Journal: Microorganisms

Article Title: LncRNA MEG3 Regulates Glaesserella parasuis -Induced Apoptosis of Porcine Alveolar Macrophages via Regulating ssc-miR-135/CASP8 Axis

doi: 10.3390/microorganisms13102287

Figure Lengend Snippet: Effects of MEG3 overexpression on G. parasuis -induced apoptosis of 3D4/21 cells. ( A ) After transfection with the MEG3 overexpression plasmid, the expression of MEG3 in G. parasuis -infected 3D4/21 cells was detected by qPCR. “**” indicates p < 0.01. ( B ) The effect of MEG3 overexpression on cell viability of G. parasuis -infected 3D4/21 cells was detected by the CCK-8 assay. “**” indicates p < 0.01. ( C ) After transfection with the MEG3 overexpression plasmid, 3D4/21 cells were infected with G. parasuis , and the apoptosis rate was detected by flow cytometry. ( D ) Statistical analysis results of the apoptosis rate. “**” indicates p < 0.01. ( E ) After transfection with the MEG3 overexpression plasmid, mRNA expression levels of apoptosis-related genes in G. parasuis -infected 3D4/21 cells were detected by qPCR. “*” indicates p < 0.05; “**” indicates p < 0.01. ( F ) After transfection with the MEG3 overexpression plasmid, the protein expression levels of apoptosis-related genes in G. parasuis -infected 3D4/21 cells were detected by Western blot. “**” indicates p < 0.01.

Article Snippet: According to the manufacturer’s instructions, 5 × 10 5 cells were incubated with Annexin V-FITC Reagent and PI Reagent (Annexin V-FITC/PI Cell Apoptosis Detection Kit, Elabscience Biotechnology, Wuhan, China) at room temperature in the dark for 20 min, and the apoptosis rate was detected using BD FACSAria III (Becton Dickinson, Franklin Lakes, NJ, USA).

Techniques: Over Expression, Transfection, Plasmid Preparation, Expressing, Infection, Real-time Polymerase Chain Reaction, CCK-8 Assay, Flow Cytometry, Western Blot

Journal: Microorganisms

Article Title: LncRNA MEG3 Regulates Glaesserella parasuis -Induced Apoptosis of Porcine Alveolar Macrophages via Regulating ssc-miR-135/CASP8 Axis

doi: 10.3390/microorganisms13102287

Figure Lengend Snippet: Effects of MEG3 overexpression on the extrinsic apoptosis pathway in G. parasuis -induced 3D4/21 cells. ( A ) After treatment with different concentrations of Z-IETD-FMK, the mRNA expression levels of apoptosis-related genes in G. parasuis -infected 3D4/21 cells were detected by qPCR. “**” indicates p < 0.01. ( B ) After transfection with the MEG3 overexpression vector and treatment with 20 μM of Z-IETD-FMK, the mRNA expression levels of apoptosis-related genes in G. parasuis -infected 3D4/21 cells were detected by qPCR. “**” indicates p < 0.01. ( C ) After transfection with the MEG3 overexpression vector and treatment with 20 μM of Z-IETD-FMK, the apoptosis rate was detected by flow cytometry. ( D ) Statistical analysis results of the apoptosis rate. “**” indicates p < 0.01.

Article Snippet: According to the manufacturer’s instructions, 5 × 10 5 cells were incubated with Annexin V-FITC Reagent and PI Reagent (Annexin V-FITC/PI Cell Apoptosis Detection Kit, Elabscience Biotechnology, Wuhan, China) at room temperature in the dark for 20 min, and the apoptosis rate was detected using BD FACSAria III (Becton Dickinson, Franklin Lakes, NJ, USA).

