Journal: Oncology Reports

Article Title: 3-Bromopyruvate sensitizes human breast cancer cells to TRAIL-induced apoptosis via the phosphorylated AMPK-mediated upregulation of DR5

doi: 10.3892/or.2018.6644

Figure Lengend Snippet: 3-BP induces AMPK phosphorylation and induces cell death. (A) The levels of CHOP, GRP78, AMPK-α and p-AMPK were detected in breast cancer MCF-7 and MDA-MB-231 cells treated with 3-BP (0, 40, 80 and 160 µmol/l) for 24 h via western blot analysis. (B) The expression levels of Bax in MCF-7 cells and caspase-3 protein in MDA-MB-231 cells were detected via western blotting in cells treated with 80 µmol/l 3-BP and 200 ng/ml TRAIL. 3-BP, 3-bomopyruvate.

Article Snippet: The rabbit polyclonal antibody against phosphorylated (p)-AMPKα (Thr172) (1:1,000 dilution; cat. no. 50081) was purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: Phospho-proteomics, Western Blot, Expressing

Journal: International Journal of Oncology

Article Title: Combined inhibition of IL-6 and IL-8 pathways suppresses ovarian cancer cell viability and migration and tumor growth

doi: 10.3892/ijo.2022.5340

Figure Lengend Snippet: Baze and SCH inhibit the expression of target genes downstream of the IL-6 and IL-8 pathways in human ovarian cancer cells. SKOV3 and CAOV3 cells were treated with DMSO, a single drug or their combination. The levels of p-STAT3, p-AKT, p-S6 and survivin were determined by western blot analysis. (A) CAOV3. (B) SKOV3. * P<0.05, ** P<0.01, *** P<0.001 and **** P<0.0001 (DMSO vs. Baze, SCH or Baze + SCH). B+S, Baze + SCH; B10, BAZE 10 µ M; B5, BAZE 5 µ M; BAZE, Baze; S50, SCH 50 µ M; p-phosphorylated; SCH, SCH.

Article Snippet: The following primary antibodies were purchased from Cell Signaling Technology, Inc. and diluted at 1:1,000 in 5% milk: Phosphorylated (p)-STAT3 (Y705; rabbit mAb; cat. no. 9131S), STAT3 (rabbit mAb; cat. no. 4904S), p-S6 (rabbit mAb; cat. no. 4858S), S6 (rabbit mAb; cat. no. 2217S), p-AKT (rabbit mAb; cat. no. 4060S), AKT (rabbit mAb; cat. no. 4691S), survivin (rabbit mAb; cat. no. 2808S) and GAPDH (rabbit mAb; cat. no. 2118S).

Techniques: Expressing, Western Blot

Journal: Experimental and Therapeutic Medicine

Article Title: miR-223 ameliorates thalamus hemorrhage-induced central poststroke pain via targeting NLRP3 in a mouse model

doi: 10.3892/etm.2022.11280

Figure Lengend Snippet: Effect of NLRP3 siRNA on NLRP3 inflammasome activity and the downstream factors IL-18, IL-1β, GFAP and p-ERK1/2. (A) Transfection with NLRP3 siRNA significantly decreased the protein expression levels of caspase-1, ASC and NLRP3. (B) NLRP3 siRNA also significantly reduced IL-18 and IL-1β protein expression levels. (C) After microinjection of NLRP3 siRNA, the protein expression levels of p-ERK1/2 and GFAP significantly decreased. n=3 mice/group. * P<0.05 vs. saline + scramble; # P<0.05 vs. Coll IV + scramble. NLRP3, NLR family pyrin domain containing 3; siRNA, small interfering RNA; GFAP, glial fibrillary acidic protein; p, phosphorylated; ASC, apoptosis-associated speck-like protein containing a CARD.

