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Effect of MFN2 on the immune function of Jurkat cells in response to <t>PMA/ionomycin.</t> Jurkat cells were transfected with LV-MFN2 and MFN2-RNAi and then stimulated with PMA (50 ng/ml) plus ionomycin (1 mmol/L) for various lengths of time (6, 12, 24, and 48 h). (a) A methyl-thiazolyl-tetrazolium cell proliferation assay was used to assess Jurkat cells activity. Levels of IL-2 (b) and the IFN-γ/IL-4 ratio (c) were measured by ELISA. Results are shown as mean ± standard deviation. * P
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1) Product Images from "Role of the Ca2+-Calcineurin-Nuclear Factor of Activated T cell Pathway in Mitofusin-2-Mediated Immune Function of Jurkat Cells"

Article Title: Role of the Ca2+-Calcineurin-Nuclear Factor of Activated T cell Pathway in Mitofusin-2-Mediated Immune Function of Jurkat Cells

Journal: Chinese Medical Journal

doi: 10.4103/0366-6999.223855

Effect of MFN2 on the immune function of Jurkat cells in response to PMA/ionomycin. Jurkat cells were transfected with LV-MFN2 and MFN2-RNAi and then stimulated with PMA (50 ng/ml) plus ionomycin (1 mmol/L) for various lengths of time (6, 12, 24, and 48 h). (a) A methyl-thiazolyl-tetrazolium cell proliferation assay was used to assess Jurkat cells activity. Levels of IL-2 (b) and the IFN-γ/IL-4 ratio (c) were measured by ELISA. Results are shown as mean ± standard deviation. * P
Figure Legend Snippet: Effect of MFN2 on the immune function of Jurkat cells in response to PMA/ionomycin. Jurkat cells were transfected with LV-MFN2 and MFN2-RNAi and then stimulated with PMA (50 ng/ml) plus ionomycin (1 mmol/L) for various lengths of time (6, 12, 24, and 48 h). (a) A methyl-thiazolyl-tetrazolium cell proliferation assay was used to assess Jurkat cells activity. Levels of IL-2 (b) and the IFN-γ/IL-4 ratio (c) were measured by ELISA. Results are shown as mean ± standard deviation. * P

Techniques Used: Transfection, Proliferation Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation

Regulation of calcineurin expression reverses MFN2-induced changes in immune function of Jurkat cells. After being transfected with MFN2-RNAi followed by LV-calcineurin, Jurkat cells were treated with PMA plus ionomycin for 24 h. Then, cell proliferative activity (a), IL-2 levels (c), and the ratio of IFN-γ/IL-4 (e) were assessed. Jurkat cells were pretreated LV-MFN2 followed by FK-506 and then stimulated with PMA plus ionomycin for 24 h. The cell proliferative activity (b), IL-2 levels (d), and the ratio of IFN-γ/IL-4 (f) were detected. * P
Figure Legend Snippet: Regulation of calcineurin expression reverses MFN2-induced changes in immune function of Jurkat cells. After being transfected with MFN2-RNAi followed by LV-calcineurin, Jurkat cells were treated with PMA plus ionomycin for 24 h. Then, cell proliferative activity (a), IL-2 levels (c), and the ratio of IFN-γ/IL-4 (e) were assessed. Jurkat cells were pretreated LV-MFN2 followed by FK-506 and then stimulated with PMA plus ionomycin for 24 h. The cell proliferative activity (b), IL-2 levels (d), and the ratio of IFN-γ/IL-4 (f) were detected. * P

Techniques Used: Expressing, Transfection, Activity Assay

Effect of MFN2 on intracellular calcium, calcineurin expression, and NFAT activity in Jurkat cells. Jurkat cells were transfected with LV-MFN2 and MFN2-RNAi and then stimulated with PMA (50 ng/ml) plus ionomycin (1 mmmol/L) for various lengths of time. (a and c) Intracellular calcium was measured by FACS with the fluorescent probe Fluo-3/AM. (b) Calcineurin activity was measured using a calcineurin assay kit. (d) NFAT activity in Jurkat cells was measured by ELISA. * P
Figure Legend Snippet: Effect of MFN2 on intracellular calcium, calcineurin expression, and NFAT activity in Jurkat cells. Jurkat cells were transfected with LV-MFN2 and MFN2-RNAi and then stimulated with PMA (50 ng/ml) plus ionomycin (1 mmmol/L) for various lengths of time. (a and c) Intracellular calcium was measured by FACS with the fluorescent probe Fluo-3/AM. (b) Calcineurin activity was measured using a calcineurin assay kit. (d) NFAT activity in Jurkat cells was measured by ELISA. * P

Techniques Used: Expressing, Activity Assay, Transfection, FACS, Enzyme-linked Immunosorbent Assay

Regulation of calcineurin expression reverses MFN2-induced NFAT activation in Jurkat cells. After various pretreatments, Jurkat cells were stimulated with PMA plus ionomycin for 24 h. Jurkat cells were pretreated with MFN2-RNAi and then LV-calcineurin, and calcineurin activity was measured by commercial assay kit (a) and NFAT activity was determined by ELISA (c). Jurkat cells were pretreated with LV-MFN2 and then FK-506, and calcineurin activity (b) and NFAT activity (d) were measured. * P
Figure Legend Snippet: Regulation of calcineurin expression reverses MFN2-induced NFAT activation in Jurkat cells. After various pretreatments, Jurkat cells were stimulated with PMA plus ionomycin for 24 h. Jurkat cells were pretreated with MFN2-RNAi and then LV-calcineurin, and calcineurin activity was measured by commercial assay kit (a) and NFAT activity was determined by ELISA (c). Jurkat cells were pretreated with LV-MFN2 and then FK-506, and calcineurin activity (b) and NFAT activity (d) were measured. * P

Techniques Used: Expressing, Activation Assay, Activity Assay, Enzyme-linked Immunosorbent Assay

2) Product Images from "Suppression of Th17 cell differentiation by misshapen/NIK-related kinase MINK1"

Article Title: Suppression of Th17 cell differentiation by misshapen/NIK-related kinase MINK1

