Journal: Scientific Reports

Article Title: Transcription factor NF-kappa B represses ANT1 transcription and leads to mitochondrial dysfunctions

doi: 10.1038/srep44708

Figure Lengend Snippet: ( A ) RT-PCR shows the ANT1, TNFα, and IκBα mRNA expression in HEK 293 cells after 24 hours transfection with IKKβ and NF-κB/p65. The histogram depicts the mean ratios of ANT1 mRNA to β-actin ± SEM (n = 3); *p < 0.05, Student’s t test. ( B ) ANT1 and IκBα mRNA expression in HEK293 were examined in the presence of NF-κB activity inhibitor JSH-23 (15 μM) and the NF-κB activator H 2 O 2 (50 μM Values represent means ± SEM (n = 3); **p < 0.01, Student’s t test. ( C ) The endogenous ANT1 protein in HEK293 was affected by NF-κB/p65 transfection and NF-κB inhibitor JSH-23 (15 μM). Cropped blots were displayed. Anti-ANT1 (ab110322, Abcam) and anti-NF-κB/p65 (#8242, CST) antibodies were used in WB. β-actin was used as loading controls. Values represent means ± SEM (n = 3); *p < 0.05, Student’s t test. ( D ) The IRDye 800-labelled consensus NRE oligo was used as probes in EMSA. Nuclear extracts in EMSA were extracted from T98G cells treated with TNFα (10 ng/ml) for 0, 90, 180 and 360 minutes. NRE, NF-κB responsive elements ( E ) ANT1 mRNA levels and NF-κB activities in TNFα (10 ng/ml) induced T98G cells for 0, 90, 180 and 360 minutes were depicted in line graph; *p < 0.05, **p < 0.01, One-way ANOVA test, as compared with initial time point. ( F ) T98G cells stimulated by TNFα (10 ng/ml) for 0, 90, 180 and 360 minutes were harvested and lysed for WB of endogenous ANT1, IκBα and COX-IV. Anti-ANT1 (ab110322; Abcam), anti-IκBα (#4814, CST) and anti-COX-IV (#4850, CST) antibodies were used in WB. β-actin was used as a loading control. Cropped blots were displayed. Values represent means ± SEM (n = 3); **p < 0.01, One-way ANOVA test, as compared with initial time point. ( G ) ANT1 over-expression plasmid (p3xflagANT1) was transfected into T98G cells and stimulated by TNFα for 0, 90, 180 and 360 minutes. Anti-flag (M2, F1804, Sigma-Aldrich) was used to detect exogenous ANT1 expression. Cropped blots were displayed. Values represent means ± SEM (n = 3); NS, no significant difference, One-way ANOVA test, as compared with initial time point.

Article Snippet: ANT1 mAb (ab110322, Abcam, Cambrige, UK); ATP Determination Kit (A22066, Invitrogen, Waltham, USA); anti-flag mAb (M2, F1804, Sigma-Aldrich, Saint Louis, USA); β -actin mAb (SAB1403520; Sigma-Aldrich, Saint Louis, USA); Bongkrekic acid (1820–100, Biovision, USA); calcein-AM (17783, Sigma-Aldrich, Saint Louis, USA); carboxyatractyloside (C4992, Sigma-Aldrich, Saint Louis, USA); Chromatin Immunoprecipitation (ChIP) Assay Kit (17–295, Milllipore, Darmstadt, Germany); cleaved caspase-3 mAb (#9664, CST, Beverly, USA); CoCl 2 (V900021, Sigma-Aldrich, Saint Louis, USA); COX-IV mAb (#4850, CST, Beverly, USA); DCFH-DA (S0033, Beyotime, Nanjing, China); Dihydroethidium (DHE, S0063, Beyotime, Nanjing, China), digitonin (D141, Sigma-Aldrich, Saint Louis, USA); Dual-Luciferase ® Reporter Assay System (E1910, Promega, Wisconsin, USA);Dulbecco’s modified Eagle’s medium (SH30243.01B, Hyclone, South Logan, USA); Fetal bovine serum (10100147, Gibco, Gaithersburg, MD); IκB-α mAb (#4814, CST, Beverly, USA); ionomycin (S1672, Beyotime, Nanjing, China); JSH-23 (S7351;Selleckchem, Houston, USA); Lipofectamine TM 2000 transfection reagent (11668027, Invitrogen, Waltham, USA); Magnesium Green™ (M3733, Invitrogen, Waltham, USA); NF-κB/p65 mAb (#8242, CST, Beverly, USA); Odyssey EMSA Buffer Kit (ABIN2169587, Li-cor, USA); Opti-MEM(51985042, Gibco, Gaithersburg, MD);PEG8000 (89510, Sigma-Aldrich, Saint Louis, USA); secondary antibodies (Jackson Immuno Research, West Grove, USA);SYBR-Green PCR Master Mix (QPK-201, Toyobo, Japan); TMRM, (T668, Invitrogen, Waltham, USA); TNFα (10602-HNAE-10, Sino Biological lnc.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection, Activity Assay, Over Expression, Plasmid Preparation

