Journal: Viruses

Article Title: Stromal Antigen 2 Deficiency Induces Interferon Responses and Restricts Porcine Deltacoronavirus Infection

doi: 10.3390/v14081783

Figure Lengend Snippet: Establishment of STAG2-depleted IPEC-J2 cells line. ( A ) Western blot analysis for STAG2 expression in IPEC-J2 cell line; ( B ) Gene sequence alignment revealed that were heterozygous for STAG2 knockout alleles generated using CRISPR-Cas9 gene editing; ( C ) Cell viability was determined using CCK-8 detection; ( D ) Western blot analysis for STAG2 expression of WT IPEC-J2, STAG2 − / − IPEC-J2 passage 5 (P. 5), passage 10 (P. 10), passage 15 (P. 15) and passage 20 (P. 20).

Article Snippet: Cell viabilities were assessed using a cell counting kit-8 (CCK-8) (Cat NO. GK10001, GLPBIO, Montclair, CA, USA).

Techniques: Western Blot, Expressing, Sequencing, Knock-Out, Generated, CRISPR, CCK-8 Assay

Journal: International journal of molecular medicine

Article Title: Benzyl isothiocyanate inhibits inflammasome activation in E. coli LPS-stimulated BV2 cells.

doi: 10.3892/ijmm.2016.2667

Figure Lengend Snippet: Figure 1. Chemical structure of benzyl isothiocyanate (BITC) and the effect of BITC on BV2 microglial cell viability. (A) Chemical structure of BITC. (B) The cells were treated with various concentrations (1-30 µM) of BITC for 24 h. Cell viability was assessed using the cell counting kit-8 (CCK-8) assay, and the results are expressed as the percentage of surviving cells relative to the untreated cells. Each value indicates the mean ± SD and is representative of results obtained from 3 independent experiments. ***p<0.001 vs. untreated cells.

Article Snippet: An antibody against IL-1β (cat. no. AF-401-NA) was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Cell Counting, CCK-8 Assay

Journal: International journal of molecular medicine

Article Title: Benzyl isothiocyanate inhibits inflammasome activation in E. coli LPS-stimulated BV2 cells.

doi: 10.3892/ijmm.2016.2667

Figure Lengend Snippet: Figure 3. Effects of benzyl isothiocyanate (BITC) on NLRP3 and caspase-1 activation in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. The cells were seeded at 2x105 cells/ml and incubated with various concentra tions (1, 5, and 10 µM) of BITC for 1 h prior to LPS stimulation (1 µg/ml). (A) Following stimulation with LPS for 3 h, the NLRP3 mRNA expression levels were determined by RT-PCR. GAPDH was used as an internal control for RT-PCR. (B) Following stimulation with LPS for 24 h, the cell lysates were resolved on sodium dodecyl sulfate (SDS)-polyacrylamide gels, transferred onto nitrocellulose membranes, and probed using antibodies against NLRP3 and caspase-1. Actin was used as an internal control for western blot analysis. Pro Cas-1, procaspase-1; Act Cas-1, active caspase-1.

Article Snippet: An antibody against IL-1β (cat. no. AF-401-NA) was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Activation Assay, Incubation, Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Western Blot

Journal: International journal of molecular medicine

Article Title: Benzyl isothiocyanate inhibits inflammasome activation in E. coli LPS-stimulated BV2 cells.

doi: 10.3892/ijmm.2016.2667

Figure Lengend Snippet: Figure 4. Effects of benzyl isothiocyanate (BITC) on adenosine triphos phate (ATP) levels in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. The cells were seeded at 2x105 cells/ml and incubated with various concentra tions (1, 5 and 10 µM) of BITC for 1 h prior to LPS stimulation (1 µg/ml) for 5 min. The cell culture supernatant was analyzed for ATP secretion. ATP levels in the medium were detected using a luminescence-based assay in accordance with the manufacturer's instructions. Each value indicates the mean ± SD and is representative of results obtained from 3 independent experiments. #p<0.05 vs. control group (no treatment); ***p<0.001 vs. LPS-stimulated group.

