for 1 h. (C) ATG8 binding to ATG13 is important for starvation-induced formation of LC3B puncta. Red bars are mean ± SEM (**** p

    Techniques Used: Binding Assay, Activity Assay, Mutagenesis, Cell Culture, Clone Assay, Stable Transfection, Plasmid Preparation

    ATG8 binding to ULK1 is important for ULK1 activity. (A) ATG8 binding to ULK1 is important for the phosphorylation of ATG14 Ser29 by ULK1 in response to starvation. HCT116 cells whose genome was modified to express LIR-mutated ULK1 (LIRmut) and unmodified HCT116 cells (WT) were cultured in EBSS for the indicated periods of time. (B) Quantitative analysis of ATG14 Ser29 phosphorylation at 60 min of starvation in (A). Bar values are mean ± SEM (** p
    Figure Legend Snippet: ATG8 binding to ULK1 is important for ULK1 activity. (A) ATG8 binding to ULK1 is important for the phosphorylation of ATG14 Ser29 by ULK1 in response to starvation. HCT116 cells whose genome was modified to express LIR-mutated ULK1 (LIRmut) and unmodified HCT116 cells (WT) were cultured in EBSS for the indicated periods of time. (B) Quantitative analysis of ATG14 Ser29 phosphorylation at 60 min of starvation in (A). Bar values are mean ± SEM (** p

    Techniques Used: Binding Assay, Activity Assay, Modification, Cell Culture

    ATG8 binding to ULK1 is important for autophagy. (A) ATG8 binding to ULK1 is important for autophagic degradation of SQSTM1 and autophagic flux of LC3B. WT or ULK1 LIR mutant HCT116 cells were cultured in full media or EBSS for 2 h in the presence or absence of 100 nM BAFA1. Blots from 2 independent experiments are shown. (B-C) Quantitative analysis of SQSTM1 and LC3B-II from (A). The protein levels were normalized based on the levels of GAPDH. Bar values are mean ± SEM (* p
    Figure Legend Snippet: ATG8 binding to ULK1 is important for autophagy. (A) ATG8 binding to ULK1 is important for autophagic degradation of SQSTM1 and autophagic flux of LC3B. WT or ULK1 LIR mutant HCT116 cells were cultured in full media or EBSS for 2 h in the presence or absence of 100 nM BAFA1. Blots from 2 independent experiments are shown. (B-C) Quantitative analysis of SQSTM1 and LC3B-II from (A). The protein levels were normalized based on the levels of GAPDH. Bar values are mean ± SEM (* p

    Techniques Used: Binding Assay, Mutagenesis, Cell Culture

    23) Product Images from "High expression level and nuclear localization of Sam68 are associated with progression and poor prognosis in colorectal cancer"

    Article Title: High expression level and nuclear localization of Sam68 are associated with progression and poor prognosis in colorectal cancer

    Journal: BMC Gastroenterology

    doi: 10.1186/1471-230X-13-126

    Analysis of Sam68 protein and mRNA expression in colorectal cancer (CRC) cell lines and normal intestine tissues. (A) Analysis of Sam68 protein expression in CRC cell lines (LS174t, Colo205, SW480, HT29, HCT116, SW620) and two cases of normal intestine tissues (N1 and N2) by Western blotting. (B) Analysis of Sam68 mRNA expression by RT-PCR. (C) Analysis of Sam68 mRNA expression in CRC cell lines and normal intestine tissues by Q-PCR, the average ratio of Sam68 expression normalized to GAPDH is shown; values are the mean ± SD of three parallel experiments.
    Figure Legend Snippet: Analysis of Sam68 protein and mRNA expression in colorectal cancer (CRC) cell lines and normal intestine tissues. (A) Analysis of Sam68 protein expression in CRC cell lines (LS174t, Colo205, SW480, HT29, HCT116, SW620) and two cases of normal intestine tissues (N1 and N2) by Western blotting. (B) Analysis of Sam68 mRNA expression by RT-PCR. (C) Analysis of Sam68 mRNA expression in CRC cell lines and normal intestine tissues by Q-PCR, the average ratio of Sam68 expression normalized to GAPDH is shown; values are the mean ± SD of three parallel experiments.

    Techniques Used: Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    24) Product Images from "SP1-induced lncRNA-ZFAS1 contributes to colorectal cancer progression via the miR-150-5p/VEGFA axis"

    Article Title: SP1-induced lncRNA-ZFAS1 contributes to colorectal cancer progression via the miR-150-5p/VEGFA axis

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-0962-6

    ZFAS1 functioned as a competing endogenous RNA (ceRNA) by sponging miR-150-5p. a The expression of miR-150-5p and miR-590–3p were detected in HCT116 and HCT8 cells after transfecting with pcDNA3.1-ZFAS1 or blank vector. b The expression of miR-150-5p was detected using qRT-PCR after the biotinylated-ZFAS1 pull down assay in HCT116 cells. c The biotinylated wild-type/mutant miR-150-5p was transfected into HCT116 cells with ZFAS1 overexpression. The expression levels of ZFAS1 were measured by qRT-PCR after streptavidin capture. d Wild and mutant ZFAS1 sequences were cloned into pmirGLO reporter, Luciferase activity in HCT116 and 293T cells cotransfected with agomiR-150-5p or agomiR-NC and pmirGLO-ZFAS1-WT or pmirGLO-ZFAS1-Mut. Luciferase activities were normalized to renilla luciferase. e Anti-AGO2 RIP was used in HCT116 cells overexpressing agomiR-150-5p, followed by qRT-PCR to evaluate the expression of ZFAS1 or H19 (control) associated with AGO2. The data are shown as the mean ± SD of three independent experiments. f miR-15-5p was downregulated in CRC tissues compared to paired adjacent normal tissues. g The expression of miR-150-5p in HCT116, HCT8, HT29, SW620, SW480, DLD-1, and FHC. h The correlation between ZFAS1 level and miR-150-5p level in CRC tissues. i CCK-8 proliferation assays in siZFAS1-1 and antagomiR-150-5p transfected HCT116 and HCT8 cells. j Wound healing assays in siZFAS1-1 and antagomiR-150-5p transfected HCT116 and HCT8 cells. k Transwell invasion assays in siZFAS1-1 and antagomiR-150-5p transfected HCT116 and HCT8 cells. l HUVECs tube formation in siZFAS1-1 and antagomiR-150-5p transfected HCT116 and HCT8 cells. Each experiment were performed three times. Data were shown as mean ± SD. * P
    Figure Legend Snippet: ZFAS1 functioned as a competing endogenous RNA (ceRNA) by sponging miR-150-5p. a The expression of miR-150-5p and miR-590–3p were detected in HCT116 and HCT8 cells after transfecting with pcDNA3.1-ZFAS1 or blank vector. b The expression of miR-150-5p was detected using qRT-PCR after the biotinylated-ZFAS1 pull down assay in HCT116 cells. c The biotinylated wild-type/mutant miR-150-5p was transfected into HCT116 cells with ZFAS1 overexpression. The expression levels of ZFAS1 were measured by qRT-PCR after streptavidin capture. d Wild and mutant ZFAS1 sequences were cloned into pmirGLO reporter, Luciferase activity in HCT116 and 293T cells cotransfected with agomiR-150-5p or agomiR-NC and pmirGLO-ZFAS1-WT or pmirGLO-ZFAS1-Mut. Luciferase activities were normalized to renilla luciferase. e Anti-AGO2 RIP was used in HCT116 cells overexpressing agomiR-150-5p, followed by qRT-PCR to evaluate the expression of ZFAS1 or H19 (control) associated with AGO2. The data are shown as the mean ± SD of three independent experiments. f miR-15-5p was downregulated in CRC tissues compared to paired adjacent normal tissues. g The expression of miR-150-5p in HCT116, HCT8, HT29, SW620, SW480, DLD-1, and FHC. h The correlation between ZFAS1 level and miR-150-5p level in CRC tissues. i CCK-8 proliferation assays in siZFAS1-1 and antagomiR-150-5p transfected HCT116 and HCT8 cells. j Wound healing assays in siZFAS1-1 and antagomiR-150-5p transfected HCT116 and HCT8 cells. k Transwell invasion assays in siZFAS1-1 and antagomiR-150-5p transfected HCT116 and HCT8 cells. l HUVECs tube formation in siZFAS1-1 and antagomiR-150-5p transfected HCT116 and HCT8 cells. Each experiment were performed three times. Data were shown as mean ± SD. * P

    Techniques Used: Expressing, Plasmid Preparation, Quantitative RT-PCR, Pull Down Assay, Mutagenesis, Transfection, Over Expression, Clone Assay, Luciferase, Activity Assay, CCK-8 Assay

    Ki8751 could suppress CRC progression through EMT process and inactivating Akt/mTOR signaling pathway. a Western blot was performed to detect the expression of VEGFR2, p-VEGFR2, Akt, p-Akt, mTOR, p-mTOR, E-cadherin, Vimentin, and N-cadherin after adding Ki8751 to HCT116, and HCT8 cells. b – d Ki8751 inhibited proliferation ( b ), migration ( c ), and invasion ( d ) of HCT116 and HCT8 cells, and suppressed HUVECs tube formation ( e ). Results are presented as mean ± SD. * P
    Figure Legend Snippet: Ki8751 could suppress CRC progression through EMT process and inactivating Akt/mTOR signaling pathway. a Western blot was performed to detect the expression of VEGFR2, p-VEGFR2, Akt, p-Akt, mTOR, p-mTOR, E-cadherin, Vimentin, and N-cadherin after adding Ki8751 to HCT116, and HCT8 cells. b – d Ki8751 inhibited proliferation ( b ), migration ( c ), and invasion ( d ) of HCT116 and HCT8 cells, and suppressed HUVECs tube formation ( e ). Results are presented as mean ± SD. * P

    Techniques Used: Western Blot, Expressing, Migration

    ZFAS1 knockdown inhibited CRC progression via inhibiting EMT process and inactivating Akt/mTOR signaling pathway. Western blot was used to measure the expression of VEGFA, VEGFR2, p-VEGFR2, Akt, p-Akt, mTOR, p-mTOR, E-cadherin, Vimentin, and N-cadherin after ZFAS1 silencing HCT116 and HCT8 cells transfected with antagomiR-150-5p and VEGFA or their negative control, GAPDH was performed as a loading control. Data were showed as mean ± SD of three independent experiments. * P
    Figure Legend Snippet: ZFAS1 knockdown inhibited CRC progression via inhibiting EMT process and inactivating Akt/mTOR signaling pathway. Western blot was used to measure the expression of VEGFA, VEGFR2, p-VEGFR2, Akt, p-Akt, mTOR, p-mTOR, E-cadherin, Vimentin, and N-cadherin after ZFAS1 silencing HCT116 and HCT8 cells transfected with antagomiR-150-5p and VEGFA or their negative control, GAPDH was performed as a loading control. Data were showed as mean ± SD of three independent experiments. * P

    Techniques Used: Western Blot, Expressing, Transfection, Negative Control

    ZFAS1 knockdown inhibited tumor growth, metastasis, and angiogenesis in vivo. a , b Subcutaneous implant model was established using HCT116 cells. The volume of xenograft tumors in four groups ( n = 8). Data are presented as the mean ± SD. c The number of metastatic nodules in the lungs of mice (three sections evaluated per lung) from four groups ( n = 8). d Chicken embryos were incubated with conditioned medium (CM) from the four groups ( n = 8) for 4 days, then photographed with a camera and quantified by vessel count. Each experiment were performed three times. Results are presented as mean ± SD. *** P
    Figure Legend Snippet: ZFAS1 knockdown inhibited tumor growth, metastasis, and angiogenesis in vivo. a , b Subcutaneous implant model was established using HCT116 cells. The volume of xenograft tumors in four groups ( n = 8). Data are presented as the mean ± SD. c The number of metastatic nodules in the lungs of mice (three sections evaluated per lung) from four groups ( n = 8). d Chicken embryos were incubated with conditioned medium (CM) from the four groups ( n = 8) for 4 days, then photographed with a camera and quantified by vessel count. Each experiment were performed three times. Results are presented as mean ± SD. *** P

    Techniques Used: In Vivo, Mouse Assay, Incubation

    VEGFA was identified as a direct target of miR-150-5p in CRC cells. a The direct target genes of miR-150-5p were predicted using PicTarSites, miRandaSites, and Tarbase databases. b Wild and mutant VEGFA-3′UTR sequences were cloned into luciferase reporter, Luciferase activity 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 renilla luciferase. c , d QRT-PCR and Western blot analysis showed that both VEGFA mRNA and protein expression levels were dramatically suppressed by siZFAS1( c ) or agomiR-150-5p ( d ) in HCT116 and HCT8 cells. ** P
    Figure Legend Snippet: VEGFA was identified as a direct target of miR-150-5p in CRC cells. a The direct target genes of miR-150-5p were predicted using PicTarSites, miRandaSites, and Tarbase databases. b Wild and mutant VEGFA-3′UTR sequences were cloned into luciferase reporter, Luciferase activity 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 renilla luciferase. c , d QRT-PCR and Western blot analysis showed that both VEGFA mRNA and protein expression levels were dramatically suppressed by siZFAS1( c ) or agomiR-150-5p ( d ) in HCT116 and HCT8 cells. ** P

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

    The transcription factor SP1 is involved in ZFAS1 upregulation. a The predicted positions of puative SP1 binding motif in −2500 bp human ZFAS1 promoter. b Quantitative ChIP assays were performed to show direct binding of SP1 to endogenous ZFAS1 promoter regions. The primers designed for ChIP were provided in supplementary materials and methods. c A luciferase reporter assay was used by cotransfecting the full ZFAS1 promoter (ZFAS1-pGL3-F) or deleted ZFAS1 promoter fragment E2 (ZFAS1-pGL3-D) with SP1 expression plasmid or blank vector in 293T cells. Luciferase activities were expressed as relative to that of the pGL3 vector. d qPCR analysis of ZFAS1 expression levels following the treatment of siSP1-1, siSP1-2 in HCT116 and HCT8 cells. Data were shown as mean ± SD of three independent experiments. ** P
    Figure Legend Snippet: The transcription factor SP1 is involved in ZFAS1 upregulation. a The predicted positions of puative SP1 binding motif in −2500 bp human ZFAS1 promoter. b Quantitative ChIP assays were performed to show direct binding of SP1 to endogenous ZFAS1 promoter regions. The primers designed for ChIP were provided in supplementary materials and methods. c A luciferase reporter assay was used by cotransfecting the full ZFAS1 promoter (ZFAS1-pGL3-F) or deleted ZFAS1 promoter fragment E2 (ZFAS1-pGL3-D) with SP1 expression plasmid or blank vector in 293T cells. Luciferase activities were expressed as relative to that of the pGL3 vector. d qPCR analysis of ZFAS1 expression levels following the treatment of siSP1-1, siSP1-2 in HCT116 and HCT8 cells. Data were shown as mean ± SD of three independent experiments. ** P

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Luciferase, Reporter Assay, Expressing, Plasmid Preparation, Real-time Polymerase Chain Reaction

    ZFAS1 knockdown inhibited CRC cells proliferation, migration, invasion, and HUVECs tube formation. a The relative expression of ZFAS1 were detected in HCT116 and HCT8 cells after transfecting with siZFAS1-1, siZFAS1-2, siZFAS1-3, siZFAS1-4 or scramble. b , c Cells proliferation were assessed in ZFAS1 knockdown HCT116 and HCT8 cells using CCK-8 ( b ) and colony formation ( c ). d , e . Wound healing ( d ) and transwell invasion ( e ) assays were used to evaluate the ability of ZFAS1 knockdown CRC cells. f HUVECs were cultured in TCM derived from ZFAS1 knockdown HCT116 and HCT8 cells. The relative number of tube branches were measured in random 8 photographic fields. Each experiment were performed three times. Data were shown as mean ± SD. * P
    Figure Legend Snippet: ZFAS1 knockdown inhibited CRC cells proliferation, migration, invasion, and HUVECs tube formation. a The relative expression of ZFAS1 were detected in HCT116 and HCT8 cells after transfecting with siZFAS1-1, siZFAS1-2, siZFAS1-3, siZFAS1-4 or scramble. b , c Cells proliferation were assessed in ZFAS1 knockdown HCT116 and HCT8 cells using CCK-8 ( b ) and colony formation ( c ). d , e . Wound healing ( d ) and transwell invasion ( e ) assays were used to evaluate the ability of ZFAS1 knockdown CRC cells. f HUVECs were cultured in TCM derived from ZFAS1 knockdown HCT116 and HCT8 cells. The relative number of tube branches were measured in random 8 photographic fields. Each experiment were performed three times. Data were shown as mean ± SD. * P

    Techniques Used: Migration, Expressing, CCK-8 Assay, Cell Culture, Derivative Assay

    ZFAS1 upregulation promoted CRC cells growth, migration, invasion, and HUVECs tube formation. a Relative ZFAS1 expression was assessed after transfection with pcDNA3.1-ZFAS1 (ZFAS1) or blank vector (vector). b , c The proliferation of HCT116 and HCT8 cells transfected with ZFAS1 or blank vector were measured using CCK-8 ( b ) and colony formation ( c ). d , e Wound healing ( d ) and transwell invasion ( e ) assays were performed to evaluate the ability of migration and invasion in HCT116 and HCT8 cells transfected with ZFAS1 or blank vector, respectively. f HUVECs were cultured in TCM derived from HCT116 and HCT8 cells transfected with pcDNA3.1-ZFAS1 or blank vector, the relative number of tube branches were measured in random 8 photographic fields. Each experiment were performed three times. Data were shown as mean ± SD. * P
    Figure Legend Snippet: ZFAS1 upregulation promoted CRC cells growth, migration, invasion, and HUVECs tube formation. a Relative ZFAS1 expression was assessed after transfection with pcDNA3.1-ZFAS1 (ZFAS1) or blank vector (vector). b , c The proliferation of HCT116 and HCT8 cells transfected with ZFAS1 or blank vector were measured using CCK-8 ( b ) and colony formation ( c ). d , e Wound healing ( d ) and transwell invasion ( e ) assays were performed to evaluate the ability of migration and invasion in HCT116 and HCT8 cells transfected with ZFAS1 or blank vector, respectively. f HUVECs were cultured in TCM derived from HCT116 and HCT8 cells transfected with pcDNA3.1-ZFAS1 or blank vector, the relative number of tube branches were measured in random 8 photographic fields. Each experiment were performed three times. Data were shown as mean ± SD. * P

    Techniques Used: Migration, Expressing, Transfection, Plasmid Preparation, CCK-8 Assay, Cell Culture, Derivative Assay

    25) Product Images from "Circular RNA circHUWE1 Is Upregulated and Promotes Cell Proliferation, Migration and Invasion in Colorectal Cancer by Sponging miR-486"

    Article Title: Circular RNA circHUWE1 Is Upregulated and Promotes Cell Proliferation, Migration and Invasion in Colorectal Cancer by Sponging miR-486

    Journal: OncoTargets and therapy

    doi: 10.2147/OTT.S233338

    Transfecting miR-486 mimics inhibit the capacity of proliferation, migration, invasion of CRC cells, and promote apoptotic rate in vitro. ( A ) CCK-8 assays showed that the proliferative ability of HCT116 and SW480 cells was significantly inhibited after transfected with miR-486 mimics. ( B ) Flow cytometry suggested HCT116 and SW480 cells showed significantly higher apoptotic rate after transfected with miR-486 mimics. ( C and D ) Transwell assays showed that the migration and invasion capacity of HCT116 and SW480 cells were significantly attenuated after transfected with miR-486 mimics. Data are showed as means ± s.d. of at least three independent experiments. *p
    Figure Legend Snippet: Transfecting miR-486 mimics inhibit the capacity of proliferation, migration, invasion of CRC cells, and promote apoptotic rate in vitro. ( A ) CCK-8 assays showed that the proliferative ability of HCT116 and SW480 cells was significantly inhibited after transfected with miR-486 mimics. ( B ) Flow cytometry suggested HCT116 and SW480 cells showed significantly higher apoptotic rate after transfected with miR-486 mimics. ( C and D ) Transwell assays showed that the migration and invasion capacity of HCT116 and SW480 cells were significantly attenuated after transfected with miR-486 mimics. Data are showed as means ± s.d. of at least three independent experiments. *p

    Techniques Used: Migration, In Vitro, CCK-8 Assay, Transfection, Flow Cytometry

    Silencing circHUWE1 inhibited the proliferation, migration and invasion capacity of CRC cells in vitro. ( A ) The expression of circHUWE1 and HUWE1 mRNA in HCT116 and SW480 cells were measured by qRT-PCR after transfected with different siRNAs targeting the back-splice junction site of circHUWE1 or negative control (si-NC). ( B ) CCK-8 assays showed that silencing circHUWE1 inhibited the proliferative ability of HCT116 and SW480 cells. ( C ) Colony formation assays indicated that the colony-forming capacity of HCT116 and SW480 cells was attenuated with silencing circHUWE1. ( D ) Flow cytometry to detect cell apoptosis suggested that HCT116 and SW480 cells with circHUWE1 silencing presented significantly higher apoptotic rate. ( E and F ) Transwell assays showed that silencing circHUWE1 significantly inhibited the migration and invasion capacity of HCT116 and SW480 cells. Data are showed as means ± s.d. of at least three independent experiments. *p
    Figure Legend Snippet: Silencing circHUWE1 inhibited the proliferation, migration and invasion capacity of CRC cells in vitro. ( A ) The expression of circHUWE1 and HUWE1 mRNA in HCT116 and SW480 cells were measured by qRT-PCR after transfected with different siRNAs targeting the back-splice junction site of circHUWE1 or negative control (si-NC). ( B ) CCK-8 assays showed that silencing circHUWE1 inhibited the proliferative ability of HCT116 and SW480 cells. ( C ) Colony formation assays indicated that the colony-forming capacity of HCT116 and SW480 cells was attenuated with silencing circHUWE1. ( D ) Flow cytometry to detect cell apoptosis suggested that HCT116 and SW480 cells with circHUWE1 silencing presented significantly higher apoptotic rate. ( E and F ) Transwell assays showed that silencing circHUWE1 significantly inhibited the migration and invasion capacity of HCT116 and SW480 cells. Data are showed as means ± s.d. of at least three independent experiments. *p