Techniques: Over Expression, Expressing, Infection, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, Flow Cytometry

Journal: Microorganisms

Article Title: LncRNA MEG3 Regulates Glaesserella parasuis -Induced Apoptosis of Porcine Alveolar Macrophages via Regulating ssc-miR-135/CASP8 Axis

doi: 10.3390/microorganisms13102287

Figure Lengend Snippet: Effect of ssc-miR-135 on G. parasuis -induced apoptosis in 3D4/21 cells. ( A ) After transfection with ssc-miR-135 mimic, cells were infected with G. parasuis , and qPCR was used to detect the expression of ssc-miR-135 in G. parasuis -infected 3D4/21 cells. “**” indicates p < 0.01. ( B ) CCK-8 assay to assess the effect of ssc-miR-135 mimic on cell viability in G. parasuis -infected 3D4/21 cells. “*” indicates p < 0.05; “**” indicates p < 0.01. ( C ) After transfecting 3D4/21 cells with ssc-miR-135 mimic, cells were infected with G. parasuis , and apoptosis rates were measured by flow cytometry. ( D ) Statistical analysis results of apoptosis rate. “**” indicates p < 0.01. ( E ) qPCR detection of mRNA expression levels of apoptosis-related genes in G. parasuis -infected 3D4/21 cells after transfection with ssc-miR-135 mimic. “**” indicates p < 0.01. ( F ) After transfection with ssc-miR-135 mimic, Western Blot was used to detect the expression levels of apoptosis-related gene proteins in G. parasuis -infected 3D4/21 cells. “**” indicates p < 0.01. ( G ) After transfection with ssc-miR-135 inhibitor, cells were infected with G. parasuis , and qPCR was used to detect the expression of ssc-miR-135 in G. parasuis -infected 3D4/21 cells. “**” indicates p < 0.01. ( H ) CCK-8 assay to assess the effect of ssc-miR-135 inhibitor on cell viability in G. parasuis -infected 3D4/21 cells. “**” indicates p < 0.01. ( I ) After transfecting 3D4/21 cells with ssc-miR-135 inhibitor, cells were infected with G. parasuis , and apoptosis rates were measured by flow cytometry. ( J ) Statistical analysis results of apoptosis rate. “**” indicates p < 0.01. ( K ) qPCR detection of mRNA expression levels of apoptosis-related genes in G. parasuis -infected 3D4/21 cells after transfection with ssc-miR-135 inhibitor. “**” indicates p < 0.01. ( L ) After transfection with ssc-miR-135 inhibitor, Western Blot was used to detect the expression levels of apoptosis-related gene proteins in G. parasuis -infected 3D4/21 cells. “**” indicates p < 0.01.

Article Snippet: According to the manufacturer’s instructions, 5 × 10 5 cells were incubated with Annexin V-FITC Reagent and PI Reagent (Annexin V-FITC/PI Cell Apoptosis Detection Kit, Elabscience Biotechnology, Wuhan, China) at room temperature in the dark for 20 min, and the apoptosis rate was detected using BD FACSAria III (Becton Dickinson, Franklin Lakes, NJ, USA).

Techniques: Transfection, Infection, Expressing, CCK-8 Assay, Flow Cytometry, Western Blot

Journal: Microorganisms

Article Title: LncRNA MEG3 Regulates Glaesserella parasuis -Induced Apoptosis of Porcine Alveolar Macrophages via Regulating ssc-miR-135/CASP8 Axis

doi: 10.3390/microorganisms13102287

Figure Lengend Snippet: Effect of MEG3 on CASP8 expression via ssc-miR-135-mediated pathway. ( A ) CCK-8 assay was performed to detect the effect of co-transfection of MEG3 overexpression plasmid and ssc-miR-135 mimic on cell viability after G. parasuis infection of 3D4/21 cells. “**” indicates p < 0.01. ( B ) After co-transfection with MEG3 overexpression plasmid and ssc-miR-135 mimic, 3D4/21 cells were infected with G. parasuis , and apoptosis rates were detected by flow cytometry. ( C ) Statistical analysis results of apoptosis rate. “**” indicates p < 0.01. ( D ) After co-transfection of MEG3 overexpression plasmid and ssc-miR-135 mimic, qPCR was used to detect the expression of MEG3 and ( E ) ssc-miR-135 in G. parasuis -infected 3D4/21 cells. “**” indicates p < 0.01. ( F ) After co-transfection with MEG3 overexpression plasmid and ssc-miR-135 mimic, qPCR was performed to determine the mRNA level of CASP8 in G. parasuis -infected 3D4/21 cells. “**” indicates p < 0.01. ( G ) After co-transfection of the MEG3 overexpression plasmid and ssc-miR-135 mimic, Western Blot analysis was conducted to detect the protein level of CASP8 in G. parasuis -infected 3D4/21 cells. “**” indicates p < 0.01.