Article Snippet: An antibody against IL-1β (cat. no. AF-401-NA) was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Incubation, Cell Culture, Luminescence Assay, Control

Journal: International journal of molecular medicine

Article Title: Benzyl isothiocyanate inhibits inflammasome activation in E. coli LPS-stimulated BV2 cells.

doi: 10.3892/ijmm.2016.2667

Figure Lengend Snippet: Figure 2. Effect of benzyl isothiocyanate (BITC) on intereukin-1β (IL‑1β) pro duction by lipopolysaccharide (LPS)-stimulated BV2 microglial cells. The cells were seeded at 2x105 cells/ml and incubated with various concentrations (1, 5 and 10 µM) of BITC for 1 h prior to LPS stimulation (1 µg/ml) (A) Following stimulation with LPS for 3 h, the IL-1β mRNA expression level was determined by RT-PCR. GAPDH was used as an internal control for RT-PCR. (B) Following incubation for 48 h, the culture supernatant was collected, and the quantity of IL-1β was measured by ELISA. Each value indicates the mean ± SD and is representative of results obtained from 3 independent experiments. #p<0.05 vs. control group (no treatment); ***p<0.001 vs. LPS-stimulated group.

Article Snippet: An antibody against IL-1β (cat. no. AF-401-NA) was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Incubation, Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Enzyme-linked Immunosorbent Assay

Journal: International journal of molecular medicine

Article Title: Benzyl isothiocyanate inhibits inflammasome activation in E. coli LPS-stimulated BV2 cells.

doi: 10.3892/ijmm.2016.2667

Figure Lengend Snippet: Figure 5. Involvement of mitochondrial reactive oxygen species (ROS) in the activation of NLRP3 and caspase-1. (A) Cells were seeded at 2x105 cells/ml and incubated with the indicated concentrations of N-Acetyl-l-cysteine (NAC), diphenyleneiodonium (DPI) and YCG 063 for 1 h prior to lipopolysaccha ride (LPS) stimulation (1 µg/ml). Following incubation for 24 h, the culture thye supernatant was collected, and the IL-1β quantity was measured by ELISA. Each value indicates the mean ± SD and is representative of results obtained from 3 independent experiments. #p<0.05 vs. control group (no treatment); *p<0.05, **p<0.01 and ***p<0.001 vs. LPS-stimulated group. (B) Following stimulation with LPS for 24 h, the cell lysates were resolved on sodium dodecyl sulfate (SDS)-polyacrylamide gels, transferred onto nitrocellulose membranes, and probed using antibodies against NLRP3 and caspase-1. Actin was used as an internal control for western blot analysis. Pro Cas-1, procaspase-1; Act Cas-1, active caspase-1.

Article Snippet: An antibody against IL-1β (cat. no. AF-401-NA) was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Activation Assay, Incubation, Enzyme-linked Immunosorbent Assay, Control, Western Blot

Journal: International journal of molecular medicine

Article Title: Benzyl isothiocyanate inhibits inflammasome activation in E. coli LPS-stimulated BV2 cells.

doi: 10.3892/ijmm.2016.2667

Figure Lengend Snippet: Figure 6. Effect of benzyl isothiocyanate (BITC) on the nuclear factor-κB (NF‑κB) translocation and binding to lipopolysaccharide (LPS)-stimulated BV2 microglial cell nuclei. The cells were treated with BITC (1, 5 and 10 µM) for 1 h and then stimulated with LPS for 2 h. (A) Nuclear extracts were prepared as described in the Materials and methods and NF-κB translocation was evalu ated by western blot analysis. (B) Nuclear protein extracts were prepared and analyzed for NF-κB DNA binding activity using an electrophoretic mobility shift assay (EMSA). Cold-κB, unlabeled NF-κB probe.

Article Snippet: An antibody against IL-1β (cat. no. AF-401-NA) was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Translocation Assay, Binding Assay, Western Blot, Activity Assay, Electrophoretic Mobility Shift Assay

Journal: Molecular medicine reports

Article Title: MicroRNA‑433 reduces cell proliferation and invasion in non‑small cell lung cancer via directly targeting E2F transcription factor 3.

doi: 10.3892/mmr.2018.9020

Figure Lengend Snippet: Figure 3. E2F3 is a direct target of miR‑433 in non‑small‑cell lung cancer. (A) Putative Wt and Mut binding sequences in the 3'‑UTR of E2F3. (B) A549 and H460 cells were cotransfected with miR‑433 mimics or miR‑NC and pMIR‑Wt‑E2F3‑3'‑UTR or pMIR‑Mut‑E2F3‑3'‑UTR. Following transfection for 48 h, relative luciferase activity was determined using a dual luciferase reporter assay system. *P<0.05 vs. miR‑NC. E2F3 (C) mRNA and (D) protein expression levels were detected by reverse transcription‑quantitative polymerase chain reaction and western blot analysis, respectively, in A549 and H460 cells transfected with miR‑433 mimics or miR‑NC. *P<0.05 vs. miR‑NC. E2F3, E2F transcription factor 3; miR, microRNA; Mut, mutant; NC, negative control; pMIR, plasmid vector; UTR, untranslated region; Wt, wild‑type.