Article Snippet: The membrane was blocked with 5% skimmed milk in TBS with 0.1% Tween-20 for 1 h. Subsequently, membranes were incubated overnight at 4˚C with the following primary antibodies: rabbit anti-caspase-1 (1:1,000; Cell Signaling Technology, Inc. cat. no. 24232), rabbit anti-ASC (1:1,000; Cell Signaling Technology, Inc.; cat. no. 67824), rabbit anti-NLRP3 (1:1,000; Cell Signaling Technology, Inc. cat. no. 15101), rabbit anti-IL-18 (1:1,000; Abcam; cat. no. ab243091), mouse anti-IL-1β (1:1,000; Cell Signaling Technology, Inc. cat. no. 63124), rabbit anti-phosphorylated (p)-ERK1/2 (1:2,000; Cell Signaling Technology, Inc. cat. no. 4370), rabbit anti-ERK1/2 (1:2,000; Cell Signaling Technology, Inc. cat. no. 4695), rabbit anti-GAPDH (1:5,000; Cell Signaling Technology, Inc. cat. no. 2118) and mouse anti-glial fibrillary acidic protein (GFAP; 1:2,000; Cell Signaling Technology, Inc. cat. no. 3670).

Techniques: Activity Assay, Transfection, Expressing, Small Interfering RNA

Journal: Experimental and Therapeutic Medicine

Article Title: miR-223 ameliorates thalamus hemorrhage-induced central poststroke pain via targeting NLRP3 in a mouse model

doi: 10.3892/etm.2022.11280

Figure Lengend Snippet: miR-223 targets the NLRP3 3'UTR and affects the protein expression levels of caspase-1, ASC, NLRP3, IL-18, IL-1β, p-ERK1/2 and GFAP. (A) Binding site of miR-223 within the NLRP3 3'-UTR. (B) miR-223 agomir significantly reduced the relative luciferase activity in PC12 cells transfected with the NLRP3 3'-UTR. (C) miR-223 agomir significantly increased miR-223 expression levels. (D) Administration of miR-223 agomir significantly reduced the protein expression levels of caspase-1, ASC and NLRP3. (E) miR-223 agomir also significantly reduced the expression of IL-18 and IL-1β. (F) After microinjection of miR-223 agomir, p-ERK1/2 and GFAP protein expression levels significantly decreased. n=3 mice/cohort. * P<0.05 vs. saline + agomir-scramble; # P<0.05 vs. Coll IV + agomir-scramble. miR, microRNA; NLRP3, NLR family pyrin domain containing 3; UTR, untranslated region; GFAP, glial fibrillary acidic protein; p, phosphorylated; ASC, apoptosis-associated speck-like protein containing a CARD; wt, wild-type; mut, mutant.

Article Snippet: The membrane was blocked with 5% skimmed milk in TBS with 0.1% Tween-20 for 1 h. Subsequently, membranes were incubated overnight at 4˚C with the following primary antibodies: rabbit anti-caspase-1 (1:1,000; Cell Signaling Technology, Inc. cat. no. 24232), rabbit anti-ASC (1:1,000; Cell Signaling Technology, Inc.; cat. no. 67824), rabbit anti-NLRP3 (1:1,000; Cell Signaling Technology, Inc. cat. no. 15101), rabbit anti-IL-18 (1:1,000; Abcam; cat. no. ab243091), mouse anti-IL-1β (1:1,000; Cell Signaling Technology, Inc. cat. no. 63124), rabbit anti-phosphorylated (p)-ERK1/2 (1:2,000; Cell Signaling Technology, Inc. cat. no. 4370), rabbit anti-ERK1/2 (1:2,000; Cell Signaling Technology, Inc. cat. no. 4695), rabbit anti-GAPDH (1:5,000; Cell Signaling Technology, Inc. cat. no. 2118) and mouse anti-glial fibrillary acidic protein (GFAP; 1:2,000; Cell Signaling Technology, Inc. cat. no. 3670).