Article Snippet: According to the manufacturer’s instructions, 5 × 10 5 cells were incubated with Annexin V-FITC Reagent and PI Reagent (Annexin V-FITC/PI Cell Apoptosis Detection Kit, Elabscience Biotechnology, Wuhan, China) at room temperature in the dark for 20 min, and the apoptosis rate was detected using BD FACSAria III (Becton Dickinson, Franklin Lakes, NJ, USA).

Techniques: Expressing, CCK-8 Assay, Cotransfection, Over Expression, Plasmid Preparation, Infection, Flow Cytometry, Western Blot

Journal: Microorganisms

Article Title: LncRNA MEG3 Regulates Glaesserella parasuis -Induced Apoptosis of Porcine Alveolar Macrophages via Regulating ssc-miR-135/CASP8 Axis

doi: 10.3390/microorganisms13102287

Figure Lengend Snippet: Functional verification of MEG3/ssc-miR-135/CASP8 axis in regulating G. parasuis -induced apoptosis in 3D4/21 cells. ( A ) Verification of CASP8 knockdown efficiency. “**” indicates p < 0.01. ( B ) CCK-8 assay was performed to detect the effect of co-transfection of MEG3 overexpression plasmid and ssc-miR-135 mimic, as well as MEG3 overexpression plasmid and si-CASP8, on cell viability after G. parasuis infection of 3D4/21 cells. “**” indicates p < 0.01. ( C ) After co-transfection with MEG3 overexpression plasmid and ssc-miR-135 mimic, as well as MEG3 overexpression plasmid and si-CASP8, 3D4/21 cells were infected with G. parasuis , and apoptosis rates were detected by flow cytometry. ( D ) Statistical analysis results of apoptosis rate. “**” indicates p < 0.01. ( E ) After co-transfection with MEG3 overexpression plasmid and ssc-miR-135 mimic, as well as MEG3 overexpression plasmid and si-CASP8, qPCR was performed to determine the mRNA level of CASP3 in G. parasuis -infected 3D4/21 cells. “**” indicates p < 0.01. ( F ) After co-transfection with MEG3 overexpression plasmid and ssc-miR-135 mimic, as well as MEG3 overexpression plasmid and si-CASP8, Western Blot analysis was conducted to detect the protein level of CASP3 in G. parasuis -infected 3D4/21 cells. “**” indicates p < 0.01.

Article Snippet: According to the manufacturer’s instructions, 5 × 10 5 cells were incubated with Annexin V-FITC Reagent and PI Reagent (Annexin V-FITC/PI Cell Apoptosis Detection Kit, Elabscience Biotechnology, Wuhan, China) at room temperature in the dark for 20 min, and the apoptosis rate was detected using BD FACSAria III (Becton Dickinson, Franklin Lakes, NJ, USA).

Techniques: Functional Assay, Knockdown, CCK-8 Assay, Cotransfection, Over Expression, Plasmid Preparation, Infection, Flow Cytometry, Western Blot

Journal: Microorganisms

Article Title: LncRNA MEG3 Regulates Glaesserella parasuis -Induced Apoptosis of Porcine Alveolar Macrophages via Regulating ssc-miR-135/CASP8 Axis

doi: 10.3390/microorganisms13102287

Figure Lengend Snippet: Schematic diagram of MEG3 regulates G. parasuis -induced apoptosis of porcine alveolar macrophages via regulating ssc-miR-135/CASP8 Axis. Red arrow: active.

Article Snippet: According to the manufacturer’s instructions, 5 × 10 5 cells were incubated with Annexin V-FITC Reagent and PI Reagent (Annexin V-FITC/PI Cell Apoptosis Detection Kit, Elabscience Biotechnology, Wuhan, China) at room temperature in the dark for 20 min, and the apoptosis rate was detected using BD FACSAria III (Becton Dickinson, Franklin Lakes, NJ, USA).