    Techniques Used: Migration, In Vitro, Expressing, Quantitative RT-PCR, Transfection, Negative Control, CCK-8 Assay, Flow Cytometry

    The tumor-supressing effect of silencing circHUWE1 could be reversed by miR-486 down-regulation. ( A ) miR-486 inhibitor significantly reversed silencing circHUWE1 mediated supression of proliferation of both HCT116 and SW480 cells. ( B and C ) miR-486 inhibitor significantly reversed silencing circHUWE1 mediated supression of migration and invasion of both HCT116 and SW480 cells. *p
    Figure Legend Snippet: The tumor-supressing effect of silencing circHUWE1 could be reversed by miR-486 down-regulation. ( A ) miR-486 inhibitor significantly reversed silencing circHUWE1 mediated supression of proliferation of both HCT116 and SW480 cells. ( B and C ) miR-486 inhibitor significantly reversed silencing circHUWE1 mediated supression of migration and invasion of both HCT116 and SW480 cells. *p

    Techniques Used: Migration

    26) Product Images from "IGF-1R inhibition induces MEK phosphorylation to promote survival in colon carcinomas"

    Article Title: IGF-1R inhibition induces MEK phosphorylation to promote survival in colon carcinomas

    Journal: Signal Transduction and Targeted Therapy

    doi: 10.1038/s41392-020-0204-0

    The combination of BMS-754807 and U0126 significantly suppresses tumor growth in the HCT116 colon xenograft model. NCr nude mice (five mice per group) were treated with vehicle [10% (v/v) DMSO in corn oil], BMS-754807 (25 mg/kg, BMS), U0126 (15 mg/kg), or BMS-754807 + U0126 (25 mg/kg+15 mg/kg) for 12 days. a The tumor volume was measured every other day with calipers. Data were analyzed by one-way ANOVA with Dunnett’s multiple comparison versus vehicle, BMS-754807, or U0126 after 12-day treatment (mean ± SD; ### P
    Figure Legend Snippet: The combination of BMS-754807 and U0126 significantly suppresses tumor growth in the HCT116 colon xenograft model. NCr nude mice (five mice per group) were treated with vehicle [10% (v/v) DMSO in corn oil], BMS-754807 (25 mg/kg, BMS), U0126 (15 mg/kg), or BMS-754807 + U0126 (25 mg/kg+15 mg/kg) for 12 days. a The tumor volume was measured every other day with calipers. Data were analyzed by one-way ANOVA with Dunnett’s multiple comparison versus vehicle, BMS-754807, or U0126 after 12-day treatment (mean ± SD; ### P

    Techniques Used: Mouse Assay

    Induction of p70S6K1 phosphorylation by IGF-1R inhibitors. a , b IGF-1R inhibitors induced p70S6K1 phosphorylation in HCT116 cells. Cells were treated with BMS-754807 (240 nM, BMS) or GSK1838705A (2 μM, GSK) for 0–72 h. a Representative western blot images are shown. b Densitometric quantification of the western blot results in ( a ). For each inhibitor, the band intensity of p-p70S6K1/GAPDH with 0 h treatment is designated as 1. Data from three independent experiments were analyzed by one-sample t -test (mean ± SD; * P
    Figure Legend Snippet: Induction of p70S6K1 phosphorylation by IGF-1R inhibitors. a , b IGF-1R inhibitors induced p70S6K1 phosphorylation in HCT116 cells. Cells were treated with BMS-754807 (240 nM, BMS) or GSK1838705A (2 μM, GSK) for 0–72 h. a Representative western blot images are shown. b Densitometric quantification of the western blot results in ( a ). For each inhibitor, the band intensity of p-p70S6K1/GAPDH with 0 h treatment is designated as 1. Data from three independent experiments were analyzed by one-sample t -test (mean ± SD; * P

    Techniques Used: Western Blot

    BMS-754807-induced p70S6K1 phosphorylation contributes to cell survival. a BMS-754807 induced p70S6K1 phosphorylation in Pdcd4 knockdown cells. Western blot analyses were performed using cell extracts from control (HT29-L) and Pdcd4 knockdown (HT29-P) cells treated with BMS-754807 (240 nM) for 0–72 h. Representative images are shown. b A combination of BMS-754807 and PF-4708671 reversed the BMS-754807-induced p70S6K1 activation and increased the cleaved caspase 3 level. Western blot analyses were performed using cell extracts from cells treated with vehicle, BMS-754807 (240 nM), PF-4708671(10 μM), and BMS-754807 (240 nM)+PF-4708671 (10 μM) for 72 h. Representative images are shown. c , d The combination of BMS-754807 and PF-4708671 inhibits the proliferation and colony formation. c HCT116 cells were treated with vehicle, BMS-754807 (240 nM), PF-4708671 (10 μM), BMS-754807 (240 nM)+PF-4708671 (10 μM) for 0–5 days. Cell viability was determined by XTT. The absorbance at day 0 is designated as 100%. Data from four replicates were analyzed one-way ANOVA with Dunnett’s multiple comparison (mean ± SD; # P
    Figure Legend Snippet: BMS-754807-induced p70S6K1 phosphorylation contributes to cell survival. a BMS-754807 induced p70S6K1 phosphorylation in Pdcd4 knockdown cells. Western blot analyses were performed using cell extracts from control (HT29-L) and Pdcd4 knockdown (HT29-P) cells treated with BMS-754807 (240 nM) for 0–72 h. Representative images are shown. b A combination of BMS-754807 and PF-4708671 reversed the BMS-754807-induced p70S6K1 activation and increased the cleaved caspase 3 level. Western blot analyses were performed using cell extracts from cells treated with vehicle, BMS-754807 (240 nM), PF-4708671(10 μM), and BMS-754807 (240 nM)+PF-4708671 (10 μM) for 72 h. Representative images are shown. c , d The combination of BMS-754807 and PF-4708671 inhibits the proliferation and colony formation. c HCT116 cells were treated with vehicle, BMS-754807 (240 nM), PF-4708671 (10 μM), BMS-754807 (240 nM)+PF-4708671 (10 μM) for 0–5 days. Cell viability was determined by XTT. The absorbance at day 0 is designated as 100%. Data from four replicates were analyzed one-way ANOVA with Dunnett’s multiple comparison (mean ± SD; # P

    Techniques Used: Western Blot, Activation Assay

    Inhibition of AKT induces phosphorylation of MEK1/2 but not ERK1/2. a , b BMS-754807 induces MEK1/2 phosphorylation in colon tumor cells. Western blot analyses of MEK1/2 and ERK1/2 phosphorylation using extracts from cells treated with BMS-754807 (240 nM) for 0–72 h. a Representative images are shown. b Densitometric quantification of the levels of phospho-MEK and phoshpo-ERK in ( a ). The ratio of phospho-MEK/GAPDH or phospho-ERK/GAPDH in HCT116 or SW480 cells with 0 h treatment is designated as 1. Data from three independent experiments were analyzed by one-sample t -test (mean ± SD; * P
    Figure Legend Snippet: Inhibition of AKT induces phosphorylation of MEK1/2 but not ERK1/2. a , b BMS-754807 induces MEK1/2 phosphorylation in colon tumor cells. Western blot analyses of MEK1/2 and ERK1/2 phosphorylation using extracts from cells treated with BMS-754807 (240 nM) for 0–72 h. a Representative images are shown. b Densitometric quantification of the levels of phospho-MEK and phoshpo-ERK in ( a ). The ratio of phospho-MEK/GAPDH or phospho-ERK/GAPDH in HCT116 or SW480 cells with 0 h treatment is designated as 1. Data from three independent experiments were analyzed by one-sample t -test (mean ± SD; * P

    Techniques Used: Inhibition, Western Blot

    27) Product Images from "Sirtuin5 contributes to colorectal carcinogenesis by enhancing glutaminolysis in a deglutarylation-dependent manner"

    Article Title: Sirtuin5 contributes to colorectal carcinogenesis by enhancing glutaminolysis in a deglutarylation-dependent manner

    Journal: Nature Communications

    doi: 10.1038/s41467-018-02951-4

    SIRT5 supports colorectal cancer growth by promoting glutamine metabolism via increased GLUD1 enzyme activity. a A diagram showing the enzymes involved in glutamine metabolism and the inhibitors used in this study. b Expression of GLUD1, GOT1/2, PSAT1, GPT2, and GLS (including kidney-type glutaminase (KGA isoform), and glutaminase C (GAC isoform)) upon SIRT5 knockdown. c GLUD1 enzyme activity was determined upon SIRT5 knockdown in HCT116 and LoVo cells. Left, representative images ( n = 3). Right, quantification of GLUD1 activity. GLUD1 inhibitor epigallocatechin gallate (EGCG; 20 μM) was used as a positive control. d – f GLUD1 ( d ), GLS ( e ), and GOT2 ( f ) enzyme activities were determined in HCT116 and LoVo cells stably expressing the control vector, SIRT5 WT, or SIRT5 H158Y, respectively; n = 3. g Proliferation rate of HCT116 and LoVo cells stably expressing the control vector or SIRT5 WT plasmid in different culture conditions. DM glutamate (10 mM), DM α-KG (1 mM), and non-essential amino acids (NEAAs; 0.1 mM aspartate and asparagine) were added to glutamine-free medium, respectively; n = 4. h CCK-8 assays of HCT116 and LoVo cells stably expressing the control vector or SIRT5 WT treated with/without the specific GLUD1 siRNA; n = 5. i GLUD1 was knocked down in HCT116 and LoVo cells stably expressing the control vector or SIRT5 WT vector. Protein levels were assessed by western blotting. j Growth curves of HCT116 and LoVo cells after transfection of control siRNA (red line) and SIRT5 siRNAs (blue line) under the indicated conditions. Cells were cultured in standard media, and DM α-KG (1 mM) was added to media as indicated. The OD 450 was measured for 6 consecutive days using the CCK-8 assay; n = 5. k HCT116 and LoVo cells were transfected with control siRNA or SIRT5 siRNAs, and then cultured in standard media with DM α-KG (1 mM). The levels of cleaved PARP were detected by western blotting. Data in c – j are presented as the mean ± SD. P values were calculated by ANOVA. ** P
    Figure Legend Snippet: SIRT5 supports colorectal cancer growth by promoting glutamine metabolism via increased GLUD1 enzyme activity. a A diagram showing the enzymes involved in glutamine metabolism and the inhibitors used in this study. b Expression of GLUD1, GOT1/2, PSAT1, GPT2, and GLS (including kidney-type glutaminase (KGA isoform), and glutaminase C (GAC isoform)) upon SIRT5 knockdown. c GLUD1 enzyme activity was determined upon SIRT5 knockdown in HCT116 and LoVo cells. Left, representative images ( n = 3). Right, quantification of GLUD1 activity. GLUD1 inhibitor epigallocatechin gallate (EGCG; 20 μM) was used as a positive control. d – f GLUD1 ( d ), GLS ( e ), and GOT2 ( f ) enzyme activities were determined in HCT116 and LoVo cells stably expressing the control vector, SIRT5 WT, or SIRT5 H158Y, respectively; n = 3. g Proliferation rate of HCT116 and LoVo cells stably expressing the control vector or SIRT5 WT plasmid in different culture conditions. DM glutamate (10 mM), DM α-KG (1 mM), and non-essential amino acids (NEAAs; 0.1 mM aspartate and asparagine) were added to glutamine-free medium, respectively; n = 4. h CCK-8 assays of HCT116 and LoVo cells stably expressing the control vector or SIRT5 WT treated with/without the specific GLUD1 siRNA; n = 5. i GLUD1 was knocked down in HCT116 and LoVo cells stably expressing the control vector or SIRT5 WT vector. Protein levels were assessed by western blotting. j Growth curves of HCT116 and LoVo cells after transfection of control siRNA (red line) and SIRT5 siRNAs (blue line) under the indicated conditions. Cells were cultured in standard media, and DM α-KG (1 mM) was added to media as indicated. The OD 450 was measured for 6 consecutive days using the CCK-8 assay; n = 5. k HCT116 and LoVo cells were transfected with control siRNA or SIRT5 siRNAs, and then cultured in standard media with DM α-KG (1 mM). The levels of cleaved PARP were detected by western blotting. Data in c – j are presented as the mean ± SD. P values were calculated by ANOVA. ** P

    Techniques Used: Activity Assay, Expressing, Positive Control, Stable Transfection, Plasmid Preparation, CCK-8 Assay, Western Blot, Transfection, Cell Culture

    The direct interaction between GLUD1 and SIRT5 causes hypoglutarylation of GLUD1. a , b HCT116 ( a ) and LoVo ( b ) cells were immunostained for FLAG-SIRT5 WT/H158Y (in green) and GLUD1 (in red); yellow in the merged magnified images (left) indicates the co-localization. Scale bars indicate 20 μm. c Fluorescence intensity of FLAG-SIRT5 WT/H158Y (green line) and GLUD1 (red line) traced along the white line in HCT116 using the line profiling function of the ImageJ software. d , e FLAG-SIRT5 WT/H158Y were immunoprecipitated with anti-FLAG antibody and followed by western blotting with an anti-GLUD1 antibody in HCT116 ( d ) and LoVo ( e ) cells. f , g The interaction between endogenous GLUD1 and SIRT5 in HCT116 ( f ) and LoVo ( g ) cells. h GST pull-down assays revealed the interaction between SIRT5 and GLUD1. In vitro-translated SIRT5 was incubated GST fusion of GLUD1 or GST, and analyzed by western blotting. The bound SIRT5 is indicated by an arrow on the right. i , j Exogenous GLUD1 proteins were purified in HCT116 ( i ) and LoVo ( j ) cells expressing the control vector, SIRT5 WT, and SIRT5 H158Y; and the glutarylation (GluK) levels of GLUD1 were determined by western blotting. Integrated density values were calculated using the ImageJ software. k Exogenous GLUD1 was purified upon SIRT5 knockdown, and the GluK level of GLUD1 was determined. l HA-tagged GLUD1 proteins were purified and incubated with different concentrations of glutaryl-CoA (0, 1, and 2 mM) at 37 °C for 60 min. The GLUD1 activity was determined. Left, representative images ( n = 6). Right, quantification of GLUD1 activity. m Wild-type GLUD1, K399R, K503R, and K545R mutants were transfected into HCT116 cells followed by SIRT5 knockdown. The GLUD1 was immunoprecipitated and the level of GluK was determined. n HCT116 cells expressing HA-tagged GLUD1 WT/K545R mutant were treated with or without SIRT5 siRNAs. The GLUD1 activity was measured and normalized against the protein levels. The results in i – n are the mean ± SD of three independent experiments. P values were calculated by ANOVA with Tukey’s test. * P
    Figure Legend Snippet: The direct interaction between GLUD1 and SIRT5 causes hypoglutarylation of GLUD1. a , b HCT116 ( a ) and LoVo ( b ) cells were immunostained for FLAG-SIRT5 WT/H158Y (in green) and GLUD1 (in red); yellow in the merged magnified images (left) indicates the co-localization. Scale bars indicate 20 μm. c Fluorescence intensity of FLAG-SIRT5 WT/H158Y (green line) and GLUD1 (red line) traced along the white line in HCT116 using the line profiling function of the ImageJ software. d , e FLAG-SIRT5 WT/H158Y were immunoprecipitated with anti-FLAG antibody and followed by western blotting with an anti-GLUD1 antibody in HCT116 ( d ) and LoVo ( e ) cells. f , g The interaction between endogenous GLUD1 and SIRT5 in HCT116 ( f ) and LoVo ( g ) cells. h GST pull-down assays revealed the interaction between SIRT5 and GLUD1. In vitro-translated SIRT5 was incubated GST fusion of GLUD1 or GST, and analyzed by western blotting. The bound SIRT5 is indicated by an arrow on the right. i , j Exogenous GLUD1 proteins were purified in HCT116 ( i ) and LoVo ( j ) cells expressing the control vector, SIRT5 WT, and SIRT5 H158Y; and the glutarylation (GluK) levels of GLUD1 were determined by western blotting. Integrated density values were calculated using the ImageJ software. k Exogenous GLUD1 was purified upon SIRT5 knockdown, and the GluK level of GLUD1 was determined. l HA-tagged GLUD1 proteins were purified and incubated with different concentrations of glutaryl-CoA (0, 1, and 2 mM) at 37 °C for 60 min. The GLUD1 activity was determined. Left, representative images ( n = 6). Right, quantification of GLUD1 activity. m Wild-type GLUD1, K399R, K503R, and K545R mutants were transfected into HCT116 cells followed by SIRT5 knockdown. The GLUD1 was immunoprecipitated and the level of GluK was determined. n HCT116 cells expressing HA-tagged GLUD1 WT/K545R mutant were treated with or without SIRT5 siRNAs. The GLUD1 activity was measured and normalized against the protein levels. The results in i – n are the mean ± SD of three independent experiments. P values were calculated by ANOVA with Tukey’s test. * P

    Techniques Used: Fluorescence, Software, Immunoprecipitation, Western Blot, In Vitro, Incubation, Purification, Expressing, Plasmid Preparation, Activity Assay, Transfection, Mutagenesis

    Oncogenic role of SIRT5 and GLUD1 is vital for the tumorigenesis capacity of SIRT5 in vivo. a HCT116 cells stably expressing the control vector, SIRT5 WT, or SIRT5 H158Y were injected subcutaneously into nude mice ( n = 9 for each group). Tumor volumes were measured at the indicated time points and the mean tumor volumes were calculated. Data are presented as the mean ± SD. b – d At the end of experiment, tumors from three groups were dissected, photographed ( b , c ), and weighed ( d ). Data are presented as the mean ± SD. e , f GLUD1 enzyme activities in tumor lysates derived from xenografts were measured ( e ). Data are presented as the mean ± SD. GLUD1 protein level and the overexpression of SIRT5 in the xenografts were confirmed by immunoblotting ( f ). g – i SIRT5-overexpressing LoVo cells infected with viruses expressing non-target control (NTC) short hairpin RNA (shRNA) or GLUD1 shRNA were injected subcutaneously into nude mice ( n = 6 for each group). Tumor growth curves were constructed ( g ). Statistical analysis of tumor weight. Each dot represents the tumor mass from one mouse ( h ). Digital photograph of the dissected tumors ( i ). Data are presented as the mean ± SD. All P values were calculated by ANOVA with Tukey’s test. ** P
    Figure Legend Snippet: Oncogenic role of SIRT5 and GLUD1 is vital for the tumorigenesis capacity of SIRT5 in vivo. a HCT116 cells stably expressing the control vector, SIRT5 WT, or SIRT5 H158Y were injected subcutaneously into nude mice ( n = 9 for each group). Tumor volumes were measured at the indicated time points and the mean tumor volumes were calculated. Data are presented as the mean ± SD. b – d At the end of experiment, tumors from three groups were dissected, photographed ( b , c ), and weighed ( d ). Data are presented as the mean ± SD. e , f GLUD1 enzyme activities in tumor lysates derived from xenografts were measured ( e ). Data are presented as the mean ± SD. GLUD1 protein level and the overexpression of SIRT5 in the xenografts were confirmed by immunoblotting ( f ). g – i SIRT5-overexpressing LoVo cells infected with viruses expressing non-target control (NTC) short hairpin RNA (shRNA) or GLUD1 shRNA were injected subcutaneously into nude mice ( n = 6 for each group). Tumor growth curves were constructed ( g ). Statistical analysis of tumor weight. Each dot represents the tumor mass from one mouse ( h ). Digital photograph of the dissected tumors ( i ). Data are presented as the mean ± SD. All P values were calculated by ANOVA with Tukey’s test. ** P

    Techniques Used: In Vivo, Stable Transfection, Expressing, Plasmid Preparation, Injection, Mouse Assay, Derivative Assay, Over Expression, Infection, shRNA, Construct

    SIRT5 is required for proliferation and regulates cell cycle and apoptosis in colorectal cancer cells. a , b Growth curves of HCT116 ( a ) and LoVo ( b ) cells transfected with two different SIRT5 short interfering RNAs (siRNAs; blue lines) or control siRNA (red line). Results are presented as mean ± SD of five independent samples. ANOVA with Tukey’s test. Western blotting was performed to validate the knockdown efficiency. c , d Flow cytometric assay based on phycoerythrin-conjugated Annexin V staining showing the increased apoptosis of HCT116 and LoVo cells at 48 h post transfection with SIRT5 siRNAs. Representative fluorescence-activated cell sorting (FACS) images are shown in c . Data in c were quantified ( d ). e , f G2-M phase and S phase arrest were detected in SIRT5 -knockdown HCT116 and LoVo cells ( e ). Data in e were quantified ( f ). Results in d and f are presented as mean ± SD of three independent samples. Student’s t -test for d . ANOVA with Tukey’s test for f . * P
    Figure Legend Snippet: SIRT5 is required for proliferation and regulates cell cycle and apoptosis in colorectal cancer cells. a , b Growth curves of HCT116 ( a ) and LoVo ( b ) cells transfected with two different SIRT5 short interfering RNAs (siRNAs; blue lines) or control siRNA (red line). Results are presented as mean ± SD of five independent samples. ANOVA with Tukey’s test. Western blotting was performed to validate the knockdown efficiency. c , d Flow cytometric assay based on phycoerythrin-conjugated Annexin V staining showing the increased apoptosis of HCT116 and LoVo cells at 48 h post transfection with SIRT5 siRNAs. Representative fluorescence-activated cell sorting (FACS) images are shown in c . Data in c were quantified ( d ). e , f G2-M phase and S phase arrest were detected in SIRT5 -knockdown HCT116 and LoVo cells ( e ). Data in e were quantified ( f ). Results in d and f are presented as mean ± SD of three independent samples. Student’s t -test for d . ANOVA with Tukey’s test for f . * P