Journal: Scientific Reports

Article Title: Transcription factor NF-kappa B represses ANT1 transcription and leads to mitochondrial dysfunctions

doi: 10.1038/srep44708

Figure Lengend Snippet: ( A , B ) T98G cells were transfected with pSiANT1 and primary neurons were infected with lentivirus LV-SiANT1 or negative control (pSiCON or LV-SiCON) for 72 hours. Cell lysates were separated by SDS-PAGE and analysed with anti-ANT1 antibody (ab110322; Abcam). Cropped blots were displayed. The histogram depicts the mean ratios of ANT1 protein to β-actin ± SEM (n = 3); ** p < 0.01, Student’s t test. ( C ) ATP/ADP exchange rates were determined in T98G cells with TNFα treatment, ANT1 knockdown (pSiANT1 transfected) and ANT1 over-expression (pANT1 transfected). ANT1 knockdown and TNFα treatment decreased ATP/ADP exchange rate while ANT1 overexpression increased the rate. The histogram depicts the means of the ATP/ADP exchange rates in treatment groups ± SEM (n = 3); * p < 0.05, ** p < 0.01, Student’s t test, as compared to their respective control groups (CON). ( D , E ) ATP levels in T98G ( D ) and neurons ( E ) were measured by a luminometer using an ATP determination kit (A22066; Invitrogen). The histogram depicts the means of the cellular ATP level in treatment groups ± SEM (n = 3); ** p < 0.01, Student’s t test, as compared to their respective control groups (CON). ( F ) Total and cleaved caspase-3 were determined by caspase-3 (#9665, CST) and cleaved caspase-3 (#9664, CST) antibodies in T98G cells treated by TNFα (10 ng/ml) for 0, 90, 180 and 360 minutes. Cropped blots were displayed. The histogram depicts the mean ratios of cleaved caspase-3 protein level to total caspase-3 protein level ± SEM (n = 3); NS, no significant difference, One-way ANOVA test, as compared with initial time point.

Article Snippet: ANT1 mAb (ab110322, Abcam, Cambrige, UK); ATP Determination Kit (A22066, Invitrogen, Waltham, USA); anti-flag mAb (M2, F1804, Sigma-Aldrich, Saint Louis, USA); β -actin mAb (SAB1403520; Sigma-Aldrich, Saint Louis, USA); Bongkrekic acid (1820–100, Biovision, USA); calcein-AM (17783, Sigma-Aldrich, Saint Louis, USA); carboxyatractyloside (C4992, Sigma-Aldrich, Saint Louis, USA); Chromatin Immunoprecipitation (ChIP) Assay Kit (17–295, Milllipore, Darmstadt, Germany); cleaved caspase-3 mAb (#9664, CST, Beverly, USA); CoCl 2 (V900021, Sigma-Aldrich, Saint Louis, USA); COX-IV mAb (#4850, CST, Beverly, USA); DCFH-DA (S0033, Beyotime, Nanjing, China); Dihydroethidium (DHE, S0063, Beyotime, Nanjing, China), digitonin (D141, Sigma-Aldrich, Saint Louis, USA); Dual-Luciferase ® Reporter Assay System (E1910, Promega, Wisconsin, USA);Dulbecco’s modified Eagle’s medium (SH30243.01B, Hyclone, South Logan, USA); Fetal bovine serum (10100147, Gibco, Gaithersburg, MD); IκB-α mAb (#4814, CST, Beverly, USA); ionomycin (S1672, Beyotime, Nanjing, China); JSH-23 (S7351;Selleckchem, Houston, USA); Lipofectamine TM 2000 transfection reagent (11668027, Invitrogen, Waltham, USA); Magnesium Green™ (M3733, Invitrogen, Waltham, USA); NF-κB/p65 mAb (#8242, CST, Beverly, USA); Odyssey EMSA Buffer Kit (ABIN2169587, Li-cor, USA); Opti-MEM(51985042, Gibco, Gaithersburg, MD);PEG8000 (89510, Sigma-Aldrich, Saint Louis, USA); secondary antibodies (Jackson Immuno Research, West Grove, USA);SYBR-Green PCR Master Mix (QPK-201, Toyobo, Japan); TMRM, (T668, Invitrogen, Waltham, USA); TNFα (10602-HNAE-10, Sino Biological lnc.