Techniques:

Journal: Journal of Inflammation Research

Article Title: Engeletin Protects Against TNF-α-Induced Apoptosis and Reactive Oxygen Species Generation in Chondrocytes and Alleviates Osteoarthritis in vivo

doi: 10.2147/JIR.S297166

Figure Lengend Snippet: Engeletin ameliorated TNF-α-induced chondrocyte apoptosis. Chondrocytes were pretreated with engeletin (10 μM and 20 μM) for two hours, and then TNF-α (20 ng/mL) was added to the culture medium and incubated for another 24 hours. ( A ) Chondrocytes were stained with Annexin V-FITC/PI and measured by flow cytometry to determine the apoptotic rate. ( B ) Statistical analysis of the apoptotic rate of chondrocytes. ( C – F ) The protein expression levels of Bax, Bcl-2, and cleaved caspase-3 were determined by Western blotting. The results were quantified using ImageJ software. Protein expression was normalized to GAPDH. The data are presented as the mean ± SD. n=3. *p < 0.05, **p < 0.01, ***p< 0.001, ****p < 0.0001.

Article Snippet: Cell Counting Kit-8 (CCK-8), protease and phosphatase inhibitor cocktails, RIPA lysis buffer, the nuclear and cytoplasmic protein extraction kit, HRP-labeled goat anti-rabbit IgG, electrochemiluminescence reagents, the Annexin V-FITC apoptosis detection kit and the ROS assay kit were obtained from Beyotime (Shanghai, China).

Techniques: Incubation, Staining, Flow Cytometry, Expressing, Western Blot, Software

Journal: Journal of Inflammation Research

Article Title: Engeletin Protects Against TNF-α-Induced Apoptosis and Reactive Oxygen Species Generation in Chondrocytes and Alleviates Osteoarthritis in vivo

doi: 10.2147/JIR.S297166

Figure Lengend Snippet: Schematic diagram of the mechanisms by which engeletin affects chondrocytes. TNF-α promotes apoptosis, ROS generation and ECM degradation in chondrocytes. Engeletin alleviates apoptosis by upregulating Bcl-2 and downregulating Bax. Engeletin scavenges ROS by activating the Nrf2 pathway and inhibits ECM degradation by inhibiting the MAPK and NF-κB pathways.

Article Snippet: Cell Counting Kit-8 (CCK-8), protease and phosphatase inhibitor cocktails, RIPA lysis buffer, the nuclear and cytoplasmic protein extraction kit, HRP-labeled goat anti-rabbit IgG, electrochemiluminescence reagents, the Annexin V-FITC apoptosis detection kit and the ROS assay kit were obtained from Beyotime (Shanghai, China).

Techniques:

Journal: Biomarker Research

Article Title: 5mC and H3K9me3 of TRAF3IP2 promoter region accelerates the progression of translocation renal cell carcinoma

doi: 10.1186/s40364-022-00402-3

Figure Lengend Snippet: High expression of TRAF3IP2 induces development of NONO-TFE3 tRCC. A-E The effects of TRAF3IP2 overexpression or knockdown on the proliferation of UOK109 and 786-O cells respectively were examined by CCK-8 assay ( A-B ), colony formation assays ( C-D ) and tumor sphere formation ( E ). F EdU assays were used to detect the proliferation rate of UOK109 and 786-O cells after transfection for 48 h. G Cell cycle was analyzed using flow cytometry after transfection for 48 h. The statistical results of the proportion of cells in S stage in correspond groups. H Cell apoptosis was analyzed via flow cytometry using an Annexin V/PI kit after transfection for 48 h. I-K Migration and invasion assays were performed with transfected cells using Transwell inserts. L-M Nude mice were injected subcutaneously with 786-O cells and tumor formation monitored over a period of several weeks. N The tumor volume was measured as indicated. O Representative H&E staining of xenograft tumors. The data are presented as the mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The cells were then incubated with reagents from an Annexin V-FITC / propidium iodide (PI) apoptosis kit (Vazyme) and analyzed using a BD Beckman cytometer (BD Biosciences) and FlowJo software.

