Journal: Molecular medicine reports

Article Title: TrkB/C-induced HOXC6 activation enhances the ADAM8-mediated metastasis of chemoresistant colon cancer cells.

doi: 10.3892/mmr.2021.12062

Figure Lengend Snippet: Figure 2. Expressions of HOXC6 and ADAM8 are significantly increased in chemoresistant colon cancer cells. OxR or FuR colon cancer and parent cells were seeded into 6‑well plates (1.5x105/well) and cultured for 24 h. (A and C) Quantitative real‑time PCR was performed to determine the relative expression of HOXC4, HOXC6, ADAM8, ADAM10, ADAM12 and ADAM17. (B and D) The total protein of each group was subjected to western blot analysis with the indicated antibodies. β‑actin served as an internal control. (E) The migration and invasion of cells were detected using tumor transendothelial migration and BME cell invasion assay kits, respectively. (F) The gelatinase activity of cells was measured using a gelatinase assay kit. Data are presented as the mean ± SD of three independent experiments. *P<0.05, #P<0.005 and ##P<0.001 vs. resistant cells. HOX, homeobox; ADAM, A disintegrin and metallopro teinase domain‑containing 8; OxR, oxaliplatin resistant; FuR, 5‑Fu‑resistant; p, phosphorylated.

Article Snippet: The following primary antibodies were used: TrkB (#4603), TrkC (#3376), phospho‐ERK1/2 (Thr202/Tyr204; #9101), ERK1/2 (#9102), MMP2 (#4022), MMP9 (#3852), E‐cadherin (#3195), N‐cadherin (#13116), Snail (#3879), and β‐actin (#4967) (Cell Signaling Technology); phospho‐TrkB (Tyr817; #NBP2‐67578), ADAM8 (#NB600‐1393), and HOXC4 (#NBP2‐56195) (Novus Biologicals); phospho‐TrkC (Tyr518; #PA5‐40271) (Thermo Fisher Scientific); and HOXC4 (#sc‐81965), HOXC6 (#sc‐376330), phospho‐MEK1/2 (Ser218/Ser222; #sc‐7995), and MEK1/2 (#sc‐436) (Santa Cruz Biotechnology).

Techniques: Cell Culture, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Control, Migration, Invasion Assay, Activity Assay

Journal: Molecular medicine reports

Article Title: TrkB/C-induced HOXC6 activation enhances the ADAM8-mediated metastasis of chemoresistant colon cancer cells.

doi: 10.3892/mmr.2021.12062

Figure Lengend Snippet: Figure 4. MEK/ERK activation induces HOXC6 and ADAM8 during chemoresistant colon cancer cell metastasis. OxR or FuR colon cancer cells were seeded into 6‑well plates (1.5x105/well) and treated with 20 µM PD98059 for 24 h. (A and B) Total cell lysates were immunoblotted with the indicated antibodies. β‑actin served as an internal control. (C) The gelatinase activity of cells was measured using the gelatinase assay kit. (D) The migration and invasion of cells was detected using tumor transendothelial migration and BME cell invasion assay kits, respectively. Data are presented as the mean ± SD of three independent experiments. *P<0.05, **P<0.01, #P<0.005 and ##P<0.001 as indicated. HOX, homeobox; ADAM, A disintegrin and metalloproteinase domain‑containing 8; OxR, oxaliplatin resistant; FuR, 5‑Fu‑resistant; Trk, tropomyosin receptor kinase; p, phosphorylated.

Article Snippet: The following primary antibodies were used: TrkB (#4603), TrkC (#3376), phospho‐ERK1/2 (Thr202/Tyr204; #9101), ERK1/2 (#9102), MMP2 (#4022), MMP9 (#3852), E‐cadherin (#3195), N‐cadherin (#13116), Snail (#3879), and β‐actin (#4967) (Cell Signaling Technology); phospho‐TrkB (Tyr817; #NBP2‐67578), ADAM8 (#NB600‐1393), and HOXC4 (#NBP2‐56195) (Novus Biologicals); phospho‐TrkC (Tyr518; #PA5‐40271) (Thermo Fisher Scientific); and HOXC4 (#sc‐81965), HOXC6 (#sc‐376330), phospho‐MEK1/2 (Ser218/Ser222; #sc‐7995), and MEK1/2 (#sc‐436) (Santa Cruz Biotechnology).