Techniques: Transfection, Infection, Negative Control, SDS Page, Over Expression

Journal: Cell Death Discovery

Article Title: 5-Iodotubercidin sensitizes cells to RIPK1-dependent necroptosis by interfering with NFκB signaling

doi: 10.1038/s41420-023-01576-x

Figure Lengend Snippet: A MK2/3-deficient MEFs transduced with MK2 expression or control vector are treated with TNF in the presence of smac mimetics (SM), a pro-apoptotic stimulus, for 60 and 90 min. B Diverse apoptotic stimuli including combinations of TNF with the IKK1/2 inhibitor BMS345541 (BMS) and with BX795 (TBK1 inhibitor) induce predominant necroptotic response in MK2/3-deficient cells. C MK2/3-deficient cells were pre-treated with RIPK3 inhibitor (GSK872) and SM for 30 min followed by TNF treatment for 2 h. A – C Numbers at the right of the blot indicate molecular mass of the marker proteins in kDa. D Sytox Green-based cytotoxicity assay was performed with MK2/3-deficient and MK2-rescued cells treated in the presence and absence of caspase inhibitor (zVAD) as indicated.

Article Snippet: The following reagents were used for cell treatments at given concentrations: LPS ( Escherichia coli O55:B5, #L2880, Sigma-Aldrich, 100 ng ml −1 ), recombinant human TNFα (rHuTNF, #50435.50, Biomol, 10 ng/ml), Birinapant/SM (#HY-16591, MedChem Express, 1 μM), pan-caspase inhibitor zVAD-fmk (#4026865.0005, Bachem, 25 μM), MK2 inhibitor PF3644022 (#4279, Tocris, 5 μM), RIPK1 inhibitor Nec-1 (#BML-AP309-0020, Enzo Life Sciences, 50 μM), RIPK1 inhibitor Nec-1s (# 10-4544-5 mg, Tebu-Bio, 50 μM), RIPK3 inhibitor GSK872 (#HY-101872, MedChem Express, 5 μM), IKKβ inhibitor BMS345541 (#Axon 1731, Axon Medchem, 5 μM), TBK1/IKKε inhibitor BX795 (#T1830, Tebu-Bio), 5-Iodotubercidin (#HY-15424, MedChem Express, 2.5–10 μM), ABT-702 dihydrochloride (#HY-103161, MedChem Express, 5 μM), Staurosporine (#81590, Cayman, 5 μM), Etoposide (#E1383, Sigma, 5 μM), Doxorubicin (#15007, Cayman, 5 μM), Gemcitabine (#S1714, Selleckchem, 100 nM).

Techniques: Transduction, Expressing, Control, Plasmid Preparation, Marker, Cytotoxicity Assay

Journal: Cell Death Discovery

Article Title: 5-Iodotubercidin sensitizes cells to RIPK1-dependent necroptosis by interfering with NFκB signaling

doi: 10.1038/s41420-023-01576-x

Figure Lengend Snippet: MK2-deficient MEFs transduced with MK2 expression or control vector were treated with TNF alone or in combination with inhibitors from the kinase inhibitor panel (at 10 µM concentration) for 6 h. Cell viability was quantified by the CCK8 colorimetric assay. Each treatment was performed in triplicates. While TNF alone is not cytotoxic, several small molecules sensitize both MEF lines to TNF (comps. 15, 42, 133, 142). Compound 14/CAY10657 (IKK2 inhibitor), comp. 32/BIO (GSK3 inhibitor), comp. 54,55/Bisindolylmaleimide VIII/IX (PKC inhibitors), comp. 94/PIK-75 (PI3 kinase inhibitor) and comp. 130/5-Iodotubercidin (5-ITu) display specific sensitizing effects dependent on MK2 deficiency. Average values of n = 3 independent wells are plotted ± SD.