Techniques: Expressing, Over Expression, CCK-8 Assay, Transfection, Flow Cytometry, Migration, Injection, Staining

Journal: Bioactive Materials

Article Title: Translational selenium nanoparticles boost GPx1 activation to reverse HAdV-14 virus-induced oxidative damage

doi: 10.1016/j.bioactmat.2024.04.034

Figure Lengend Snippet: Intracellular localization of HAdV-14 and the inhibitory effect of SeNPs@LNT on apoptosis induced by HAdV-14 infection. (A) DAPI was used for nuclear staining and Lyso-tracker was used for lysosome staining. The A549 cells were infected by rAD14 EGFP for different periods and were observed under the fluorescent microscope; (B) Schematic diagram of the infection process of HAdV-14 in A549 cells; (C) HAdV-14 infection of A549 cells were treated with SeNPs@LNT and the caspase-3 activity was detected by the absorbance of pNA; (D) DNA fragmentation and nuclear deformation in A549 cells were detected by TUNEL-DAPI co-staining assay; (E and F) A549 cells in each group were stained with Annexin V-FITC and PI to detect phosphatidylserine on the cell membrane surface. The results of fluorescent labeling were observed by flow cytometry and fluorescence microscopy. The A549 cells were treated with 8 μM SeNPs@LNT for 48 h. Bars with different characters are statistically different at P ＜0.05 (* or # ) level. * represents comparison with the Control group, # represented comparison with the Virus group.

Article Snippet: Chemiluminescence reagents include Caspase-3 activity detection kit and enhanced cell counting kit-8 (CCK-8) purchased from Beyotime (China).

Techniques: Infection, Staining, Microscopy, Activity Assay, TUNEL Assay, Membrane, Labeling, Flow Cytometry, Fluorescence, Comparison, Virus

Journal: Bioactive Materials

Article Title: Translational selenium nanoparticles boost GPx1 activation to reverse HAdV-14 virus-induced oxidative damage

doi: 10.1016/j.bioactmat.2024.04.034

Figure Lengend Snippet: SeNPs@LNT resists adenovirus pneumonia induced by HAdV-14 in mice. (A) Schematic showing the in vivo experimental design for the treatment of HAdV-14 infection; (B) Analysis of lung index in mice treated with SeNPs@LNT. After the mice in the virus group were infected with HAdV-14 through nose drops, the lung index was significantly higher than that in the control group; (C) Survival and body weight of mice in different groups at 14 d; (D) The lung tissue sections of mice in different treatment groups were stained with HE and the number of cells in each field was counted and analyzed by Image J. TUNEL test was performed on the lung tissue of mice to detect the DNA fragmentation; (E) Immunohistochemistry of the mice in different groups. The dark brown particles representing T-p38, T-p53, T-JNK, p -JNK (Thr183/Tyr185), T-Caspase-3, T-Akt, and T-Erk proteins expression in the lung tissue of the mice. The therapeutic concentration of SeNPs@LNT was 0.2 mg Se/kg. Bars with different characters are statistically different at P ＜0.05 (* or # ) or P ＜0.01 (**) level. * represents comparison with the Control group, # represented comparison with the Virus group.

Article Snippet: Chemiluminescence reagents include Caspase-3 activity detection kit and enhanced cell counting kit-8 (CCK-8) purchased from Beyotime (China).

Techniques: In Vivo, Infection, Virus, Staining, TUNEL Assay, Immunohistochemistry, Expressing, Concentration Assay, Comparison

Journal: PeerJ

Article Title: Titanium dioxide dental implants surfaces related oxidative stress in bone remodeling: a systematic review

doi: 10.7717/peerj.12951

Figure Lengend Snippet: A descriptive summary of the findings are tabulated based on the focused question.

Article Snippet: 5 , ( ) ) China , Study on potential toxic of titanium oxide nanoparticles on osteoblasts , Cell culture study , Osteoblast cells , Titanium oxide nanoparticles (TiO2-NPs) , less than 25 nm. , * The cellular and molecular cross talk in bone remodeling were not identified. * The effect of TiO 2 NPs induced oxidative stress on the osteogenesis-angiogenesis coupling in bone remodeling were not identified. , MTS reagent kit for cytoactive detection. LDH reagent kit for cytotoxicity detection. ROS detection reagent kit. RIPA cell lysis. Transmission electron microscopy (TEM). Zetasizer particle size analyzer Malvern Instrument, Ultrasonic oscillation instrument. Inverted phase contrast microscope. , ND , Osteogenesis , TiO 2 -NPs can cause the decrease of the survival rate of the osteoblast and increase of the content of the LDH released by the cell, and with dosage dependency effect , 71.66 Strong Evidence.

