Journal: Chinese Science Bulletin

Article Title: C-terminal domain of Chk1 regulates its subcellular location and kinase activity for DNA repair

doi: 10.1007/s11434-011-4538-4

Figure Lengend Snippet: Figure 14 Relative kinase activities of the different C-terminal truncation mutants. Cdc25C was used as an internal reference to adjust the gray val- ues of the target protein. The ratio of Cdc25C Ser216 phosphorylation in cells without HU treatment was set as 1. Values are mean ± SD for 3 re- peated experiments with stable results per cell type. HU = hydroxyurea. Compare with control 2, *, P<0.05; **, P<0.01; #, compare with control 3, P<0.05.

Article Snippet: Phospho-cdc25C (Ser216)(63F9) rabbit mAb and cdc25C (5H9) rabbit mAb were purchased from Cell signaling Technology (Cell signaling Technology, Danvers, MA) and anti-rabbit IRDye800-linked IgG was obtained from Rockland Immunochemicals (Rockland Immunochemicals, Inc., Gilbertsville, PA).

Techniques: Phospho-proteomics, Control

Journal: Journal of Thoracic Disease

Article Title: Aumolertinib combined with targeting ETV4 in the treatment of non-small cell lung cancer

doi: 10.21037/jtd-2025-aw-2071

Figure Lengend Snippet: Aumolertinib inhibits proliferation, migration, and induces apoptosis in PC-9 cells. (A) CCK-8 assay was used to evaluate the inhibitory effects of aumolertinib on cell viability of PC-9 cells. (B) CCK-8 assay was used to evaluate the inhibitory effects of aumolertinib on proliferation of PC-9 cells (P=0.003). (C,D) In vitro wound healing assay was performed to assess the effects of aumolertinib on migration ability of PC-9 cells (P=0.02), magnification 100×. (E,F) Flow cytometry with Annexin V/PI staining was used to determine the effects of aumolertinib on apoptosis of PC-9 cells (P<0.001). (G,H) Flow cytometric analysis of the cell cycle in PC-9 cells treated with aumolertinib (P=0.049). *, P<0.05; **, P<0.01; ***, P<0.001. CCK-8, Cell Counting Kit-8; ETV4 , ETS variant transcription factor 4; FITC, fluorescein isothiocyanate; PI, propidium iodide; si ETV4 , ETV4 siRNA; siNC, negative control siRNA; siRNA, small interfering RNA.

Article Snippet: 5% skim milk was used for blocking nonspecific binding by incubation for 1 h. Subsequently, the membrane was incubated with ETV4 antibodies (1:1,000, 10684-1-AP, Proteintech, Wuhan, China) overnight at 4 °C.

Techniques: Migration, CCK-8 Assay, In Vitro, Wound Healing Assay, Flow Cytometry, Staining, Cell Counting, Variant Assay, Negative Control, Small Interfering RNA

Journal: Journal of Thoracic Disease

Article Title: Aumolertinib combined with targeting ETV4 in the treatment of non-small cell lung cancer

doi: 10.21037/jtd-2025-aw-2071

Figure Lengend Snippet: Silencing ETV4 potentiates aumolertinib’s growth inhibition in PC-9 cells. (A) PC-9 cells were transfected with si ETV4 (si ETV4 -1, si ETV4 -2, and si ETV4 -3) or siNC. Knockdown efficiency using si ETV4 in PC-9 cells was analyzed by qRT-PCR (P<0.001). (B,C) Knockdown efficiency using si ETV4 in PC-9 cells was analyzed by immunoblotting. GAPDH was used as the control (P<0.001). (D) CCK-8 assay was used to evaluate the effects of si ETV4 on proliferation of PC-9 cells that treated with aumolertinib (P=0.001). **, P<0.01; ***, P<0.001. CCK-8, Cell Counting Kit-8; ETV4 , ETS variant transcription factor 4; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; mRNA, messenger RNA; qRT-PCR, quantitative real-time polymerase chain reaction; si ETV4 , ETV4 siRNA; siNC, negative control siRNA; siRNA, small interfering RNA.

Article Snippet: 5% skim milk was used for blocking nonspecific binding by incubation for 1 h. Subsequently, the membrane was incubated with ETV4 antibodies (1:1,000, 10684-1-AP, Proteintech, Wuhan, China) overnight at 4 °C.