Article Snippet: The following reagents were used for cell treatments at given concentrations: LPS ( Escherichia coli O55:B5, #L2880, Sigma-Aldrich, 100 ng ml −1 ), recombinant human TNFα (rHuTNF, #50435.50, Biomol, 10 ng/ml), Birinapant/SM (#HY-16591, MedChem Express, 1 μM), pan-caspase inhibitor zVAD-fmk (#4026865.0005, Bachem, 25 μM), MK2 inhibitor PF3644022 (#4279, Tocris, 5 μM), RIPK1 inhibitor Nec-1 (#BML-AP309-0020, Enzo Life Sciences, 50 μM), RIPK1 inhibitor Nec-1s (# 10-4544-5 mg, Tebu-Bio, 50 μM), RIPK3 inhibitor GSK872 (#HY-101872, MedChem Express, 5 μM), IKKβ inhibitor BMS345541 (#Axon 1731, Axon Medchem, 5 μM), TBK1/IKKε inhibitor BX795 (#T1830, Tebu-Bio), 5-Iodotubercidin (#HY-15424, MedChem Express, 2.5–10 μM), ABT-702 dihydrochloride (#HY-103161, MedChem Express, 5 μM), Staurosporine (#81590, Cayman, 5 μM), Etoposide (#E1383, Sigma, 5 μM), Doxorubicin (#15007, Cayman, 5 μM), Gemcitabine (#S1714, Selleckchem, 100 nM).

Techniques: Transduction, Expressing, Control, Plasmid Preparation, Concentration Assay, Colorimetric Assay

Journal: Cell Death Discovery

Article Title: 5-Iodotubercidin sensitizes cells to RIPK1-dependent necroptosis by interfering with NFκB signaling

doi: 10.1038/s41420-023-01576-x

Figure Lengend Snippet: A MK2-deficient MEFs transduced with MK2 expression or control vector were treated with different doses of 5-ITu in the presence or absence of TNF (10 ng/ml) for 6 h. B Cells of the indicated genotype were treated with 10 µM 5-ITu for 6 and 24 h and cell viability was quantified. C MK2-deficient MEFs transduced with MK2 expression or control vector were treated with indicated small molecules (5 µM ABT-702, 100 nM gemcitabine, 5 µM etoposid, 5 µM doxorubicin and 5 µM staurosporine) in the presence or absence of TNF for 6 h and cell viability was assessed. D MK2-deficient MEFs transduced with MK2 expression or control vector were treated as indicated and viability was quantified after 6 h treatment. E , F RAW264.7 cells were treated as indicated to monitor the effect of 5-ITu in LPS-induced necroptosis ( E ) and SM-mediated autocrine TNF-dependent death ( F ). Average values of n = 3 independent wells are plotted ± SD (** p ≤ 0.001, *** p ≤ 0.0001, **** p ≤ 0.0001).

Article Snippet: The following reagents were used for cell treatments at given concentrations: LPS ( Escherichia coli O55:B5, #L2880, Sigma-Aldrich, 100 ng ml −1 ), recombinant human TNFα (rHuTNF, #50435.50, Biomol, 10 ng/ml), Birinapant/SM (#HY-16591, MedChem Express, 1 μM), pan-caspase inhibitor zVAD-fmk (#4026865.0005, Bachem, 25 μM), MK2 inhibitor PF3644022 (#4279, Tocris, 5 μM), RIPK1 inhibitor Nec-1 (#BML-AP309-0020, Enzo Life Sciences, 50 μM), RIPK1 inhibitor Nec-1s (# 10-4544-5 mg, Tebu-Bio, 50 μM), RIPK3 inhibitor GSK872 (#HY-101872, MedChem Express, 5 μM), IKKβ inhibitor BMS345541 (#Axon 1731, Axon Medchem, 5 μM), TBK1/IKKε inhibitor BX795 (#T1830, Tebu-Bio), 5-Iodotubercidin (#HY-15424, MedChem Express, 2.5–10 μM), ABT-702 dihydrochloride (#HY-103161, MedChem Express, 5 μM), Staurosporine (#81590, Cayman, 5 μM), Etoposide (#E1383, Sigma, 5 μM), Doxorubicin (#15007, Cayman, 5 μM), Gemcitabine (#S1714, Selleckchem, 100 nM).