Techniques: In Vivo, In Vitro, Titanium Dioxide, Cell Culture, Lactate Dehydrogenase Assay, Flow Cytometry, Membrane, Permeability, RNA Extraction, Quantitative RT-PCR, Clinical Proteomics, Mass Spectrometry, Peroxidation Assay, Activity Assay, Glutathione Assay, FRAP Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Micro-CT, Staining, Immunohistochemical staining, Immunohistochemistry, Expressing, Electrophoresis, Colorimetric Assay, Lysis, Transmission Assay, Electron Microscopy, Microscopy, WST-1 Assay, Nitration, Immunoenzymatic Assay, Immunoprecipitation, Biomarker Discovery, Real-time Polymerase Chain Reaction, MTT Assay, Tube Formation Assay, Wound Healing Assay, ROS Assay, ATP Assay, X-ray Diffraction, BIA-KA, Confocal Laser Scanning Microscopy, Laser-Scanning Microscopy, Viability Assay, Immunofluorescence, Control, Inhibition, CCK-8 Assay, ALP Assay, Modification, Protein-Protein interactions

Journal: Clinical and Translational Medicine

Article Title: APE1 shRNA‐loaded cancer stem cell‐derived extracellular vesicles reverse Erlotinib resistance in non‐small cell lung cancer via the IL‐6/STAT3 signalling

doi: 10.1002/ctm2.876

Figure Lengend Snippet: Effect of HCC827P‐CSC‐EVs and HCC827R‐CSC‐EVs on the resistance of NSCLC cells to Erlotinib. (A) Expression of Erlotinib resistance‐related genes MDR1, MRP, LRP and ABCG2 in HCC827P and PC9P cells following co‐culture with HCC827P‐CSC‐EVs or HCC827R‐CSC‐EVs, detected by qRT‐PCR and Western blot. (B) Viability of HCC827P and PC9P cells co‐cultured with HCC827P‐CSC‐EVs or HCC827R‐CSC‐EVs and further treated with different concentrations of Erlotinib, detected by CCK‐8 assay. Quantification of the migration (C) and invasion (D) of Erlotinib (5 μM)‐treated HCC827P and PC9P cells in response to co‐culture with HCC827P‐CSC‐EVs or HCC827R‐CSC‐EVs, observed by Transwell assay. (E) Apoptosis of Erlotinib (5 μM)‐treated HCC827P and PC9P cells in response to co‐culture with HCC827P‐CSC‐EVs or HCC827R‐CSC‐EVs, detected by flow cytometry. (F) Protein expression of anti‐apoptotic Bcl‐2 and pro‐apoptotic Bax and cleaved caspase‐3 in Western blot of Erlotinib (5 μM)‐treated HCC827P and PC9P cells in response to co‐culture with HCC827P‐CSC‐EVs or HCC827R‐CSC‐EVs. * p < .05. Each cell experiment was conducted in triplicate. CSC, cancer stem cell; EV, extracellular vesicle; NSCLC, non‐small cell lung cancer

Article Snippet: An Annexin V‐FITC/PI staining reagent was prepared following the protocols of the Annexin‐V‐FITC apoptosis kit (K201‐100, BioVision, USA).

Techniques: Expressing, Co-Culture Assay, Quantitative RT-PCR, Western Blot, Cell Culture, CCK-8 Assay, Migration, Transwell Assay, Flow Cytometry

Journal: Clinical and Translational Medicine

Article Title: APE1 shRNA‐loaded cancer stem cell‐derived extracellular vesicles reverse Erlotinib resistance in non‐small cell lung cancer via the IL‐6/STAT3 signalling

doi: 10.1002/ctm2.876

Figure Lengend Snippet: APE1 affects the Erlotinib resistance of NSCLC cells through mediating the IL‐6/STAT3 signalling. (A) The protein expression of APE1 and p‐STAT3/STAT3 in HCC827R and PC9R cells treated by Erlotinib alone or in combination with shAPE1, measured by the Western blot. (B) The content of IL‐6 in the supernatant of HCC827R and PC9R cells treated by Erlotinib alone or in combination with shAPE1, detected by ELISA. (C) Cell viability in HCC827R and PC9R cells treated by Erlotinib alone or in combination with shAPE1, detected by CCK‐8 assay. Quantification of the migration (D) and invasion (E) in HCC827R and PC9R cells treated by Erlotinib alone or in combination with shAPE1, observed in the Transwell assay. (F) The apoptosis of HCC827R and PC9R cells treated by Erlotinib alone or in combination with shAPE1, tested by flow cytometry. (G) The protein expression of Bcl‐2, Bax and cleaved caspase‐3 in HCC827R and PC9R cells treated by Erlotinib alone or in combination with shAPE1, measured by the Western blot. (H) The mRNA and protein expression of MDR1, MRP, LRP and ABCG2 in CC827R and PC9R cells treated by Erlotinib alone or in combination with shAPE1, measured by qRT‐PCR and Western blot. * p < .05. Each cell experiment was conducted in triplicate. APE1, apurinic endonuclease 1; IL‐6, interleukin‐6; NSCLC, non‐small cell lung cancer