    Techniques Used: Transfection, Western Blot, Flow Cytometry, Staining, Fluorescence, FACS

    SIRT5 enhances glutamine-driven TCA cycle metabolite abundances in colorectal cancer cells. a Heat map representing significantly different metabolites after SIRT5 deletion in HCT116 cells. Blue (red) indicates the relative down (up) regulation levels of TCA cycle and glutaminolysis intermediates compared with cells treated with the control siRNA; n = 6. b Schematic model of glutamine metabolism in cancer cells. Red circles represent carbons derived from [U- 13 C 5 ] glutamine, and black circles are unlabeled. The black arrows indicate oxidative carboxylation flux from glutamine. c Glutamine uptake was determined in HCT116 and LoVo cells. At 48 h post transfection with SIRT5 siRNAs, the cells were placed in fresh medium. Metabolite levels were measured after 9 h of culture and normalized to the cell number. The results were normalized to the control siRNA. Data are the mean ± SD of five independent samples. Student’s t -test. N.S. = not significant for the indicated comparison. d – h Mass isotopologue distributions of glutamate ( d ); TCA cycle metabolites including α-KG ( e ), succinate, fumarate, malate, citrate, and isocitrate ( f ); glutamine-derived aspartate and asparagine ( g ); and pyruvate and lactate ( h ) in LoVo cells treated with the control siRNA or SIRT5 siRNAs. Cells were cultured in [U- 13 C 5 ] glutamine for 24 h before metabolites extraction and GC-MS analysis; n = 3, data in d – h are shown as the mean ± SD. Student’s t -test. * P
    Figure Legend Snippet: SIRT5 enhances glutamine-driven TCA cycle metabolite abundances in colorectal cancer cells. a Heat map representing significantly different metabolites after SIRT5 deletion in HCT116 cells. Blue (red) indicates the relative down (up) regulation levels of TCA cycle and glutaminolysis intermediates compared with cells treated with the control siRNA; n = 6. b Schematic model of glutamine metabolism in cancer cells. Red circles represent carbons derived from [U- 13 C 5 ] glutamine, and black circles are unlabeled. The black arrows indicate oxidative carboxylation flux from glutamine. c Glutamine uptake was determined in HCT116 and LoVo cells. At 48 h post transfection with SIRT5 siRNAs, the cells were placed in fresh medium. Metabolite levels were measured after 9 h of culture and normalized to the cell number. The results were normalized to the control siRNA. Data are the mean ± SD of five independent samples. Student’s t -test. N.S. = not significant for the indicated comparison. d – h Mass isotopologue distributions of glutamate ( d ); TCA cycle metabolites including α-KG ( e ), succinate, fumarate, malate, citrate, and isocitrate ( f ); glutamine-derived aspartate and asparagine ( g ); and pyruvate and lactate ( h ) in LoVo cells treated with the control siRNA or SIRT5 siRNAs. Cells were cultured in [U- 13 C 5 ] glutamine for 24 h before metabolites extraction and GC-MS analysis; n = 3, data in d – h are shown as the mean ± SD. Student’s t -test. * P

    Techniques Used: Derivative Assay, Transfection, Cell Culture, Gas Chromatography-Mass Spectrometry

    SIRT5 promotes colorectal cancer cell growth dependent on its catalytic activity. a , b Western blotting confirming the stable expression of the control vector, FLAG-SIRT5 WT, or FLAG-SIRT5 H158Y in HCT116 ( a ) and LoVo ( b ) cells. c , d Growth curve of HCT116 ( c ) and LoVo ( d ) cells stably expressing the control vector (blue line), SIRT5 WT (red line), or SIRT5 H158Y (purple line). Data are the mean ± SD of five independent samples. ANOVA with Tukey’s test. *** P
    Figure Legend Snippet: SIRT5 promotes colorectal cancer cell growth dependent on its catalytic activity. a , b Western blotting confirming the stable expression of the control vector, FLAG-SIRT5 WT, or FLAG-SIRT5 H158Y in HCT116 ( a ) and LoVo ( b ) cells. c , d Growth curve of HCT116 ( c ) and LoVo ( d ) cells stably expressing the control vector (blue line), SIRT5 WT (red line), or SIRT5 H158Y (purple line). Data are the mean ± SD of five independent samples. ANOVA with Tukey’s test. *** P

    Techniques Used: Activity Assay, Western Blot, Expressing, Plasmid Preparation, Stable Transfection

    28) Product Images from "Inhibition of Ubiquitin Specific Protease 1 Sensitizes Colorectal Cancer Cells to DNA-Damaging Chemotherapeutics"

    Article Title: Inhibition of Ubiquitin Specific Protease 1 Sensitizes Colorectal Cancer Cells to DNA-Damaging Chemotherapeutics

    Journal: Frontiers in Oncology

    doi: 10.3389/fonc.2019.01406

    Knockdown of ubiquitin specific protease 1 (USP1) induces growth arrest in colorectal cancer cells. (A) HCT116 cells were stably infected with shNC or shUSP1-expressing lentivirus and prepared for immunoblotting against USP1. GAPDH was used as a loading control. (B) HCT116 cells were stably infected with shNC or shUSP1-expressing lentivirus, and examined with CCK-8 staining at day 0, 1, 3, or 5. (C) HCT116 cells were stably infected with shNC or shUSP1-expressing lentivirus and subjected to cell cycle by Propidium Iodide (PI) staining and flow cytometer analysis. (D,E) HCT116 cells stably infected with shNC or shUSP1-expressing lentivirus were prepared for immunoblotting against CDK4, CDK6, p-P53, P21, USP1, FANCD2, ID1, and PCNA. GAPDH and β-actin were used as a loading control. * p
    Figure Legend Snippet: Knockdown of ubiquitin specific protease 1 (USP1) induces growth arrest in colorectal cancer cells. (A) HCT116 cells were stably infected with shNC or shUSP1-expressing lentivirus and prepared for immunoblotting against USP1. GAPDH was used as a loading control. (B) HCT116 cells were stably infected with shNC or shUSP1-expressing lentivirus, and examined with CCK-8 staining at day 0, 1, 3, or 5. (C) HCT116 cells were stably infected with shNC or shUSP1-expressing lentivirus and subjected to cell cycle by Propidium Iodide (PI) staining and flow cytometer analysis. (D,E) HCT116 cells stably infected with shNC or shUSP1-expressing lentivirus were prepared for immunoblotting against CDK4, CDK6, p-P53, P21, USP1, FANCD2, ID1, and PCNA. GAPDH and β-actin were used as a loading control. * p

    Techniques Used: Stable Transfection, Infection, Expressing, CCK-8 Assay, Staining, Flow Cytometry, Cytometry

    Ubiquitin specific protease 1 (USP1) regulated the expression of cell cycle and anti-apoptosis proteins. (A) Knockdown of USP1 decreased the expression of cyclins. siNC and siUSP1 were transfected into HCT116 cells for 24 h, followed by qRT-PCR against USP1, CyclinA1, CyclinD1, and CyclinE1. β-actin was used as an internal control. (B) Knockdown of USP1 reduced the expression of Bcl-2 and Mcl-1. HCT116 cells transfected with siNC or siUSP1 for 24 h were prepared for qRT-PCR against USP1, Bcl2, and Mcl1. β-actin was used as an internal control. (C,D) Expression of wild-type but not mutated USP1 upregulated cyclins. Empty vector (EV), Flag-USP1-WT or Flag-USP1-C90S plasmids were transfected into HCT116 cells for 48 h, followed by qRT-PCR against CyclinA1, CyclinD1, CyclinE1, Bcl2, and Mcl1 (C) , and immunoblotting against Flag and ID1. (D) β-actin was used as an internal control. (E) siNC and siUSP1 were transfected into HCT116 cells for 24 h. After treatment with doxorubicin (DOX) for 24 h, the cells were prepared for analyzing the expression of USP1, cleaved-poly(ADP-ribose) polymerase (PARP) and GAPDH by immunoblotting. (F) HCT116 cells were transfected with empty vector or Flag-USP1 for 24 h. After treatment with DOX for 24 h, the cells were analyzed by immunoblotting against Flag, cleaved-PARP and GAPDH. * p
    Figure Legend Snippet: Ubiquitin specific protease 1 (USP1) regulated the expression of cell cycle and anti-apoptosis proteins. (A) Knockdown of USP1 decreased the expression of cyclins. siNC and siUSP1 were transfected into HCT116 cells for 24 h, followed by qRT-PCR against USP1, CyclinA1, CyclinD1, and CyclinE1. β-actin was used as an internal control. (B) Knockdown of USP1 reduced the expression of Bcl-2 and Mcl-1. HCT116 cells transfected with siNC or siUSP1 for 24 h were prepared for qRT-PCR against USP1, Bcl2, and Mcl1. β-actin was used as an internal control. (C,D) Expression of wild-type but not mutated USP1 upregulated cyclins. Empty vector (EV), Flag-USP1-WT or Flag-USP1-C90S plasmids were transfected into HCT116 cells for 48 h, followed by qRT-PCR against CyclinA1, CyclinD1, CyclinE1, Bcl2, and Mcl1 (C) , and immunoblotting against Flag and ID1. (D) β-actin was used as an internal control. (E) siNC and siUSP1 were transfected into HCT116 cells for 24 h. After treatment with doxorubicin (DOX) for 24 h, the cells were prepared for analyzing the expression of USP1, cleaved-poly(ADP-ribose) polymerase (PARP) and GAPDH by immunoblotting. (F) HCT116 cells were transfected with empty vector or Flag-USP1 for 24 h. After treatment with DOX for 24 h, the cells were analyzed by immunoblotting against Flag, cleaved-PARP and GAPDH. * p

    Techniques Used: Expressing, Transfection, Quantitative RT-PCR, Plasmid Preparation

    Ubiquitin specific protease 1 (USP1) inhibitor sensitized colorectal cancer cells to DNA-targeting agents doxorubicin, PARP inhibitor, etoposide, TOPI, and TOPII inhibitor in colorectal cancer cells. (A,B) HCT116 and SW480 cells were treated with doxorubicin (DOX) for 24 h in the presence or absence of USP1 inhibitor ML323, and then evaluated with CCK-8 staining (A) and immunoblotting against cleaved-PARP, USP1, and GAPDH (B) . (C–G) CCK-8 staining was used to evaluate HCT116 cells treated with 20 μM of PARP inhibitor (Olaparib) (C) , 10 μM of Camptothecin (CAM) (D) , 10 μM of etoposide (E) , 10 μM of Amonafide (AMO) (F) , 50 μg/ml of 5-FU (G) for 24 h in the presence or absence of 50 μM ML323. *** p
    Figure Legend Snippet: Ubiquitin specific protease 1 (USP1) inhibitor sensitized colorectal cancer cells to DNA-targeting agents doxorubicin, PARP inhibitor, etoposide, TOPI, and TOPII inhibitor in colorectal cancer cells. (A,B) HCT116 and SW480 cells were treated with doxorubicin (DOX) for 24 h in the presence or absence of USP1 inhibitor ML323, and then evaluated with CCK-8 staining (A) and immunoblotting against cleaved-PARP, USP1, and GAPDH (B) . (C–G) CCK-8 staining was used to evaluate HCT116 cells treated with 20 μM of PARP inhibitor (Olaparib) (C) , 10 μM of Camptothecin (CAM) (D) , 10 μM of etoposide (E) , 10 μM of Amonafide (AMO) (F) , 50 μg/ml of 5-FU (G) for 24 h in the presence or absence of 50 μM ML323. *** p

    Techniques Used: CCK-8 Assay, Staining, Chick Chorioallantoic Membrane Assay

    29) Product Images from "microR-4449 Promotes Colorectal Cancer Cell Proliferation via Regulation of SOCS3 and Activation of STAT3 Signaling"

    Article Title: microR-4449 Promotes Colorectal Cancer Cell Proliferation via Regulation of SOCS3 and Activation of STAT3 Signaling

    Journal: Cancer Management and Research

    doi: 10.2147/CMAR.S266153

    miR-4449 regulated STAT3 signaling via targeting SOCS3. ( A, B ) The SOCS3 protein expression was detected in HCT116 ( A ) and SW480 ( B ) cells with transfection of control siRNA or SOCS3 siRNA. ( C ) Protein expression of p-STAT3, STAT3 and β-actin was detected in HCT116 cells with transfection of miR-NC or miR-4449 inhibitor in combination with control siRNA or SOCS3 siRNA by Western blotting. ( D ) The mRNA levels of VEGF, c-Myc and BIRC5 were detected in HCT116 cells with transfection of miR-NC or miR-4449 inhibitor in combination with control siRNA or SOCS3 siRNA by RT-qPCR. ( E ) Protein expression of p-STAT3, STAT3 and β-actin was detected in SW480 cells with transfection of miR-NC or miR-4449 inhibitor in combination with control siRNA or SOCS3 siRNA by Western blotting. ( F ) The mRNA levels of VEGF, c-Myc and BIRC5 were detected in SW480 cells with transfection of miR-NC or miR-4449 inhibitor in combination with control siRNA or SOCS3 siRNA by RT-qPCR. * vs control p
    Figure Legend Snippet: miR-4449 regulated STAT3 signaling via targeting SOCS3. ( A, B ) The SOCS3 protein expression was detected in HCT116 ( A ) and SW480 ( B ) cells with transfection of control siRNA or SOCS3 siRNA. ( C ) Protein expression of p-STAT3, STAT3 and β-actin was detected in HCT116 cells with transfection of miR-NC or miR-4449 inhibitor in combination with control siRNA or SOCS3 siRNA by Western blotting. ( D ) The mRNA levels of VEGF, c-Myc and BIRC5 were detected in HCT116 cells with transfection of miR-NC or miR-4449 inhibitor in combination with control siRNA or SOCS3 siRNA by RT-qPCR. ( E ) Protein expression of p-STAT3, STAT3 and β-actin was detected in SW480 cells with transfection of miR-NC or miR-4449 inhibitor in combination with control siRNA or SOCS3 siRNA by Western blotting. ( F ) The mRNA levels of VEGF, c-Myc and BIRC5 were detected in SW480 cells with transfection of miR-NC or miR-4449 inhibitor in combination with control siRNA or SOCS3 siRNA by RT-qPCR. * vs control p

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

    miR-4449 was aberrantly expressed in colorectal cancer. ( A ). The expression of miR-4449 was analyzed in microarray data of GSE115513 (411 colon tumors and 381 normal colon mucosa). ( B ). The expression of miR-4449 in 50 pairs of colorectal tumors and the matched normal tissues were detected by RT-qPCR. ( C ). Comparison of miR-4449 expression in normal colon tissues and colorectal tumors of different stages (Stage 1, Stage 2, Stage 3 and Stage 4). ( D ). The expression of miR-4449 in the immortalized colon cell line NCM460 and colorectal cancer cell lines HCT116, SW480 and HT29 was detected by RT-qPCR. **p
    Figure Legend Snippet: miR-4449 was aberrantly expressed in colorectal cancer. ( A ). The expression of miR-4449 was analyzed in microarray data of GSE115513 (411 colon tumors and 381 normal colon mucosa). ( B ). The expression of miR-4449 in 50 pairs of colorectal tumors and the matched normal tissues were detected by RT-qPCR. ( C ). Comparison of miR-4449 expression in normal colon tissues and colorectal tumors of different stages (Stage 1, Stage 2, Stage 3 and Stage 4). ( D ). The expression of miR-4449 in the immortalized colon cell line NCM460 and colorectal cancer cell lines HCT116, SW480 and HT29 was detected by RT-qPCR. **p

    Techniques Used: Expressing, Microarray, Quantitative RT-PCR

    miR-4449 regulated SOCS3 expression in colorectal cancer cells. ( A, B ). The SOCS3 protein expression was detected in HCT116 ( A ) and SW480 ( B ) cells with transfection of miR-NC or miR-4449 inhibitor. ( C ). The SOCS3 mRNA levels were detected in HCT116 and SW480 cells with transfection of miR-NC or miR-4449 inhibitor. ( D ). The miR-4449 levels were detected in HCT116 and SW480 cells with transfection of miR-NC or miR-4449 mimic. ( E, F ). SOCS3, p-STAT3 and STAT3 protein expression were detected in HCT116 ( E ) and SW480 ( F ) cells with transfection of miR-NC or miR-4449 mimic. ( G ). The sequence alignment among miR-4449, SOCS3 3ʹUTR wild type and SOCS3 3ʹUTR mutant (3ʹUTR-M) was presented. ( H ). The luciferase activity was detected in HCT116 and SW480 cells with transfection of miR-NC or miR-4449 mimic in combination with SOCS3 3ʹUTR. ( I ). The luciferase activity was detected in HCT116 and SW480 cells with transfection of miR-NC or miR-4449 mimic in combination with SOCS3 3ʹUTR-M. **p
    Figure Legend Snippet: miR-4449 regulated SOCS3 expression in colorectal cancer cells. ( A, B ). The SOCS3 protein expression was detected in HCT116 ( A ) and SW480 ( B ) cells with transfection of miR-NC or miR-4449 inhibitor. ( C ). The SOCS3 mRNA levels were detected in HCT116 and SW480 cells with transfection of miR-NC or miR-4449 inhibitor. ( D ). The miR-4449 levels were detected in HCT116 and SW480 cells with transfection of miR-NC or miR-4449 mimic. ( E, F ). SOCS3, p-STAT3 and STAT3 protein expression were detected in HCT116 ( E ) and SW480 ( F ) cells with transfection of miR-NC or miR-4449 mimic. ( G ). The sequence alignment among miR-4449, SOCS3 3ʹUTR wild type and SOCS3 3ʹUTR mutant (3ʹUTR-M) was presented. ( H ). The luciferase activity was detected in HCT116 and SW480 cells with transfection of miR-NC or miR-4449 mimic in combination with SOCS3 3ʹUTR. ( I ). The luciferase activity was detected in HCT116 and SW480 cells with transfection of miR-NC or miR-4449 mimic in combination with SOCS3 3ʹUTR-M. **p

    Techniques Used: Expressing, Transfection, Sequencing, Mutagenesis, Luciferase, Activity Assay

    miR-4449 regulated STAT3 pathway in colorectal cancer cells. ( A ). The expression of miR-4449 in HCT116 and SW480 cells with transfection of miR-NC or miR-4449 inhibitor. ( B–C ). Protein expression of p-STAT3, STAT3 and β-actin were detected by Western blotting in HCT116 ( B ) and SW480 ( C ) cells with transfection of miR-NC or miR-4449 inhibitor. ( D, E ). The mRNA levels of VEGF, c-Myc and BIRC5 were detected in HCT116 ( D ) and SW480 ( E ) cells with transfection of miR-NC or miR-4449 inhibitor by RT-qPCR. **p
    Figure Legend Snippet: miR-4449 regulated STAT3 pathway in colorectal cancer cells. ( A ). The expression of miR-4449 in HCT116 and SW480 cells with transfection of miR-NC or miR-4449 inhibitor. ( B–C ). Protein expression of p-STAT3, STAT3 and β-actin were detected by Western blotting in HCT116 ( B ) and SW480 ( C ) cells with transfection of miR-NC or miR-4449 inhibitor. ( D, E ). The mRNA levels of VEGF, c-Myc and BIRC5 were detected in HCT116 ( D ) and SW480 ( E ) cells with transfection of miR-NC or miR-4449 inhibitor by RT-qPCR. **p

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

    The miR-4449/SOCS3 axis regulated colorectal cancer cell proliferation and apoptosis. ( A, B ) The CCK-8 assay was used to detect cell proliferation of HCT116 ( A ) and SW480 ( B ) cells with transfection of miR-NC or miR-4449 inhibitor in combination with control siRNA or SOCS3 siRNA. C-D. The flow cytometry was used to detect cell apoptosis of HCT116 ( C ) and SW480 ( D ) cells with transfection of miR-NC or miR-4449 inhibitor in combination with control siRNA or SOCS3 siRNA. *** vs control p
    Figure Legend Snippet: The miR-4449/SOCS3 axis regulated colorectal cancer cell proliferation and apoptosis. ( A, B ) The CCK-8 assay was used to detect cell proliferation of HCT116 ( A ) and SW480 ( B ) cells with transfection of miR-NC or miR-4449 inhibitor in combination with control siRNA or SOCS3 siRNA. C-D. The flow cytometry was used to detect cell apoptosis of HCT116 ( C ) and SW480 ( D ) cells with transfection of miR-NC or miR-4449 inhibitor in combination with control siRNA or SOCS3 siRNA. *** vs control p

    Techniques Used: CCK-8 Assay, Transfection, Flow Cytometry

    30) Product Images from "Systematic Identification of MACC1-Driven Metabolic Networks in Colorectal Cancer"