Article Snippet: The primary antibodies used in the present study were acquired from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) and included mouse anti-human monoclonal E2F3 (1:1,000; cat. no. sc-28308) and mouse anti-human monoclonal GAPDH (1:1,000; cat. no. sc-365062) antibodies.

Techniques: Binding Assay, Transfection, Luciferase, Activity Assay, Reporter Assay, Expressing, Polymerase Chain Reaction, Western Blot, Mutagenesis, Negative Control, Plasmid Preparation

Journal: Molecular medicine reports

Article Title: MicroRNA‑433 reduces cell proliferation and invasion in non‑small cell lung cancer via directly targeting E2F transcription factor 3.

doi: 10.3892/mmr.2018.9020

Figure Lengend Snippet: Figure 4. E2F3 downregulation suppresses cell proliferation and invasion in non‑small‑cell lung cancer. (A) E2F3 siRNA or NC siRNA was transfected into A549 and H460 cells. A total of 72 h post‑transfection, western blot analysis was performed to detect E2F3 protein expression levels. *P<0.05 vs. NC siRNA. The effect of E2F3 knockdown on A549 and H460 cell proliferation and invasion was determined by a (B) Cell Counting kit‑8 and (C) cell invasion assays, respectively (magnification, x200). *P<0.05 vs. NC siRNA. E2F3, E2F transcription factor 3; NC, negative control; OD, optical density; siRNA, small interfering RNA.

Article Snippet: The primary antibodies used in the present study were acquired from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) and included mouse anti-human monoclonal E2F3 (1:1,000; cat. no. sc-28308) and mouse anti-human monoclonal GAPDH (1:1,000; cat. no. sc-365062) antibodies.

Techniques: Transfection, Western Blot, Expressing, Knockdown, CCK-8 Assay, Negative Control, Small Interfering RNA

Journal: Molecular medicine reports

Article Title: MicroRNA‑433 reduces cell proliferation and invasion in non‑small cell lung cancer via directly targeting E2F transcription factor 3.

doi: 10.3892/mmr.2018.9020

Figure Lengend Snippet: Figure 5. E2F3 restoration counteracts the effects of miR‑433 on non‑small‑cell lung cancer cell proliferation and invasion. A549 and H460 cells were trans fected with miR‑433 mimics with pcDNA3.1‑E2F3 or pcDNA3.1, or miR‑NC alone. (A) Western blot analysis was employed to detect E2F3 protein expression levels at 72 h following transfection. *P<0.05 vs. miR‑NC. #P<0.05 vs. miR‑433 mimics+pcDNA3.1‑E2F3. (B) Cell Counting kit‑8 and (C) cell invasion assays were applied to determine the proliferation and invasion in variably treated cells. *P<0.05 vs. miR‑NC. #P<0.05 vs. miR‑433 mimics+pcDNA3.1‑E2F3 (magnification, x200). E2F3, E2F transcription factor 3; miR, microRNA; NC, negative control; pcDNA3.1, plasmid vector; OD, optical density.

Article Snippet: The primary antibodies used in the present study were acquired from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) and included mouse anti-human monoclonal E2F3 (1:1,000; cat. no. sc-28308) and mouse anti-human monoclonal GAPDH (1:1,000; cat. no. sc-365062) antibodies.

Techniques: Western Blot, Expressing, Transfection, CCK-8 Assay, Negative Control, Plasmid Preparation