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20161120

MINK1 suppress Th17 cell differentiation through a cell-intrinsic mechanism. (A, left) Splenocytes from Mink1 −/− and WT mice were stimulated with PMA + ionomycin for 5 h, and the CD44 + CD4 + population was gated and then analyzed for IL-17A + , IFN-γ + , and Foxp3 + cells as indicated. Numbers in quadrants indicate the percentages of cells in each throughout. (Right) Summary of IL-17A + , IFN-γ + , and Foxp3 + cells from the CD44 + CD4 + population of Mink1 −/− and WT mice. (B, left) FACS analysis of LP CD4 + T cells in the small intestine for IL-17A, Foxp3, and IFN-γ expression. (Right) Summary of CD4 + IL-17A + , CD4 + Foxp3 + T, and CD4 + IFN-γ + cells in Mink1 −/− and WT small intestine. (C) Splenic γδT cells derived from Mink1 −/− or WT animals were stimulated with PMA + ionomycin for 5 h and stained for IL-17A production. (D) Rag1 −/− mice were reconstituted with mixed bone marrow cells from B6.SJL (CD45.1 + ) and Mink1 −/− (CD45.2 + ) mice or from B6.SJL (CD45.1 + ) and WT (CD45.2 + ) mice at a 1:1 ratio. Total splenocytes in the recipient mice were analyzed 8 wk later for IL-17A + CD4 + and Foxp3 + CD4 + T cells. The data shown were gated on CD4 + CD45.1 + or CD4 + CD45.2 + ( Mink1 −/− or WT) populations. (E, top) Total splenocytes in the recipient mice as in Fig. 2 D were analyzed for CD62L and CD44 expression. (Bottom) Percentages of naive (CD4 + CD62L + ) and memory (CD4 + CD44 + ) T cells in the spleen of host mice. The numbers in the flow cytometry graphs show the percentages of the gated populations. Error bars show mean ± SD. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. n = 3–6 in each group; Student’s t test. Data are representative of three (A and B) or two (C–E) experiments.
Figure Legend Snippet: MINK1 suppress Th17 cell differentiation through a cell-intrinsic mechanism. (A, left) Splenocytes from Mink1 −/− and WT mice were stimulated with PMA + ionomycin for 5 h, and the CD44 + CD4 + population was gated and then analyzed for IL-17A + , IFN-γ + , and Foxp3 + cells as indicated. Numbers in quadrants indicate the percentages of cells in each throughout. (Right) Summary of IL-17A + , IFN-γ + , and Foxp3 + cells from the CD44 + CD4 + population of Mink1 −/− and WT mice. (B, left) FACS analysis of LP CD4 + T cells in the small intestine for IL-17A, Foxp3, and IFN-γ expression. (Right) Summary of CD4 + IL-17A + , CD4 + Foxp3 + T, and CD4 + IFN-γ + cells in Mink1 −/− and WT small intestine. (C) Splenic γδT cells derived from Mink1 −/− or WT animals were stimulated with PMA + ionomycin for 5 h and stained for IL-17A production. (D) Rag1 −/− mice were reconstituted with mixed bone marrow cells from B6.SJL (CD45.1 + ) and Mink1 −/− (CD45.2 + ) mice or from B6.SJL (CD45.1 + ) and WT (CD45.2 + ) mice at a 1:1 ratio. Total splenocytes in the recipient mice were analyzed 8 wk later for IL-17A + CD4 + and Foxp3 + CD4 + T cells. The data shown were gated on CD4 + CD45.1 + or CD4 + CD45.2 + ( Mink1 −/− or WT) populations. (E, top) Total splenocytes in the recipient mice as in Fig. 2 D were analyzed for CD62L and CD44 expression. (Bottom) Percentages of naive (CD4 + CD62L + ) and memory (CD4 + CD44 + ) T cells in the spleen of host mice. The numbers in the flow cytometry graphs show the percentages of the gated populations. Error bars show mean ± SD. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. n = 3–6 in each group; Student’s t test. Data are representative of three (A and B) or two (C–E) experiments.

Techniques Used: Cell Differentiation, Mouse Assay, FACS, Expressing, Derivative Assay, Staining, Flow Cytometry, Cytometry

MINK1 suppresses Th17 cell polarization in vitro. (A, left) Naive CD4 + T cells (CD4 + CD62L hi CD44 lo CD25 − ) from Mink1 −/− and WT mice were differentiated into Th17 cells with 10 ng/ml IL-6 and 3 ng/ml TGF-β1. On day 5, the differentiated cells were stimulated with PMA + ionomycin for 5 h. The frequencies of IL-17A + cells were determined by flow cytometry. (Right) Percentages of IL-17A + CD4 + T cells generated from in vitro polarization. (B) On day 5 after in vitro polarization, the supernatants of cell cultures were collected, and the secreted amounts of IL-17A were measured by ELISA. (C–H) Naive CD4 + T cells from Mink1 −/− and WT mice were polarized under Th1 (C), Th2 (E), and iT reg (G) cell conditions and analyzed by flow cytometry 5 d after differentiation. Cytokines secreted into the cell culture supernatant (D, F, and H) were measured by ELISA. (I) Naive CD4 + T cells from Mink1 −/− and WT mice were differentiated into Th17 cells with 10 ng/ml IL-6 and 3 ng/ml TGF-β for 5 d. The indicated genes’ expressions were analyzed by real-time PCR. (J, top) CFSE staining of naive T cells after 3 d of stimulation in the presence of plate-bound anti-CD3 antibody. (Bottom) Representative intracellular staining of IL-17A against CFSE on day 5 as in Fig. 3 A . (K) Naive CD4 + T cells from Mink1 −/− and WT mice were differentiated into Th17 cells with the indicated cytokines. Induction of IL-17A + cells was analyzed 5 d after differentiation. When indicated, the TGF-β antibody was added throughout the culture. The numbers in the flow cytometry graphs show the percentages of the gated populations. Error bars show mean ± SD. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. n = 3–6 in each group; Student’s t test. Data are representative of three experiments.
Figure Legend Snippet: MINK1 suppresses Th17 cell polarization in vitro. (A, left) Naive CD4 + T cells (CD4 + CD62L hi CD44 lo CD25 − ) from Mink1 −/− and WT mice were differentiated into Th17 cells with 10 ng/ml IL-6 and 3 ng/ml TGF-β1. On day 5, the differentiated cells were stimulated with PMA + ionomycin for 5 h. The frequencies of IL-17A + cells were determined by flow cytometry. (Right) Percentages of IL-17A + CD4 + T cells generated from in vitro polarization. (B) On day 5 after in vitro polarization, the supernatants of cell cultures were collected, and the secreted amounts of IL-17A were measured by ELISA. (C–H) Naive CD4 + T cells from Mink1 −/− and WT mice were polarized under Th1 (C), Th2 (E), and iT reg (G) cell conditions and analyzed by flow cytometry 5 d after differentiation. Cytokines secreted into the cell culture supernatant (D, F, and H) were measured by ELISA. (I) Naive CD4 + T cells from Mink1 −/− and WT mice were differentiated into Th17 cells with 10 ng/ml IL-6 and 3 ng/ml TGF-β for 5 d. The indicated genes’ expressions were analyzed by real-time PCR. (J, top) CFSE staining of naive T cells after 3 d of stimulation in the presence of plate-bound anti-CD3 antibody. (Bottom) Representative intracellular staining of IL-17A against CFSE on day 5 as in Fig. 3 A . (K) Naive CD4 + T cells from Mink1 −/− and WT mice were differentiated into Th17 cells with the indicated cytokines. Induction of IL-17A + cells was analyzed 5 d after differentiation. When indicated, the TGF-β antibody was added throughout the culture. The numbers in the flow cytometry graphs show the percentages of the gated populations. Error bars show mean ± SD. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. n = 3–6 in each group; Student’s t test. Data are representative of three experiments.

Techniques Used: In Vitro, Mouse Assay, Flow Cytometry, Cytometry, Generated, Enzyme-linked Immunosorbent Assay, Cell Culture, Real-time Polymerase Chain Reaction, Staining