Techniques: Transduction, Expressing, Control, Plasmid Preparation

Journal: Cell Death Discovery

Article Title: 5-Iodotubercidin sensitizes cells to RIPK1-dependent necroptosis by interfering with NFκB signaling

doi: 10.1038/s41420-023-01576-x

Figure Lengend Snippet: A MK2-deficient MEFs transduced with MK2 expression or control vector were treated with IKK1/2 inhibitor BMS and 5-ITu in combination with TNF for 2 h. MLKL and RIPK1 phosphorylation were monitored with additional control blots. The RIPK1 band-shift induced by MK2-mediated phosphorylation is absent in MK2-KO cells. EF2 is shown as loading control. B MK2/3-deficient MEFs transduced with MK2 expression or control vector were treated with 5-ITu alone or in combination with TNF for 3 h. Cells were pre-treated for 30 min with the MK2 inhibitor PF3604422, the RIPK1 inhibitor Nec-1 or the RIPK3 inhibitor GSK872, as indicated. C Wild-type (WT) MEFs were treated with 5-ITu or TNF alone or in combination for short-term kinetics (10–180 min). D RAW264.7 cells were treated with 5-ITu or LPS alone or in combination for 10–180 min and signaling was monitored by immunoblotting as indicated. A – D Numbers at the right of the blot indicate molecular mass of the marker proteins in kDa.

Article Snippet: The following reagents were used for cell treatments at given concentrations: LPS ( Escherichia coli O55:B5, #L2880, Sigma-Aldrich, 100 ng ml −1 ), recombinant human TNFα (rHuTNF, #50435.50, Biomol, 10 ng/ml), Birinapant/SM (#HY-16591, MedChem Express, 1 μM), pan-caspase inhibitor zVAD-fmk (#4026865.0005, Bachem, 25 μM), MK2 inhibitor PF3644022 (#4279, Tocris, 5 μM), RIPK1 inhibitor Nec-1 (#BML-AP309-0020, Enzo Life Sciences, 50 μM), RIPK1 inhibitor Nec-1s (# 10-4544-5 mg, Tebu-Bio, 50 μM), RIPK3 inhibitor GSK872 (#HY-101872, MedChem Express, 5 μM), IKKβ inhibitor BMS345541 (#Axon 1731, Axon Medchem, 5 μM), TBK1/IKKε inhibitor BX795 (#T1830, Tebu-Bio), 5-Iodotubercidin (#HY-15424, MedChem Express, 2.5–10 μM), ABT-702 dihydrochloride (#HY-103161, MedChem Express, 5 μM), Staurosporine (#81590, Cayman, 5 μM), Etoposide (#E1383, Sigma, 5 μM), Doxorubicin (#15007, Cayman, 5 μM), Gemcitabine (#S1714, Selleckchem, 100 nM).

Techniques: Transduction, Expressing, Control, Plasmid Preparation, Phospho-proteomics, Electrophoretic Mobility Shift Assay, Western Blot, Marker

Journal: International Journal of Molecular Sciences

Article Title: Rheumatoid Arthritis-Associated MicroRNA-155 Targets SOCS1 and Upregulates TNF-α and IL-1β in PBMCs

doi: 10.3390/ijms141223910

Figure Lengend Snippet: Demographic, clinical, and laboratory data of patients with rheumatoid arthritis, osteoarthritis, and controls.