Article Snippet: An Annexin V‐FITC/PI staining reagent was prepared following the protocols of the Annexin‐V‐FITC apoptosis kit (K201‐100, BioVision, USA).

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Migration, Transwell Assay, Flow Cytometry, Quantitative RT-PCR

Journal: Clinical and Translational Medicine

Article Title: APE1 shRNA‐loaded cancer stem cell‐derived extracellular vesicles reverse Erlotinib resistance in non‐small cell lung cancer via the IL‐6/STAT3 signalling

doi: 10.1002/ctm2.876

Figure Lengend Snippet: Effects of HCC827R‐CSC‐EVs loaded with APE1 shRNA on the Erlotinib resistance of NSCLC cells in vitro. (A) The enrichment of APEX1 (APE1) in EVs of different origins, analysed using the ExoRBase database. (B) The mRNA expression of APE1 in HCC827P‐CSCs, HCC827R‐CSCs, HCC827P‐CSC‐EVs and HCC827R‐CSC‐EVs determined by qRT‐PCR assay. (C) The mRNA expression of APE1 in HCC827R‐CSCs and HCC827R‐CSC‐EVs in response to shAPE1 treatment, determined by qRT‐PCR assay. (D) The mRNA expression of APE1 in HCC827P and PC9P cells in response to co‐culture with HCC827R‐CSCs EVs shAPE1 determined by qRT‐PCR assay. (E) Western blot measurement of the protein expression of p‐STAT3/STAT3 in HCC827P and PC9P cells in response to co‐culture with HCC827R‐CSCs EVs shAPE1. (F) ELISA detection of the content of IL‐6 in the supernatant of HCC827P and PC9P cells in response to co‐culture with HCC827R‐CSCs EVs shAPE1. (G) Cell viability in HCC827P and PC9P cells in response to co‐culture with HCC827R‐CSCs EVs shAPE1 and further treatment with Erlotinib, detected by CCK‐8 assay. Quantification of the migration (H) and invasion (I) in HCC827P and PC9P cells in response to co‐culture with HCC827R‐CSCs EVs shAPE1 and further treatment with Erlotinib (5 μM), detected by the Transwell assay. (J) Cell apoptosis in HCC827P and PC9P cells in response to co‐culture with HCC827R‐CSCs EVs shAPE1 and further treatment with Erlotinib (5 μM), detected by flow cytometry. (K) Protein expression of anti‐apoptotic Bcl‐2 and pro‐apoptotic Bax and cleaved caspase‐3 in the Western blot of Erlotinib (5 μM)‐treated HCC827P and PC9P cells in response to co‐culture with HCC827R‐CSCs EVs shAPE1. (L) The expression of MDR1, MRP, LRP and ABCG2 in Erlotinib (5 μM)‐treated HCC827P and PC9P cells in response to co‐culture with HCC827R‐CSCs EVs shAPE1 measured by qRT‐PCR and the Western blot. * p < .05. Each cell experiment was conducted in triplicate. APE1, apurinic endonuclease 1; CSC, cancer stem cell; EV, extracellular vesicle; IL‐6, interleukin‐6; NSCLC, non‐small cell lung cancer; shRNA, short hairpin RNA

Article Snippet: An Annexin V‐FITC/PI staining reagent was prepared following the protocols of the Annexin‐V‐FITC apoptosis kit (K201‐100, BioVision, USA).

Techniques: shRNA, In Vitro, Expressing, Quantitative RT-PCR, Co-Culture Assay, Western Blot, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Migration, Transwell Assay, Flow Cytometry