Loss of MINK1 in T cells results in the accumulation of Th17 cells in vivo. (A) Surface staining of CD4 and CD8 on Mink1 −/− and WT thymocytes. Numbers in or adjacent to outlined areas (or in quadrants) indicate the percentages of cells in each throughout. (B) Splenocytes from Mink1 −/− and WT mice stained for CD4 and CD8. Numbers in quadrants indicate the percentages of cells in each throughout. (C, left) Splenocytes from Mink1 −/− and WT mice were stained for CD4, CD44, and CD62L and analyzed by flow cytometry. The gated CD4 + T cells were analyzed for CD44 and CD62L expression. Numbers in quadrants indicate the percentages of cells in each throughout. (Right) Percentages of naive (CD4 + CD62L + ) and memory (CD4 + CD44 + ) T cells in the spleen of Mink1 −/− and WT mice. (D) Splenocytes from Mink1 −/− and WT mice were stimulated ex vivo with PMA + ionomycin for 5 h and analyzed for IL-17A–, IFN-γ–, and Foxp3-expressing CD4 + T cells by flow cytometry. The data shown were gated on CD4 + splenocytes, and numbers in quadrants indicate the percentages of cells in each throughout. (E) Percentages of splenic IL-17A + , IFN-γ + , IL-4 + , and Foxp3 + CD4 + T cells in Mink1 −/− and WT mice. (F) Suppression of CFSE-labeled CD4 + T cells by Mink1 −/− and WT T reg cells, presented as CFSE dilution in responding T cells cultured at a ratio of 2:1 or 4:1 with T reg cells. (G) Real-time PCR analysis of the indicated genes’ expression in purified Mink1 −/− and WT peripheral CD4 + T cells. Error bars show mean ± SD. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. n = 3–6 in each group; Student’s t test. Data are representative of three experiments.
Figure Legend Snippet: Loss of MINK1 in T cells results in the accumulation of Th17 cells in vivo. (A) Surface staining of CD4 and CD8 on Mink1 −/− and WT thymocytes. Numbers in or adjacent to outlined areas (or in quadrants) indicate the percentages of cells in each throughout. (B) Splenocytes from Mink1 −/− and WT mice stained for CD4 and CD8. Numbers in quadrants indicate the percentages of cells in each throughout. (C, left) Splenocytes from Mink1 −/− and WT mice were stained for CD4, CD44, and CD62L and analyzed by flow cytometry. The gated CD4 + T cells were analyzed for CD44 and CD62L expression. Numbers in quadrants indicate the percentages of cells in each throughout. (Right) Percentages of naive (CD4 + CD62L + ) and memory (CD4 + CD44 + ) T cells in the spleen of Mink1 −/− and WT mice. (D) Splenocytes from Mink1 −/− and WT mice were stimulated ex vivo with PMA + ionomycin for 5 h and analyzed for IL-17A–, IFN-γ–, and Foxp3-expressing CD4 + T cells by flow cytometry. The data shown were gated on CD4 + splenocytes, and numbers in quadrants indicate the percentages of cells in each throughout. (E) Percentages of splenic IL-17A + , IFN-γ + , IL-4 + , and Foxp3 + CD4 + T cells in Mink1 −/− and WT mice. (F) Suppression of CFSE-labeled CD4 + T cells by Mink1 −/− and WT T reg cells, presented as CFSE dilution in responding T cells cultured at a ratio of 2:1 or 4:1 with T reg cells. (G) Real-time PCR analysis of the indicated genes’ expression in purified Mink1 −/− and WT peripheral CD4 + T cells. Error bars show mean ± SD. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. n = 3–6 in each group; Student’s t test. Data are representative of three experiments.

Techniques Used: In Vivo, Staining, Mouse Assay, Flow Cytometry, Cytometry, Expressing, Ex Vivo, Labeling, Cell Culture, Real-time Polymerase Chain Reaction, Purification

MINK1 mediates Th17 cell differentiation by mediating SMAD2-α–helix 1 phosphorylation. (A) Sorted Mink1 −/− and WT naive T cells were polarized under Th17 cell conditions as described in Fig. 3 A , except that the cells were first infected with the indicated retrovirus, and the cytokines were added 24 h later. The infection was repeated once at 48 h. 5 d later, the cells were analyzed for IL-17A + cells after restimulation with PMA + ionomycin for 5 h. The infection efficiency was determined by GFP expression. The numbers in the graphs show the percentages of the gated populations. (B) Summary of retrovirus-infected Th17 cells as described in Fig. 6 A . Error bars show mean ± SD. *, P ≤ 0.05. n = 3 in each group; Student’s t test. Data are representative of two independent experiments. IRES, internal ribosomal entry site.
Figure Legend Snippet: MINK1 mediates Th17 cell differentiation by mediating SMAD2-α–helix 1 phosphorylation. (A) Sorted Mink1 −/− and WT naive T cells were polarized under Th17 cell conditions as described in Fig. 3 A , except that the cells were first infected with the indicated retrovirus, and the cytokines were added 24 h later. The infection was repeated once at 48 h. 5 d later, the cells were analyzed for IL-17A + cells after restimulation with PMA + ionomycin for 5 h. The infection efficiency was determined by GFP expression. The numbers in the graphs show the percentages of the gated populations. (B) Summary of retrovirus-infected Th17 cells as described in Fig. 6 A . Error bars show mean ± SD. *, P ≤ 0.05. n = 3 in each group; Student’s t test. Data are representative of two independent experiments. IRES, internal ribosomal entry site.

Techniques Used: Cell Differentiation, Infection, Expressing

3) Product Images from "Dissection and integration of the autophagy signaling network initiated by bluetongue virus infection: crucial candidates ERK1/2, Akt and AMPK"

Article Title: Dissection and integration of the autophagy signaling network initiated by bluetongue virus infection: crucial candidates ERK1/2, Akt and AMPK

Journal: Scientific Reports

doi: 10.1038/srep23130

[Ca 2+ ] Cyto level affects autophagy through CaMKKβ and AMPK. ( a ) Effects of BTV1 infection on intracellular Ca 2+ level in BSR cells. The BSR cells infected with BTV1 at the indicated times were incubated with Fluo-3 AM as a probe and the intracellular Ca 2+ was measured using a flow cytometer. The shifts of fluorescence peaks were recorded. ( b ) BSR cells were left untreated (Mock) or treated with BTV1 (MOI = 1) or 4 μM ionomycin (Iono, positive control) for 30 h. Then the intracellular Ca 2+ was measured using a flow cytometer as described above. ( c ) Proteins from BSR cells left untreated (Mock) or treated with BTV1 or 4 μM ionomycin (Iono, positive control) for 30 h were analyzed by immunoblotting for the activities of CaMKKβ, AMPK , mTOR and LC3. ( d ) After BTV1 adsorption for 1 h, BSR cells were cultured in the absence or presence of BAPTA-AM (25 μM). At 30 hpi, [Ca 2+ ] Cyto level were analyzed as in ( b , e ) The levels of related proteins were detected by Western blotting after mock-infection or BTV1-infection in the presence or absence of 25 μM BAPTA-AM for 30 h.
Figure Legend Snippet: [Ca 2+ ] Cyto level affects autophagy through CaMKKβ and AMPK. ( a ) Effects of BTV1 infection on intracellular Ca 2+ level in BSR cells. The BSR cells infected with BTV1 at the indicated times were incubated with Fluo-3 AM as a probe and the intracellular Ca 2+ was measured using a flow cytometer. The shifts of fluorescence peaks were recorded. ( b ) BSR cells were left untreated (Mock) or treated with BTV1 (MOI = 1) or 4 μM ionomycin (Iono, positive control) for 30 h. Then the intracellular Ca 2+ was measured using a flow cytometer as described above. ( c ) Proteins from BSR cells left untreated (Mock) or treated with BTV1 or 4 μM ionomycin (Iono, positive control) for 30 h were analyzed by immunoblotting for the activities of CaMKKβ, AMPK , mTOR and LC3. ( d ) After BTV1 adsorption for 1 h, BSR cells were cultured in the absence or presence of BAPTA-AM (25 μM). At 30 hpi, [Ca 2+ ] Cyto level were analyzed as in ( b , e ) The levels of related proteins were detected by Western blotting after mock-infection or BTV1-infection in the presence or absence of 25 μM BAPTA-AM for 30 h.