Techniques: Expressing, Binding Assay, Luciferase, Activity Assay, Transfection, Mutagenesis

Journal: Experimental and Therapeutic Medicine

Article Title: miR-223 ameliorates thalamus hemorrhage-induced central poststroke pain via targeting NLRP3 in a mouse model

doi: 10.3892/etm.2022.11280

Figure Lengend Snippet: Effect of miR-223 antagomir on the nociceptive threshold in naïve mice. Administration of miR-223 antagomir contributed to a significant increase in the paw withdrawal frequency in response to (A) 0.07 g and (B) 0.4 g von Frey filaments and a significant decrease in paw withdrawal latency to (C) thermal and (D) cold stimuli on day 3, 5 and 7 on the contralateral side. (E-G) Administration of miR-223 antagomir or antagomir-scramble did not contribute to behavioral changes on the ipsilateral side. n=8 mice/group. (H) miR-223 antagomir significantly reduced miR-223 expression levels. (I) Administration of miR-223 antagomir significantly elevated caspase-1, ASC and NLRP3 protein expression levels. (J) miR-223 antagomir also significantly elevated IL-18 and IL-1β protein expression levels. (K) Following microinjection of miR-223 antagomir, p-ERK1/2 and GFAP protein expression levels increased. n=3 mice/group. * P<0.05 vs. naïve group. miR, microRNA; NLRP3, NLR family pyrin domain containing 3; GFAP, glial fibrillary acidic protein; p, phosphorylated; ASC, apoptosis-associated speck-like protein containing a CARD.

Article Snippet: The membrane was blocked with 5% skimmed milk in TBS with 0.1% Tween-20 for 1 h. Subsequently, membranes were incubated overnight at 4˚C with the following primary antibodies: rabbit anti-caspase-1 (1:1,000; Cell Signaling Technology, Inc. cat. no. 24232), rabbit anti-ASC (1:1,000; Cell Signaling Technology, Inc.; cat. no. 67824), rabbit anti-NLRP3 (1:1,000; Cell Signaling Technology, Inc. cat. no. 15101), rabbit anti-IL-18 (1:1,000; Abcam; cat. no. ab243091), mouse anti-IL-1β (1:1,000; Cell Signaling Technology, Inc. cat. no. 63124), rabbit anti-phosphorylated (p)-ERK1/2 (1:2,000; Cell Signaling Technology, Inc. cat. no. 4370), rabbit anti-ERK1/2 (1:2,000; Cell Signaling Technology, Inc. cat. no. 4695), rabbit anti-GAPDH (1:5,000; Cell Signaling Technology, Inc. cat. no. 2118) and mouse anti-glial fibrillary acidic protein (GFAP; 1:2,000; Cell Signaling Technology, Inc. cat. no. 3670).

Techniques: Expressing

Journal: Experimental and Therapeutic Medicine

Article Title: miR-223 ameliorates thalamus hemorrhage-induced central poststroke pain via targeting NLRP3 in a mouse model

doi: 10.3892/etm.2022.11280

Figure Lengend Snippet: CY 09 affects IL-18, IL-1β, p-ERK1/2 and GFAP protein expression levels. (A) CY 09 injection caused no alterations in miR-223 expression levels. CY 09 injection (B) significantly reduced IL-18 and IL-1β protein expression levels in the naïve + miR-223 antagomir cohort and (C) significantly lowered the protein expression levels of GFAP and p-ERK1/2. n=3 mice/cohort. * P<0.05 vs. naïve group; # P<0.05 vs. miR-223 antagomir + vehicle. GFAP, glial fibrillary acidic protein; p, phosphorylated; miR, microRNA.