    Article Title: Systematic Identification of MACC1-Driven Metabolic Networks in Colorectal Cancer

    Journal: Cancers

    doi: 10.3390/cancers13050978

    Surface GLUT1 is enhanced in MACC1 high cells. ( A ) MACC1 wt. promoter activity or activity with indicated mutated Sp1, Ap1, or cEBP binding sites was measured in HCT116 cells, treated with 0 mM, 2 mM and 10 mM glucose. ( B ) MACC1 mRNA and protein expression in SW620, HCT116, HT29 and HCT15 cells treated with 0 mM, 2 mM and 10 mM glucose. ( C ) Cell viability of SW620 shcntl and MACC1 shMACC1 (knockdown) cells treated with 0 mM, 2 mM and 10 mM of glucose. ( D , E ) GC-MS screening of growth medium metabolites after 5 days of culture of SW620 shcntl and SW620 shMACC1 cells grown in basal medium (supplemented with 10 mM glucose and 2 mM glutamine). ( D ) Levels of top differential metabolites, expressed relative to day 0 with fold-change > 2 and p
    Figure Legend Snippet: Surface GLUT1 is enhanced in MACC1 high cells. ( A ) MACC1 wt. promoter activity or activity with indicated mutated Sp1, Ap1, or cEBP binding sites was measured in HCT116 cells, treated with 0 mM, 2 mM and 10 mM glucose. ( B ) MACC1 mRNA and protein expression in SW620, HCT116, HT29 and HCT15 cells treated with 0 mM, 2 mM and 10 mM glucose. ( C ) Cell viability of SW620 shcntl and MACC1 shMACC1 (knockdown) cells treated with 0 mM, 2 mM and 10 mM of glucose. ( D , E ) GC-MS screening of growth medium metabolites after 5 days of culture of SW620 shcntl and SW620 shMACC1 cells grown in basal medium (supplemented with 10 mM glucose and 2 mM glutamine). ( D ) Levels of top differential metabolites, expressed relative to day 0 with fold-change > 2 and p

    Techniques Used: Activity Assay, Binding Assay, Expressing, Gas Chromatography-Mass Spectrometry

    MACC1 impact on glutamine use. ( A ) Schematic representation of metabolic pathways fueled by indicated nutrients and workflow outline for identification of MACC1-dependent nutrient conditions influencing cell proliferation. MACC1-dependent cell proliferation is investigated under different nutrient conditions. Conditions by which MACC1 does not increase proliferation are considered to be negative hits. Conditions by which MACC1 supports cell proliferation are considered positive hits, for which then further uptake and 13 C labeled substrate studies are performed. Depending on the results of uptake and 13 C studies, targets and their drugs are selected and applied to positive hit conditions in vitro, followed by animal experiments. Cell viability of SW620 shcntl and SW620 shMACC1 cells treated with various glutamine concentrations ( B ) in high, ( C ) in low and ( D ) in no glucose conditions. ( E – G ) Cell viability of HCT116 shcntl and HCT116 shMACC1 cells treated with various glutamine concentrations ( E ) in high, ( F ) in low and ( G ) in no glucose conditions. Glutamine depletion from cell growth medium of SW620 shcntl and SW620 shMACC1 cells after ( H ) 5 days of culture in nutrient replete medium and ( I ) after 48 h of culture at indicated conditions. ( J ) Cell viability of SW620 shcntl and SW620 shMACC1 cells grown in basal medium and treated with DON, an inhibitor of glutamine-using enzymes. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Data represent mean values ± SEM of at least three independent experiments.
    Figure Legend Snippet: MACC1 impact on glutamine use. ( A ) Schematic representation of metabolic pathways fueled by indicated nutrients and workflow outline for identification of MACC1-dependent nutrient conditions influencing cell proliferation. MACC1-dependent cell proliferation is investigated under different nutrient conditions. Conditions by which MACC1 does not increase proliferation are considered to be negative hits. Conditions by which MACC1 supports cell proliferation are considered positive hits, for which then further uptake and 13 C labeled substrate studies are performed. Depending on the results of uptake and 13 C studies, targets and their drugs are selected and applied to positive hit conditions in vitro, followed by animal experiments. Cell viability of SW620 shcntl and SW620 shMACC1 cells treated with various glutamine concentrations ( B ) in high, ( C ) in low and ( D ) in no glucose conditions. ( E – G ) Cell viability of HCT116 shcntl and HCT116 shMACC1 cells treated with various glutamine concentrations ( E ) in high, ( F ) in low and ( G ) in no glucose conditions. Glutamine depletion from cell growth medium of SW620 shcntl and SW620 shMACC1 cells after ( H ) 5 days of culture in nutrient replete medium and ( I ) after 48 h of culture at indicated conditions. ( J ) Cell viability of SW620 shcntl and SW620 shMACC1 cells grown in basal medium and treated with DON, an inhibitor of glutamine-using enzymes. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Data represent mean values ± SEM of at least three independent experiments.

    Techniques Used: Labeling, In Vitro

    31) Product Images from "PRMT5 functionally associates with EZH2 to promote colorectal cancer progression through epigenetically repressing CDKN2B expression"

    Article Title: PRMT5 functionally associates with EZH2 to promote colorectal cancer progression through epigenetically repressing CDKN2B expression

    Journal: Theranostics

    doi: 10.7150/thno.53023

    CDKN2B is a direct transcriptional target of EZH2. ( A ) Immunoblot analysis of CDKN2B (p15 INK4b ) expression in HCT116 and SW480 cells stably expressing control, shEZH2-1 and shEZH2-2. GAPDH was used as loading control. ( B ) Relative mRNA expression of EZH2 was determined by real-time PCR in HCT116 and SW480 cells stably expressing control shRNA (negative control), shEZH2-1 and shEZH2-2. GAPDH was used as an internal control. ** P
    Figure Legend Snippet: CDKN2B is a direct transcriptional target of EZH2. ( A ) Immunoblot analysis of CDKN2B (p15 INK4b ) expression in HCT116 and SW480 cells stably expressing control, shEZH2-1 and shEZH2-2. GAPDH was used as loading control. ( B ) Relative mRNA expression of EZH2 was determined by real-time PCR in HCT116 and SW480 cells stably expressing control shRNA (negative control), shEZH2-1 and shEZH2-2. GAPDH was used as an internal control. ** P

    Techniques Used: Expressing, Stable Transfection, Real-time Polymerase Chain Reaction, shRNA, Negative Control

    PRMT5 epigenetically represses CDKN2B expression. ( A ) Relative mRNA expression of genes involved in the regulation of cell proliferation and cell cycle was determined by real-time PCR in shPRMT5-1 and shPRMT5-2-infected HCT116 and SW480 cells compared with control cells. GAPDH was used as an internal control. * P
    Figure Legend Snippet: PRMT5 epigenetically represses CDKN2B expression. ( A ) Relative mRNA expression of genes involved in the regulation of cell proliferation and cell cycle was determined by real-time PCR in shPRMT5-1 and shPRMT5-2-infected HCT116 and SW480 cells compared with control cells. GAPDH was used as an internal control. * P

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

    Depletion of CDKN2B expression abrogates the proliferation inhibition caused by PRMT5 knockdown. ( A ) Immunoblot analysis of PRMT5 and CDKN2B (p15 INK4b ) expression in HCT116 and SW480 cells treated with control shRNA (negative control), shPRMT5 and shPRMT5 + shCDKN2B. GAPDH was used as loading control. ( B ) Cell proliferation of HCT116 and SW480 cells infected with control shRNA, shPRMT5 and shPRMT5 + shCDKN2B was evaluated by CCK-8 assays at the same time point of each day. ** P
    Figure Legend Snippet: Depletion of CDKN2B expression abrogates the proliferation inhibition caused by PRMT5 knockdown. ( A ) Immunoblot analysis of PRMT5 and CDKN2B (p15 INK4b ) expression in HCT116 and SW480 cells treated with control shRNA (negative control), shPRMT5 and shPRMT5 + shCDKN2B. GAPDH was used as loading control. ( B ) Cell proliferation of HCT116 and SW480 cells infected with control shRNA, shPRMT5 and shPRMT5 + shCDKN2B was evaluated by CCK-8 assays at the same time point of each day. ** P

    Techniques Used: Expressing, Inhibition, shRNA, Negative Control, Infection, CCK-8 Assay

    PRMT5 associates with EZH2 in CRC cells. ( A ) Co-immunoprecipitation of endogenous EZH2 from HCT116 and SW480 cells overexpressing Flag-tagged PRMT5. IgG was used as the negative control. ( B ) Western blot analysis of EZH2 binding to purified GST and GST-tagged PRMT5 using EZH2 antibody (top). GST and GST-tagged PRMT5 from E. coli BL21 (DE3) strain were visualized by staining with Coomassie brilliant blue R-250 (bottom). The red asterisk and black asterisk were GST and GST-tagged PRMT5, respectively. ( C ) The subcellular location of endogenous PRMT5 and EZH2 proteins was analyzed in HCT116 and SW480 cells by immunofluorescence microscopy. Scale bar: 10 µm.
    Figure Legend Snippet: PRMT5 associates with EZH2 in CRC cells. ( A ) Co-immunoprecipitation of endogenous EZH2 from HCT116 and SW480 cells overexpressing Flag-tagged PRMT5. IgG was used as the negative control. ( B ) Western blot analysis of EZH2 binding to purified GST and GST-tagged PRMT5 using EZH2 antibody (top). GST and GST-tagged PRMT5 from E. coli BL21 (DE3) strain were visualized by staining with Coomassie brilliant blue R-250 (bottom). The red asterisk and black asterisk were GST and GST-tagged PRMT5, respectively. ( C ) The subcellular location of endogenous PRMT5 and EZH2 proteins was analyzed in HCT116 and SW480 cells by immunofluorescence microscopy. Scale bar: 10 µm.

    Techniques Used: Immunoprecipitation, Negative Control, Western Blot, Binding Assay, Purification, Staining, Immunofluorescence, Microscopy

    Synergistic effect of combined treatment with PRMT5i and EZH2i. (A) Drug dose-response matrix for proliferation inhibition of HCT116 and SW480 cells treated with PRMT5i (GSK591) and EZH2i (GSK126). Color gradation indicates percentage viability at the indicated dose combination. Combination index (CI) plots for 100 nM PRMT5i (GSK591) (top) or EZH2i (GSK126) (bottom) with graded doses of EZH2i (GSK126) or PRMT5i (GSK591) in HCT116 and SW480 cells. ( B ) Immunoblots of PRMT5, EZH2 and CDKN2B (p15 INK4b ) protein levels in HCT116 and SW480 cells following treatment with PRMT5i (GSK591; 100 nM), EZH2i (GSK126; 100 nM), or PRMT5i (GSK591; 100 nM) + EZH2i (GSK126; 100 nM). GAPDH was used as a loading control. ( C ) Cell proliferation of HCT116 and SW480 cells treated with PRMT5i (GSK591; 100 nM), EZH2i (GSK126; 100 nM), or PRMT5i (GSK591; 100 nM) + EZH2i (GSK126; 100 nM) by CCK-8 assays at the same time point of each day. ** P
    Figure Legend Snippet: Synergistic effect of combined treatment with PRMT5i and EZH2i. (A) Drug dose-response matrix for proliferation inhibition of HCT116 and SW480 cells treated with PRMT5i (GSK591) and EZH2i (GSK126). Color gradation indicates percentage viability at the indicated dose combination. Combination index (CI) plots for 100 nM PRMT5i (GSK591) (top) or EZH2i (GSK126) (bottom) with graded doses of EZH2i (GSK126) or PRMT5i (GSK591) in HCT116 and SW480 cells. ( B ) Immunoblots of PRMT5, EZH2 and CDKN2B (p15 INK4b ) protein levels in HCT116 and SW480 cells following treatment with PRMT5i (GSK591; 100 nM), EZH2i (GSK126; 100 nM), or PRMT5i (GSK591; 100 nM) + EZH2i (GSK126; 100 nM). GAPDH was used as a loading control. ( C ) Cell proliferation of HCT116 and SW480 cells treated with PRMT5i (GSK591; 100 nM), EZH2i (GSK126; 100 nM), or PRMT5i (GSK591; 100 nM) + EZH2i (GSK126; 100 nM) by CCK-8 assays at the same time point of each day. ** P

    Techniques Used: Inhibition, Western Blot, CCK-8 Assay

    PRMT5 depletion retards CRC cell proliferation in vitro . ( A ) Immunoblot analysis of PRMT5 expression in control, shPRMT5-1 and shPRMT5-2-infected HCT116 and SW480 cells. GAPDH was used as loading control. ( B ) Cell proliferation of HCT116 and SW480 cells treated with control, shPRMT5-1 and shPRMT5-2 was evaluated by CCK-8 assay at the same time point of each day. ** P
    Figure Legend Snippet: PRMT5 depletion retards CRC cell proliferation in vitro . ( A ) Immunoblot analysis of PRMT5 expression in control, shPRMT5-1 and shPRMT5-2-infected HCT116 and SW480 cells. GAPDH was used as loading control. ( B ) Cell proliferation of HCT116 and SW480 cells treated with control, shPRMT5-1 and shPRMT5-2 was evaluated by CCK-8 assay at the same time point of each day. ** P

    Techniques Used: In Vitro, Expressing, Infection, CCK-8 Assay

    32) Product Images from "The research on the mechanism of Tsoong inhibiting for colon cancer"

    Article Title: The research on the mechanism of Tsoong inhibiting for colon cancer

    Journal: Saudi Journal of Biological Sciences

    doi: 10.1016/j.sjbs.2018.11.007

    Effect of Tsoong on the proliferation and cell cycle of HCT116 and SW480 cancer cells. The cells were treated with Tsoong-containing serum solutions prepared from mice treated by gastrogavage with saline (control), or 5 (low), 10 (mid) or 20 (high) g/kg of Tsoong for indicated durations. (A) cell proliferation was detected using a cell counting kit‐8 assay, and (B) cell cycle was detected using flow cytometry. (C) Cell apoptosis was analyzed using flow cytometry. Oxaliplatin was used as a control. P
    Figure Legend Snippet: Effect of Tsoong on the proliferation and cell cycle of HCT116 and SW480 cancer cells. The cells were treated with Tsoong-containing serum solutions prepared from mice treated by gastrogavage with saline (control), or 5 (low), 10 (mid) or 20 (high) g/kg of Tsoong for indicated durations. (A) cell proliferation was detected using a cell counting kit‐8 assay, and (B) cell cycle was detected using flow cytometry. (C) Cell apoptosis was analyzed using flow cytometry. Oxaliplatin was used as a control. P

    Techniques Used: Mouse Assay, Cell Counting, Flow Cytometry, Cytometry

    33) Product Images from "MiR-216b functions as a tumor suppressor by targeting HMGB1-mediated JAK2/STAT3 signaling way in colorectal cancer"

    Article Title: MiR-216b functions as a tumor suppressor by targeting HMGB1-mediated JAK2/STAT3 signaling way in colorectal cancer

    Journal: American Journal of Cancer Research

    doi:

    Upregulation of HMGB1 reversed the tumor suppressive effect of miR-216b in CRC. (A) HMGB1 protein expression was assessed in HCT116 and HT29 cells transfected with mimic and pcDNA3.1-HMGB1 or blank vector. (B-D) Cell proliferation, migration, invasion and angiogenesis were detected in HCT116 and HT29 cells transfected with mimic and pcDNA3.1-HMGB1 or blank vector by CCK-8 (B), wound healing (C), transwell invasion (D) and tube formation (E) assays. * P
    Figure Legend Snippet: Upregulation of HMGB1 reversed the tumor suppressive effect of miR-216b in CRC. (A) HMGB1 protein expression was assessed in HCT116 and HT29 cells transfected with mimic and pcDNA3.1-HMGB1 or blank vector. (B-D) Cell proliferation, migration, invasion and angiogenesis were detected in HCT116 and HT29 cells transfected with mimic and pcDNA3.1-HMGB1 or blank vector by CCK-8 (B), wound healing (C), transwell invasion (D) and tube formation (E) assays. * P

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Migration, CCK-8 Assay

    miR-216b promoted CRC malignant progression through JAK2/STAT3 signaling pathway. A. Representative western blotting results for JAK2, p-JAK2, STAT3 and p-STAT3 from HCT116 and HT29 cells transfected with mimic or mimic-NC, respectively. B. Representative western blotting results for JAK2, p-JAK2, STAT3 and p-STAT3 from HCT116 and HT29 cells transfected with mimic and pcDNA3.1-HMGB1 or blank vector, respectively. ** P
    Figure Legend Snippet: miR-216b promoted CRC malignant progression through JAK2/STAT3 signaling pathway. A. Representative western blotting results for JAK2, p-JAK2, STAT3 and p-STAT3 from HCT116 and HT29 cells transfected with mimic or mimic-NC, respectively. B. Representative western blotting results for JAK2, p-JAK2, STAT3 and p-STAT3 from HCT116 and HT29 cells transfected with mimic and pcDNA3.1-HMGB1 or blank vector, respectively. ** P

    Techniques Used: Western Blot, Transfection, Plasmid Preparation

    (A) Predicted miR-216b target sequence in HMGB1 3’-UTR was shown. (B) The predicted binding sites for miR-216b in the 3’-UTR of HMGB1 and the mutations in the binding sites are shown. (C) HT29 cells were cotransfected with miR-216b mimic or mimic-NC and wild-type (WT) or mutant-type (MT) HMGB1 3’-UTR reporter plasmid. Luciferase activity was measured 48 h after transfection. (D, E) HMGB1 mRNA (D) and protein (E) expression levels were measured in HCT116 and HT29 cells after transfection with miR-216b mimic or mimic-NC by qRT-PCR and western blot, respectively. β-actin was used as internal control, ** P
    Figure Legend Snippet: (A) Predicted miR-216b target sequence in HMGB1 3’-UTR was shown. (B) The predicted binding sites for miR-216b in the 3’-UTR of HMGB1 and the mutations in the binding sites are shown. (C) HT29 cells were cotransfected with miR-216b mimic or mimic-NC and wild-type (WT) or mutant-type (MT) HMGB1 3’-UTR reporter plasmid. Luciferase activity was measured 48 h after transfection. (D, E) HMGB1 mRNA (D) and protein (E) expression levels were measured in HCT116 and HT29 cells after transfection with miR-216b mimic or mimic-NC by qRT-PCR and western blot, respectively. β-actin was used as internal control, ** P

    Techniques Used: Sequencing, Binding Assay, Mutagenesis, Plasmid Preparation, Luciferase, Activity Assay, Transfection, Expressing, Quantitative RT-PCR, Western Blot

    (A, B) HMGB1 protein (A) and mRNA (B) expression levels were analyzed in HCT116, HCT8, HT29 and FHC by qRT-PCR, * P
    Figure Legend Snippet: (A, B) HMGB1 protein (A) and mRNA (B) expression levels were analyzed in HCT116, HCT8, HT29 and FHC by qRT-PCR, * P

    Techniques Used: Expressing, Quantitative RT-PCR

    (A) Relative miR-216b levels were assessed in HCT116 and HT29 cells after transfection with miR-216b mimic or mimic-NC. miR-216b inhibits proliferation, migration, invasion and angiogenesis in CRC cells. (B-E) Overexpression of miR-216b significantly reduced proliferation (B), migration (C), invasion (D) and angiogenesis (E) in HCT116 and HT29 cells. ** P
    Figure Legend Snippet: (A) Relative miR-216b levels were assessed in HCT116 and HT29 cells after transfection with miR-216b mimic or mimic-NC. miR-216b inhibits proliferation, migration, invasion and angiogenesis in CRC cells. (B-E) Overexpression of miR-216b significantly reduced proliferation (B), migration (C), invasion (D) and angiogenesis (E) in HCT116 and HT29 cells. ** P

    Techniques Used: Transfection, Migration, Over Expression

    34) Product Images from "Long noncoding RNA MAPKAPK5-AS1 promotes colorectal cancer progression by cis-regulating the nearby gene MK5 and acting as a let-7f-1-3p sponge"

    Article Title: Long noncoding RNA MAPKAPK5-AS1 promotes colorectal cancer progression by cis-regulating the nearby gene MK5 and acting as a let-7f-1-3p sponge