Techniques Used: Infection, Incubation, Flow Cytometry, Cytometry, Fluorescence, Positive Control, Adsorption, Cell Culture, Western Blot

4) Product Images from "Transcription factor NF-kappa B represses ANT1 transcription and leads to mitochondrial dysfunctions"

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

Journal: Scientific Reports

doi: 10.1038/srep44708

NF-κB decreased Ca 2+ -induced mPTP opening, increased Δψ m and ROS. ( A , B ) To determine mPTP opening, cells in different treatment groups were loaded with calcein-AM for 30 minutes and subsequently incubated with ionomycin (5 μM), BKA (5 μM) or CATR (1 μM) for another 30 minutes. The mPTP opening level was measured as calcein loss by Varioskan flash instruments (Thermo Scientific, USA). The histogram depicts the means of remaining calcein fluorescences of treatment groups ± SEM (n = 4); * p = 0.028 ** p
Figure Legend Snippet: NF-κB decreased Ca 2+ -induced mPTP opening, increased Δψ m and ROS. ( A , B ) To determine mPTP opening, cells in different treatment groups were loaded with calcein-AM for 30 minutes and subsequently incubated with ionomycin (5 μM), BKA (5 μM) or CATR (1 μM) for another 30 minutes. The mPTP opening level was measured as calcein loss by Varioskan flash instruments (Thermo Scientific, USA). The histogram depicts the means of remaining calcein fluorescences of treatment groups ± SEM (n = 4); * p = 0.028 ** p

Techniques Used: Incubation

5) Product Images from "Duck enteritis virus activates CaMKKβ-AMPK to trigger autophagy in duck embryo fibroblast cells via increased cytosolic calcium"

Article Title: Duck enteritis virus activates CaMKKβ-AMPK to trigger autophagy in duck embryo fibroblast cells via increased cytosolic calcium

Journal: Virology Journal

doi: 10.1186/s12985-018-1029-0

DEV infection activated CaMKKβ and its substrate AMPK, as well as increased the extent of LC3I transformation to LC3II. a DEF cells infected with DEV (MOI = 1) or mock-infected cells were lysed and blotted with antibody against CaMKKβ, p-AMPK, AMPK, LC3, and β-actin at the indicated times. Ionomycin treated cells as a postive control ( b ) The ratios of CaMKKβ/β-actin. p-AMPK/AMPK, and LC3II/LC3I in DEF cells from three independent experiments, expressed as means ± SD. * p
Figure Legend Snippet: DEV infection activated CaMKKβ and its substrate AMPK, as well as increased the extent of LC3I transformation to LC3II. a DEF cells infected with DEV (MOI = 1) or mock-infected cells were lysed and blotted with antibody against CaMKKβ, p-AMPK, AMPK, LC3, and β-actin at the indicated times. Ionomycin treated cells as a postive control ( b ) The ratios of CaMKKβ/β-actin. p-AMPK/AMPK, and LC3II/LC3I in DEF cells from three independent experiments, expressed as means ± SD. * p

Techniques Used: Infection, Transformation Assay

6) Product Images from "Role of the Ca2+-Calcineurin-Nuclear Factor of Activated T cell Pathway in Mitofusin-2-Mediated Immune Function of Jurkat Cells"

Article Title: Role of the Ca2+-Calcineurin-Nuclear Factor of Activated T cell Pathway in Mitofusin-2-Mediated Immune Function of Jurkat Cells

Journal: Chinese Medical Journal

doi: 10.4103/0366-6999.223855

Effect of MFN2 on the immune function of Jurkat cells in response to PMA/ionomycin. Jurkat cells were transfected with LV-MFN2 and MFN2-RNAi and then stimulated with PMA (50 ng/ml) plus ionomycin (1 mmol/L) for various lengths of time (6, 12, 24, and 48 h). (a) A methyl-thiazolyl-tetrazolium cell proliferation assay was used to assess Jurkat cells activity. Levels of IL-2 (b) and the IFN-γ/IL-4 ratio (c) were measured by ELISA. Results are shown as mean ± standard deviation. * P
Figure Legend Snippet: Effect of MFN2 on the immune function of Jurkat cells in response to PMA/ionomycin. Jurkat cells were transfected with LV-MFN2 and MFN2-RNAi and then stimulated with PMA (50 ng/ml) plus ionomycin (1 mmol/L) for various lengths of time (6, 12, 24, and 48 h). (a) A methyl-thiazolyl-tetrazolium cell proliferation assay was used to assess Jurkat cells activity. Levels of IL-2 (b) and the IFN-γ/IL-4 ratio (c) were measured by ELISA. Results are shown as mean ± standard deviation. * P

Techniques Used: Transfection, Proliferation Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation

Regulation of calcineurin expression reverses MFN2-induced changes in immune function of Jurkat cells. After being transfected with MFN2-RNAi followed by LV-calcineurin, Jurkat cells were treated with PMA plus ionomycin for 24 h. Then, cell proliferative activity (a), IL-2 levels (c), and the ratio of IFN-γ/IL-4 (e) were assessed. Jurkat cells were pretreated LV-MFN2 followed by FK-506 and then stimulated with PMA plus ionomycin for 24 h. The cell proliferative activity (b), IL-2 levels (d), and the ratio of IFN-γ/IL-4 (f) were detected. * P
Figure Legend Snippet: Regulation of calcineurin expression reverses MFN2-induced changes in immune function of Jurkat cells. After being transfected with MFN2-RNAi followed by LV-calcineurin, Jurkat cells were treated with PMA plus ionomycin for 24 h. Then, cell proliferative activity (a), IL-2 levels (c), and the ratio of IFN-γ/IL-4 (e) were assessed. Jurkat cells were pretreated LV-MFN2 followed by FK-506 and then stimulated with PMA plus ionomycin for 24 h. The cell proliferative activity (b), IL-2 levels (d), and the ratio of IFN-γ/IL-4 (f) were detected. * P

Techniques Used: Expressing, Transfection, Activity Assay

Effect of MFN2 on intracellular calcium, calcineurin expression, and NFAT activity in Jurkat cells. Jurkat cells were transfected with LV-MFN2 and MFN2-RNAi and then stimulated with PMA (50 ng/ml) plus ionomycin (1 mmmol/L) for various lengths of time. (a and c) Intracellular calcium was measured by FACS with the fluorescent probe Fluo-3/AM. (b) Calcineurin activity was measured using a calcineurin assay kit. (d) NFAT activity in Jurkat cells was measured by ELISA. * P
Figure Legend Snippet: Effect of MFN2 on intracellular calcium, calcineurin expression, and NFAT activity in Jurkat cells. Jurkat cells were transfected with LV-MFN2 and MFN2-RNAi and then stimulated with PMA (50 ng/ml) plus ionomycin (1 mmmol/L) for various lengths of time. (a and c) Intracellular calcium was measured by FACS with the fluorescent probe Fluo-3/AM. (b) Calcineurin activity was measured using a calcineurin assay kit. (d) NFAT activity in Jurkat cells was measured by ELISA. * P

Techniques Used: Expressing, Activity Assay, Transfection, FACS, Enzyme-linked Immunosorbent Assay

Regulation of calcineurin expression reverses MFN2-induced NFAT activation in Jurkat cells. After various pretreatments, Jurkat cells were stimulated with PMA plus ionomycin for 24 h. Jurkat cells were pretreated with MFN2-RNAi and then LV-calcineurin, and calcineurin activity was measured by commercial assay kit (a) and NFAT activity was determined by ELISA (c). Jurkat cells were pretreated with LV-MFN2 and then FK-506, and calcineurin activity (b) and NFAT activity (d) were measured. * P
Figure Legend Snippet: Regulation of calcineurin expression reverses MFN2-induced NFAT activation in Jurkat cells. After various pretreatments, Jurkat cells were stimulated with PMA plus ionomycin for 24 h. Jurkat cells were pretreated with MFN2-RNAi and then LV-calcineurin, and calcineurin activity was measured by commercial assay kit (a) and NFAT activity was determined by ELISA (c). Jurkat cells were pretreated with LV-MFN2 and then FK-506, and calcineurin activity (b) and NFAT activity (d) were measured. * P

Techniques Used: Expressing, Activation Assay, Activity Assay, Enzyme-linked Immunosorbent Assay

7) Product Images from "Duck enteritis virus activates CaMKKβ-AMPK to trigger autophagy in duck embryo fibroblast cells via increased cytosolic calcium"

Article Title: Duck enteritis virus activates CaMKKβ-AMPK to trigger autophagy in duck embryo fibroblast cells via increased cytosolic calcium