Article Snippet: The membrane was blocked with 5% skimmed milk in TBS with 0.1% Tween-20 for 1 h. Subsequently, membranes were incubated overnight at 4˚C with the following primary antibodies: rabbit anti-caspase-1 (1:1,000; Cell Signaling Technology, Inc. cat. no. 24232), rabbit anti-ASC (1:1,000; Cell Signaling Technology, Inc.; cat. no. 67824), rabbit anti-NLRP3 (1:1,000; Cell Signaling Technology, Inc. cat. no. 15101), rabbit anti-IL-18 (1:1,000; Abcam; cat. no. ab243091), mouse anti-IL-1β (1:1,000; Cell Signaling Technology, Inc. cat. no. 63124), rabbit anti-phosphorylated (p)-ERK1/2 (1:2,000; Cell Signaling Technology, Inc. cat. no. 4370), rabbit anti-ERK1/2 (1:2,000; Cell Signaling Technology, Inc. cat. no. 4695), rabbit anti-GAPDH (1:5,000; Cell Signaling Technology, Inc. cat. no. 2118) and mouse anti-glial fibrillary acidic protein (GFAP; 1:2,000; Cell Signaling Technology, Inc. cat. no. 3670).

Techniques: Expressing, Injection

Journal: Antioxidants

Article Title: Ellagic Acid Reduces Cadmium Exposure-Induced Apoptosis in HT22 Cells via Inhibiting Oxidative Stress and Mitochondrial Dysfunction and Activating Nrf2/HO-1 Pathway

doi: 10.3390/antiox13111296

Figure Lengend Snippet: The expression of Nrf2, HO-1, p-Erk1/2, p-JNK, p-p38, Bax/Bcl-2 ratio, cleaved caspase-3, and cleaved PARP-1 proteins. Cells were pre-treated with ellagic acid (EA) at 5, 10, or 20 μM for 2 h, followed by co-treatment with CdSO 4 at 10 μM for an additional 24 h. Protein expressions were assessed using the Western Blotting method. All results were presented as Mean ± SD (n = 3). Statistical significance levels were delineated as follows: ** p < 0.01 compared to the untreated control group; # p < 0.05 and ## p < 0.01 compared to the CdSO 4 only-treated group.

Article Snippet: Primary rabbit polyclonal antibodies targeting phosphorylated (p)-p38 (Thr180/Tyr182), p-JNK (Thr183/Tyr185), p-ERK1/2 (Thr202/Tyr204) (1:1000 dilution; CST company, Beverly, MA, USA), Nrf2, HO-1, Bax, Bcl-2, and caspase-3 (1:1000 dilution; Proteintech, Chicago, IL, USA), and mouse monoclonal antibody against β-actin (1:1000 dilution; Santa Cruz, CA, USA) were used.

Techniques: Expressing, Western Blot, Control

Journal: Experimental and Therapeutic Medicine

Article Title: Elevated estrogen receptor β expression in triple negative breast cancer cells is associated with sensitivity to doxorubicin by inhibiting the PI3K/AKT/mTOR signaling pathway

doi: 10.3892/etm.2020.8809

Figure Lengend Snippet: Liq treatment enhances the protein expression of ERβ and inhibits the activity of the PI3K/AKT/mTOR signaling pathway in TNBC cells. MDA-MB-231 cells were treated with DOX (1 ng/ml), Liq (40 µg/ml) or a combination of DOX (1 ng/ml) and Liq (40 µg/ml). Subsequently, the protein expression levels of ERβ, p-AKT, AKT, p-mTOR and mTOR were (A) determined by western blotting and (B) quantified. ERβ expression levels were increased in MDA-MB-231 cells treated with Liq (40 µg/ml) or the combined treatment, whereas the ratio of p-AKT/AKT and p-mTOR/mTOR was decreased, compared with the control group. * P<0.05 vs. the negative control group. # P<0.05 vs. the DOX-treated group. Liq, liquiritigenin; ERβ, estrogen receptor β; DOX, doxorubicin; p, phosphorylated; Ctrl, control.

