Journal: Clinical Science (London, England : 1979)

Article Title: Glutamine alleviates immunosuppression in polymicrobial sepsis by augmenting bacterial phagocytosis through sustaining the GFAT-DRP1 dependent mitochondrial calcium dynamics

doi: 10.1042/CS20256651

Figure Lengend Snippet: ( A ) LPS-tolerant BMDMs were untreated or treated with LPS or together with Gln, or further treated with DON for 24 h. The protein expression of DRP1 or phosphorylated DRP1 at site Ser-616 or Ser-637 [p-DRP1 (S616) and p-DRP1 (S637)] was detected. ( B ) LPS-tolerant DRP1 WT and DRP1 MU J774A.1 cells were treated with LPS or together with Gln, or further treated with DON for 24 h. The protein expression of DRP1 and p-DRP1(S616) was detected. ( C-D ) LPS-tolerant BMDMs were untreated or treated with LPS or together with Gln for 24 h. The protein expression of CAMKK2, phosphorylated CAMKK2 (p-CAMKK2), P38, phosphorylated P38 ( p-P38 ), ERK1/2, phosphorylated ERK1/2 (p-ERK1/2) ( C ) and PINK1, GSK3β ( D ) was detected. ( E-F ) LPS-tolerant BMDMs were treated as in ( A ). The mRNA ( E ) and protein ( F ) expression of CDK1 was detected. ( G ) LPS-tolerant BMDMs were untreated or treated with LPS alone or together with Gln, or further treated with DON, OSMI1, or Thiamet G (Th G) for 24 h. The protein expression of CDK1, DRP1, and p-DRP1 (S616) was detected. ( H ) LPS-tolerant BMDMs were untreated or treated with LPS alone or together with Gln, or further treated with PUGNAc (PU) for 24 h. The protein expression of CDK1, DRP1, and p-DRP1 (S616) was detected. The concentration of LPS, Gln, DON, OSMI1, Thiamet G, and PUGNAc was 100 ng/ml, 2 mM, 50 μM, 10 μM, 10 μM, and 100 μM, respectively. The MOI for E. coli and S. aureus was 1. M: LPS-tolerant macrophages that have not been treated with Gln. **: P <0.01, *: P <0.05, N.S .: no significance. n =3.

Article Snippet: Primary antibody against p-CDK1 (Thr-161) was purchased from ABclonal (AP0324, Wuhan, China).

Techniques: Expressing, Concentration Assay

Journal: Clinical Science (London, England : 1979)

Article Title: Glutamine alleviates immunosuppression in polymicrobial sepsis by augmenting bacterial phagocytosis through sustaining the GFAT-DRP1 dependent mitochondrial calcium dynamics

doi: 10.1042/CS20256651

Figure Lengend Snippet: ( A ) LPS-tolerant BMDMs were untreated or treated with LPS or together with Gln or further treated with DON for 24 h. The protein expression of CDK1, phosphorylated CDK1 at site Thr-161 [p-CDK1 (T161)] DRP1 or p-DRP1(S616) was detected. ( B ) LPS-tolerant J774A.1 cells were transfected with control siRNA (siNC) or GFAT1 and two siRNA (siGFAT) for 48 h and then untreated or treated with LPS alone, or together with Gln for 24 h. The protein expression of GFAT1, GFAT2, CDK1, DRP1, or p-DRP1 (S616) was detected. ( C ) LPS-tolerant BMDMs were untreated or treated with LPS or together with Gln or further treated with Avotaciclib (Av) for 24 h. The protein expression of CDK1, DRP1, or p-DRP1 (S616) was detected. ( D-H ) LPS-tolerant J774A.1 cells were transfected with control siRNA (siNC) or CDK1 siRNA (siCDK1) for 48 h and then untreated or treated with LPS alone, or together with Gln for 24 h. The protein expressions of CDK1, DRP1, or p-DRP1(S616) ( D ), GTP-binding activity of DRP1 ( E ), mitochondrial morphology ( F ), cytosolic calcium ions, mitochondrial calcium ions ( G ), and fluorescence images of intracellular F-actin were detected ( H ). ( I ) LPS-tolerant J774A.1 cells were transfected with control siRNA (siNC) or CDK1 siRNA (siCDK1) for 48 h and then untreated or treated with LPS alone, or together with Gln for 24 h, and then infected with E. coli and S. aureus for 1 h. Phagocytosis was analyzed. The concentration of LPS, Gln, DON, and Avotaciclib was 100 ng/ml, 2 mM, 50 μM and 5 μM, respectively. The MOI for E. coli and S. aureus was 1. M: LPS-tolerant macrophages that have not been treated with Gln. **: P <0.01, *: P <0.05, N.S .: no significance. n =3. Scale bar = 5 µm.