Journal: Virology Journal

doi: 10.1186/s12985-018-1029-0

DEV infection activated CaMKKβ and its substrate AMPK, as well as increased the extent of LC3I transformation to LC3II. a DEF cells infected with DEV (MOI = 1) or mock-infected cells were lysed and blotted with antibody against CaMKKβ, p-AMPK, AMPK, LC3, and β-actin at the indicated times. Ionomycin treated cells as a postive control ( b ) The ratios of CaMKKβ/β-actin. p-AMPK/AMPK, and LC3II/LC3I in DEF cells from three independent experiments, expressed as means ± SD. * p
Figure Legend Snippet: DEV infection activated CaMKKβ and its substrate AMPK, as well as increased the extent of LC3I transformation to LC3II. a DEF cells infected with DEV (MOI = 1) or mock-infected cells were lysed and blotted with antibody against CaMKKβ, p-AMPK, AMPK, LC3, and β-actin at the indicated times. Ionomycin treated cells as a postive control ( b ) The ratios of CaMKKβ/β-actin. p-AMPK/AMPK, and LC3II/LC3I in DEF cells from three independent experiments, expressed as means ± SD. * p

Techniques Used: Infection, Transformation Assay

Related Articles

Transfection:

Article Title: Transcription factor NF-kappa B represses ANT1 transcription and leads to mitochondrial dysfunctions
Article Snippet: .. Materials 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); CoCl2 (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); LipofectamineTM 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. .. Beijing, China); total caspase-3 mAb (#9665, CST, Beverly, USA); TRI reagent (T9424, Sigma-Aldrich, Saint Louis, USA).

Reporter Assay:

Article Title: Transcription factor NF-kappa B represses ANT1 transcription and leads to mitochondrial dysfunctions
Article Snippet: .. Materials 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); CoCl2 (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); LipofectamineTM 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. .. Beijing, China); total caspase-3 mAb (#9665, CST, Beverly, USA); TRI reagent (T9424, Sigma-Aldrich, Saint Louis, USA).

Cytotoxicity Assay:

Article Title: Dissection and integration of the autophagy signaling network initiated by bluetongue virus infection: crucial candidates ERK1/2, Akt and AMPK
Article Snippet: .. Rapamycin (Rapa), U0126, insulin, AICAR, Fluo-3 AM, Ionomycin and WST-1 cell proliferation and cytotoxicity assay kits were purchased from Beyotime (Beijing, China). .. Compound C was purchased from Calbiochem (Merck-Millipore, Darmstadt, Germany).

Modification:

Article Title: Transcription factor NF-kappa B represses ANT1 transcription and leads to mitochondrial dysfunctions
Article Snippet: .. Materials 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); CoCl2 (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); LipofectamineTM 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. .. Beijing, China); total caspase-3 mAb (#9665, CST, Beverly, USA); TRI reagent (T9424, Sigma-Aldrich, Saint Louis, USA).

Polymerase Chain Reaction:

Article Title: Transcription factor NF-kappa B represses ANT1 transcription and leads to mitochondrial dysfunctions
Article Snippet: .. Materials 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); CoCl2 (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); LipofectamineTM 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. .. Beijing, China); total caspase-3 mAb (#9665, CST, Beverly, USA); TRI reagent (T9424, Sigma-Aldrich, Saint Louis, USA).

Staining:

Article Title: Suppression of Th17 cell differentiation by misshapen/NIK-related kinase MINK1
Article Snippet: .. For intracellular staining, cells were stimulated for 4–5 h with 50 ng/ml PMA and 500 ng/ml ionomycin (both from Beyotime) in the presence of brefeldin A (BioLegend), fixed, made permeable (Fix/Perm; eBioscience) according to the manufacturer’s instructions, and stained with the respective antibodies for 20–30 min for intracellular cytokine detection. .. Before fixation, cells were stained with Fixable Viability Dye (eBioscience) to exclude dead cells.

Chromatin Immunoprecipitation:

Article Title: Transcription factor NF-kappa B represses ANT1 transcription and leads to mitochondrial dysfunctions
Article Snippet: .. Materials 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); CoCl2 (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); LipofectamineTM 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. .. Beijing, China); total caspase-3 mAb (#9665, CST, Beverly, USA); TRI reagent (T9424, Sigma-Aldrich, Saint Louis, USA).

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    Beyotime ionomycin
    Effect of MFN2 on the immune function of Jurkat cells in response to <t>PMA/ionomycin.</t> Jurkat cells were transfected with LV-MFN2 and MFN2-RNAi and then stimulated with PMA (50 ng/ml) plus ionomycin (1 mmol/L) for various lengths of time (6, 12, 24, and 48 h). (a) A methyl-thiazolyl-tetrazolium cell proliferation assay was used to assess Jurkat cells activity. Levels of IL-2 (b) and the IFN-γ/IL-4 ratio (c) were measured by ELISA. Results are shown as mean ± standard deviation. * P
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    Effect of MFN2 on the immune function of Jurkat cells in response to PMA/ionomycin. Jurkat cells were transfected with LV-MFN2 and MFN2-RNAi and then stimulated with PMA (50 ng/ml) plus ionomycin (1 mmol/L) for various lengths of time (6, 12, 24, and 48 h). (a) A methyl-thiazolyl-tetrazolium cell proliferation assay was used to assess Jurkat cells activity. Levels of IL-2 (b) and the IFN-γ/IL-4 ratio (c) were measured by ELISA. Results are shown as mean ± standard deviation. * P

    Journal: Chinese Medical Journal

    Article Title: Role of the Ca2+-Calcineurin-Nuclear Factor of Activated T cell Pathway in Mitofusin-2-Mediated Immune Function of Jurkat Cells

    doi: 10.4103/0366-6999.223855

    Figure Lengend Snippet: Effect of MFN2 on the immune function of Jurkat cells in response to PMA/ionomycin. Jurkat cells were transfected with LV-MFN2 and MFN2-RNAi and then stimulated with PMA (50 ng/ml) plus ionomycin (1 mmol/L) for various lengths of time (6, 12, 24, and 48 h). (a) A methyl-thiazolyl-tetrazolium cell proliferation assay was used to assess Jurkat cells activity. Levels of IL-2 (b) and the IFN-γ/IL-4 ratio (c) were measured by ELISA. Results are shown as mean ± standard deviation. * P

    Article Snippet: Phorbol myristate acetate (PMA) and ionomycin were purchased from the Beyotime Institute (Nanjing, China).

    Techniques: Transfection, Proliferation Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation

    Regulation of calcineurin expression reverses MFN2-induced changes in immune function of Jurkat cells. After being transfected with MFN2-RNAi followed by LV-calcineurin, Jurkat cells were treated with PMA plus ionomycin for 24 h. Then, cell proliferative activity (a), IL-2 levels (c), and the ratio of IFN-γ/IL-4 (e) were assessed. Jurkat cells were pretreated LV-MFN2 followed by FK-506 and then stimulated with PMA plus ionomycin for 24 h. The cell proliferative activity (b), IL-2 levels (d), and the ratio of IFN-γ/IL-4 (f) were detected. * P

    Journal: Chinese Medical Journal

    Article Title: Role of the Ca2+-Calcineurin-Nuclear Factor of Activated T cell Pathway in Mitofusin-2-Mediated Immune Function of Jurkat Cells

    doi: 10.4103/0366-6999.223855

    Figure Lengend Snippet: Regulation of calcineurin expression reverses MFN2-induced changes in immune function of Jurkat cells. After being transfected with MFN2-RNAi followed by LV-calcineurin, Jurkat cells were treated with PMA plus ionomycin for 24 h. Then, cell proliferative activity (a), IL-2 levels (c), and the ratio of IFN-γ/IL-4 (e) were assessed. Jurkat cells were pretreated LV-MFN2 followed by FK-506 and then stimulated with PMA plus ionomycin for 24 h. The cell proliferative activity (b), IL-2 levels (d), and the ratio of IFN-γ/IL-4 (f) were detected. * P

    Article Snippet: Phorbol myristate acetate (PMA) and ionomycin were purchased from the Beyotime Institute (Nanjing, China).