Article Snippet: Subsequently, the membranes were incubated for 24 h at 4˚C with primary antibodies targeted against: Phosphorylated (p)-mTOR (cat. no. 2974; 1:1,000; Cell Signaling Technology, Inc.), mTOR (cat. no. 2983; 1:1,000; Cell Signaling Technology, Inc.), ERβ (cat. no. sc-8974; 1:2,000; Santa Cruz Biotechnology, Inc.), β-actin (cat. no. 4970; 1:2,000; Cell Signaling Technology, Inc.), phosphorylated (p)-AKT (cat. no. 4060; 1:2,000; Cell Signaling Technology, Inc.) and total AKT (cat. no. 4691; 1:2,000; Cell Signaling Technology, Inc.).

Techniques: Expressing, Activity Assay, Western Blot, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: Elevated estrogen receptor β expression in triple negative breast cancer cells is associated with sensitivity to doxorubicin by inhibiting the PI3K/AKT/mTOR signaling pathway

doi: 10.3892/etm.2020.8809

Figure Lengend Snippet: ERβ KD increases the activity of the PI3K/AKT/mTOR signaling pathway in MDA-MB-231 cells. Protein expression levels of p-AKT, AKT, p-mTOR and mTOR in NC-KD and ERβ-KD cells were (A) determined by western blotting and (B) the ratio of phosphorylated/total protein was quantified. * P<0.05 vs. the NC-KD group. ERβ, estrogen receptor β; p, phosphorylated; NC, negative control; KD, knockdown; Ctrl, control; Liq, liquiritigenin; DOX, doxorubicin.

Article Snippet: Subsequently, the membranes were incubated for 24 h at 4˚C with primary antibodies targeted against: Phosphorylated (p)-mTOR (cat. no. 2974; 1:1,000; Cell Signaling Technology, Inc.), mTOR (cat. no. 2983; 1:1,000; Cell Signaling Technology, Inc.), ERβ (cat. no. sc-8974; 1:2,000; Santa Cruz Biotechnology, Inc.), β-actin (cat. no. 4970; 1:2,000; Cell Signaling Technology, Inc.), phosphorylated (p)-AKT (cat. no. 4060; 1:2,000; Cell Signaling Technology, Inc.) and total AKT (cat. no. 4691; 1:2,000; Cell Signaling Technology, Inc.).

Techniques: Activity Assay, Expressing, Western Blot, Negative Control

Journal: Oncology Reports

Article Title: miR-100-5p and miR-203a-3p suppress esophageal squamous cell carcinoma progression by targeting FKBP5

doi: 10.3892/or.2025.9003

Figure Lengend Snippet: Characterization of (A) epithelial-to-mesenchymal transition markers (E-cadherin and Vimentin) and (B) PI3K/ AKT signaling pathway components, including p-PI3K, p-AKT and total (PI3K and AKT) proteins, in response to FKBP5 regulation by western blotting in KYSE70 cells. Quantification of (C) p-PI3K/ PI3K and (D) p-AKT/ AKT ratios expressed as fold change relative to NC. *P<0.05. p-, phosphorylated; NC, negative control; si-, small interfering.

Article Snippet: After blocking with StartingBlock Blocking Buffer (Thermo Fisher Scientific, Inc.) at room temperature for 1 h, the membranes were incubated overnight at 4°C with the following antibodies: E-cadherin (1:1,000; cat. no. 20874-1-AP; Proteintech Group, Inc.), Vimentin (1:2,000; cat. no. 10366-1-AP; Proteintech Group, Inc.), phosphorylated (p-)PI3K (1:1,000; cat. no. 17366; Cell Signaling Technology, Inc.), PI3K (1:1,000; cat. no. 11889; Cell Signaling Technology, Inc.), p-AKT (1:1,000; cat. no. 4060; Cell Signaling Technology, Inc.), AKT (1:1,000; cat. no. 4691; Cell Signaling Technology, Inc.), GAPDH (1:50,000; cat. no. 60004-1-lg; Proteintech Group, Inc.).

Techniques: Western Blot, Negative Control