Article Snippet: The levels of TNF-α and IL-1β in plasma and PBMCs supernatant were measured by an enzyme immunoassay using the human TNF-α and IL-1β ELISA Kit (Sino Biological Inc., Beijing, China).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Rheumatoid Arthritis-Associated MicroRNA-155 Targets SOCS1 and Upregulates TNF-α and IL-1β in PBMCs

doi: 10.3390/ijms141223910

Figure Lengend Snippet: miR-155 upregulation correlates with increased production of TNF-α and IL-1β in Rheumatoid arthritis (RA) patients. ( A ) and ( B ): Increased expression of TNF-α and IL-1β in RA plasma. Results are expressed as pg/mL by an ELISA test; ( C ): MiR-155 is upregulated in PBMCs of active RA patients. Results in patients with RA ( n = 45) and OA ( n = 32) are shown as fold increase relative to the control group ( n = 25); ( D ) and ( E ): Correlations between Relative miR-155 expression with the TNF-α or IL-1β expression. RA, rheumatoid arthritis; OA, osteoarthritis. Statistical significance was considered with a p value < 0.05.

Article Snippet: The levels of TNF-α and IL-1β in plasma and PBMCs supernatant were measured by an enzyme immunoassay using the human TNF-α and IL-1β ELISA Kit (Sino Biological Inc., Beijing, China).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Rheumatoid Arthritis-Associated MicroRNA-155 Targets SOCS1 and Upregulates TNF-α and IL-1β in PBMCs

doi: 10.3390/ijms141223910

Figure Lengend Snippet: Correlations between miR-155 expression and the various clinical and laboratory data of patients with rheumatoid arthritis ( n = 45).

Article Snippet: The levels of TNF-α and IL-1β in plasma and PBMCs supernatant were measured by an enzyme immunoassay using the human TNF-α and IL-1β ELISA Kit (Sino Biological Inc., Beijing, China).

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: Rheumatoid Arthritis-Associated MicroRNA-155 Targets SOCS1 and Upregulates TNF-α and IL-1β in PBMCs

doi: 10.3390/ijms141223910

Figure Lengend Snippet: miR-155 promotes production of TNF-α and IL-1β in human PBMCs. ( A ) Fold changes of miR-155 expression were determined by qRT-PCR after the transfection of miR-control, or miR-155 mimics at 25 or 50 nM for 24 h; ( B ) and ( C ) Expression of TNF-α and IL-1β in supernatant of PBMCs 24 h post LPS treatment, or 24 h post transfection with 25 nM miR-155 mimics, 50 nM miR-155 mimics or 50 nM miRNA control. Results were expressed as pg/mL by an ELISA test; and ( D ) mRNA expression of TNF-α (group 1) and IL-1β (group 2) in PBMCs 24 h after LPS treatment or 24 h post transfection with 25 nM miR-155 mimics, 50 nM miR-155 mimics or 50 nM miRNA control. All results are the average of three independent experiments. Statistical significance is shown.

Article Snippet: The levels of TNF-α and IL-1β in plasma and PBMCs supernatant were measured by an enzyme immunoassay using the human TNF-α and IL-1β ELISA Kit (Sino Biological Inc., Beijing, China).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Rheumatoid Arthritis-Associated MicroRNA-155 Targets SOCS1 and Upregulates TNF-α and IL-1β in PBMCs

doi: 10.3390/ijms141223910

Figure Lengend Snippet: miR-155 inhibitor reduces the production of proinflammatory cytokines induced by LPS. ( A ) Fold changes in miR-155 expression were determined by RT-qPCR 24 h post transfection with 50 nM miRNA inhibitor control, 25 or 50 nM miR-155 inhibitor; ( B ) and ( C ) Expression of TNF-α and IL-1β in supernatant of PBMCs 24 h after LPS treatment, or (and) 24 h post transfection with 5 nM miR-155 inhibitor or 10 nM miR-155 inhibitor. Results were expressed as pg/mL by an ELISA test; and ( D ) mRNA expression of TNF-α (group 1) and IL-1β (group 2) in PBMCs 24 h after LPS treatment, or (and) 24 h post transfection with 10 nM miRNA inhibitor control, 5 nM miR-155 inhibitor or 10 nM miRNA inhibitor. All results are the average of three independent experiments. Statistical significance is shown.

Article Snippet: The levels of TNF-α and IL-1β in plasma and PBMCs supernatant were measured by an enzyme immunoassay using the human TNF-α and IL-1β ELISA Kit (Sino Biological Inc., Beijing, China).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Enzyme-linked Immunosorbent Assay