    Techniques: Expressing, Transfection, Activity Assay

    Effect of MFN2 on intracellular calcium, calcineurin expression, and NFAT activity in Jurkat cells. Jurkat cells were transfected with LV-MFN2 and MFN2-RNAi and then stimulated with PMA (50 ng/ml) plus ionomycin (1 mmmol/L) for various lengths of time. (a and c) Intracellular calcium was measured by FACS with the fluorescent probe Fluo-3/AM. (b) Calcineurin activity was measured using a calcineurin assay kit. (d) NFAT activity in Jurkat cells was measured by ELISA. * P

    Journal: Chinese Medical Journal

    Article Title: Role of the Ca2+-Calcineurin-Nuclear Factor of Activated T cell Pathway in Mitofusin-2-Mediated Immune Function of Jurkat Cells

    doi: 10.4103/0366-6999.223855

    Figure Lengend Snippet: Effect of MFN2 on intracellular calcium, calcineurin expression, and NFAT activity in Jurkat cells. Jurkat cells were transfected with LV-MFN2 and MFN2-RNAi and then stimulated with PMA (50 ng/ml) plus ionomycin (1 mmmol/L) for various lengths of time. (a and c) Intracellular calcium was measured by FACS with the fluorescent probe Fluo-3/AM. (b) Calcineurin activity was measured using a calcineurin assay kit. (d) NFAT activity in Jurkat cells was measured by ELISA. * P

    Article Snippet: Phorbol myristate acetate (PMA) and ionomycin were purchased from the Beyotime Institute (Nanjing, China).

    Techniques: Expressing, Activity Assay, Transfection, FACS, Enzyme-linked Immunosorbent Assay

    Regulation of calcineurin expression reverses MFN2-induced NFAT activation in Jurkat cells. After various pretreatments, Jurkat cells were stimulated with PMA plus ionomycin for 24 h. Jurkat cells were pretreated with MFN2-RNAi and then LV-calcineurin, and calcineurin activity was measured by commercial assay kit (a) and NFAT activity was determined by ELISA (c). Jurkat cells were pretreated with LV-MFN2 and then FK-506, and calcineurin activity (b) and NFAT activity (d) were measured. * P

    Journal: Chinese Medical Journal

    Article Title: Role of the Ca2+-Calcineurin-Nuclear Factor of Activated T cell Pathway in Mitofusin-2-Mediated Immune Function of Jurkat Cells

    doi: 10.4103/0366-6999.223855

    Figure Lengend Snippet: Regulation of calcineurin expression reverses MFN2-induced NFAT activation in Jurkat cells. After various pretreatments, Jurkat cells were stimulated with PMA plus ionomycin for 24 h. Jurkat cells were pretreated with MFN2-RNAi and then LV-calcineurin, and calcineurin activity was measured by commercial assay kit (a) and NFAT activity was determined by ELISA (c). Jurkat cells were pretreated with LV-MFN2 and then FK-506, and calcineurin activity (b) and NFAT activity (d) were measured. * P

    Article Snippet: Phorbol myristate acetate (PMA) and ionomycin were purchased from the Beyotime Institute (Nanjing, China).

    Techniques: Expressing, Activation Assay, Activity Assay, Enzyme-linked Immunosorbent Assay

    MINK1 suppress Th17 cell differentiation through a cell-intrinsic mechanism. (A, left) Splenocytes from Mink1 −/− and WT mice were stimulated with PMA + ionomycin for 5 h, and the CD44 + CD4 + population was gated and then analyzed for IL-17A + , IFN-γ + , and Foxp3 + cells as indicated. Numbers in quadrants indicate the percentages of cells in each throughout. (Right) Summary of IL-17A + , IFN-γ + , and Foxp3 + cells from the CD44 + CD4 + population of Mink1 −/− and WT mice. (B, left) FACS analysis of LP CD4 + T cells in the small intestine for IL-17A, Foxp3, and IFN-γ expression. (Right) Summary of CD4 + IL-17A + , CD4 + Foxp3 + T, and CD4 + IFN-γ + cells in Mink1 −/− and WT small intestine. (C) Splenic γδT cells derived from Mink1 −/− or WT animals were stimulated with PMA + ionomycin for 5 h and stained for IL-17A production. (D) Rag1 −/− mice were reconstituted with mixed bone marrow cells from B6.SJL (CD45.1 + ) and Mink1 −/− (CD45.2 + ) mice or from B6.SJL (CD45.1 + ) and WT (CD45.2 + ) mice at a 1:1 ratio. Total splenocytes in the recipient mice were analyzed 8 wk later for IL-17A + CD4 + and Foxp3 + CD4 + T cells. The data shown were gated on CD4 + CD45.1 + or CD4 + CD45.2 + ( Mink1 −/− or WT) populations. (E, top) Total splenocytes in the recipient mice as in Fig. 2 D were analyzed for CD62L and CD44 expression. (Bottom) Percentages of naive (CD4 + CD62L + ) and memory (CD4 + CD44 + ) T cells in the spleen of host mice. The numbers in the flow cytometry graphs show the percentages of the gated populations. Error bars show mean ± SD. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. n = 3–6 in each group; Student’s t test. Data are representative of three (A and B) or two (C–E) experiments.

    Journal: The Journal of Experimental Medicine

    Article Title: Suppression of Th17 cell differentiation by misshapen/NIK-related kinase MINK1

    doi: 10.1084/jem.20161120

    Figure Lengend Snippet: MINK1 suppress Th17 cell differentiation through a cell-intrinsic mechanism. (A, left) Splenocytes from Mink1 −/− and WT mice were stimulated with PMA + ionomycin for 5 h, and the CD44 + CD4 + population was gated and then analyzed for IL-17A + , IFN-γ + , and Foxp3 + cells as indicated. Numbers in quadrants indicate the percentages of cells in each throughout. (Right) Summary of IL-17A + , IFN-γ + , and Foxp3 + cells from the CD44 + CD4 + population of Mink1 −/− and WT mice. (B, left) FACS analysis of LP CD4 + T cells in the small intestine for IL-17A, Foxp3, and IFN-γ expression. (Right) Summary of CD4 + IL-17A + , CD4 + Foxp3 + T, and CD4 + IFN-γ + cells in Mink1 −/− and WT small intestine. (C) Splenic γδT cells derived from Mink1 −/− or WT animals were stimulated with PMA + ionomycin for 5 h and stained for IL-17A production. (D) Rag1 −/− mice were reconstituted with mixed bone marrow cells from B6.SJL (CD45.1 + ) and Mink1 −/− (CD45.2 + ) mice or from B6.SJL (CD45.1 + ) and WT (CD45.2 + ) mice at a 1:1 ratio. Total splenocytes in the recipient mice were analyzed 8 wk later for IL-17A + CD4 + and Foxp3 + CD4 + T cells. The data shown were gated on CD4 + CD45.1 + or CD4 + CD45.2 + ( Mink1 −/− or WT) populations. (E, top) Total splenocytes in the recipient mice as in Fig. 2 D were analyzed for CD62L and CD44 expression. (Bottom) Percentages of naive (CD4 + CD62L + ) and memory (CD4 + CD44 + ) T cells in the spleen of host mice. The numbers in the flow cytometry graphs show the percentages of the gated populations. Error bars show mean ± SD. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. n = 3–6 in each group; Student’s t test. Data are representative of three (A and B) or two (C–E) experiments.

    Article Snippet: For intracellular staining, cells were stimulated for 4–5 h with 50 ng/ml PMA and 500 ng/ml ionomycin (both from Beyotime) in the presence of brefeldin A (BioLegend), fixed, made permeable (Fix/Perm; eBioscience) according to the manufacturer’s instructions, and stained with the respective antibodies for 20–30 min for intracellular cytokine detection.