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-020-01633-8

    MK5 upregulated SNAI1 expression by phosphorylating c-Jun. a. Immunoblotting was confirmed the protein levels of c-Jun, p-c-Jun(S63) and SNAI1 after MK5 overexpression and silencing in HCT116 and SW620 cells. b. Immunoblotting analysis of MK5, c-Jun, p-c-Jun(S63) and SNAI1 after MK5-AS1 overexpression and knockdown in HCT116 and SW620 cells. c. Coimmunoprecipitation was used to identify interaction between MK5 and c-Jun in HCT116 cells. d. After cotransfection with pcDNA3.1 MK5, si-c-Jun or control siRNA, the proteins levels of MK5, c-Jun, p-c-Jun (S63) and SNAI1 were determined by immunoblotting. e. Immunoblotting of MK5, c-Jun, p-c-Jun(S63) and SNAI1 proteins levels from pcDNA3.1 MK5, pcDNA3.1 c-Jun, pcDNA3.1 c-Jun (S63A) transfection samples treated with DMSO or c-Jun phosphorylation inhibitor SR11302 in HCT116 and SW620 cells. f. Dual luciferase reporter plasmids containing SNAI1 promoter or pGL3 basic were co-transfected into HCT116 cells with c-Jun or mutant c-Jun (S63A) in parallel. g. Dual luciferase reporter plasmids of SNAI1 promoter were designed to contain c-Jun binding sequences or not. h. Dual luciferase reporter plasmids containing SNAI1 promoter, SNAI1 promoter MUT or pGL3 basic were cotransfected into HCT116 cells with c-Jun plasmid in parallel. i. Identification of the c-Jun binding sequences in SNAI1 promoters by ChIP-PCR. Data were shown as mean ± SD for three independent experiments. * P
    Figure Legend Snippet: MK5 upregulated SNAI1 expression by phosphorylating c-Jun. a. Immunoblotting was confirmed the protein levels of c-Jun, p-c-Jun(S63) and SNAI1 after MK5 overexpression and silencing in HCT116 and SW620 cells. b. Immunoblotting analysis of MK5, c-Jun, p-c-Jun(S63) and SNAI1 after MK5-AS1 overexpression and knockdown in HCT116 and SW620 cells. c. Coimmunoprecipitation was used to identify interaction between MK5 and c-Jun in HCT116 cells. d. After cotransfection with pcDNA3.1 MK5, si-c-Jun or control siRNA, the proteins levels of MK5, c-Jun, p-c-Jun (S63) and SNAI1 were determined by immunoblotting. e. Immunoblotting of MK5, c-Jun, p-c-Jun(S63) and SNAI1 proteins levels from pcDNA3.1 MK5, pcDNA3.1 c-Jun, pcDNA3.1 c-Jun (S63A) transfection samples treated with DMSO or c-Jun phosphorylation inhibitor SR11302 in HCT116 and SW620 cells. f. Dual luciferase reporter plasmids containing SNAI1 promoter or pGL3 basic were co-transfected into HCT116 cells with c-Jun or mutant c-Jun (S63A) in parallel. g. Dual luciferase reporter plasmids of SNAI1 promoter were designed to contain c-Jun binding sequences or not. h. Dual luciferase reporter plasmids containing SNAI1 promoter, SNAI1 promoter MUT or pGL3 basic were cotransfected into HCT116 cells with c-Jun plasmid in parallel. i. Identification of the c-Jun binding sequences in SNAI1 promoters by ChIP-PCR. Data were shown as mean ± SD for three independent experiments. * P

    Techniques Used: Expressing, Over Expression, Cotransfection, Transfection, Luciferase, Mutagenesis, Binding Assay, Plasmid Preparation, Chromatin Immunoprecipitation, Polymerase Chain Reaction

    MK5-AS1 negatively regulated let-7f-1-3p expression. a. The possible binding sites among MK5-AS1 and microRNAs were predicted by DIANA Tools and miRcode. b. The expressions of miR-1284 and let-7f-1-3p were detected by qPCR in HCT116 and SW620 cells after intervening MK5-AS1, respectively. c. Let-7f-1-3p was downregulated in 42 pairs of CRC tissues compared with normal tissues. d. Correlation analysis of the expression of MK5-AS1 and let-7f-1-3p in 42 pairs of CRC tissues. e. Upper panel, the potential binding sites between MK5-AS1 and let-7f-1-3p. Lower panel, the luciferase reporter plasmids containing wild type (WT) or mutant (MUT) MK5-AS1 were cotransfected into HCT116 cells with let-7f-1-3p. f, g. CCK8 assays and colony formations were used to determine the proliferation of HCT116 and SW620 after transfection. The data represented the mean ± SD from three independent experiments. * P
    Figure Legend Snippet: MK5-AS1 negatively regulated let-7f-1-3p expression. a. The possible binding sites among MK5-AS1 and microRNAs were predicted by DIANA Tools and miRcode. b. The expressions of miR-1284 and let-7f-1-3p were detected by qPCR in HCT116 and SW620 cells after intervening MK5-AS1, respectively. c. Let-7f-1-3p was downregulated in 42 pairs of CRC tissues compared with normal tissues. d. Correlation analysis of the expression of MK5-AS1 and let-7f-1-3p in 42 pairs of CRC tissues. e. Upper panel, the potential binding sites between MK5-AS1 and let-7f-1-3p. Lower panel, the luciferase reporter plasmids containing wild type (WT) or mutant (MUT) MK5-AS1 were cotransfected into HCT116 cells with let-7f-1-3p. f, g. CCK8 assays and colony formations were used to determine the proliferation of HCT116 and SW620 after transfection. The data represented the mean ± SD from three independent experiments. * P

    Techniques Used: Expressing, Binding Assay, Real-time Polymerase Chain Reaction, Luciferase, Mutagenesis, Transfection

    MK5-AS1/let-7f-1-3p/SNAI1 ceRNA network. a. Transwell assays were used to perform the invasion and migration abilities in HCT116 and SW620 cells after transfection. Scale bar, 100 μm for A. b. The potential binding sites among MK5-AS1, let-7f-1-3p and SNAI1. c. Upper panel, the potential binding sites between let-7f-1-3p and SNAI1. Lower panel, the luciferase reporter plasmids containing wild type (WT) or mutant (MUT) SNAI1 3′ UTR were cotransfected into HCT116 cells with let-7f-1-3p. d. MK5, c-Jun, p-c-Jun(S63) and SNAI1 expressions were detected in HCT116 and SW620 cells by immunoblotting after transfection with let-7f-1-3p inhibitor or let-7f-1-3p mimics. e. Left panel, the effects of si-MK5-AS1, let-7f-1-3p inhibitor and si-MK5-AS1 + let-7f-1-3p inhibitor on protein levels of SNAI1 in HCT116 and SW620 cells. Right panel, the effects of pcDNA3.1 MK5-AS1, let-7f-1-3p mimics, and pcDNA3.1 MK5-AS1 + let-7f-1-3p mimics on protein levels of SNAI1 in HCT116 and SW620 cells. Data were shown as mean ± SD for three independent experiments. * P
    Figure Legend Snippet: MK5-AS1/let-7f-1-3p/SNAI1 ceRNA network. a. Transwell assays were used to perform the invasion and migration abilities in HCT116 and SW620 cells after transfection. Scale bar, 100 μm for A. b. The potential binding sites among MK5-AS1, let-7f-1-3p and SNAI1. c. Upper panel, the potential binding sites between let-7f-1-3p and SNAI1. Lower panel, the luciferase reporter plasmids containing wild type (WT) or mutant (MUT) SNAI1 3′ UTR were cotransfected into HCT116 cells with let-7f-1-3p. d. MK5, c-Jun, p-c-Jun(S63) and SNAI1 expressions were detected in HCT116 and SW620 cells by immunoblotting after transfection with let-7f-1-3p inhibitor or let-7f-1-3p mimics. e. Left panel, the effects of si-MK5-AS1, let-7f-1-3p inhibitor and si-MK5-AS1 + let-7f-1-3p inhibitor on protein levels of SNAI1 in HCT116 and SW620 cells. Right panel, the effects of pcDNA3.1 MK5-AS1, let-7f-1-3p mimics, and pcDNA3.1 MK5-AS1 + let-7f-1-3p mimics on protein levels of SNAI1 in HCT116 and SW620 cells. Data were shown as mean ± SD for three independent experiments. * P

    Techniques Used: Migration, Transfection, Binding Assay, Luciferase, Mutagenesis

    MK5-AS1 regulated CRC invasion and migration in vitro and vivo . a. Transwell assays were used to determine the invasion and migration abilities of HCT116 and SW620 cells after MK5-AS1 overexpression and knockdown. Scale bar, 100 μm for A. b. Immunoblotting of EMT-related markers after transfection in CRC cells. c. Nude mice were injected with HCT116 cell after MK5-AS1 knockdown into tail vein. The number of metastatic nodules of lung was shown and counted. The black arrow marked metastatic nodules. d. Tumor progression was monitored using a small animal imaging system, HE-stained lung sections (Scale bar, 100 μm) and antibody vimentin of metastatic nodules were shown (× 400 magnification). The black arrow marked metastatic nodules. e. The Ensembl Genome browser ( http://asia.ensembl.org/ ) showed that MK5 was the nearby gene of MK5-AS1. f. Immunoblotting was used to investigate the level of MK5 of HCT116 and SW620 cells after intervening MK5-AS1, respectively. The data represented the mean ± SD from three independent experiments. * P
    Figure Legend Snippet: MK5-AS1 regulated CRC invasion and migration in vitro and vivo . a. Transwell assays were used to determine the invasion and migration abilities of HCT116 and SW620 cells after MK5-AS1 overexpression and knockdown. Scale bar, 100 μm for A. b. Immunoblotting of EMT-related markers after transfection in CRC cells. c. Nude mice were injected with HCT116 cell after MK5-AS1 knockdown into tail vein. The number of metastatic nodules of lung was shown and counted. The black arrow marked metastatic nodules. d. Tumor progression was monitored using a small animal imaging system, HE-stained lung sections (Scale bar, 100 μm) and antibody vimentin of metastatic nodules were shown (× 400 magnification). The black arrow marked metastatic nodules. e. The Ensembl Genome browser ( http://asia.ensembl.org/ ) showed that MK5 was the nearby gene of MK5-AS1. f. Immunoblotting was used to investigate the level of MK5 of HCT116 and SW620 cells after intervening MK5-AS1, respectively. The data represented the mean ± SD from three independent experiments. * P

    Techniques Used: Migration, In Vitro, Over Expression, Transfection, Mouse Assay, Injection, Imaging, Staining

    MK5-AS1 recruited RBM4/eIF4A1. a. RNA FISH analysis of the location of MK5-AS1 (red) in the cytoplasm of HCT116 cells. Original magnifications, × 400 for A. b. RNA pull-down assay showed that MK5-AS1 could not retrieve MK5 from HCT116 cells lysates. c, d . Analysis of the interaction propensities between MK5-AS1 and RBM4 by public databases catRAPID. e. RNA pull-down assays followed by immunoblotting showed that MK5-AS1 bound RBM4 and eIF4A1 in HCT116 cells. f. RIP experiments for RBM4 and eIF4A1 were performed and the coprecipitated RNA was subjected to qPCR for MK5-AS1. g. Coimmunoprecipitation was used to identify interaction between RBM4 and eIF4A1 in HCT116 cells. Data were shown as mean ± SD for three independent experiments. * P
    Figure Legend Snippet: MK5-AS1 recruited RBM4/eIF4A1. a. RNA FISH analysis of the location of MK5-AS1 (red) in the cytoplasm of HCT116 cells. Original magnifications, × 400 for A. b. RNA pull-down assay showed that MK5-AS1 could not retrieve MK5 from HCT116 cells lysates. c, d . Analysis of the interaction propensities between MK5-AS1 and RBM4 by public databases catRAPID. e. RNA pull-down assays followed by immunoblotting showed that MK5-AS1 bound RBM4 and eIF4A1 in HCT116 cells. f. RIP experiments for RBM4 and eIF4A1 were performed and the coprecipitated RNA was subjected to qPCR for MK5-AS1. g. Coimmunoprecipitation was used to identify interaction between RBM4 and eIF4A1 in HCT116 cells. Data were shown as mean ± SD for three independent experiments. * P

    Techniques Used: Fluorescence In Situ Hybridization, Pull Down Assay, Real-time Polymerase Chain Reaction

    MK5 promoted CRC cells invasion and migration in vitro and vivo . a. Immunoblotting of MK5 expression in 4 pairs of human CRC tissues (T) and adjacent non-tumor tissues (N). b. Expression of MK5 in the normal colorectal epithelium cell line (FHC) and CRC cells by immunoblotting. c. Statistical analysis of MK5 expression in TCGA database. d. Correlation analysis of the expression of MK5-AS1 and MK5 in GEPIA. e. Transwell assays were used to determine the changes in invasion and migration abilities of HCT116 and SW620 cells after transfection. Scale bar, 100 μm for E. f. Immunoblotting analysis of EMT-related markers after transfection in CRC cells, respectively. g. The number of metastatic pulmonary nodules was shown and counted. The black arrow marked metastatic nodules. h. After HCT116 cells with knockdown MK5 were injected into the tail vein of nude mice, In vivo fluorescence imaging, the gross lesion in lung tissues and H. E staining and antibody vimentin of metastatic nodules in the lungs were observed (× 400 magnification). Scale bar, 100 μm for H.E. The black arrow marked metastatic nodules. The data represented the mean ± SD from three independent experiments. * P
    Figure Legend Snippet: MK5 promoted CRC cells invasion and migration in vitro and vivo . a. Immunoblotting of MK5 expression in 4 pairs of human CRC tissues (T) and adjacent non-tumor tissues (N). b. Expression of MK5 in the normal colorectal epithelium cell line (FHC) and CRC cells by immunoblotting. c. Statistical analysis of MK5 expression in TCGA database. d. Correlation analysis of the expression of MK5-AS1 and MK5 in GEPIA. e. Transwell assays were used to determine the changes in invasion and migration abilities of HCT116 and SW620 cells after transfection. Scale bar, 100 μm for E. f. Immunoblotting analysis of EMT-related markers after transfection in CRC cells, respectively. g. The number of metastatic pulmonary nodules was shown and counted. The black arrow marked metastatic nodules. h. After HCT116 cells with knockdown MK5 were injected into the tail vein of nude mice, In vivo fluorescence imaging, the gross lesion in lung tissues and H. E staining and antibody vimentin of metastatic nodules in the lungs were observed (× 400 magnification). Scale bar, 100 μm for H.E. The black arrow marked metastatic nodules. The data represented the mean ± SD from three independent experiments. * P

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

    35) Product Images from "Loss of CCDC6 Affects Cell Cycle through Impaired Intra-S-Phase Checkpoint Control"

    Article Title: Loss of CCDC6 Affects Cell Cycle through Impaired Intra-S-Phase Checkpoint Control

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0031007

    Normal Cell cycle progression is altered upon CCDC6 knock down. Cell cycle analysis was performed using propidium iodide (PI) staining and measuring the DNA content, at the indicated time points, starting 48 hours after transduction. Cell cycle was analyzed with FlowJo software and Jean-Fox algorithm. In all time points both in HCT116 ( A ), ( B ) and HeLa ( C ), ( D ) the percentage of cells in the S phase is reduced upon knock down of CCDC6 in comparison to the control (mock). One, out of three, representative experiment is shown. ( E ) HCT116 cells were synchronized by serum starvation for 48 hours followed by restimulation with 5% of FCS. CCDC6 knock down resulted in incomplete arrest at G 1 and not total synchronization, as the control cells. 14 hours after serum stimulation the majority of the control cells are in S phase while CCDC6 knock down cells demonstrated a delay in S phase entering. 4 hours later, control and CCDC6 knock down cells showed the same profile, suggesting shorter duration of S upon CCDC6 knock down. 24 hours later, control cells were cycling normally and CCDC6 knock down cells exhibited a delay in completing G 2 phase and re-entering G 1 .
    Figure Legend Snippet: Normal Cell cycle progression is altered upon CCDC6 knock down. Cell cycle analysis was performed using propidium iodide (PI) staining and measuring the DNA content, at the indicated time points, starting 48 hours after transduction. Cell cycle was analyzed with FlowJo software and Jean-Fox algorithm. In all time points both in HCT116 ( A ), ( B ) and HeLa ( C ), ( D ) the percentage of cells in the S phase is reduced upon knock down of CCDC6 in comparison to the control (mock). One, out of three, representative experiment is shown. ( E ) HCT116 cells were synchronized by serum starvation for 48 hours followed by restimulation with 5% of FCS. CCDC6 knock down resulted in incomplete arrest at G 1 and not total synchronization, as the control cells. 14 hours after serum stimulation the majority of the control cells are in S phase while CCDC6 knock down cells demonstrated a delay in S phase entering. 4 hours later, control and CCDC6 knock down cells showed the same profile, suggesting shorter duration of S upon CCDC6 knock down. 24 hours later, control cells were cycling normally and CCDC6 knock down cells exhibited a delay in completing G 2 phase and re-entering G 1 .

    Techniques Used: Cell Cycle Assay, Staining, Transduction, Software

    CCDC6 knock down results in altered cellular localization of CDC25C and accelerated G 2 /S transition upon etoposide-mediated genotoxic stress. Control and CCDC6 knock down HCT116 cells were treated with etoposide (20 µM). ( A ) Cell lysates of HCT116 cells treated with etoposide (20 µM) for 2, 4, 8, 12 and 24 hours and mock control treated with DMSO vehicle were resolved on a SDS-PAGE and probed for 14-3-3σ and CDC25C. 14-3-3σ protein levels were down-regulated in the absence of CCDC6 protein expression and the CDC25C protein level regulation was altered. ( B ) Cells grown on cover slips were exposed to etoposide for 4, 8, 12, 24 hours, fixed and stained for CDC25C. In mock cells, CDC25C is kept in the cytosol upon etoposide treatment at 8 and 12 hours but is localized in the nucleus in the absence of CCDC6. ( C ) Cells exposed to etoposide for 12 hours were co-stained for CDC25C and 14-3-3σ. CDC25C is kept in the cytosol upon etoposide treatment and exhibits co-localization with 14-3-3σ (seen in yellow) but enters the nucleus in the absence of CCDC6.
    Figure Legend Snippet: CCDC6 knock down results in altered cellular localization of CDC25C and accelerated G 2 /S transition upon etoposide-mediated genotoxic stress. Control and CCDC6 knock down HCT116 cells were treated with etoposide (20 µM). ( A ) Cell lysates of HCT116 cells treated with etoposide (20 µM) for 2, 4, 8, 12 and 24 hours and mock control treated with DMSO vehicle were resolved on a SDS-PAGE and probed for 14-3-3σ and CDC25C. 14-3-3σ protein levels were down-regulated in the absence of CCDC6 protein expression and the CDC25C protein level regulation was altered. ( B ) Cells grown on cover slips were exposed to etoposide for 4, 8, 12, 24 hours, fixed and stained for CDC25C. In mock cells, CDC25C is kept in the cytosol upon etoposide treatment at 8 and 12 hours but is localized in the nucleus in the absence of CCDC6. ( C ) Cells exposed to etoposide for 12 hours were co-stained for CDC25C and 14-3-3σ. CDC25C is kept in the cytosol upon etoposide treatment and exhibits co-localization with 14-3-3σ (seen in yellow) but enters the nucleus in the absence of CCDC6.

    Techniques Used: SDS Page, Expressing, Staining

    Knock down of CCDC6 increases vulnerability to genotoxic stress by UV induced DNA damage. HCT116 cells were transduced with CCDC6 shRNA expressing lentivirus or empty control followed by UV irradiation (0.002 J/cm 2 ), 48 hours after transduction. Cells were harvested at 2, 6 and 24 hours after irradiation and analyzed for the cell cycle using flow cytometry. The percentages of the cell populations in each phase of the cell cycle for every time point are depicted as bars in the diagram ( A ). In control cells, UV irradiation results in G 1 and S phase increase while in CCDC6 knock down cells UV irradiation causes a reduction of cell population in S phase and an increase in G 2 phase. A representative experiment is shown. ( B ) The apoptotic levels were measured by flow cytometric assessment of the subG 0 /G 1 population. Knock down of CCDC6 increase UV-mediated cell death. ( C ) Cell survival analysis by Po-PRO and 7-ADD staining (excluding the double positive cells) revealed a significantly decreased cell survival of cells lacking CCDC6. Error Bars represent 3 independent experiments. ( D ) Cell lysates of HCT116 cells treated with etoposide (20 µM) or radiated with UV (0.002 J/cm 2 ) for 2, 6 or 24 hours and the untreated controls were resolved on a SDS-PAGE and probed for pH2Ax Ser139. Upon UV irradiation, pH2Ax Ser139 levels arise earlier and to a higher extent in CCDC6 knock down cells compared to the control. UV irradiation is causing high levels of pH2Ax Ser139 even in 2 hours after irradiation in CCDC6 knock down cells. The effect is similar upon etoposide treatment, although less dramatic. ( E ) Increased basal levels and nuclear foci of pH2Ax Ser139 are present in CCDC6 knock down cells, even in the absence of any additional treatment.
    Figure Legend Snippet: Knock down of CCDC6 increases vulnerability to genotoxic stress by UV induced DNA damage. HCT116 cells were transduced with CCDC6 shRNA expressing lentivirus or empty control followed by UV irradiation (0.002 J/cm 2 ), 48 hours after transduction. Cells were harvested at 2, 6 and 24 hours after irradiation and analyzed for the cell cycle using flow cytometry. The percentages of the cell populations in each phase of the cell cycle for every time point are depicted as bars in the diagram ( A ). In control cells, UV irradiation results in G 1 and S phase increase while in CCDC6 knock down cells UV irradiation causes a reduction of cell population in S phase and an increase in G 2 phase. A representative experiment is shown. ( B ) The apoptotic levels were measured by flow cytometric assessment of the subG 0 /G 1 population. Knock down of CCDC6 increase UV-mediated cell death. ( C ) Cell survival analysis by Po-PRO and 7-ADD staining (excluding the double positive cells) revealed a significantly decreased cell survival of cells lacking CCDC6. Error Bars represent 3 independent experiments. ( D ) Cell lysates of HCT116 cells treated with etoposide (20 µM) or radiated with UV (0.002 J/cm 2 ) for 2, 6 or 24 hours and the untreated controls were resolved on a SDS-PAGE and probed for pH2Ax Ser139. Upon UV irradiation, pH2Ax Ser139 levels arise earlier and to a higher extent in CCDC6 knock down cells compared to the control. UV irradiation is causing high levels of pH2Ax Ser139 even in 2 hours after irradiation in CCDC6 knock down cells. The effect is similar upon etoposide treatment, although less dramatic. ( E ) Increased basal levels and nuclear foci of pH2Ax Ser139 are present in CCDC6 knock down cells, even in the absence of any additional treatment.

