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: Experimental and Therapeutic Medicine

Article Title: Adenosine A 1 receptor agonism protection mechanism in intestinal ischemia/reperfusion injury via activation of PI3K/Akt signaling

doi: 10.3892/etm.2022.11740

Figure Lengend Snippet: A 1 R agonists activate the PI3K/Akt signaling pathway during intestinal ischemia/reperfusion injury. The protein expression of PI3K, p-Akt/Akt and p-p53/p53 was measured using western blotting. Data are shown as the mean ± SEM and experiments were performed three times. * P<0.05, ** P<0.01 vs. OGD/R. OGD/R, oxygen-glucose deprivation/reoxygenation; PI3K, phosphatidylinositol 3-kinase; Akt, activated protein kinase B; p-, phosphorylated.

Article Snippet: The membranes were blocked with 5% BSA (Cell Signaling Technology) for 2 h at room temperature, before incubation at 4˚C overnight with the following primary antibodies (all from Cell Signaling Technology) at a 1:1,000 dilution: PI3K (cat. no. 3811S), Akt (cat. no. 9272S), phosphorylated (p)-Akt (cat. no. 9275S), p53 (cat. no. 9282S), p-p53 (cat. no. 9284S) and GAPDH (cat. no. 5174S; Cell Signaling Technology, Inc.).

Techniques: Expressing, Western Blot

Journal: Frontiers in Cellular Neuroscience

Article Title: Glutathione Peroxidase 1 Protects Against Peroxynitrite-Induced Spiral Ganglion Neuron Damage Through Attenuating NF-κB Pathway Activation

doi: 10.3389/fncel.2022.841731

Figure Lengend Snippet: The activated nuclear factor-kappa B (NF-κB) pathway is inhibited by GPX1 in SGNs after peroxynitrite damage. (A) The cultured SGNs from P3 WT mice were incubated with 100 μM peroxynitrite for 48 h with or without the pretreatment of 30 μM ebselen for 1 h, or the SGNs from gpx1 –/– mice were exposed to 100 μM peroxynitrite for 48 h. (B) The anti-NeuN antibody was used to label the nucleus of SGN, and the immunostaining results showed that the fluorescence of NF-κB p65 was mainly detected in the cytoplasm of SGNs in the control group. Peroxynitrite induced obvious nuclear distribution of NF-κB p65 in SGNs and pretreatment with ebselen significantly reduced it, whereas the lack of GPX1 intensified the nuclear fluorescence of NF-κB p65 in SGNs of the peroxynitrite + gpx1 –/– group. Scale bar = 12.5 μm. (C) Western blot results verified that the expression of the p-NF-kB p65 protein was upregulated in peroxynitrite-exposed SGNs compared with the untreated controls, and the pretreatment with ebselen significantly reduced it while the lack of GPX1 increased it. (D) Real-time PCR (RT-PCR) results revealed that peroxynitrite treatment caused significant increases in the microRNA (mRNA) expression of proapoptotic genes Bax and P53 , and decreased the expression of antiapoptotic genes Bcl2 , Bcl-xL compared with controls. The lower mRNA expression levels of Bax and P53 and the higher expression levels of Bcl2 and Bcl-xL were found in the peroxynitrite + ebselen group compared with the peroxynitrite-only group, while the lack of GPX1 in SGNs from gpx1 –/– mice led to an opposite pattern. * p < 0.05, ** p < 0.01, and *** p < 0.001, n = 3 for each group.

Article Snippet: The primary antibodies were anti-GPX1 (1:1,000 dilution, GeneTex, GTX116040), anti-C-caspase 3 (1:500 dilution, Cell Signaling Technology, 9664), anti-4-HNE (1:1000, Abcam, ab46545), anti-phosphorylated (p)-NFκB p65 (1:1,000 dilution, Cell Signaling Technology, 3033), and anti-β-actin (1:2,000 dilution, ZSGB-BIO, TA-09).

Techniques: Cell Culture, Incubation, Immunostaining, Fluorescence, Control, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

Journal: Frontiers in Cellular Neuroscience

Article Title: Glutathione Peroxidase 1 Protects Against Peroxynitrite-Induced Spiral Ganglion Neuron Damage Through Attenuating NF-κB Pathway Activation

doi: 10.3389/fncel.2022.841731

Figure Lengend Snippet: Glutathione peroxidase inhibits the NF-κB pathway to protect against peroxynitrite-induced SGNs damage. (A) The cultured SGN explants from WT or gpx1 – / – mice were treated with peroxynitrite and 10 μM BAY 11-7082 for 48 h. (B,C) Immunostaining and RT-PCR results showed that BAY 11-7082 effectively inhibited the nuclear translocation of NF-κB p65 and the mRNA expression of Bax and P53 , but increased the mRNA expression of Bcl2 and Bcl-xL , in the peroxynitrite + BAY 11-7082 group compared with the peroxynitrite-only group, as well as in the peroxynitrite + gpx1 –/– + BAY 11-7082 group compared with the peroxynitrite + gpx1 –/– group. Scale bar = 12.5 μm. (D–F) Immunostaining, cell counting, and western blot results showed that the inhibition of NF-κB by BAY 11-7082 significantly increased the survived SGN numbers, while decreased the C-CASP3 positive SGNs numbers and the C-CASP3 expression in the peroxynitrite + BAY 11-7082 group compared with the peroxynitrite group. Moreover, BAY 11-7082 treatment also increased the number of SGNs, whereas reduced the C-CASP3 expression in the peroxynitrite + gpx1 –/– + BAY 11-7082 group vs. the peroxynitrite + gpx1 –/– group. Scale bar = 25 μm. * p < 0.05, ** p < 0.01, and *** p < 0.001, n = 3 for each group.

Article Snippet: The primary antibodies were anti-GPX1 (1:1,000 dilution, GeneTex, GTX116040), anti-C-caspase 3 (1:500 dilution, Cell Signaling Technology, 9664), anti-4-HNE (1:1000, Abcam, ab46545), anti-phosphorylated (p)-NFκB p65 (1:1,000 dilution, Cell Signaling Technology, 3033), and anti-β-actin (1:2,000 dilution, ZSGB-BIO, TA-09).

Techniques: Cell Culture, Immunostaining, Reverse Transcription Polymerase Chain Reaction, Translocation Assay, Expressing, Cell Counting, Western Blot, Inhibition