Journal: Scientific Reports

Article Title: Mechanisms of FOXK1-regulated glycolipid metabolism in mediating TOX-induced histone lactylation to promote CD8⁺ T cell exhaustion in high-grade serous ovarian cancer

doi: 10.1038/s41598-025-32938-3

Figure Lengend Snippet: Molecular mechanism of FOXK1 regulation of glucose and lipid metabolism in SKOV3 cells transfected with sh-NC or sh-FOXK1 plasmids, and treated with insulin. ( A ) Western blot analysis of FOXK1 protein expression levelss. Original blots/gels are presented in Supplementary Fig. 4. ( B ) CCK-8 assay measuring cell viability. ( C ) Glucose consumption assay by ELISA to assess the glucose consumption levels. ( D ) Lactic acid measurement by ELISA to evaluate the lactic acid levels. ( E ) Western blot analysis of key metabolic proteins: HK2, PKM2, LDHA, Ppara, Acadm, and Decr1. Original blots/gels are presented in Supplementary Fig. 5. ( F ) Oil Red O staining to visualize lipid accumulation in SKOV3 cells under different experimental conditions (Scar bar: 50 µM). ( G ) Cell invasion assay to evaluate the invasive rate of SKOV3 cells (Scar bar: 50 µM). ( H ) Cell migration assay to measure the migration rate of SKOV3 cells. ( I ) ELISA assay for intracellular triglycerides (TG), free fatty acids (FFA), and total cholesterol (TC) levels. ( J ) Western blot analysis of E-cadherin, N-cadherin, and Vimentin expression in SKOV3 cells to assess epithelial-mesenchymal transition (EMT) under different conditions. Original blots/gels are presented in Supplementary Fig. 6. Experimental groups: Ctrl: SKOV3 cells; sh-NC: SKOV3 cells transfected with sh-NC plasmid; sh-FOXK1: SKOV3 cells transfected with sh-FOXK1 plasmid; insulin: SKOV3 cells treated with 10 µg/mL insulin for 24 h; sh-FOXK1 + insulin: SKOV3 cells transfected with sh-FOXK1 plasmid and treated with 10 µg/mL insulin for 24 h. Statistical significance: * P < 0.05, ** P < 0.01, *** P < 0.001, n = 3.

Article Snippet: After 24 h of incubation, 10 μL of CCK8 solution (cat no. 40203ES60, Yeasen, China) was added to each well 2 h before the end of the incubation period.

Techniques: Transfection, Western Blot, Expressing, CCK-8 Assay, Enzyme-linked Immunosorbent Assay, Staining, Invasion Assay, Cell Migration Assay, Migration, Plasmid Preparation

Journal: Scientific Reports

Article Title: Mechanisms of FOXK1-regulated glycolipid metabolism in mediating TOX-induced histone lactylation to promote CD8⁺ T cell exhaustion in high-grade serous ovarian cancer

doi: 10.1038/s41598-025-32938-3

Figure Lengend Snippet: Mechanism of FOXK1 regulating glucose and lipid metabolism, mediating TOX histone lactylation modification, and inducing CD8 + T cell exhaustion. ( A ) Cell viability was detected by CCK-8. ( B ) TOX constructed in PLVX-AcGFP-N1 vector was transient transfected into 293 T cells and immunoprecipitated by HA antibody. Interaction between Pan lac and HA was detected. After the TOX-HA-Tag vector was transferred into CD8 + T cells, TOX lactonization modification was observed by Co-IP assay. ( C ) Detection of histone pan-lactic acidification levels by flow cytometry. ( D ) Apoptosis was detected by flow cytometry. ( E ) Detection of immune checkpoint positive rate by flow cytometry. ( F ) Detect the expression levels of cytokines granzyme B (GzmB), IFN-γ, PRF1 and TNF-α by ELISA. Experimental grouping: Ctrl (Co-culture of CD8 + T cells and SKOV3 cells), sh-FOXK1 (Co-culture of CD8 + T cells and SKOV3 cells transfected with sh-FOXK1 plasmid), NaL (Co-culture of CD8 + T cells and SKOV3 cells treated with 20 mmol/L sodium lactate), sh-FOXK1 + NaL (Co-culture of CD8 + T cells with SKOV3 cells transfected with sh-FOXK1 and treated with 20 mmol/L sodium lactate). * P < 0.05, **: P < 0.01, ***: P < 0.001, n = 3.

Article Snippet: After 24 h of incubation, 10 μL of CCK8 solution (cat no. 40203ES60, Yeasen, China) was added to each well 2 h before the end of the incubation period.

