Journal: FEBS letters

Article Title: RuvBl2 cooperates with Ets2 to transcriptionally regulate hTERT in colon cancer.

doi: 10.1016/j.febslet.2011.07.005

Figure Lengend Snippet: Fig. 2. RuvBl2 can cooperate with Ets-2 to regulate hTERT. (A) Cartoon of the hTERT promoter with the Myc binding site (E-box) and the Ets binding site illustrated, along with the proposed Ets2 binding on treatment with EGF. ChIP was performed on 6G cells treated with vehicle or EGF as described. Cell fractions were immunoprecipitated with either rabbit anti-Ets2 (sc-351, Santa Cruz) or H4 antibody. Proteins were uncrosslinked and primers were used to amplify the DNA 366 to 142 of the hTERT transcriptional start site. PCR image is representative of four separate experiments. (B) The effect of knock-down RuvBl2 expression on hTERT promoter activity relative to Ets2 knockdowns in 6G cells (i) colon cancer HCA-7 cells (ii). Luciferase assays were carried out using a Dual-Glo luciferase assay system, luciferase reporter activity was normalised to Renilla luciferase, results are expressed as mean ± SD (n = 3). (C) Knock down of RuvBl2 and Ets-2 expression with siRNA reduced hTERT protein expression in HCA-7 and 6G cells as determined by western blot. Actin was used as a loading control. Blots shown are representative of three separate experiments. (D) Over-expression of Ets2 with pCGN Ets2 induced an increase in hTERT expression compared to empty vector control (pCGN). Concomitant knock down of RuvBl2 with siRNA inhibited Ets2 induced hTERT protein expression as determined by western blot. Actin was used as a loading control. Blot shown is representative of three separate experiments. Confirmation of successful knockdown of RuvBl2 and over-expression of Ets2 in HCA-7 cells. Blots shown are representative of three separate experiments.

Article Snippet: Antibodies were as follows: hTERT 1:500 (Abcam, ab5181), RuvBl2 1:500 (Abcam, 36569), Ets2 1:200 (Santa Cruz Biotechnology, sc-351), beta-actin 1:7500 (Sigma, AI978).

Techniques: Binding Assay, Immunoprecipitation, Knockdown, Expressing, Activity Assay, Luciferase, Western Blot, Control, Over Expression, Plasmid Preparation

Journal: FEBS letters

Article Title: RuvBl2 cooperates with Ets2 to transcriptionally regulate hTERT in colon cancer.

doi: 10.1016/j.febslet.2011.07.005

Figure Lengend Snippet: Fig. 3. RuvBl2 impacts hTERT activity and associates with pEts2 and hTERT expression in human colon cancer patients. (A) Knock down of RuvBl2 or Ets2 induces a reduction in telomerase activity in 6G and HCA-7 colon cancer cells. TRAP assay was used to assess telomerase activity. siRNA-mediated depletion of RuvBl2 or Ets2 significantly reduced telomerase activity in 6G and HCA-7 cell lines compared to respective controls (F-statistic = 2,146, P < 0.001). 1-3 cells where used as hTERT negative control cells. Results are expressed as mean ± SD (n = 3). (B) RuvBl2, pEts2 and hTERT (200 inset 600) were localised in tumour tissue from colon cancer patients (n = 170). Staining was scored using the Allred scoring system as previously described [37]. Significant associations were observed between nuclear expression of RuvBl2 and pEts2 (P = 0.05, Fisher’s exact test) and between nuclear expression of RuvBl2 and hTERT (P = 0.004, Fisher’s exact test).

Article Snippet: Antibodies were as follows: hTERT 1:500 (Abcam, ab5181), RuvBl2 1:500 (Abcam, 36569), Ets2 1:200 (Santa Cruz Biotechnology, sc-351), beta-actin 1:7500 (Sigma, AI978).

Techniques: Activity Assay, Expressing, Knockdown, TRAP Assay, Negative Control, Staining

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