Techniques: Inhibition, Transfection, Knockdown, Quantitative RT-PCR, Western Blot, Control, CCK-8 Assay, Cell Counting, Variant Assay, Real-time Polymerase Chain Reaction, Negative Control, Small Interfering RNA

Journal: Journal of Thoracic Disease

Article Title: Aumolertinib combined with targeting ETV4 in the treatment of non-small cell lung cancer

doi: 10.21037/jtd-2025-aw-2071

Figure Lengend Snippet: ETV4 knockdown enhances aumolertinib’s anti-tumor effects in PC-9 cells. (A,B) Wound healing assay was performed to evaluate the effect of ETV4 knockdown on migration ability of PC-9 cells that treated with aumolertinib (P=0.03), magnification 100×. (C,D) Flow cytometry with Annexin V/PI staining was used to assess the influence of ETV4 knockdown on apoptosis of PC-9 cells that treated with aumolertinib (P<0.001). (E,F) Flow cytometric analysis was used to assess the influence of ETV4 knockdown on cell cycle of PC-9 cells that treated with aumolertinib. *, P<0.05; **, P<0.01; ***, P<0.001. ETV4 , ETS variant transcription factor 4; FITC, fluorescein isothiocyanate; PI, propidium iodide; si ETV4 , ETV4 siRNA; siNC, negative control siRNA; siRNA, small interfering RNA.

Article Snippet: 5% skim milk was used for blocking nonspecific binding by incubation for 1 h. Subsequently, the membrane was incubated with ETV4 antibodies (1:1,000, 10684-1-AP, Proteintech, Wuhan, China) overnight at 4 °C.

Techniques: Knockdown, Wound Healing Assay, Migration, Flow Cytometry, Staining, Variant Assay, Negative Control, Small Interfering RNA

Journal: Journal of Thoracic Disease

Article Title: Aumolertinib combined with targeting ETV4 in the treatment of non-small cell lung cancer

doi: 10.21037/jtd-2025-aw-2071

Figure Lengend Snippet: ETV4 enhances the inhibitory effects of aumolertinib on tumor growth in vivo . (A) Photographs of mice models treated with ETV4 inhibitors and/or aumolertinib. (B) Tumor growth curves of mice models treated with ETV4 inhibitors and/or aumolertinib (P<0.001). Tumor volume (mm 3 ) was measured every 3 days. (C,D) Comparison of tumor weights (g) treated with ETV4 inhibitors and/or aumolertinib (P<0.001). *, P<0.05; ***, P<0.001. ALM, aumolertinib administration; ETV4 , ETS variant transcription factor 4; NC, negative control; si ETV4 , ETV4 siRNA; siRNA, small interfering RNA.

Article Snippet: 5% skim milk was used for blocking nonspecific binding by incubation for 1 h. Subsequently, the membrane was incubated with ETV4 antibodies (1:1,000, 10684-1-AP, Proteintech, Wuhan, China) overnight at 4 °C.

Techniques: In Vivo, Comparison, Variant Assay, Negative Control, Small Interfering RNA

Journal: Cancers

Article Title: Rabdosianone I, a Bitter Diterpene from an Oriental Herb, Suppresses Thymidylate Synthase Expression by Directly Binding to ANT2 and PHB2

doi: 10.3390/cancers13050982

Figure Lengend Snippet: Rabdosianone I directly binds to ANT2 and PHB2. ( A ) Scheme of fixation of rabdosianone I onto magnetic FG beads and the two predicted structures of rabdosianone I-fixed beads. α, β-Unsaturated ketones are shown by the red lines. ( B ) Identification of rabdosianone I-binding proteins. Three rabdosianone I-binding proteins were purified from whole-cell extracts of HT-29 cells with rabdosianone I-fixed FG beads and detected by silver staining. Mass spectrometry analysis identified three rabdosianone I-binding proteins, as shown in the right table. ( C ) Confirmation of the interaction of rabdosianone I with ANT2 and PHB2. Bound ANT2 and PHB2 were detected by Western blotting with either anti-ANT2 or PHB2 antibody. ( D ) Confirmation of the interaction of rabdosianone I with recombinant ANT2. Purified recombinant FLAG-ANT2 was incubated with rabdosianone I-fixed FG beads, and bound FLAG-ANT2 was detected by Western blotting with the anti-FLAG antibody. ( E ) Confirmation of the interaction of rabdosianone I with recombinant PHB2. Purified recombinant His-PHB2 was incubated with rabdosianone I-fixed FG beads, and bound His-PHB2 was detected by Western blotting with the anti-His antibody.

Article Snippet: Purified recombinant human ANT2 (Homo sapiens solute carrier family 25, member 5, catalog No: TP308949) and PHB2 (Human prohibitin 2, catalog No: TP760501) were obtained from OriGene (Rockville, MD, USA).

Techniques: Binding Assay, Purification, Silver Staining, Mass Spectrometry, Western Blot, Recombinant, Incubation

Journal: Cancers

Article Title: Rabdosianone I, a Bitter Diterpene from an Oriental Herb, Suppresses Thymidylate Synthase Expression by Directly Binding to ANT2 and PHB2

doi: 10.3390/cancers13050982

Figure Lengend Snippet: Molecular binding models for the interaction between rabdosianone I with ANT2 in the membrane. ( A ) Most populated complex structure of rabdosianone I with ANT2. ( B ) Emphasis of aromatic residues in A . ( C ) Secondary populated complex structure of rabdosianone I with ANT2. ( D ) Emphasis of aromatic residues in C . The structure of bovine mitochondrial ADP/ATP carrier (PDBcode:1OKC) with high sequence identity (90.07%) was used as a template for homology modeling. The Figures were made by PyMOL (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC.). Color shows ANT2 (green), aromatic residues (yellow), rabdosianone I (red), and POPC lipid (gray).

