Journal: Cell Death & Disease

Article Title: HCRP-1 regulates EGFR–AKT–BIM-mediated anoikis resistance and serves as a prognostic marker in human colon cancer

doi: 10.1038/s41419-018-1217-2

Figure Lengend Snippet: a , b HCT116 and SW620 cells were transfected with si-HCRP-1 or control siRNA, and then cells were harvested and submitted to Western blot detection for the protein expression of BIM, FoxO3a, EGFR, AKT, ERK, Mcl-1, Bcl-2, and β-actin. Phosphorylated forms of EGFR, AKT, FoxO3a, and ERK were also detected by western blot with the corresponding antibodies. c–f Cells were treatedwith CHX for 0, 0.5 , 1, and 2 h after transfection with si-HCRP-1 or control siRNA for 48 h, lysates obtained from these cells were submitted to western blot detection for the protein expression of EGFR. Error bars indicate mean ± SD. Every experiment was repeated at least three times. * P < 0.05 vs. siCtrl group

Article Snippet: Antibodies against EGFR (sc-03-G), ERK (sc-271269), and p-ERK (Tyr204, sc-7382) were purchased from Santa Cruz (CA, USA).

Techniques: Transfection, Control, Western Blot, Expressing

Journal:

Article Title: Epidermal growth factor-induced nuclear factor ?B activation: A major pathway of cell-cycle progression in estrogen-receptor negative breast cancer cells

doi:

Figure Lengend Snippet: A proposed pathway of EGF-induced cell proliferation of ER− breast cancer cells. The interaction of EGF with EGFR and the proposed downstream events of NF-κB activation and cell-cycle progression are schematically shown. The shaded molecules are monitored. The sites of action of agents used to block individual steps of this pathway, such as EGF–EGFR interaction by EGFR-Ab, PI3 kinase (phosphotidylinositol 3-kinase) activity by Ly294–002, PKC (protein kinase C) activity by G06976, and IKK activity by IKK-M (dominant-negative mutant of IKK) are shown. The role of G1-specific cell-cycle regulatory proteins, ccD1 and phosphorylated retinoblastoma (pRb), and unphosphorylated Rb is illustrated.

Article Snippet: Anti-human ER-α antibody (SC543), anti-EGFR antibody (SC-03-G), anti-human Neu antibody (SC-284-G), and anti-human cyclin D1 (ccD1) antibody (Sc 8396) were obtained from Santa Cruz Biotechnology.

Techniques: Activation Assay, Blocking Assay, Activity Assay, Dominant Negative Mutation

Journal:

Article Title: Epidermal growth factor-induced nuclear factor ?B activation: A major pathway of cell-cycle progression in estrogen-receptor negative breast cancer cells

doi:

Figure Lengend Snippet: Levels of ER and EGFR family receptors and activation and inhibition of NF-κB in breast cancer cells. A shows the levels of ER, EGFR, Neu, and actin proteins in whole-cell extracts of ER− MDA-MB435 and MDA-MB231 and ER+ T47D and MCF-7 breast cancer cells in culture, as measured by Western blot analysis. Cells were grown in rich (R) medium to 90–95% confluency, whole-cell extracts were prepared (6), and 50 μg of protein in samples (in duplicate, designated by numerals under each cell line) was subjected to Western blot analysis and immunodetected with anti-ER-antibody Sc-543 (row 1) (42). The same blot was stripped and reused for detection of EGFR with anti-EGFR antibody Sc-03-G (row 2). Row 3 shows the level of Neu detected similarly with the anti-Neu antibody (Sc 284 G), and row 4 shows the levels of actin in the same samples as determined by reprobing the same blot with antiactin antibody, which serves as a loading control. These determinations were made three times, and results of one experiment are shown here. B shows the level of 32P-DNA-binding activity of NF-κB in the indicated amounts (protein) of nuclear extracts from ER− MDA-MB231 and ER+ MCF-7 cells grown in rich medium (R) or basal medium (B), as measured by EMSA (38–39). The retarded specific NF-κB-32P-DNA complex is indicated by the upper arrow, and the free 32P-DNA (NF-κB–oligonucleotide) is indicated by the bottom arrow. C shows stimulation of NF-κB-32P-DNA-binding activity by E2 and EGF. The binding activity (numerals on the y axis) represents integrated intensity of the autoradiographic signals quantitated, as described in Materials and Methods. The ER− MDA-MB231 and MDA-MB435 and ER+ MCF-7 cells were plated in 25 ml of rich medium in 150-mm tissue culture dishes. Forty-eight hours later, the medium was removed, and cells were washed with basal medium (B) and replenished with 25 ml of the same medium. Seventy-two hours later, the medium was removed and replenished with 25 ml of basal medium, and cells were grown for an additional 12 h in the presence of either E2 (10−6 M) or EGF (12 ng/ml). Nuclear extracts from the treated and control cells were prepared (40), and NF-κB-32P-DNA-binding activities in 5 μg of nuclear extracts of these samples were measured by EMSA. One of four such experiments is reported here. D shows the NF-κB-32P-DNA-binding activity in nuclear extracts (5 μg) from the four breast cancer cell lines grown in basal medium plus EGF (12 ng/m) and indicated amounts of anti-EGFR-antibody per 10 ml of basal medium for 12 h. Growth and treatment conditions of the cells were the same as described in C. NF-κB-32P-DNA-binding activity was determined in nuclear extracts from two ER− MDA-MB-435 and MDA-MB231 and two ER+, MCF-7, T47D cells by EMSA and quantitated, as described above. E shows similar analysis for the determination of NF-κB-32P-DNA-binding activity in nuclear extracts of cells treated with indicated concentrations of G06976. Growth and treatment conditions of cells are the same as described in C. Nuclear extracts from three ER− and two ER+ cells were prepared and subjected to EMSA. Quantitation of the autoradiographic signals of the NF-κB-32P-DNA complex was the same as described above.