Techniques: Activation Assay, Control, Activity Assay, Migration, Invasion Assay

Journal: Molecular medicine reports

Article Title: TrkB/C-induced HOXC6 activation enhances the ADAM8-mediated metastasis of chemoresistant colon cancer cells.

doi: 10.3892/mmr.2021.12062

Figure Lengend Snippet: Figure 5. HOXC6 knockdown in OxR or FuR colon cancer cells decreases cell invasion and downregulates ADAM8 activation and gelatinase activity. OxR or FuR colon cancer cells were seeded into 6‑well plates (1.5x105/well) and grown overnight. Cells were transfected with small interfering RNA against HOXC6 or control for 48 h. (A) Total cell lysates were immunoblotted with the indicated antibodies. β‑actin served as an internal control. (B) The gelatinase activity of cells was measured by the gelatinase assay kit. (C) The migration and invasion of cells was detected using tumor transendothelial migration and the BME cell invasion assay kits, respectively. Data are presented as the mean ± SD of the three independent experiments. *P<0.05, **P<0.01, #P<0.005 and ##P<0.001 as indicated. HOX, homeobox; OxR, oxaliplatin resistant; FuR, 5‑Fu‑resistant; ADAM, A disintegrin and metalloproteinase domain‑containing 8; Trk, tropomyosin receptor kinase; si, small interfering RNA; ctrl, control; p, phosphorylated.

Article Snippet: The following primary antibodies were used: TrkB (#4603), TrkC (#3376), phospho‐ERK1/2 (Thr202/Tyr204; #9101), ERK1/2 (#9102), MMP2 (#4022), MMP9 (#3852), E‐cadherin (#3195), N‐cadherin (#13116), Snail (#3879), and β‐actin (#4967) (Cell Signaling Technology); phospho‐TrkB (Tyr817; #NBP2‐67578), ADAM8 (#NB600‐1393), and HOXC4 (#NBP2‐56195) (Novus Biologicals); phospho‐TrkC (Tyr518; #PA5‐40271) (Thermo Fisher Scientific); and HOXC4 (#sc‐81965), HOXC6 (#sc‐376330), phospho‐MEK1/2 (Ser218/Ser222; #sc‐7995), and MEK1/2 (#sc‐436) (Santa Cruz Biotechnology).

Techniques: Knockdown, Activation Assay, Activity Assay, Transfection, Small Interfering RNA, Control, Migration, Invasion Assay

Journal: Molecular medicine reports

Article Title: TrkB/C-induced HOXC6 activation enhances the ADAM8-mediated metastasis of chemoresistant colon cancer cells.

doi: 10.3892/mmr.2021.12062

Figure Lengend Snippet: Figure 6. Activation of ADAM8 but not ADAM10 or ADAM17 regulates the invasion and gelatinase activity of metastatic drug‑resistant colon cancer cells. OxR or FuR colon cancer cells were seeded into 6‑well plates (1.5x105/well) and grown overnight. Cells were transfected with small interfering RNA against ADAM8 or control for 48 h, or treated with GI (10 µM) and MA (50 nM) for 24 h. (A and B) Total cell lysates were immunoblotted with the indicated antibodies. β‑actin served as an internal control. (C) The gelatinase activity of cells was measured using the gelatinase assay kit. (D) The migration and invasion of cells were detected using tumor transendothelial migration and BME cell invasion assay kits, respectively. Data are presented as the mean ± SD of three independent exper iments. *P<0.05 and ##P<0.001 as indicated. (E) Schematic diagram of the intracellular signaling pathway in drug‑resistant human colon cancer cells. ADAM8 expression contributed to the increased resistance and migratory activity of chemoresistant colon cancer cells through the activation of the TrkB/C‑HOXC6 axis. ADAM, A disintegrin and metalloproteinase domain‑containing 8; OxR, oxaliplatin resistant; FuR, 5‑Fu‑resistant; GI, ADAM10 inhibitor GI254023X; MA, ADAM17 inhibitor marimastat; Trk, tropomyosin receptor kinase; HOX, homeobox; si, small interfering RNA; Ctrl, control; p, phosphorylated.