    Techniques Used: Transduction, shRNA, Expressing, Irradiation, Flow Cytometry, Cytometry, Staining, SDS Page

    Deficient S phase checkpoint regulation upon etoposide treatment in the absence of CCDC6. ( A ) HCT116 cells were treated with 20 µM etoposide and cells were harvested at predetermined time points for cell cycle analysis. In the absence of CCDC6, no S phase accumulation is observed and the transition to G 2 phase is accelerated. One representative experiment is shown, out of three performed. ( B ) Concomitant apoptotic cell death was quantified by measuring the subG 0 /G 1 DNA content. CCDC6 knock down cells showed higher levels of apoptosis, at earlier time point, in comparison to the control, in response to genotoxic stress upon etoposide treatment. ( C ) The percentage of cell survival was assessed by gating for PoPRO and 7-AAD negative cells. CCDC6 knock down resulted in lower cell survival upon etoposide induced genotoxic stress. The assays were performed in triplicates.
    Figure Legend Snippet: Deficient S phase checkpoint regulation upon etoposide treatment in the absence of CCDC6. ( A ) HCT116 cells were treated with 20 µM etoposide and cells were harvested at predetermined time points for cell cycle analysis. In the absence of CCDC6, no S phase accumulation is observed and the transition to G 2 phase is accelerated. One representative experiment is shown, out of three performed. ( B ) Concomitant apoptotic cell death was quantified by measuring the subG 0 /G 1 DNA content. CCDC6 knock down cells showed higher levels of apoptosis, at earlier time point, in comparison to the control, in response to genotoxic stress upon etoposide treatment. ( C ) The percentage of cell survival was assessed by gating for PoPRO and 7-AAD negative cells. CCDC6 knock down resulted in lower cell survival upon etoposide induced genotoxic stress. The assays were performed in triplicates.

    Techniques Used: Cell Cycle Assay

    36) Product Images from "Downregulation of the tumor suppressor HSPB7, involved in the p53 pathway, in renal cell carcinoma by hypermethylation"

    Article Title: Downregulation of the tumor suppressor HSPB7, involved in the p53 pathway, in renal cell carcinoma by hypermethylation

    Journal: International Journal of Oncology

    doi: 10.3892/ijo.2014.2314

    HSPB7 is regulated by p53. (A and B) HSPB7 expression in NCI-H1299 cells with or without p53 induction (A) dose- and (B) time-dependently. Cells were infected with replication-deficient recombinant adenovirus encoding p53 (Ad-p53) or LacZ (Ad-LacZ) at indicated doses, and the cells were collected 48 h later and qPCR analysis was performed (A). The cells were infected at 8 MOI and collected at different time points (B). (C and D) HSPB7 expression in HCT116 (p53 −/− ) and HCT116 (p53 +/+ ) cells treated with adriamycin at indicated doses for 2 h and the cells were harvested at 48 h (C). The cells were treated with adriamycin at 2 μ g/ml for 2 h and then harvested at different time points (D). B2M was used for normalization of expression levels. Values are expressed as the mean ± SD.
    Figure Legend Snippet: HSPB7 is regulated by p53. (A and B) HSPB7 expression in NCI-H1299 cells with or without p53 induction (A) dose- and (B) time-dependently. Cells were infected with replication-deficient recombinant adenovirus encoding p53 (Ad-p53) or LacZ (Ad-LacZ) at indicated doses, and the cells were collected 48 h later and qPCR analysis was performed (A). The cells were infected at 8 MOI and collected at different time points (B). (C and D) HSPB7 expression in HCT116 (p53 −/− ) and HCT116 (p53 +/+ ) cells treated with adriamycin at indicated doses for 2 h and the cells were harvested at 48 h (C). The cells were treated with adriamycin at 2 μ g/ml for 2 h and then harvested at different time points (D). B2M was used for normalization of expression levels. Values are expressed as the mean ± SD.

    Techniques Used: Expressing, Infection, Recombinant, Real-time Polymerase Chain Reaction

    37) Product Images from "Mycobacterium bovis BCG promotes tumor cell survival from tumor necrosis factor-α-induced apoptosis"

    Article Title: Mycobacterium bovis BCG promotes tumor cell survival from tumor necrosis factor-α-induced apoptosis

    Journal: Molecular Cancer

    doi: 10.1186/1476-4598-13-210

    BCG inhibits TNF-α-mediated apoptosis. (A and B) HCT116, HCT116 p53 -/- , T24, MNT-1, HepG2, HELA and MDA-MB-231 cells were infected with BCG for 12 h prior to treatment with TNF-α. Representative immunofluorescence images (A) and MFI (B) for Annexin V-FITC staining. Data is representative of mean ± SEM of at least 3 different experiments. *p
    Figure Legend Snippet: BCG inhibits TNF-α-mediated apoptosis. (A and B) HCT116, HCT116 p53 -/- , T24, MNT-1, HepG2, HELA and MDA-MB-231 cells were infected with BCG for 12 h prior to treatment with TNF-α. Representative immunofluorescence images (A) and MFI (B) for Annexin V-FITC staining. Data is representative of mean ± SEM of at least 3 different experiments. *p

    Techniques Used: Multiple Displacement Amplification, Infection, Immunofluorescence, Staining

    38) Product Images from "Angiogenic factor-driven inflammation promotes extravasation of human proangiogenic monocytes to tumours"

    Article Title: Angiogenic factor-driven inflammation promotes extravasation of human proangiogenic monocytes to tumours

    Journal: Nature Communications

    doi: 10.1038/s41467-017-02610-0

    Blocking VEGF-A signalling inhibits homing of proangiogenic monocytes to tumours. a Analysis of Cx3cl1 protein expression in xenografts of SKBR7, DLD1 and HCT116 tumours. b Quantification of Cx3cl1 band intensity in a after normalisation with same sample Actin level. c Scheme of the study of VEGF-A implication in human monocyte recruitment in DLD1 xenograft. DLD1 xenograft-bearing mice were treated either with the mixture of DC101 and bevacizumab (D/B mix) or control IgG for 24 h before adoptive transfer of human pan monocytes. The mice were perfused with PBS-EDTA to wash out circulating and non-extravasated leucocytes before tumour collect and analysis. d Analysis of Cx3cl1 protein level in DLD1 tumour after 24 h of D/B mix treatment by western blotting. e Quantification of Cx3cl1 band intensity in d after normalisation with Pecam1 level in same samples. f Expression level of Cx3cl1 in tumour vasculature by immunofluorescence of DLD1 xenografts. Scale bar = 80 µm. g Quantification of Cx3cl1 in blood vessels. Data are presented as scatter dot plots with medians. Each dot represent the mean intensity of Cx3cl1 per µm 2 blood vessels from 10 sections of the same tumour. n = 5 tumours per group were used for this quantification. ** p
    Figure Legend Snippet: Blocking VEGF-A signalling inhibits homing of proangiogenic monocytes to tumours. a Analysis of Cx3cl1 protein expression in xenografts of SKBR7, DLD1 and HCT116 tumours. b Quantification of Cx3cl1 band intensity in a after normalisation with same sample Actin level. c Scheme of the study of VEGF-A implication in human monocyte recruitment in DLD1 xenograft. DLD1 xenograft-bearing mice were treated either with the mixture of DC101 and bevacizumab (D/B mix) or control IgG for 24 h before adoptive transfer of human pan monocytes. The mice were perfused with PBS-EDTA to wash out circulating and non-extravasated leucocytes before tumour collect and analysis. d Analysis of Cx3cl1 protein level in DLD1 tumour after 24 h of D/B mix treatment by western blotting. e Quantification of Cx3cl1 band intensity in d after normalisation with Pecam1 level in same samples. f Expression level of Cx3cl1 in tumour vasculature by immunofluorescence of DLD1 xenografts. Scale bar = 80 µm. g Quantification of Cx3cl1 in blood vessels. Data are presented as scatter dot plots with medians. Each dot represent the mean intensity of Cx3cl1 per µm 2 blood vessels from 10 sections of the same tumour. n = 5 tumours per group were used for this quantification. ** p

    Techniques Used: Blocking Assay, Expressing, Mouse Assay, Adoptive Transfer Assay, Western Blot, Immunofluorescence

    Human tumour xenografts recruit human proangiogenic monocytes. a Time course protocol of the assay of human monocyte recruitment to human tumour xenograft (colorectal adenocarcinoma DLD1, colorectal carcinoma HCT116 and breast carcinoma SKBR7) in vivo. Human tumour xenografts were injected subcutaneously at day di and let grow to reach 0.5 cm 3 of tumour volume. The mice were then i.v. injected at day dt with freshly isolated human monocytes. After 4 h mice were perfused with PBS-EDTA and killed to collect tumours that were then dissociated for FACS analysis of recruited human monocytes. b FACS analysis of recruited human proangiogenic monocytes (HLA-DR+CD16+). Human HLA-DR staining identified human monocytes recruited to DLD1 xenograft. Gating on HLA-DR+ cells allowed quantification of human proangiogenic monocytes (CD16+). c Quantification of the percentage of total human monocytes within tumour cell suspension (left) and percentage of HPMo within recruited human monocytes ( n = 12 tumours from six mice per group injected with monocytes from the same donor in each experiment, the data were combined from two independent experiments). About 20–60% of recruited human monocytes were from proangiogenic subset with the highest proportion for xenografts of colorectal adenocarcinoma cells DLD1 and the lowest for xenografts of breast carcinoma SKBR7. The data are presented as boxplot with median (horizontal line in boxes), 25–75th percentiles (box limits) and Min–Max indicated by whiskers. * p
    Figure Legend Snippet: Human tumour xenografts recruit human proangiogenic monocytes. a Time course protocol of the assay of human monocyte recruitment to human tumour xenograft (colorectal adenocarcinoma DLD1, colorectal carcinoma HCT116 and breast carcinoma SKBR7) in vivo. Human tumour xenografts were injected subcutaneously at day di and let grow to reach 0.5 cm 3 of tumour volume. The mice were then i.v. injected at day dt with freshly isolated human monocytes. After 4 h mice were perfused with PBS-EDTA and killed to collect tumours that were then dissociated for FACS analysis of recruited human monocytes. b FACS analysis of recruited human proangiogenic monocytes (HLA-DR+CD16+). Human HLA-DR staining identified human monocytes recruited to DLD1 xenograft. Gating on HLA-DR+ cells allowed quantification of human proangiogenic monocytes (CD16+). c Quantification of the percentage of total human monocytes within tumour cell suspension (left) and percentage of HPMo within recruited human monocytes ( n = 12 tumours from six mice per group injected with monocytes from the same donor in each experiment, the data were combined from two independent experiments). About 20–60% of recruited human monocytes were from proangiogenic subset with the highest proportion for xenografts of colorectal adenocarcinoma cells DLD1 and the lowest for xenografts of breast carcinoma SKBR7. The data are presented as boxplot with median (horizontal line in boxes), 25–75th percentiles (box limits) and Min–Max indicated by whiskers. * p

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

    Human tumours express a panel of cytokines and angiogenic factors. a Screening of human tumour cells-expressed cytokines (screened with human primers) in the xenografts ( n = 6 tumours per group pooled from two independent experiments). All molecule expression was normalised against the expression of housekeeping genes, namely GAPDH and ACTB . The expression levels of cytokines in DLD1 and SKBR7 xenografts are presented as relative (fold change) to their expression levels in HCT116 xenograft, which showed intermediate recruitment levels of proangiogenic monocytes. b Confirmation of cytokine expression level in tumour xenografts by ELISA. The protein levels of TNF, IFNγ and VEGF-A were quantified in protein extract of DLD1, HCT116 and SKBR7 xenografts ( n = 5 samples per tumour type) by ELISA. The data are presented as mean ± s.d., ** p -value
    Figure Legend Snippet: Human tumours express a panel of cytokines and angiogenic factors. a Screening of human tumour cells-expressed cytokines (screened with human primers) in the xenografts ( n = 6 tumours per group pooled from two independent experiments). All molecule expression was normalised against the expression of housekeeping genes, namely GAPDH and ACTB . The expression levels of cytokines in DLD1 and SKBR7 xenografts are presented as relative (fold change) to their expression levels in HCT116 xenograft, which showed intermediate recruitment levels of proangiogenic monocytes. b Confirmation of cytokine expression level in tumour xenografts by ELISA. The protein levels of TNF, IFNγ and VEGF-A were quantified in protein extract of DLD1, HCT116 and SKBR7 xenografts ( n = 5 samples per tumour type) by ELISA. The data are presented as mean ± s.d., ** p -value

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay

    39) Product Images from "hsa_circRNA_000166 Facilitated Cell Growth and Limited Apoptosis through Targeting miR-326/LASP1 Axis in Colorectal Cancer"

    Article Title: hsa_circRNA_000166 Facilitated Cell Growth and Limited Apoptosis through Targeting miR-326/LASP1 Axis in Colorectal Cancer

    Journal: Gastroenterology Research and Practice

    doi: 10.1155/2020/8834359

    Decreased hsa_circRNA_000166 expression affected cell growth and apoptosis in CRC cells. (a) The transcriptional level of hsa_circRNA_000166 was measured by qRT-PCR in both HCT116 and SW480 cells after transfection with siRNAs. (b) Cell proliferation assay showed that decreased hsa_circRNA_000166 expression limited the growth of HCT116 and SW480 cells after siRNA-1 or siRNA-2 transfection compared with the controls. (c) Clone formation assay demonstrated that the number of colonies was significantly decreased after siRNA-1 or siRNA-2 transfection compared with the controls in HCT116 and SW480 cells. (d) Flow cytometric analysis displayed that the number of apoptotic cells was noticeably increased in siRNA-1- or siRNA-2-transfected groups compared with the controls. The Student t -test was used for statistics.
    Figure Legend Snippet: Decreased hsa_circRNA_000166 expression affected cell growth and apoptosis in CRC cells. (a) The transcriptional level of hsa_circRNA_000166 was measured by qRT-PCR in both HCT116 and SW480 cells after transfection with siRNAs. (b) Cell proliferation assay showed that decreased hsa_circRNA_000166 expression limited the growth of HCT116 and SW480 cells after siRNA-1 or siRNA-2 transfection compared with the controls. (c) Clone formation assay demonstrated that the number of colonies was significantly decreased after siRNA-1 or siRNA-2 transfection compared with the controls in HCT116 and SW480 cells. (d) Flow cytometric analysis displayed that the number of apoptotic cells was noticeably increased in siRNA-1- or siRNA-2-transfected groups compared with the controls. The Student t -test was used for statistics.

    Techniques Used: Expressing, Quantitative RT-PCR, Transfection, Proliferation Assay, Tube Formation Assay

    hsa_circRNA_000166 regulated CRC progression by targeting the miR-326/LASP1 axis. (a) The Venn analysis implied that 3 miRNAs, including miR-326, were involved in CRC. The blue represented 9 predicted miRNAs analyzed by starBase v2.0, and the yellow represented 9 predicted miRNAs analyzed by CircInteractome. (b) Binding site of hsa_circRNA_000166 in miR-326 was predicted by starBase. (c) qRT-PCR analysis showed that the transcriptional level of miR-326 was dramatically decreased in CRC tissues ( n = 40) compared to the matched normal tissues ( n = 40). (d) qRT-PCR analysis showed that miR-326 was notably downregulated in CRC cells compared with the normal colonic cells. (e) qRT-PCR assay displayed that miR-326 was significantly upregulated in siRNA-1-transfected groups compared with the controls in both HCT116 and SW480 cells. (f) Luciferase reporter assay indicated miR-326 dramatically repressed the WT hsa_circRNA_000166 luciferase activity but not Mut hsa_circRNA_000166 in HCT116 cells. (g, h) qRT-PCR and western blot assay showed that the transcriptional and translational levels of LASP1 were obviously downregulated in siRNA-1-transfected groups compared with the controls in both HCT116 and SW480 cells. (i) Luciferase reporter assay indicated miR-326 dramatically repressed the WT LASP1 luciferase activity but not Mut LASP1 in HCT116 cells. The Student t -test was used for statistics.
    Figure Legend Snippet: hsa_circRNA_000166 regulated CRC progression by targeting the miR-326/LASP1 axis. (a) The Venn analysis implied that 3 miRNAs, including miR-326, were involved in CRC. The blue represented 9 predicted miRNAs analyzed by starBase v2.0, and the yellow represented 9 predicted miRNAs analyzed by CircInteractome. (b) Binding site of hsa_circRNA_000166 in miR-326 was predicted by starBase. (c) qRT-PCR analysis showed that the transcriptional level of miR-326 was dramatically decreased in CRC tissues ( n = 40) compared to the matched normal tissues ( n = 40). (d) qRT-PCR analysis showed that miR-326 was notably downregulated in CRC cells compared with the normal colonic cells. (e) qRT-PCR assay displayed that miR-326 was significantly upregulated in siRNA-1-transfected groups compared with the controls in both HCT116 and SW480 cells. (f) Luciferase reporter assay indicated miR-326 dramatically repressed the WT hsa_circRNA_000166 luciferase activity but not Mut hsa_circRNA_000166 in HCT116 cells. (g, h) qRT-PCR and western blot assay showed that the transcriptional and translational levels of LASP1 were obviously downregulated in siRNA-1-transfected groups compared with the controls in both HCT116 and SW480 cells. (i) Luciferase reporter assay indicated miR-326 dramatically repressed the WT LASP1 luciferase activity but not Mut LASP1 in HCT116 cells. The Student t -test was used for statistics.

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

    miR-326 downregulation or LASP1 upregulation rescued the phenotype dominated by hsa_circRNA_000166. (a, b) CCK-8 and colony formation assay demonstrated that cell codepletion of both siRNA-1 and miR-326 I or depletion of siRNA-1 while overexpression of LASP1 promoted cell growth compared with control groups in HCT116 cells. (c) The number of apoptotic cells in codepletion of both siRNA-1 and miR-326 I or depletion of siRNA-1 while overexpression of LASP1 groups was less than the number in control group HCT116 cells. The Student t -test was used for statistics.
    Figure Legend Snippet: miR-326 downregulation or LASP1 upregulation rescued the phenotype dominated by hsa_circRNA_000166. (a, b) CCK-8 and colony formation assay demonstrated that cell codepletion of both siRNA-1 and miR-326 I or depletion of siRNA-1 while overexpression of LASP1 promoted cell growth compared with control groups in HCT116 cells. (c) The number of apoptotic cells in codepletion of both siRNA-1 and miR-326 I or depletion of siRNA-1 while overexpression of LASP1 groups was less than the number in control group HCT116 cells. The Student t -test was used for statistics.