Article Snippet: Primary antibody against p-CDK1 (Thr-161) was purchased from ABclonal (AP0324, Wuhan, China).

Techniques: Expressing, Transfection, Control, Binding Assay, Activity Assay, Fluorescence, Infection, Concentration Assay

Journal: Clinical Science (London, England : 1979)

Article Title: Glutamine alleviates immunosuppression in polymicrobial sepsis by augmenting bacterial phagocytosis through sustaining the GFAT-DRP1 dependent mitochondrial calcium dynamics

doi: 10.1042/CS20256651

Figure Lengend Snippet: ( A-H ) BALB/c mice were infected with P. aeruginosa (PA, 1 × 109 CFU/kg body weight) 24 h after CLP surgery, and the mice were treated with normal saline (NS) or Gln (0.1 g/kg body weight) or together with DON (3 µg/kg body weight) every 24 h ( A ). After 48 h, the intracellular Gln concentration of peritoneal macrophages ( B ), protein expression of CDK1, DRP1 and p-DRP1 (S616) in peritoneal macrophages ( C ), intracellular co-localization of DRP1 and TOMM20 in peritoneal macrophages ( D ), cytosolic calcium ions and mitochondrial calcium ions ( E ), PA load ( F ) and TNF-α and IL-6 in serum ( G ) were analyzed. The survival rate of mice was observed ( H ) ( n =10). **: P <0.01, *: P <0.05, N.S .: no significance. n =3. Scale bar = 5 µm.

Article Snippet: Primary antibody against p-CDK1 (Thr-161) was purchased from ABclonal (AP0324, Wuhan, China).

Techniques: Infection, Saline, Concentration Assay, Expressing

Journal: PLoS ONE

Article Title: Trichostatin A Targets the Mitochondrial Respiratory Chain, Increasing Mitochondrial Reactive Oxygen Species Production to Trigger Apoptosis in Human Breast Cancer Cells

doi: 10.1371/journal.pone.0091610

Figure Lengend Snippet: ( A ) Histograms depict cell cycle distributions in MCF-10A, MDA-MB-231 and MCF-7 cells after a 24 h treatment with DMSO (control) or TSA (1000 nmol/L, 500 nmol/L or 500 nmol/L). ( B ) Western blot of CyclinB1, Phospho-cdc2 (Thr161) and H3P in MCF-10A, MDA-MB-231 and MCF-7. Representative of 3 experiments. ( C ) Cell cycle distribution in MDA-MB-231 cells after exposure to 500 nmol/L of TSA at the indicated time points. ( D ) A time-course study of the effects of 1000 nmol/L, 500 nmol/L and 500 nmol/L TSA on the percentages of apoptotic cells in the MCF-10A, MDA-MB-231 and MCF-7 cell lines, respectively. Apoptosis was determined by flow cytometric analysis of the PI-positive and Annexin-V-positive cells. Values were shown as the means ± SD, n = 3. **P<0.01 versus the 0 h group using Student's t-test. A pan-caspase inhibitor, zVAD-fmk, was used to protect MDA-MB-231 cells from apoptosis; graphs showed the percentage of apoptotic cells under different treatments ( E ) and the cell cycle distributions ( F ). **P<0.01 compared to the TSA-treated alone group using Student's t-test.