Article Snippet: The following primary antibodies were used: TrkB (#4603), TrkC (#3376), phospho‐ERK1/2 (Thr202/Tyr204; #9101), ERK1/2 (#9102), MMP2 (#4022), MMP9 (#3852), E‐cadherin (#3195), N‐cadherin (#13116), Snail (#3879), and β‐actin (#4967) (Cell Signaling Technology); phospho‐TrkB (Tyr817; #NBP2‐67578), ADAM8 (#NB600‐1393), and HOXC4 (#NBP2‐56195) (Novus Biologicals); phospho‐TrkC (Tyr518; #PA5‐40271) (Thermo Fisher Scientific); and HOXC4 (#sc‐81965), HOXC6 (#sc‐376330), phospho‐MEK1/2 (Ser218/Ser222; #sc‐7995), and MEK1/2 (#sc‐436) (Santa Cruz Biotechnology).

Techniques: Activation Assay, Activity Assay, Transfection, Small Interfering RNA, Control, Migration, Invasion Assay, Expressing

Journal: Frontiers in Immunology

Article Title: Combined p14ARF and Interferon-β Gene Transfer to the Human Melanoma Cell Line SK-MEL-147 Promotes Oncolysis and Immune Activation

doi: 10.3389/fimmu.2020.576658

Figure Lengend Snippet: Emission of immunogenic cell death markers induced by combined adenoviral p14ARF + IFNβ gene transfer. SK-MEL-147 cells transduced as previously described, incubated for 48h h before cells and supernatants were collected for ICD assays. (A) Calreticulin exposure was assessed by flow cytometry after specific antibody staining. Representative dot plots and a bar graph showing the mean and standard deviation from three independent tests with three technical replicates each. (B) Supernatant from the same cultures were collected and evaluated for ATP secretion using a luciferase-based assay (RLU, relative light units). Data represent the mean and standard deviation from at least three independent experiments. (C) Detection of secreted IFNβ protein from cell supernatant by ELISA. Data represent the mean and standard deviation from at least three independent experiments. For both (A–C) , statistical analyses were performed using one-way ANOVA test followed by the Bonferroni post-test. *p < 0.05, **p < 0.005, and ***p < 0.0005.

Article Snippet: For the evaluation of calreticulin exposure through flow cytometry, cells were probed with rabbit anti-calreticulin antibody (Novus Biologicals, Littleton, CO, USA), followed by the Alexa488-conjugated anti-rabbit secondary antibody (Thermo Fisher Scientific).

Techniques: Incubation, Flow Cytometry, Staining, Standard Deviation, Luciferase, Enzyme-linked Immunosorbent Assay

Journal: International journal of biological macromolecules

Article Title: Disulfide bond and crosslinking analyses reveal inter-domain interactions that contribute to the rigidity of placental malaria VAR2CSA structure and formation of CSA binding channel.

doi: 10.1016/j.ijbiomac.2022.11.258

Figure Lengend Snippet: Fig. 8. The binding capacity of full-length VAR2CSA ectodomain is lower than the ectodomain lacking NTS. The binding of NTS-DBL6ε and DBL1x-DBL6ε to CSPG was measured in triplicates by ELISA. The wells of 96-well microtiter plates were coated with CSPG (200 ng/ml) and blocked with BSA and then incubated with 1:2 serially diluted solutions of NTS-DBL6ε or DBL1x-DBL6ε. The levels of bound proteins were measured using anti-cMyc antibody and HRP- conjugated secondary antibody. Data are a representative of three independent experiments using different batches of purified proteins. KD values were determined by plotting OD vs protein concentration using GraphPad Prism v9.4.1. KD ± SE: 41.5 ± 3.9 (DBBL1x-DBL6ε); 59.9 ± 6.8 (NTS-DBL6ε).

Article Snippet: Unbound proteins in the coated wells were removed by washing with 100 μl of PBST and the plates were incubated with 1:1000 diluted mouse anti-cMyc monoclonal antibody (Cat No. NB600-302, NOVUS Biologicals, Centennial, CO), and washed three times with 100 μl PBST.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation, Purification, Protein Concentration