    Techniques Used: CCK-8 Assay, Colony Assay, Over Expression

    40) Product Images from "miR-143 or miR-145 overexpression increases cetuximab-mediated antibody-dependent cellular cytotoxicity in human colon cancer cells"

    Article Title: miR-143 or miR-145 overexpression increases cetuximab-mediated antibody-dependent cellular cytotoxicity in human colon cancer cells

    Journal: Oncotarget

    doi: 10.18632/oncotarget.7010

    Bcl-2 is involved in cetuximab sensitization induced by miR-143 or miR-145 overexpression, increasing cell susceptibility to cetuximab-mediated ADCC ( A ) HCT116-Empty, HCT116-miR-143 and HCT116-miR-145 cells were exposed to 100 μg/ml cetuximab or PBMCs (10:1) alone, or co-treatment of cetuximab together with PBMCs (10:1), or vehicle (control) for 48 h to evaluate Bcl-2 protein expression, by immunoblot. HCT116 cells were transfected with Bcl-2 siRNA or siRNA control, and next treated with cetuximab for 72 h, and used for: ( B ) Bcl-2 protein expression evaluation by immunoblot (upper panel), and evaluation of cell viability and general cell death, respectively by MTS and LDH assay (lower panel); ( C ) cetuximab-mediated ADCC evaluation, where siRNA transfected cells were exposed to cetuximab and/or PBMCs (20:1) and the growth inhibitory effects were assayed using the xCELLigence system as described above. Quantification of growth inhibitory effects are presented as the percentage of cell kill for 100 μg/ml or 250 μg/ml cetuximab treatment, after 72 h of exposure. Representative blots from at least three independent experiments are shown. Results are expressed as (A) mean ± SEM fold-change to untreated control cells, (B) the mean ± SEM fold change to respective untreated cells, or (C) mean ± SEM, from at least three independent experiments. ** p
    Figure Legend Snippet: Bcl-2 is involved in cetuximab sensitization induced by miR-143 or miR-145 overexpression, increasing cell susceptibility to cetuximab-mediated ADCC ( A ) HCT116-Empty, HCT116-miR-143 and HCT116-miR-145 cells were exposed to 100 μg/ml cetuximab or PBMCs (10:1) alone, or co-treatment of cetuximab together with PBMCs (10:1), or vehicle (control) for 48 h to evaluate Bcl-2 protein expression, by immunoblot. HCT116 cells were transfected with Bcl-2 siRNA or siRNA control, and next treated with cetuximab for 72 h, and used for: ( B ) Bcl-2 protein expression evaluation by immunoblot (upper panel), and evaluation of cell viability and general cell death, respectively by MTS and LDH assay (lower panel); ( C ) cetuximab-mediated ADCC evaluation, where siRNA transfected cells were exposed to cetuximab and/or PBMCs (20:1) and the growth inhibitory effects were assayed using the xCELLigence system as described above. Quantification of growth inhibitory effects are presented as the percentage of cell kill for 100 μg/ml or 250 μg/ml cetuximab treatment, after 72 h of exposure. Representative blots from at least three independent experiments are shown. Results are expressed as (A) mean ± SEM fold-change to untreated control cells, (B) the mean ± SEM fold change to respective untreated cells, or (C) mean ± SEM, from at least three independent experiments. ** p

    Techniques Used: Over Expression, Expressing, Transfection, Lactate Dehydrogenase Assay

    miR-143 or miR-145 overexpression sensitizes HCT116 mutant KRAS colon cancer cells to cetuximab HCT116-Empty, HCT116-miR-143 and HCT116-miR-145 stably transduced cells were plated onto a 96-well E-Plate of xCELLigence System. 24 h after plating cells were exposed to 0-1600 μg/ml cetuximab, and allowed to growth for 72 h. ( A ) Cell index was measured every 5 min, which allowed IC 50 determination from the time of incubation. ( B ) Cell viability was also evaluated by MTS metabolism assay. The results are expressed as (A) the mean ± SEM fold change to control cells, or (B) the mean ± SEM fold change to respective untreated cells, from at least three independent experiments. ** p
    Figure Legend Snippet: miR-143 or miR-145 overexpression sensitizes HCT116 mutant KRAS colon cancer cells to cetuximab HCT116-Empty, HCT116-miR-143 and HCT116-miR-145 stably transduced cells were plated onto a 96-well E-Plate of xCELLigence System. 24 h after plating cells were exposed to 0-1600 μg/ml cetuximab, and allowed to growth for 72 h. ( A ) Cell index was measured every 5 min, which allowed IC 50 determination from the time of incubation. ( B ) Cell viability was also evaluated by MTS metabolism assay. The results are expressed as (A) the mean ± SEM fold change to control cells, or (B) the mean ± SEM fold change to respective untreated cells, from at least three independent experiments. ** p

    Techniques Used: Over Expression, Mutagenesis, Stable Transfection, Incubation

    miR-143 or miR-145 overexpression increases cetuximab-mediated ADCC in HCT116 cells HCT116-Empty, HCT116-miR-143 and HCT116-miR-145 cells were plated on 96-well E-Plate and used on xCELLigence System, allowed to grow for 96 h. Cells were grown in medium alone or treated with increasing concentrations of cetuximab, and or PBMCs. ( A ) Red (NA) represents cells grown in medium alone, green represents growth with 100 μg/ml cetuximab, orange with 250 μg/ml cetuximab, and blue with 1600 μg/ml cetuximab. ( B ) Cells were grown in medium alone (red), or treated with PBMCs at (10:1) green or (20:1) orange. ( C ) Cells were grown in medium alone (red), or treated with 100 μg/ml cetuximab and PBMCs at (10:1) green or (20:1) orange. Cell index values were normalized at the time of the addition. Normalized cell index values are plotted in 1 h increments as the average of two replicates together with standard deviation. ( D ) Quantification of normalized cell index was performed at 72 h, by measuring the change in area under the curve compared to non-treated controls, and are presented as percentage of cell kill for 100 μg/ml cetuximab treatment or 100 μg/ml rituximab (control antibody), alone or with PBMCs (20:1). ( E ) Quantification of cytotoxicity was performed at 48 h, by measuring the amount of LDH released into the culture supernatant, and is presented as percentage of cytotoxicity for 100 μg/ml cetuximab treatment or 100 μg/ml rituximab (control antibody), alone or with PBMCs (20:1), compared with non-treated controls. The results are expressed as the mean ± SEM, from at least three independent experiments. ** p
    Figure Legend Snippet: miR-143 or miR-145 overexpression increases cetuximab-mediated ADCC in HCT116 cells HCT116-Empty, HCT116-miR-143 and HCT116-miR-145 cells were plated on 96-well E-Plate and used on xCELLigence System, allowed to grow for 96 h. Cells were grown in medium alone or treated with increasing concentrations of cetuximab, and or PBMCs. ( A ) Red (NA) represents cells grown in medium alone, green represents growth with 100 μg/ml cetuximab, orange with 250 μg/ml cetuximab, and blue with 1600 μg/ml cetuximab. ( B ) Cells were grown in medium alone (red), or treated with PBMCs at (10:1) green or (20:1) orange. ( C ) Cells were grown in medium alone (red), or treated with 100 μg/ml cetuximab and PBMCs at (10:1) green or (20:1) orange. Cell index values were normalized at the time of the addition. Normalized cell index values are plotted in 1 h increments as the average of two replicates together with standard deviation. ( D ) Quantification of normalized cell index was performed at 72 h, by measuring the change in area under the curve compared to non-treated controls, and are presented as percentage of cell kill for 100 μg/ml cetuximab treatment or 100 μg/ml rituximab (control antibody), alone or with PBMCs (20:1). ( E ) Quantification of cytotoxicity was performed at 48 h, by measuring the amount of LDH released into the culture supernatant, and is presented as percentage of cytotoxicity for 100 μg/ml cetuximab treatment or 100 μg/ml rituximab (control antibody), alone or with PBMCs (20:1), compared with non-treated controls. The results are expressed as the mean ± SEM, from at least three independent experiments. ** p

    Techniques Used: Over Expression, Standard Deviation

    miR-143 or miR-145 overexpressing cells are more sensitive to cetuximab-mediated ADCC-induced apoptosis HCT116-Empty, HCT116-miR-143 and HCT116-miR-145 cells were exposed to 100 μg/ml cetuximab, 100 μg/ml rituximab or PBMCs (10:1 or 20:1) alone, or co-treatment of cetuximab or rituximab together with PBMCs (10:1 or 20:1), or vehicle (control). When indicated HCT116-derived cells were pretreated for 1 h with 50 μM z-VAD-fmk. ( A ) Nuclear morphology was evaluated by fluorescence microscopy after Hoechst staining, 24 h after treatment. Representative images of Hoechst staining at 400x magnification are presented. Arrows indicate nuclear fragmentation and chromatin condensation. Scale bar corresponds to 15 μm. ( B ) Apoptosis was quantified by flow cytometry using Guava Nexin assay, 48 h after treatment. (upper panel) Representative flow cytometry plots and percentage of cells positive for Annexin V and/or 7-AAD of one representative experiment. (lower panel) Quantification of apoptotic cells, positive for Annexin V and/or 7-AAD. ( C ) Caspase-3/7 activity was determined at 16 h after treatment, and ( D ) PARP cleavage was evaluated by immunoblot at 48 h after treatment. ( E ) Cetuximab-mediated ADCC was assayed using the xCELLigence system (upper panel) and LDH release assay (lower panel) as described above. Results are expressed as (A) percentage of apoptotic cells per field ± SEM, (B) percentage of apoptotic cells ± SEM, (C, D) mean ± SEM fold change to untreated control cells, and (E) mean ± SEM, from at least three independent experiments. FL, full lengh. ** p
    Figure Legend Snippet: miR-143 or miR-145 overexpressing cells are more sensitive to cetuximab-mediated ADCC-induced apoptosis HCT116-Empty, HCT116-miR-143 and HCT116-miR-145 cells were exposed to 100 μg/ml cetuximab, 100 μg/ml rituximab or PBMCs (10:1 or 20:1) alone, or co-treatment of cetuximab or rituximab together with PBMCs (10:1 or 20:1), or vehicle (control). When indicated HCT116-derived cells were pretreated for 1 h with 50 μM z-VAD-fmk. ( A ) Nuclear morphology was evaluated by fluorescence microscopy after Hoechst staining, 24 h after treatment. Representative images of Hoechst staining at 400x magnification are presented. Arrows indicate nuclear fragmentation and chromatin condensation. Scale bar corresponds to 15 μm. ( B ) Apoptosis was quantified by flow cytometry using Guava Nexin assay, 48 h after treatment. (upper panel) Representative flow cytometry plots and percentage of cells positive for Annexin V and/or 7-AAD of one representative experiment. (lower panel) Quantification of apoptotic cells, positive for Annexin V and/or 7-AAD. ( C ) Caspase-3/7 activity was determined at 16 h after treatment, and ( D ) PARP cleavage was evaluated by immunoblot at 48 h after treatment. ( E ) Cetuximab-mediated ADCC was assayed using the xCELLigence system (upper panel) and LDH release assay (lower panel) as described above. Results are expressed as (A) percentage of apoptotic cells per field ± SEM, (B) percentage of apoptotic cells ± SEM, (C, D) mean ± SEM fold change to untreated control cells, and (E) mean ± SEM, from at least three independent experiments. FL, full lengh. ** p

    Techniques Used: Derivative Assay, Fluorescence, Microscopy, Staining, Flow Cytometry, Cytometry, Activity Assay, Lactate Dehydrogenase Assay

    Granzyme B inhibition abrogates cetuximab-mediated ADCC in HCT116 cells HCT116-Empty, HCT116-miR-143 or HCT116-miR-145 cells were exposed to 100 μg/ml cetuximab or PBMCs (20:1) alone, or co-treatment of cetuximab together with PBMCs, or vehicle (control). Cetuximab-mediated ADCC was evaluated using the xCELLigence system and LDH release assay in ( A ) cells pre-treated for 2 h with 500 ng/ml of neutralizing antibody to Fas (ZB4), or in ( B ) cells exposed to PBMCs pretreated for 30 min with 100 μM Ac-IETD-CHO. ( C ) Caspase -3/7 activity was determined as described above. Results are expressed as (A, B) mean ± SEM, or (C) mean ± SEM fold-change to respective untreated cells. ** p
    Figure Legend Snippet: Granzyme B inhibition abrogates cetuximab-mediated ADCC in HCT116 cells HCT116-Empty, HCT116-miR-143 or HCT116-miR-145 cells were exposed to 100 μg/ml cetuximab or PBMCs (20:1) alone, or co-treatment of cetuximab together with PBMCs, or vehicle (control). Cetuximab-mediated ADCC was evaluated using the xCELLigence system and LDH release assay in ( A ) cells pre-treated for 2 h with 500 ng/ml of neutralizing antibody to Fas (ZB4), or in ( B ) cells exposed to PBMCs pretreated for 30 min with 100 μM Ac-IETD-CHO. ( C ) Caspase -3/7 activity was determined as described above. Results are expressed as (A, B) mean ± SEM, or (C) mean ± SEM fold-change to respective untreated cells. ** p

    Techniques Used: Inhibition, Lactate Dehydrogenase Assay, Activity Assay

    miR-143 or miR-145 overexpression reduces HCT116 colon cancer cell doubling time and migration miR-143 or miR-145 overexpressing cells were produced by transducing HCT116 cell line with viral particles containing MSCV-Neo constructs expressing miR-143, miR-145 or empty vector, as control. ( A ) miR expression was assayed by northern blot. Gel loading controls are shown from ethidium bromide (EtBr) staining of RNA. ( B ) HCT116-Empty, HCT116-miR-143, and HCT116-miR-145 cells were plated onto a 96-well E-Plate of xCELLigence System. Cell index was measured every 5 min for 24 h and used to plot and calculate cell doubling time. ( C ) Cell migration was assessed by transwell migration assay, with cells allowed to migrate for 9 h after cell platting; ( D ) and by wound healing assay at 24, 48 and 72 h after wound formation. Results are expressed as (B, C) mean ± SEM fold change to control cells, or (D) percentage of wound closure ± SEM, from at least three independent experiments. ** p
    Figure Legend Snippet: miR-143 or miR-145 overexpression reduces HCT116 colon cancer cell doubling time and migration miR-143 or miR-145 overexpressing cells were produced by transducing HCT116 cell line with viral particles containing MSCV-Neo constructs expressing miR-143, miR-145 or empty vector, as control. ( A ) miR expression was assayed by northern blot. Gel loading controls are shown from ethidium bromide (EtBr) staining of RNA. ( B ) HCT116-Empty, HCT116-miR-143, and HCT116-miR-145 cells were plated onto a 96-well E-Plate of xCELLigence System. Cell index was measured every 5 min for 24 h and used to plot and calculate cell doubling time. ( C ) Cell migration was assessed by transwell migration assay, with cells allowed to migrate for 9 h after cell platting; ( D ) and by wound healing assay at 24, 48 and 72 h after wound formation. Results are expressed as (B, C) mean ± SEM fold change to control cells, or (D) percentage of wound closure ± SEM, from at least three independent experiments. ** p

    Techniques Used: Over Expression, Migration, Produced, Construct, Expressing, Plasmid Preparation, Northern Blot, Staining, Transwell Migration Assay, Wound Healing Assay

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    Protein Extraction:

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    Activity Assay:

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    Plasmid Preparation:

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    Luciferase:

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    Negative Control:

    Article Title: MiR-590-3p promotes proliferation and metastasis of colorectal cancer via Hippo pathway
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    Transfection:

    Article Title: MiR-590-3p promotes proliferation and metastasis of colorectal cancer via Hippo pathway
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    shRNA:

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    Incubation:

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    Infection:

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    Stable Transfection:

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    Cell Culture:

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    Thermo Fisher hct116 cells
    TCONS_00026334 regulates miR‐548n expression negatively in CRC. (A) Bioinformatics software showed the predicted sequence of the TCONS_00026334 binding site on the 3ʹ‐UTR of miR‐548n mRNA. (B) MiR‐548n expression was analyzed using qRT‐PCR in 30 paired CRC tissues and adjacent normal tissues. (C) Correlations between the expression levels of miR‐548n and TCONS_00026334 in 30 paired CRC tissues. (D) The expression level of miR‐548n was detected in the presence of pcDNA3.1‐TCONS_00026334 using qRT‐PCR. (E) Luciferase reporter assay was performed after co‐transfection with miR‐548n and luciferase reporters containing TCONS_00026334 or mutant transcripts in SW1116 and <t>HCT116</t> cells. (F, G) The cell viabilities of SW1116 and HCT116 were detected after transfection with pcDNA3.1‐TCONS_00026334, or pcDNA3.1‐TCONS_00026334 and miR‐548n mimics, or pcDNA3.1‐control vector and control mimics using a CCK‐8 assay. (H, I) In the presence of pcDNA3.1‐TCONS_00026334, the efficiency of miR‐548n mimics on the invasive ability of CRC cells was detected using Transwell method. The data are shown as the mean ± SD (* p
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    TCONS_00026334 regulates miR‐548n expression negatively in CRC. (A) Bioinformatics software showed the predicted sequence of the TCONS_00026334 binding site on the 3ʹ‐UTR of miR‐548n mRNA. (B) MiR‐548n expression was analyzed using qRT‐PCR in 30 paired CRC tissues and adjacent normal tissues. (C) Correlations between the expression levels of miR‐548n and TCONS_00026334 in 30 paired CRC tissues. (D) The expression level of miR‐548n was detected in the presence of pcDNA3.1‐TCONS_00026334 using qRT‐PCR. (E) Luciferase reporter assay was performed after co‐transfection with miR‐548n and luciferase reporters containing TCONS_00026334 or mutant transcripts in SW1116 and HCT116 cells. (F, G) The cell viabilities of SW1116 and HCT116 were detected after transfection with pcDNA3.1‐TCONS_00026334, or pcDNA3.1‐TCONS_00026334 and miR‐548n mimics, or pcDNA3.1‐control vector and control mimics using a CCK‐8 assay. (H, I) In the presence of pcDNA3.1‐TCONS_00026334, the efficiency of miR‐548n mimics on the invasive ability of CRC cells was detected using Transwell method. The data are shown as the mean ± SD (* p

    Journal: Cancer Medicine

    Article Title: Long noncoding RNA TCONS_00026334 is involved in suppressing the progression of colorectal cancer by regulating miR‑548n/TP53INP1 signaling pathway, et al. Long noncoding RNA TCONS_00026334 is involved in suppressing the progression of colorectal cancer by regulating miR‑548n/TP53INP1 signaling pathway

    doi: 10.1002/cam4.3473

    Figure Lengend Snippet: TCONS_00026334 regulates miR‐548n expression negatively in CRC. (A) Bioinformatics software showed the predicted sequence of the TCONS_00026334 binding site on the 3ʹ‐UTR of miR‐548n mRNA. (B) MiR‐548n expression was analyzed using qRT‐PCR in 30 paired CRC tissues and adjacent normal tissues. (C) Correlations between the expression levels of miR‐548n and TCONS_00026334 in 30 paired CRC tissues. (D) The expression level of miR‐548n was detected in the presence of pcDNA3.1‐TCONS_00026334 using qRT‐PCR. (E) Luciferase reporter assay was performed after co‐transfection with miR‐548n and luciferase reporters containing TCONS_00026334 or mutant transcripts in SW1116 and HCT116 cells. (F, G) The cell viabilities of SW1116 and HCT116 were detected after transfection with pcDNA3.1‐TCONS_00026334, or pcDNA3.1‐TCONS_00026334 and miR‐548n mimics, or pcDNA3.1‐control vector and control mimics using a CCK‐8 assay. (H, I) In the presence of pcDNA3.1‐TCONS_00026334, the efficiency of miR‐548n mimics on the invasive ability of CRC cells was detected using Transwell method. The data are shown as the mean ± SD (* p

    Article Snippet: 0.5 × 105 cells/ml of SW1116 and HCT116 cells were seeded into six‐well plates and subsequently incubated for 24 h before being transfected with the above‐mentioned oligonucleotides and plasmids with Lipofectamine 2000 (Invitrogen).

    Techniques: Expressing, Software, Sequencing, Binding Assay, Quantitative RT-PCR, Luciferase, Reporter Assay, Cotransfection, Mutagenesis, Transfection, Plasmid Preparation, CCK-8 Assay

    Genome-wide DNA methylation profile of miR-155-5p transfected cells. ( A ) Scatter plot displaying differentially methylated regions in the genome after HCT116 cells were transfected with miR-155-5p or random 23-mers control RNA. The methylation difference (miR-155-5p minus random 23-mers transfected, average value of two biological replicates) was plotted against the –log 10 P- value. Green dots represent significantly changed regions. Numbers of differentially methylated regions were annotated in the plot. ( B ) Box plot showing the methylation level in replicates across the bins with increasing methylation level averages. ( C ) CpG site density curve per 10 kb in all regions, hypermethylated regions and hypomethylated regions. ( D ) Average differential methylation in genomic repetitive elements.

    Journal: Nucleic Acids Research

    Article Title: Small RNA-mediated DNA (cytosine-5) methyltransferase 1 inhibition leads to aberrant DNA methylation

    doi: 10.1093/nar/gkv518

    Figure Lengend Snippet: Genome-wide DNA methylation profile of miR-155-5p transfected cells. ( A ) Scatter plot displaying differentially methylated regions in the genome after HCT116 cells were transfected with miR-155-5p or random 23-mers control RNA. The methylation difference (miR-155-5p minus random 23-mers transfected, average value of two biological replicates) was plotted against the –log 10 P- value. Green dots represent significantly changed regions. Numbers of differentially methylated regions were annotated in the plot. ( B ) Box plot showing the methylation level in replicates across the bins with increasing methylation level averages. ( C ) CpG site density curve per 10 kb in all regions, hypermethylated regions and hypomethylated regions. ( D ) Average differential methylation in genomic repetitive elements.

    Article Snippet: Cell culture and transfection HEK293T and HCT116 cells were cultured in DMEM and McCoy's 5A (Gibco) media supplemented with 10% fetal bovine serum, respectively.

    Techniques: Genome Wide, DNA Methylation Assay, Transfection, Methylation

    Fascaplysin sensitizes the anti-cancer effect of TRAIL. ( A ) Cell viability was measured in HCT116, A375, and H1975 under a combination of fascaplysin and TRAIL. Cells were incubated with 0.5 µM of fascaplysin and 20 ng/mL of TRAIL for 24 h. Values represent mean ± SD of three independent experiments performed in triplicate; * p

    Journal: International Journal of Molecular Sciences

    Article Title: Fascaplysin Exerts Anti-Cancer Effects through the Downregulation of Survivin and HIF-1α and Inhibition of VEGFR2 and TRKA

    doi: 10.3390/ijms18102074

    Figure Lengend Snippet: Fascaplysin sensitizes the anti-cancer effect of TRAIL. ( A ) Cell viability was measured in HCT116, A375, and H1975 under a combination of fascaplysin and TRAIL. Cells were incubated with 0.5 µM of fascaplysin and 20 ng/mL of TRAIL for 24 h. Values represent mean ± SD of three independent experiments performed in triplicate; * p

    Article Snippet: The lentiviral particles were infected into A375 or HCT116 cells, and survivin-overexpressing cells were selected by 2 µg/mL of puromycin (Invitrogen, Carlsbad, CA, USA) for six days.

    Techniques: Incubation

    Fascaplysin induced apoptosis by suppressing survivin expression. ( A ) Multiple types of cancer cells were incubated with 1 µM of fascaplysin for 12 h, and then the survivin protein was measured by western blotting; ( B , C ) A375 and A2058 cells were treated with fascaplysin in a time- or dose-dependent manner as indicated. The levels of survivin were measured by Western blotting; ( D ) A375 and HCT116 cells were incubated with 1 µM of CDK4 inhibitors for 8 h; ( E ) The cell viability was measured in A375 or HCT116 cells that were overexpressing an empty vector or HA-tagged survivin upon fascaplysin treatment as indicated. Crystal violet staining images are shown. Values represent the mean ± SD of three independent experiments performed in triplicate; * p

    Journal: International Journal of Molecular Sciences

    Article Title: Fascaplysin Exerts Anti-Cancer Effects through the Downregulation of Survivin and HIF-1α and Inhibition of VEGFR2 and TRKA

    doi: 10.3390/ijms18102074

    Figure Lengend Snippet: Fascaplysin induced apoptosis by suppressing survivin expression. ( A ) Multiple types of cancer cells were incubated with 1 µM of fascaplysin for 12 h, and then the survivin protein was measured by western blotting; ( B , C ) A375 and A2058 cells were treated with fascaplysin in a time- or dose-dependent manner as indicated. The levels of survivin were measured by Western blotting; ( D ) A375 and HCT116 cells were incubated with 1 µM of CDK4 inhibitors for 8 h; ( E ) The cell viability was measured in A375 or HCT116 cells that were overexpressing an empty vector or HA-tagged survivin upon fascaplysin treatment as indicated. Crystal violet staining images are shown. Values represent the mean ± SD of three independent experiments performed in triplicate; * p

    Article Snippet: The lentiviral particles were infected into A375 or HCT116 cells, and survivin-overexpressing cells were selected by 2 µg/mL of puromycin (Invitrogen, Carlsbad, CA, USA) for six days.

