Journal: Frontiers in Oncology

Article Title: Resveratrol Sensitizes Colorectal Cancer Cells to Cetuximab by Connexin 43 Upregulation-Induced Akt Inhibition

doi: 10.3389/fonc.2020.00383

Figure Lengend Snippet: Cx43 expression and GJ function of HCT116 and CT26 cells, including parental, control-shRNA, and Cx43-shRNA transfected cells. RES, resveratrol-treated cells. All other groups were compared with the Parental group using one-way ANOVA. * P < 0.05 represents a significant difference from values in the Parental group. SEMs are shown as error bars. (A1) Western blot images of Cx43 and phospho-Cx43 (Ser-368). β-Actin is used as a loading control. The full-length blots are shown as . (A2,A3) Bar diagrams of densitometric analysis. Protein expression level is normalized by β-actin. (B) GJ function analysis by “Parachute” dye-coupling assay. Scale bars are 20 μm. Columns show the mean ± SEM. All images of “Parachute” dye-coupling assay are shown as .

Article Snippet: Cx43 shRNA plasmid (sc-29276-SH and sc-35091-SH), control shRNA plasmid, Plasmid Transfection Reagent, Plasmid Transfection Medium, mouse anti-Cx43 antibody, and protein A/G PLUS-Agarose were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). pTARGET Mammalian Expression Vector system was purchased from Promega Corporation (Madison, WI, USA).

Techniques: Expressing, Control, shRNA, Transfection, Western Blot

Journal: Frontiers in Oncology

Article Title: Resveratrol Sensitizes Colorectal Cancer Cells to Cetuximab by Connexin 43 Upregulation-Induced Akt Inhibition

doi: 10.3389/fonc.2020.00383

Figure Lengend Snippet: Results of the MTT assay and CalcuSyn software analysis for parental, shRNA transfected, and Cx43 transfected HCT116 and CT26 cells. vector, pTARGET vector; CTX, cetuximab treatment; RES, resveratrol treatment; High, high density cultured cells (80%); Low, low density cultured cells (5%). (A) MTT assay results. The vertical axis represents growth inhibition rate, which is compared with that of untreated cells. The horizontal axis represents agent concentrations. Points represent mean ± SEM. Panels (A13–A16) are the results of high density culture cells. The raw data of MTT are shown as . (B) CalcuSyn software analysis of the MTT assay results. CI, combination index. CI is calculated by calcusyn analysis based on the inhibition rate of different concentration of resveratrol and cetuximab. CI > 1 represents antagonistic cytotoxicity; CI = 1 represents addictive cytotoxicity; CI < 1 represents synergistic cytotoxicity. The data generated by CalcuSyn are shown as . (C) IC50 (μg/ml) of cells to resveratrol and/or cetuximab. * P < 0.05 represents a significant difference between parental and transfected cells compared by one-way ANOVA. There is no significant difference between parental and control-shRNA in all conditions. The statistical difference of growth inhibition between parental and Cx43-shRNA transfected cells in different concentration is shown as .

Article Snippet: Cx43 shRNA plasmid (sc-29276-SH and sc-35091-SH), control shRNA plasmid, Plasmid Transfection Reagent, Plasmid Transfection Medium, mouse anti-Cx43 antibody, and protein A/G PLUS-Agarose were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). pTARGET Mammalian Expression Vector system was purchased from Promega Corporation (Madison, WI, USA).

Techniques: MTT Assay, Software, shRNA, Transfection, Plasmid Preparation, Cell Culture, Inhibition, Concentration Assay, Generated, Control

Journal: Frontiers in Oncology

Article Title: Resveratrol Sensitizes Colorectal Cancer Cells to Cetuximab by Connexin 43 Upregulation-Induced Akt Inhibition

doi: 10.3389/fonc.2020.00383

Figure Lengend Snippet: Immunoprecipitation analysis of Akt and Cx43 in parental HCT116 and CT26 cells. Cell lysates were subjected to immunoprecipitation with anti-Akt or anti-Cx43 antibodies. Then immunoprecipitated proteins were examined by western blotting with anti-Cx43 or anti-Akt antibodies, respectively. The input of cell total protein was used as a positive control and rabbit IgG was used as a negative control. Molecular weight of Akt and IgG heavy chain is similar, so they are hardly to be discriminated. SDS-PAGE of immunoprecipitation analysis is shown as . (A) IB: Cx43; IP: Akt. (A1) Non-treated cells. (A2) Resveratrol-treated cells. (A3) Cetuximab-treated cells. (A4) Cetuximab + Resveratrol-treated cells. (B) IB: Akt; IP: Cx43. (B1) Non-treated cells. (B2) Resveratrol-treated cells. (B3) Cetuximab-treated cells. (B4) Cetuximab + Resveratrol-treated cells.