    Techniques: Cell Differentiation, Mouse Assay, FACS, Expressing, Derivative Assay, Staining, Flow Cytometry, Cytometry

    MINK1 suppresses Th17 cell polarization in vitro. (A, left) Naive CD4 + T cells (CD4 + CD62L hi CD44 lo CD25 − ) from Mink1 −/− and WT mice were differentiated into Th17 cells with 10 ng/ml IL-6 and 3 ng/ml TGF-β1. On day 5, the differentiated cells were stimulated with PMA + ionomycin for 5 h. The frequencies of IL-17A + cells were determined by flow cytometry. (Right) Percentages of IL-17A + CD4 + T cells generated from in vitro polarization. (B) On day 5 after in vitro polarization, the supernatants of cell cultures were collected, and the secreted amounts of IL-17A were measured by ELISA. (C–H) Naive CD4 + T cells from Mink1 −/− and WT mice were polarized under Th1 (C), Th2 (E), and iT reg (G) cell conditions and analyzed by flow cytometry 5 d after differentiation. Cytokines secreted into the cell culture supernatant (D, F, and H) were measured by ELISA. (I) Naive CD4 + T cells from Mink1 −/− and WT mice were differentiated into Th17 cells with 10 ng/ml IL-6 and 3 ng/ml TGF-β for 5 d. The indicated genes’ expressions were analyzed by real-time PCR. (J, top) CFSE staining of naive T cells after 3 d of stimulation in the presence of plate-bound anti-CD3 antibody. (Bottom) Representative intracellular staining of IL-17A against CFSE on day 5 as in Fig. 3 A . (K) Naive CD4 + T cells from Mink1 −/− and WT mice were differentiated into Th17 cells with the indicated cytokines. Induction of IL-17A + cells was analyzed 5 d after differentiation. When indicated, the TGF-β antibody was added throughout the culture. The numbers in the flow cytometry graphs show the percentages of the gated populations. Error bars show mean ± SD. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. n = 3–6 in each group; Student’s t test. Data are representative of three experiments.

    Journal: The Journal of Experimental Medicine

    Article Title: Suppression of Th17 cell differentiation by misshapen/NIK-related kinase MINK1

    doi: 10.1084/jem.20161120

    Figure Lengend Snippet: MINK1 suppresses Th17 cell polarization in vitro. (A, left) Naive CD4 + T cells (CD4 + CD62L hi CD44 lo CD25 − ) from Mink1 −/− and WT mice were differentiated into Th17 cells with 10 ng/ml IL-6 and 3 ng/ml TGF-β1. On day 5, the differentiated cells were stimulated with PMA + ionomycin for 5 h. The frequencies of IL-17A + cells were determined by flow cytometry. (Right) Percentages of IL-17A + CD4 + T cells generated from in vitro polarization. (B) On day 5 after in vitro polarization, the supernatants of cell cultures were collected, and the secreted amounts of IL-17A were measured by ELISA. (C–H) Naive CD4 + T cells from Mink1 −/− and WT mice were polarized under Th1 (C), Th2 (E), and iT reg (G) cell conditions and analyzed by flow cytometry 5 d after differentiation. Cytokines secreted into the cell culture supernatant (D, F, and H) were measured by ELISA. (I) Naive CD4 + T cells from Mink1 −/− and WT mice were differentiated into Th17 cells with 10 ng/ml IL-6 and 3 ng/ml TGF-β for 5 d. The indicated genes’ expressions were analyzed by real-time PCR. (J, top) CFSE staining of naive T cells after 3 d of stimulation in the presence of plate-bound anti-CD3 antibody. (Bottom) Representative intracellular staining of IL-17A against CFSE on day 5 as in Fig. 3 A . (K) Naive CD4 + T cells from Mink1 −/− and WT mice were differentiated into Th17 cells with the indicated cytokines. Induction of IL-17A + cells was analyzed 5 d after differentiation. When indicated, the TGF-β antibody was added throughout the culture. The numbers in the flow cytometry graphs show the percentages of the gated populations. Error bars show mean ± SD. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. n = 3–6 in each group; Student’s t test. Data are representative of three experiments.

    Article Snippet: For intracellular staining, cells were stimulated for 4–5 h with 50 ng/ml PMA and 500 ng/ml ionomycin (both from Beyotime) in the presence of brefeldin A (BioLegend), fixed, made permeable (Fix/Perm; eBioscience) according to the manufacturer’s instructions, and stained with the respective antibodies for 20–30 min for intracellular cytokine detection.

    Techniques: In Vitro, Mouse Assay, Flow Cytometry, Cytometry, Generated, Enzyme-linked Immunosorbent Assay, Cell Culture, Real-time Polymerase Chain Reaction, Staining

    Loss of MINK1 in T cells results in the accumulation of Th17 cells in vivo. (A) Surface staining of CD4 and CD8 on Mink1 −/− and WT thymocytes. Numbers in or adjacent to outlined areas (or in quadrants) indicate the percentages of cells in each throughout. (B) Splenocytes from Mink1 −/− and WT mice stained for CD4 and CD8. Numbers in quadrants indicate the percentages of cells in each throughout. (C, left) Splenocytes from Mink1 −/− and WT mice were stained for CD4, CD44, and CD62L and analyzed by flow cytometry. The gated CD4 + T cells were analyzed for CD44 and CD62L expression. Numbers in quadrants indicate the percentages of cells in each throughout. (Right) Percentages of naive (CD4 + CD62L + ) and memory (CD4 + CD44 + ) T cells in the spleen of Mink1 −/− and WT mice. (D) Splenocytes from Mink1 −/− and WT mice were stimulated ex vivo with PMA + ionomycin for 5 h and analyzed for IL-17A–, IFN-γ–, and Foxp3-expressing CD4 + T cells by flow cytometry. The data shown were gated on CD4 + splenocytes, and numbers in quadrants indicate the percentages of cells in each throughout. (E) Percentages of splenic IL-17A + , IFN-γ + , IL-4 + , and Foxp3 + CD4 + T cells in Mink1 −/− and WT mice. (F) Suppression of CFSE-labeled CD4 + T cells by Mink1 −/− and WT T reg cells, presented as CFSE dilution in responding T cells cultured at a ratio of 2:1 or 4:1 with T reg cells. (G) Real-time PCR analysis of the indicated genes’ expression in purified Mink1 −/− and WT peripheral CD4 + T cells. Error bars show mean ± SD. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. n = 3–6 in each group; Student’s t test. Data are representative of three experiments.

    Journal: The Journal of Experimental Medicine

    Article Title: Suppression of Th17 cell differentiation by misshapen/NIK-related kinase MINK1

    doi: 10.1084/jem.20161120

    Figure Lengend Snippet: Loss of MINK1 in T cells results in the accumulation of Th17 cells in vivo. (A) Surface staining of CD4 and CD8 on Mink1 −/− and WT thymocytes. Numbers in or adjacent to outlined areas (or in quadrants) indicate the percentages of cells in each throughout. (B) Splenocytes from Mink1 −/− and WT mice stained for CD4 and CD8. Numbers in quadrants indicate the percentages of cells in each throughout. (C, left) Splenocytes from Mink1 −/− and WT mice were stained for CD4, CD44, and CD62L and analyzed by flow cytometry. The gated CD4 + T cells were analyzed for CD44 and CD62L expression. Numbers in quadrants indicate the percentages of cells in each throughout. (Right) Percentages of naive (CD4 + CD62L + ) and memory (CD4 + CD44 + ) T cells in the spleen of Mink1 −/− and WT mice. (D) Splenocytes from Mink1 −/− and WT mice were stimulated ex vivo with PMA + ionomycin for 5 h and analyzed for IL-17A–, IFN-γ–, and Foxp3-expressing CD4 + T cells by flow cytometry. The data shown were gated on CD4 + splenocytes, and numbers in quadrants indicate the percentages of cells in each throughout. (E) Percentages of splenic IL-17A + , IFN-γ + , IL-4 + , and Foxp3 + CD4 + T cells in Mink1 −/− and WT mice. (F) Suppression of CFSE-labeled CD4 + T cells by Mink1 −/− and WT T reg cells, presented as CFSE dilution in responding T cells cultured at a ratio of 2:1 or 4:1 with T reg cells. (G) Real-time PCR analysis of the indicated genes’ expression in purified Mink1 −/− and WT peripheral CD4 + T cells. Error bars show mean ± SD. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. n = 3–6 in each group; Student’s t test. Data are representative of three experiments.