    Techniques: Expressing, Incubation, Western Blot, Plasmid Preparation, Staining

    Fascaplysin inhibited 4EBP1-mediated cap-dependent de novo protein synthesis. ( A ) The cells were pre-treated with 20 µM of MG132 for 1 h, and then further incubated in the absence or presence of 1 µM of fascaplysin for 8 h. The protein levels of survivin were measured by Western blotting; ( B ) Protein synthesis in A375 or HCT116 cells was blocked and synchronized using 100 nM of cycloheximide (CHX) for 12 h. After removing CHX, the cells were further incubated with 20 µM of MG132 in the absence or presence of 1 µM of fascaplysin for the indicated time prior to lysis. Relative band intensities are shown; ( C , D ) The cells were incubated with various concentrations of fascaplysin for 6 h, and then the levels of the indicated proteins were measured by Western blotting; ( E ) Cytoplasmic proteins (1 mg) from fascaplysin-treated A375 cells were incubated with 1 μg of the indicated antibodies. The precipitated proteins–mRNA complexes were treated with proteinase K to elute mRNAs, and then the indicated mRNA levels were measured by quantitative real time-PCR. Values represent mean ± SD of two independent experiments performed in triplicate; * p

    Journal: International Journal of Molecular Sciences

    Article Title: Fascaplysin Exerts Anti-Cancer Effects through the Downregulation of Survivin and HIF-1α and Inhibition of VEGFR2 and TRKA

    doi: 10.3390/ijms18102074

    Figure Lengend Snippet: Fascaplysin inhibited 4EBP1-mediated cap-dependent de novo protein synthesis. ( A ) The cells were pre-treated with 20 µM of MG132 for 1 h, and then further incubated in the absence or presence of 1 µM of fascaplysin for 8 h. The protein levels of survivin were measured by Western blotting; ( B ) Protein synthesis in A375 or HCT116 cells was blocked and synchronized using 100 nM of cycloheximide (CHX) for 12 h. After removing CHX, the cells were further incubated with 20 µM of MG132 in the absence or presence of 1 µM of fascaplysin for the indicated time prior to lysis. Relative band intensities are shown; ( C , D ) The cells were incubated with various concentrations of fascaplysin for 6 h, and then the levels of the indicated proteins were measured by Western blotting; ( E ) Cytoplasmic proteins (1 mg) from fascaplysin-treated A375 cells were incubated with 1 μg of the indicated antibodies. The precipitated proteins–mRNA complexes were treated with proteinase K to elute mRNAs, and then the indicated mRNA levels were measured by quantitative real time-PCR. Values represent mean ± SD of two independent experiments performed in triplicate; * p

    Article Snippet: The lentiviral particles were infected into A375 or HCT116 cells, and survivin-overexpressing cells were selected by 2 µg/mL of puromycin (Invitrogen, Carlsbad, CA, USA) for six days.

    Techniques: Incubation, Western Blot, Lysis, Real-time Polymerase Chain Reaction

    PR130 upregulation by MS-275 represses checkpoint kinase signalling. a HCT116 cells were stimulated with 1 mM hydroxyurea (HU) and/or 2 µM MS-275. Wedges signify increasing incubation times (6, 10 and 14 h). Asterisks signify pre-incubation with MS-275 for 1 h. Indicated (phosphorylated) proteins were analysed by western blot. Vinculin served as loading control. b HCT116 cells were cultured with 2 µM MS-275 and/or 1 mM HU for 24 h and incubated with the PP2A inhibitor ocadaic acid (25 nM) for additional 4 h. Phosphorylated forms of ATM, CHK1 and mSIN3A (loading control) were detected by immunoblot. Numbers indicate densitometric analysis of western blot signals relative to HU-treated samples and normalised to loading control. c Relative mRNA levels of PPP2R3A in HCT116 cells evaluated by quantitative RT-PCR. Cells were treated with 1 mM HU and/or 2 µM MS-275 (6–24 h). Results show the mean ± SD ( n = 3; one-way ANOVA, *** P

    Journal: Nature Communications

    Article Title: HDAC1 and HDAC2 integrate checkpoint kinase phosphorylation and cell fate through the phosphatase-2A subunit PR130

    doi: 10.1038/s41467-018-03096-0

    Figure Lengend Snippet: PR130 upregulation by MS-275 represses checkpoint kinase signalling. a HCT116 cells were stimulated with 1 mM hydroxyurea (HU) and/or 2 µM MS-275. Wedges signify increasing incubation times (6, 10 and 14 h). Asterisks signify pre-incubation with MS-275 for 1 h. Indicated (phosphorylated) proteins were analysed by western blot. Vinculin served as loading control. b HCT116 cells were cultured with 2 µM MS-275 and/or 1 mM HU for 24 h and incubated with the PP2A inhibitor ocadaic acid (25 nM) for additional 4 h. Phosphorylated forms of ATM, CHK1 and mSIN3A (loading control) were detected by immunoblot. Numbers indicate densitometric analysis of western blot signals relative to HU-treated samples and normalised to loading control. c Relative mRNA levels of PPP2R3A in HCT116 cells evaluated by quantitative RT-PCR. Cells were treated with 1 mM HU and/or 2 µM MS-275 (6–24 h). Results show the mean ± SD ( n = 3; one-way ANOVA, *** P

    Article Snippet: HCT116 cells were co-transfected with both plasmids (0.45 µg each) and a plasmid carrying puromycin resistance (0.1 µg) using the transfection reagent TurboFect (ThermoFisher Scientific) according to the manufacturer’s protocol.

    Techniques: Mass Spectrometry, Incubation, Western Blot, Cell Culture, Quantitative RT-PCR

    PR130 influences cell fate decisions. a HCT116 ΔgRNA (upper panel) and HCT116 ΔPR130 (clone #16; lower panel) cells were incubated with 1 mM hydroxyurea (HU) and 2 µM MS-275 for 24 h. Cells were fixed and incubated with RPA-specific primary and Alexa Fluor-488-coupled secondary antibody. TO-PRO3 was used to visualise nuclei. Representative images are shown; scale bar, 10 µm. b Analysis of RPA foci per cell using ImageJ software. Treatment and staining were performed as described in a . Data presented as mean ± SD ( n = 3; two-way ANOVA; ** P

    Journal: Nature Communications

    Article Title: HDAC1 and HDAC2 integrate checkpoint kinase phosphorylation and cell fate through the phosphatase-2A subunit PR130

    doi: 10.1038/s41467-018-03096-0

    Figure Lengend Snippet: PR130 influences cell fate decisions. a HCT116 ΔgRNA (upper panel) and HCT116 ΔPR130 (clone #16; lower panel) cells were incubated with 1 mM hydroxyurea (HU) and 2 µM MS-275 for 24 h. Cells were fixed and incubated with RPA-specific primary and Alexa Fluor-488-coupled secondary antibody. TO-PRO3 was used to visualise nuclei. Representative images are shown; scale bar, 10 µm. b Analysis of RPA foci per cell using ImageJ software. Treatment and staining were performed as described in a . Data presented as mean ± SD ( n = 3; two-way ANOVA; ** P

    Article Snippet: HCT116 cells were co-transfected with both plasmids (0.45 µg each) and a plasmid carrying puromycin resistance (0.1 µg) using the transfection reagent TurboFect (ThermoFisher Scientific) according to the manufacturer’s protocol.

    Techniques: Incubation, Mass Spectrometry, Recombinase Polymerase Amplification, Software, Staining

    PR130 expression antagonises ATM phosphorylation. a Schematic of PPP2R3A knockout using CRISPR-Cas9 technology. Humanised S. pyogenes Cas9 nuclease was expressed in HCT116 cells with two guide RNAs (gRNA) to introduce two DSB into the PPP2R3A gene (684 bp apart) as described in the Methods section. The usage of two cleavage sites increased the chances to create a successful knockout. b HCT116 ΔgRNA and HCT116 ΔPR130 (clone #16) cells were cultured with 2 µM MS-275 and/or 1 mM hydroxyurea (HU) for 24 h. ATM, pATM (S1981), p53, pp53 (S15) and PR130 were detected by western blot. β-Actin served as loading control. Numbers indicate densitometric analysis of pATM signal relative to HU-treated sample of each cell line and normalised to β-Actin signal ( n = 3). c HCT116 cells were transiently transfected with indicated siRNAs. After 24 h, cells were treated with 1 mM HU and 2 µM MS-275 (24 h). Protein detection was performed by immunoblot ( n = 2). d After 20 h of treatment with HU (1 mM) and MS-275 (2 µM), okadaic acid (OA, 25 nM) was added for further 4 h. Co-immunoprecipitations (IP) with anti-pS1981-ATM antibody, rabbit pre-immune serum (pre) or no antibody (no AB) were analysed for pATM and PR130 by western blot. Input (IN) represents 6% of the lysates used for IP. The right panel shows results that were obtained with the same strategy, including individual treatment with HU and MS-275. Data represent results from three independent experiments that were carried out in a similar manner. e IP performed with PR130 antibody using lysates from untreated and MS-275-treated HCT116. Precipitates were probed with pan-acetyl-lysine (ac-Lys) and PR130 antibody. Input (IN) is 6% of the lysate used for immunoprecipitation ( n = 2). Numbers indicate densitometric analysis of ac-Lys and PR130 signal relative to untreated cells. f HCT116 cells were transfected with HA-PR130 or vector control (pcDNA3.1) for 24 h and subsequently treated with MS-275 (2 µM, 24 h). Phosphatase activity of the HA-precipitates against phospho-S1981-ATM peptide or a threonine phosphopeptide (positive control) were measured in vitro as liberated pmol phosphate/min. Data are presented as mean ± SEM ( n = 3). The western blot verifies precipitation of HA-tagged PR130

    Journal: Nature Communications

    Article Title: HDAC1 and HDAC2 integrate checkpoint kinase phosphorylation and cell fate through the phosphatase-2A subunit PR130

    doi: 10.1038/s41467-018-03096-0

    Figure Lengend Snippet: PR130 expression antagonises ATM phosphorylation. a Schematic of PPP2R3A knockout using CRISPR-Cas9 technology. Humanised S. pyogenes Cas9 nuclease was expressed in HCT116 cells with two guide RNAs (gRNA) to introduce two DSB into the PPP2R3A gene (684 bp apart) as described in the Methods section. The usage of two cleavage sites increased the chances to create a successful knockout. b HCT116 ΔgRNA and HCT116 ΔPR130 (clone #16) cells were cultured with 2 µM MS-275 and/or 1 mM hydroxyurea (HU) for 24 h. ATM, pATM (S1981), p53, pp53 (S15) and PR130 were detected by western blot. β-Actin served as loading control. Numbers indicate densitometric analysis of pATM signal relative to HU-treated sample of each cell line and normalised to β-Actin signal ( n = 3). c HCT116 cells were transiently transfected with indicated siRNAs. After 24 h, cells were treated with 1 mM HU and 2 µM MS-275 (24 h). Protein detection was performed by immunoblot ( n = 2). d After 20 h of treatment with HU (1 mM) and MS-275 (2 µM), okadaic acid (OA, 25 nM) was added for further 4 h. Co-immunoprecipitations (IP) with anti-pS1981-ATM antibody, rabbit pre-immune serum (pre) or no antibody (no AB) were analysed for pATM and PR130 by western blot. Input (IN) represents 6% of the lysates used for IP. The right panel shows results that were obtained with the same strategy, including individual treatment with HU and MS-275. Data represent results from three independent experiments that were carried out in a similar manner. e IP performed with PR130 antibody using lysates from untreated and MS-275-treated HCT116. Precipitates were probed with pan-acetyl-lysine (ac-Lys) and PR130 antibody. Input (IN) is 6% of the lysate used for immunoprecipitation ( n = 2). Numbers indicate densitometric analysis of ac-Lys and PR130 signal relative to untreated cells. f HCT116 cells were transfected with HA-PR130 or vector control (pcDNA3.1) for 24 h and subsequently treated with MS-275 (2 µM, 24 h). Phosphatase activity of the HA-precipitates against phospho-S1981-ATM peptide or a threonine phosphopeptide (positive control) were measured in vitro as liberated pmol phosphate/min. Data are presented as mean ± SEM ( n = 3). The western blot verifies precipitation of HA-tagged PR130

    Article Snippet: HCT116 cells were co-transfected with both plasmids (0.45 µg each) and a plasmid carrying puromycin resistance (0.1 µg) using the transfection reagent TurboFect (ThermoFisher Scientific) according to the manufacturer’s protocol.

    Techniques: Expressing, Knock-Out, CRISPR, Introduce, Cell Culture, Mass Spectrometry, Western Blot, Transfection, Immunoprecipitation, Plasmid Preparation, Activity Assay, Positive Control, In Vitro

    PR130 modulates cell cycle control during replicative stress. a HCT116 ΔgRNA and HCT116 ΔPR130 (clone #3) cells were treated with 2 µM MS-275 and/or 1 mM hydroxyurea (HU) for 24 h. Indicated proteins and their phosphorylation were analysed by western blot; β-Actin served as loading control. b Densitometric analysis of CHK1 phosphorylation (S317) and total CHK1 levels after 24 h following protein detection via western blot. Data were normalised to the respective loading controls. The amount of (phosphorylated) proteins was normalised to that of HU-treated cells. Results represent the mean ± SD ( n = 8; two-way ANOVA, **** P

    Journal: Nature Communications

    Article Title: HDAC1 and HDAC2 integrate checkpoint kinase phosphorylation and cell fate through the phosphatase-2A subunit PR130

    doi: 10.1038/s41467-018-03096-0

    Figure Lengend Snippet: PR130 modulates cell cycle control during replicative stress. a HCT116 ΔgRNA and HCT116 ΔPR130 (clone #3) cells were treated with 2 µM MS-275 and/or 1 mM hydroxyurea (HU) for 24 h. Indicated proteins and their phosphorylation were analysed by western blot; β-Actin served as loading control. b Densitometric analysis of CHK1 phosphorylation (S317) and total CHK1 levels after 24 h following protein detection via western blot. Data were normalised to the respective loading controls. The amount of (phosphorylated) proteins was normalised to that of HU-treated cells. Results represent the mean ± SD ( n = 8; two-way ANOVA, **** P

    Article Snippet: HCT116 cells were co-transfected with both plasmids (0.45 µg each) and a plasmid carrying puromycin resistance (0.1 µg) using the transfection reagent TurboFect (ThermoFisher Scientific) according to the manufacturer’s protocol.

    Techniques: Mass Spectrometry, Western Blot

    HDACi impairs checkpoint signalling upon replicative stress. a HCT116 cells were stimulated with 1 mM hydroxyurea (HU) and 2 µM MS-275 for 24 h. Western blot analyses of whole-cell lysates were performed to detect ATM and ATR as well as their phosphorylated forms (pATM S1981; pATR T1989). β-Actin served as loading control. Numbers indicate densitometric analysis of signals relative to HU-treated samples and normalised to β-Actin ( n = 3). b Densitometric evaluation of ATM phosphorylation at S1981 and ATM levels (after 24 h) following protein detection via immunoblot. Data were normalised to the loading controls. The respective amounts (phosphorylated) proteins are compared to those of HU-treated cells. Results represent the mean ± SD ( n = 3; one-way ANOVA; **** P

    Journal: Nature Communications

    Article Title: HDAC1 and HDAC2 integrate checkpoint kinase phosphorylation and cell fate through the phosphatase-2A subunit PR130

    doi: 10.1038/s41467-018-03096-0

    Figure Lengend Snippet: HDACi impairs checkpoint signalling upon replicative stress. a HCT116 cells were stimulated with 1 mM hydroxyurea (HU) and 2 µM MS-275 for 24 h. Western blot analyses of whole-cell lysates were performed to detect ATM and ATR as well as their phosphorylated forms (pATM S1981; pATR T1989). β-Actin served as loading control. Numbers indicate densitometric analysis of signals relative to HU-treated samples and normalised to β-Actin ( n = 3). b Densitometric evaluation of ATM phosphorylation at S1981 and ATM levels (after 24 h) following protein detection via immunoblot. Data were normalised to the loading controls. The respective amounts (phosphorylated) proteins are compared to those of HU-treated cells. Results represent the mean ± SD ( n = 3; one-way ANOVA; **** P

    Article Snippet: HCT116 cells were co-transfected with both plasmids (0.45 µg each) and a plasmid carrying puromycin resistance (0.1 µg) using the transfection reagent TurboFect (ThermoFisher Scientific) according to the manufacturer’s protocol.

    Techniques: Mass Spectrometry, Western Blot

    Loss of checkpoint control in the presence of MS-275. a HCT116 cells were treated with 2 µM MS-275 and/or 1 mM hydroxyurea (HU) for 24 h. Cell cycle analysis shown as mean ± SD ( n = 3). Control cells have G1 54%, S 22%, G2 24%; HU leads to G1 27%, S 55%, G2 18%; MS-275 leads to G1 69%, S 9%, G2 22%; HU/MS-275 leads to G1 30%, S 44%, G2 26%. Statistical significance is displayed for G2 cells (one-way ANOVA, * P

    Journal: Nature Communications

    Article Title: HDAC1 and HDAC2 integrate checkpoint kinase phosphorylation and cell fate through the phosphatase-2A subunit PR130

    doi: 10.1038/s41467-018-03096-0

    Figure Lengend Snippet: Loss of checkpoint control in the presence of MS-275. a HCT116 cells were treated with 2 µM MS-275 and/or 1 mM hydroxyurea (HU) for 24 h. Cell cycle analysis shown as mean ± SD ( n = 3). Control cells have G1 54%, S 22%, G2 24%; HU leads to G1 27%, S 55%, G2 18%; MS-275 leads to G1 69%, S 9%, G2 22%; HU/MS-275 leads to G1 30%, S 44%, G2 26%. Statistical significance is displayed for G2 cells (one-way ANOVA, * P

    Article Snippet: HCT116 cells were co-transfected with both plasmids (0.45 µg each) and a plasmid carrying puromycin resistance (0.1 µg) using the transfection reagent TurboFect (ThermoFisher Scientific) according to the manufacturer’s protocol.

    Techniques: Mass Spectrometry, Cell Cycle Assay

    Checkpoint kinases ensure cell survival upon replicative stress. a HCT116 cells were pretreated with 3 µM KU-60019 for 1 h followed by stimulation with 1 mM hydroxyurea (HU) for 24–48 h. Cell death was detected using PI staining and flow cytometry. Results are displayed as mean ± SD ( n 24 h = 3, n 48 h = 4; one-way ANOVA, *** P

    Journal: Nature Communications

    Article Title: HDAC1 and HDAC2 integrate checkpoint kinase phosphorylation and cell fate through the phosphatase-2A subunit PR130

    doi: 10.1038/s41467-018-03096-0

    Figure Lengend Snippet: Checkpoint kinases ensure cell survival upon replicative stress. a HCT116 cells were pretreated with 3 µM KU-60019 for 1 h followed by stimulation with 1 mM hydroxyurea (HU) for 24–48 h. Cell death was detected using PI staining and flow cytometry. Results are displayed as mean ± SD ( n 24 h = 3, n 48 h = 4; one-way ANOVA, *** P

    Article Snippet: HCT116 cells were co-transfected with both plasmids (0.45 µg each) and a plasmid carrying puromycin resistance (0.1 µg) using the transfection reagent TurboFect (ThermoFisher Scientific) according to the manufacturer’s protocol.

    Techniques: Staining, Flow Cytometry, Cytometry

    DNA damage and apoptosis in the presence of hydroxyurea and MS-275. a Flow cytometry analysis of HCT116 cells using Annexin-V-FITC staining. Cells were treated with 1 mM hydroxyurea (HU), 2 µM MS-275 or both for 24–48 h. Results represent the mean ± SD ( n = 3; one-way ANOVA, ** P

    Journal: Nature Communications

    Article Title: HDAC1 and HDAC2 integrate checkpoint kinase phosphorylation and cell fate through the phosphatase-2A subunit PR130

    doi: 10.1038/s41467-018-03096-0

    Figure Lengend Snippet: DNA damage and apoptosis in the presence of hydroxyurea and MS-275. a Flow cytometry analysis of HCT116 cells using Annexin-V-FITC staining. Cells were treated with 1 mM hydroxyurea (HU), 2 µM MS-275 or both for 24–48 h. Results represent the mean ± SD ( n = 3; one-way ANOVA, ** P

    Article Snippet: HCT116 cells were co-transfected with both plasmids (0.45 µg each) and a plasmid carrying puromycin resistance (0.1 µg) using the transfection reagent TurboFect (ThermoFisher Scientific) according to the manufacturer’s protocol.

    Techniques: Mass Spectrometry, Flow Cytometry, Cytometry, Staining