Article Snippet: Anti-human ER-α antibody (SC543), anti-EGFR antibody (SC-03-G), anti-human Neu antibody (SC-284-G), and anti-human cyclin D1 (ccD1) antibody (Sc 8396) were obtained from Santa Cruz Biotechnology.

Techniques: Activation Assay, Inhibition, Western Blot, Binding Assay, Activity Assay, Quantitation Assay

Journal:

Article Title: Epidermal growth factor-induced nuclear factor ?B activation: A major pathway of cell-cycle progression in estrogen-receptor negative breast cancer cells

doi:

Figure Lengend Snippet: Level of ccD1 in EGF-treated cells and inhibition by EGFR-Ab and Go6976. A shows the levels of ccD1 in EGF (12 ng/ml) and E2 (10−6 M)-treated ER− and ER+ breast cancer cells. Growth conditions of the cells were the same as described in the legend to Fig. ​Fig.2.2. The ER− MDA-MB231 and MDA-MB435 and ER+ MCF-7 cells were grown in basal medium in the presence of EGF (12 ng/ml) or E2 (10−6 M) for 12 h. Whole-cell extracts were prepared, and their level of ccD1 protein in 50 μg was determined by Western blot analysis. The membranes with blot-transferred fractionated proteins were then subjected to detection with anti-ccD1-antibody (Sc 8396) and enhanced chemiluminescence immunodetection system, as described (42). The same blot was stripped and reprobed with antiactin antibody, which serves as loading control. These experiments were repeated three times. The levels of ccD1 (row 1) in EGF-stimulated ER+ MCF-7 (B), ER− MDA-MB-231 (C), and ER− MDA-MB-435 (D) cells, simultaneously treated with indicated amounts of EGFR-Ab (micrograms per 10 ml) or Go6976 (μM) are shown. Fifty micrograms of whole cell extract proteins is used in each lane. Duplicate samples were analyzed in the case ER− MDA-MB-435 cells (D). Lanes designated by Actin (row 2) (A–D) represent the results of reprobing of each of these membranes with antiactin-Ab, which served as loading controls.

Article Snippet: Anti-human ER-α antibody (SC543), anti-EGFR antibody (SC-03-G), anti-human Neu antibody (SC-284-G), and anti-human cyclin D1 (ccD1) antibody (Sc 8396) were obtained from Santa Cruz Biotechnology.

Techniques: Inhibition, Western Blot, Immunodetection

Journal:

Article Title: Epidermal growth factor-induced nuclear factor ?B activation: A major pathway of cell-cycle progression in estrogen-receptor negative breast cancer cells

doi:

Figure Lengend Snippet: (A) Level of pRb protein in EGF, EGFR-Ab, and Go6976-treated ER− MDA-MB435 cells. Cells were grown in basal medium in the presence of EGF (12 ng/ml) and EGFR-Ab or Go6976 at the indicated concentrations for 12 h. The level of pRb and underphosphorylated Rb (Rb) in 50 μg of whole-cell extracts was detected by Western blot analysis and immunodetection with anti-Rb-antibody, as described above. (B) NF-κB DNA-binding activity and ccD1 level in IKK-M-transfected ER− MDA-MB435 cells. Results presented demonstrate the levels of NF-κB activation and ccD1 in EGF-treated ER− MDA-MB435 cells transiently transfected with dominant-negative IKK-α (α), IKK-β (β) mutants (IKK-M), and vector control (v) (53). Growth conditions and EGF treatment are as described in A. Cells were transfected with indicated plasmids (10 μg) by using Superfect Transfection Reagent (Qiagen, Chatsworth, CA) following the protocol of the supplier. Cells in fresh basal medium (B) were grown for an additional 48 h after transfection. EGF was added 4 h before harvesting, nuclear extracts were prepared, and the level of NF-κB-binding activity and ccD1 was measured as described above.

Article Snippet: Anti-human ER-α antibody (SC543), anti-EGFR antibody (SC-03-G), anti-human Neu antibody (SC-284-G), and anti-human cyclin D1 (ccD1) antibody (Sc 8396) were obtained from Santa Cruz Biotechnology.

Techniques: Western Blot, Immunodetection, Binding Assay, Activity Assay, Transfection, Activation Assay, Dominant Negative Mutation, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: HDAC6-Selective Inhibitor Overcomes Bortezomib Resistance in Multiple Myeloma

doi: 10.3390/ijms22031341

Figure Lengend Snippet: Working model for HDAC6 inhibitor and PI for overcoming BTZ resistance of MM. HDAC6 inhibition by A452 synergize with BTZ or CFZ to inhibit the activation of NF-κB and STAT3, resulting in decreased expressions of LMP2 and LMP7. Additionally, combination treatment of A452 and BTZ or CFZ synergistically inactivates the AKT and ERK MAPK pathways. Combining A452 with BTZ or CFZ leads to synergistic cancer cell growth inhibition and viability decreases. This combination induces apoptosis and enhances PI sensitivity in the BTZ-resistant MM cells.

Article Snippet: Antibodies against AKT (sc-8312), p-AKT (sc-7985-R), Bak (sc-832), ERK (sc-03-G), HDAC6 (sc-11420), LMP2 (sc-514345), p65 (sc-8008), STAT3 (sc-482), and α-tubulin (sc-32293) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Inhibition, Activation Assay