    Article Snippet: For intracellular staining, cells were stimulated for 4–5 h with 50 ng/ml PMA and 500 ng/ml ionomycin (both from Beyotime) in the presence of brefeldin A (BioLegend), fixed, made permeable (Fix/Perm; eBioscience) according to the manufacturer’s instructions, and stained with the respective antibodies for 20–30 min for intracellular cytokine detection.

    Techniques: In Vivo, Staining, Mouse Assay, Flow Cytometry, Cytometry, Expressing, Ex Vivo, Labeling, Cell Culture, Real-time Polymerase Chain Reaction, Purification

    MINK1 mediates Th17 cell differentiation by mediating SMAD2-α–helix 1 phosphorylation. (A) Sorted Mink1 −/− and WT naive T cells were polarized under Th17 cell conditions as described in Fig. 3 A , except that the cells were first infected with the indicated retrovirus, and the cytokines were added 24 h later. The infection was repeated once at 48 h. 5 d later, the cells were analyzed for IL-17A + cells after restimulation with PMA + ionomycin for 5 h. The infection efficiency was determined by GFP expression. The numbers in the graphs show the percentages of the gated populations. (B) Summary of retrovirus-infected Th17 cells as described in Fig. 6 A . Error bars show mean ± SD. *, P ≤ 0.05. n = 3 in each group; Student’s t test. Data are representative of two independent experiments. IRES, internal ribosomal entry site.

    Journal: The Journal of Experimental Medicine

    Article Title: Suppression of Th17 cell differentiation by misshapen/NIK-related kinase MINK1

    doi: 10.1084/jem.20161120

    Figure Lengend Snippet: MINK1 mediates Th17 cell differentiation by mediating SMAD2-α–helix 1 phosphorylation. (A) Sorted Mink1 −/− and WT naive T cells were polarized under Th17 cell conditions as described in Fig. 3 A , except that the cells were first infected with the indicated retrovirus, and the cytokines were added 24 h later. The infection was repeated once at 48 h. 5 d later, the cells were analyzed for IL-17A + cells after restimulation with PMA + ionomycin for 5 h. The infection efficiency was determined by GFP expression. The numbers in the graphs show the percentages of the gated populations. (B) Summary of retrovirus-infected Th17 cells as described in Fig. 6 A . Error bars show mean ± SD. *, P ≤ 0.05. n = 3 in each group; Student’s t test. Data are representative of two independent experiments. IRES, internal ribosomal entry site.

    Article Snippet: For intracellular staining, cells were stimulated for 4–5 h with 50 ng/ml PMA and 500 ng/ml ionomycin (both from Beyotime) in the presence of brefeldin A (BioLegend), fixed, made permeable (Fix/Perm; eBioscience) according to the manufacturer’s instructions, and stained with the respective antibodies for 20–30 min for intracellular cytokine detection.

    Techniques: Cell Differentiation, Infection, Expressing

    [Ca 2+ ] Cyto level affects autophagy through CaMKKβ and AMPK. ( a ) Effects of BTV1 infection on intracellular Ca 2+ level in BSR cells. The BSR cells infected with BTV1 at the indicated times were incubated with Fluo-3 AM as a probe and the intracellular Ca 2+ was measured using a flow cytometer. The shifts of fluorescence peaks were recorded. ( b ) BSR cells were left untreated (Mock) or treated with BTV1 (MOI = 1) or 4 μM ionomycin (Iono, positive control) for 30 h. Then the intracellular Ca 2+ was measured using a flow cytometer as described above. ( c ) Proteins from BSR cells left untreated (Mock) or treated with BTV1 or 4 μM ionomycin (Iono, positive control) for 30 h were analyzed by immunoblotting for the activities of CaMKKβ, AMPK , mTOR and LC3. ( d ) After BTV1 adsorption for 1 h, BSR cells were cultured in the absence or presence of BAPTA-AM (25 μM). At 30 hpi, [Ca 2+ ] Cyto level were analyzed as in ( b , e ) The levels of related proteins were detected by Western blotting after mock-infection or BTV1-infection in the presence or absence of 25 μM BAPTA-AM for 30 h.

    Journal: Scientific Reports

    Article Title: Dissection and integration of the autophagy signaling network initiated by bluetongue virus infection: crucial candidates ERK1/2, Akt and AMPK

    doi: 10.1038/srep23130

    Figure Lengend Snippet: [Ca 2+ ] Cyto level affects autophagy through CaMKKβ and AMPK. ( a ) Effects of BTV1 infection on intracellular Ca 2+ level in BSR cells. The BSR cells infected with BTV1 at the indicated times were incubated with Fluo-3 AM as a probe and the intracellular Ca 2+ was measured using a flow cytometer. The shifts of fluorescence peaks were recorded. ( b ) BSR cells were left untreated (Mock) or treated with BTV1 (MOI = 1) or 4 μM ionomycin (Iono, positive control) for 30 h. Then the intracellular Ca 2+ was measured using a flow cytometer as described above. ( c ) Proteins from BSR cells left untreated (Mock) or treated with BTV1 or 4 μM ionomycin (Iono, positive control) for 30 h were analyzed by immunoblotting for the activities of CaMKKβ, AMPK , mTOR and LC3. ( d ) After BTV1 adsorption for 1 h, BSR cells were cultured in the absence or presence of BAPTA-AM (25 μM). At 30 hpi, [Ca 2+ ] Cyto level were analyzed as in ( b , e ) The levels of related proteins were detected by Western blotting after mock-infection or BTV1-infection in the presence or absence of 25 μM BAPTA-AM for 30 h.

    Article Snippet: Rapamycin (Rapa), U0126, insulin, AICAR, Fluo-3 AM, Ionomycin and WST-1 cell proliferation and cytotoxicity assay kits were purchased from Beyotime (Beijing, China).

    Techniques: Infection, Incubation, Flow Cytometry, Cytometry, Fluorescence, Positive Control, Adsorption, Cell Culture, Western Blot

    NF-κB decreased Ca 2+ -induced mPTP opening, increased Δψ m and ROS. ( A , B ) To determine mPTP opening, cells in different treatment groups were loaded with calcein-AM for 30 minutes and subsequently incubated with ionomycin (5 μM), BKA (5 μM) or CATR (1 μM) for another 30 minutes. The mPTP opening level was measured as calcein loss by Varioskan flash instruments (Thermo Scientific, USA). The histogram depicts the means of remaining calcein fluorescences of treatment groups ± SEM (n = 4); * p = 0.028 ** p

    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: NF-κB decreased Ca 2+ -induced mPTP opening, increased Δψ m and ROS. ( A , B ) To determine mPTP opening, cells in different treatment groups were loaded with calcein-AM for 30 minutes and subsequently incubated with ionomycin (5 μM), BKA (5 μM) or CATR (1 μM) for another 30 minutes. The mPTP opening level was measured as calcein loss by Varioskan flash instruments (Thermo Scientific, USA). The histogram depicts the means of remaining calcein fluorescences of treatment groups ± SEM (n = 4); * p = 0.028 ** p

    Article Snippet: Materials 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); CoCl2 (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); LipofectamineTM 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: Incubation