Techniques: Modification, CCK-8 Assay, Construct, Plasmid Preparation, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay, Co-Culture Assay

Journal: Acta biochimica Polonica

Article Title: Silencing lncRNA EZR‑AS1 induces apoptosis and attenuates the malignant properties of lung adenocarcinoma cells.

doi: 10.18388/abp.2020_6754

Figure Lengend Snippet: Figure 1. Determination of expression level of long non‑coding RNA EZR‑AS1 (ezrin antisense RNA 1) in lung ADC tissues and cells by RT-qPCR. (A) EZR‑AS1 levels in lung ADC and healthy tissues. (B) EZR‑AS1 levels in lung ADC cell lines (H1437 and H1975) and non-cancer lines (HBEC3-KT and NL20). (C) EZR‑AS1 levels in ADC cell lines H1437 and H1975 transfected with siEZR‑AS1 (si, small interfering RNA), siNC (NC, negative control). All assays were repeated three times. **denotes p<0.01 compared to si‑NC or non-transfected cells using one-way ANOVA.

Article Snippet: The human lung adenocarcinoma cell line H1437 (cat. no. CRL-5872), H1975 cells (cat. no. CRL-5908), lung epithelial cell line HBEC3-KT (cat. no. CRL-4051) and NL20 (cat. no. CRL-2503) were obtained from ATCC, Manassas, VA, USA.

Techniques: Expressing, Quantitative RT-PCR, Transfection, Small Interfering RNA, Negative Control

Journal: Acta biochimica Polonica

Article Title: Silencing lncRNA EZR‑AS1 induces apoptosis and attenuates the malignant properties of lung adenocarcinoma cells.

doi: 10.18388/abp.2020_6754

Figure Lengend Snippet: Figure 2. Cell viability and apoptosis of lung ADC cells after transfection with siEZR-AS1. (A) Cell viability was assessed using the CCK-8 assay at different culture times in lung ADC cell lines H1437 and H1975; (B) apoptosis by flow cytometry 48 h after transfection with siEZR-AS1 in lung ADC cell lines H1437 and H1975. Right panel: flow cytometry, left panel: statistical analysis of flow cytometry data. (si, small interfering RNA), siNC (NC, negative control). All assays were repeated three times. **denotes p<0.01 compared to si‑NC or non-transfected cells using one-way ANOVA.

Article Snippet: The human lung adenocarcinoma cell line H1437 (cat. no. CRL-5872), H1975 cells (cat. no. CRL-5908), lung epithelial cell line HBEC3-KT (cat. no. CRL-4051) and NL20 (cat. no. CRL-2503) were obtained from ATCC, Manassas, VA, USA.

Techniques: Transfection, CCK-8 Assay, Flow Cytometry, Small Interfering RNA, Negative Control

Journal: Acta biochimica Polonica

Article Title: Silencing lncRNA EZR‑AS1 induces apoptosis and attenuates the malignant properties of lung adenocarcinoma cells.

doi: 10.18388/abp.2020_6754

Figure Lengend Snippet: Figure 3. Migration of lung ADC cell lines H1437 and H1975 after transfected with siEZR-AS1. (A) cell migrations in wound healing experiment and (B) statistical analysis of migration rate. Scale bar, 100 μm. (si, small interfering RNA), siNC (NC, negative control) and pEZR-AS1 (positive control). All assays were repeated three times. **denotes p<0.01 compared to si‑NC or non-transfected cells using one-way ANOVA.

Article Snippet: The human lung adenocarcinoma cell line H1437 (cat. no. CRL-5872), H1975 cells (cat. no. CRL-5908), lung epithelial cell line HBEC3-KT (cat. no. CRL-4051) and NL20 (cat. no. CRL-2503) were obtained from ATCC, Manassas, VA, USA.

Techniques: Migration, Transfection, Small Interfering RNA, Negative Control, Positive Control

Journal: Acta biochimica Polonica

Article Title: Silencing lncRNA EZR‑AS1 induces apoptosis and attenuates the malignant properties of lung adenocarcinoma cells.

doi: 10.18388/abp.2020_6754

Figure Lengend Snippet: Figure 4. Level of metastasis-related proteins in lung ADC cell lines H1437 and H1975 after transfected with siEZR-AS1. Right panel: typical Western blots, left panel: statistical analysis of relative protein content. All assays were repeated three times. **de notes p<0.01 compared to si‑NC or non-transfected cells using one-way ANOVA.

Article Snippet: The human lung adenocarcinoma cell line H1437 (cat. no. CRL-5872), H1975 cells (cat. no. CRL-5908), lung epithelial cell line HBEC3-KT (cat. no. CRL-4051) and NL20 (cat. no. CRL-2503) were obtained from ATCC, Manassas, VA, USA.

Techniques: Transfection, Western Blot