Article Snippet: Cx43 shRNA plasmid (sc-29276-SH and sc-35091-SH), control shRNA plasmid, Plasmid Transfection Reagent, Plasmid Transfection Medium, mouse anti-Cx43 antibody, and protein A/G PLUS-Agarose were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). pTARGET Mammalian Expression Vector system was purchased from Promega Corporation (Madison, WI, USA).

Techniques: Immunoprecipitation, Western Blot, Positive Control, Negative Control, Molecular Weight, SDS Page

Journal: Frontiers in Oncology

Article Title: Resveratrol Sensitizes Colorectal Cancer Cells to Cetuximab by Connexin 43 Upregulation-Induced Akt Inhibition

doi: 10.3389/fonc.2020.00383

Figure Lengend Snippet: Western blot analysis of proteins related to the EGFR signal pathway, including EGFR, p-EGFR, PI3K, p-PI3K, Akt, p-Akt, mTOR, p-mTOR, IKKα, IκBα, NFκB p65, MAPK, and p-MAPK. (A) Western blot images. β-Actin is used as a loading control for total protein, and histone H3 is used as a loading control for nuclear proteins. CTX, cetuximab treatment; RES, resveratrol treatment. The full-length blots are shown as . (B) Relationship between the EGFR signaling pathway and Cx43 ( , , ). Cetuximab inhibits the binding reaction of EGF to EGFR, which inhibits the whole EGFR signaling pathway. Akt, a key molecule in this pathway, interacts with Cx43. Resveratrol increases Cx43 expression and phosphorylation.

Article Snippet: Cx43 shRNA plasmid (sc-29276-SH and sc-35091-SH), control shRNA plasmid, Plasmid Transfection Reagent, Plasmid Transfection Medium, mouse anti-Cx43 antibody, and protein A/G PLUS-Agarose were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). pTARGET Mammalian Expression Vector system was purchased from Promega Corporation (Madison, WI, USA).

Techniques: Western Blot, Control, Binding Assay, Expressing, Phospho-proteomics

Journal: Frontiers in Oncology

Article Title: Resveratrol Sensitizes Colorectal Cancer Cells to Cetuximab by Connexin 43 Upregulation-Induced Akt Inhibition

doi: 10.3389/fonc.2020.00383

Figure Lengend Snippet: Inhibition of tumor growth by cetuximab and resveratrol. CTX, cetuximab-treated mice; RES, resveratrol-treated mice. (A) The subcutaneous tumor model was created by parental, control-shRNA, or Cx43-shRNA-transfected cells. Each group contains five Balb/c mice for CT26 or nu/nu nude mice for HCT116. These mice were then treated with cetuximab and/or resveratrol for 14 days before sacrificed. Tumors were removed, and weight was measured using electronic balance. (B) Columns represent mean ± SEM volume from five tumors. Groups “CTX,” “RES,” and “CTX+RES” were compared with group “No treated” using one-way ANOVA. Blue * P < 0.05 represents a significant difference. Group “CTX+RES” was compared with group “CTX” and “RES” using one-way ANOVA. Yellow * P < 0.05 represents a significant difference.

Article Snippet: Cx43 shRNA plasmid (sc-29276-SH and sc-35091-SH), control shRNA plasmid, Plasmid Transfection Reagent, Plasmid Transfection Medium, mouse anti-Cx43 antibody, and protein A/G PLUS-Agarose were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). pTARGET Mammalian Expression Vector system was purchased from Promega Corporation (Madison, WI, USA).

Techniques: Inhibition, Control, shRNA, Transfection