Article Snippet: Purified recombinant human ANT2 (Homo sapiens solute carrier family 25, member 5, catalog No: TP308949) and PHB2 (Human prohibitin 2, catalog No: TP760501) were obtained from OriGene (Rockville, MD, USA).

Techniques: Binding Assay, Membrane, Sequencing

Journal: Cancers

Article Title: Rabdosianone I, a Bitter Diterpene from an Oriental Herb, Suppresses Thymidylate Synthase Expression by Directly Binding to ANT2 and PHB2

doi: 10.3390/cancers13050982

Figure Lengend Snippet: Depletion of ANT2 or PHB2 reduces TS expression and suppresses cell growth. ( A ) Effects of ANT2 depletion on TS protein expression. TS was analyzed by Western blotting in cells treated with siRNAs (#1 and #2) targeting different sequences of ANT2 gene or siNeg for 48 h (HT-29 cells, left panel) or 72 h (HCT116 cells, right panel). α-Tubulin was used as a loading control. ( B ) Effects of PHB2 depletion on TS protein expression. TS was analyzed by Western blotting in cells treated with siRNAs (#1 and #2) targeting different sequences of PHB2 gene or siNeg for 48 h (HT-29 cells, left panel) or 72 h (HCT116 cells, right panel). α-Tubulin was used as a loading control. ( C ) Effects of ANT2 depletion on colony formation. HT-29 cells were treated with siANT2 or siNeg. After further incubation, colonies were fixed and stained with crystal violet. The representative images of stained colonies are shown (upper panel). Colony formation rates are shown in the graph (lower panel). Columns, means ( n = 3); bars, SD, * p < 0.05, ** p < 0.01, significantly different from the siNeg-treated control. ( D ) Effects of PHB2 depletion on colony formation. HT-29 cells were treated with siPHB2 or siNeg. After further incubation, colonies were fixed and stained by crystal violet. The representative images of stained colonies are shown (upper panel). Colony formation rates are shown in the graph (lower panel). Columns, means ( n = 3); bars, SD, ** p < 0.01, significantly different from the siNeg-treated control.

Article Snippet: Purified recombinant human ANT2 (Homo sapiens solute carrier family 25, member 5, catalog No: TP308949) and PHB2 (Human prohibitin 2, catalog No: TP760501) were obtained from OriGene (Rockville, MD, USA).

Techniques: Expressing, Western Blot, Control, Incubation, Staining

Journal: Cancers

Article Title: Rabdosianone I, a Bitter Diterpene from an Oriental Herb, Suppresses Thymidylate Synthase Expression by Directly Binding to ANT2 and PHB2

doi: 10.3390/cancers13050982

Figure Lengend Snippet: ANT2 and PHB2 may prevent proteasomal degradation of TS protein, whereas PHB2 may also increase TS mRNA expression. ( A ) Effects of ANT2 depletion on TS mRNA expression. The expression of TS mRNA was measured by quantitative PCR in HT-29 cells treated with siANT2 or siNeg for 48 h. TS mRNA was normalized to GAPDH mRNA, and the results with siNeg were taken as 1. Columns, means ( n = 3); bars, SD. ( B ) Effects of ANT2 depletion on TS protein expression with or without MG132 treatment. HT-29 cells were treated with siANT2 or siNeg for 24 h, and the medium was then replaced with that containing 10 μM MG132 or DMSO. After incubation for 24 h, ANT2 and TS were analyzed by Western blotting. α-Tubulin was used as a loading control. ( C ) Effects of PHB2 depletion on TS mRNA expression. The expression of TS mRNA was measured by quantitative PCR in HT-29 cells treated with siPHB2 or siNeg for 48 h. TS mRNA was normalized to GAPDH mRNA, and the results with siNeg were taken as 1. Columns, means ( n = 3); bars, SD. ( D ) Effects of PHB2 depletion on TS protein expression with or without MG132. HT-29 cells were treated with siPHB2 or siNeg for 24 h, and the medium was then replaced with that containing 10 μM MG132 or DMSO. After incubation for 24 h, PHB2 and TS were analyzed by Western blotting. α-Tubulin was used as a loading control. ( E ) Schematic representation of the pleiotropic regulation of TS by ANT2 and PHB2. ANT2 and PHB2 may stabilize TS at the protein level, whereas PHB2 may also increase TS mRNA expression (e.g., promoting transcription). Rabdosianone I binding to ANT2 and PHB2 may promote proteasomal degradation of TS protein, whereas its binding to PHB2 may suppress TS transcription.

Article Snippet: Purified recombinant human ANT2 (Homo sapiens solute carrier family 25, member 5, catalog No: TP308949) and PHB2 (Human prohibitin 2, catalog No: TP760501) were obtained from OriGene (Rockville, MD, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Incubation, Western Blot, Control, Binding Assay