Article Snippet: Gels were analyzed by immunoblotting with antibodies against Cytochrome C, COX IV, Bcl-xL, Bcl-2, NDUFS1, NDUFS6, UQCRFS1, CYC1 (10993-1-AP, 11242-1-AP, 10783-1-AP, 12789-1-AP, 19532-1-AP, 12444-1-AP, 14417-1-AP, 18443-1-AP, 10242-1-AP, respectively; Proteintech; 1∶1000 dilutions), GAPDH, Bax, cleaved PARP, CyclinB1, Phospho-cdc2(Thr161) (2118S, 5023, 9541, 4138, 9114 respectively; Cell Signaling; 1∶1000 dilutions), H3P (ab5176, 1∶2000 dilutions) and acetylated histone H3 (06-599; Millipore; 1∶1000 dilutions).

Techniques: Control, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Thr-161 Phosphorylation of Monomeric Cdc2

doi: 10.1074/jbc.m202742200

Figure Lengend Snippet: FIG. 5. Purified Xenopus PP2C dephosphorylates the Thr-160 residue of Cdk2. A, Xenopus PP2C was purified according the proce- dure described in Table I. The active fractions of various purification steps (P60, DEAE-Sepharose, Phenyl-Superose: PS; Hi-Trap Blue: Blue) were pooled, electrophoresed, and Western-blotted with the anti- PP2C antibody (left panel). Proteins from the active fraction of the last purification step (Hi-Trap Blue column) were visualized by Amido Black staining (right panel). MWM, molecular weight markers. B, re- combinant Cdk2 was first phosphorylated by Cak1 in the presence of [-32P]ATP, then incubated for 20 min in the presence of 0.1 g of the active fraction of the last purification step (Hi-Trap Blue column) and increasing concentrations of Mg2, and the release of 32P from Cdk2 was measured.

Article Snippet: Polyclonal rabbit anti-phospho-Cdc2 (Tyr-15) and anti-phospho-Cdc2 (Thr-161) antibodies were purchased from Cell Signaling Technology, and polyclonal rabbit anti-PSTAIR antibody and polyclonal sheep antihuman PP2C were purchased from Upstate Biotechnology.

Techniques: Purification, Residue, Western Blot, Staining, Molecular Weight, Incubation

Journal: Journal of Biological Chemistry

Article Title: Thr-161 Phosphorylation of Monomeric Cdc2

doi: 10.1074/jbc.m202742200

Figure Lengend Snippet: FIG. 6. Xenopus recombinant PP2C dephosphorylates the Thr- 160 residue of Cdk2. A, recombinant Cdk2 and / casein were first phosphorylated in the presence of [-32P]ATP by Cak1 and PKA, re- spectively. Phosphorylated Cdk2 or casein were then incubated for 20 min in the presence of 2 g Xenopus recombinant PP2C protein, in the presence or in the absence of Mg2 and 106 M okadaic acid, and the percentage of dephosphorylated substrate was measured. B, recombi- nant Cdk2 (5 M) was first phosphorylated by Cak1 in the presence of cold ATP and then incubated for 30 min at 30 °C with increasing concentrations (1.2, 0.24, 0.12, 0.024, and 0.012 mg/ml) of Xenopus recombinant PP2C. Proteins were Western-blotted with an antibody recognizing specifically the phospho-Thr-160 residue of Cdk2 (lower panel) and an anti-PSTAIR antibody recognizing all forms of Cdk2 (upper panel). C, recombinant Cdk2 was first phosphorylated by Cak1 in the presence of [-32P]ATP. Phosphorylated Cdk2 was incubated for 30 min in the presence of increasing concentrations of recombinant cyclin A. Xenopus recombinant PP2C (2 g) was then added in the presence of Mg2, and the release of 32P from Cdk2 was measured.

Article Snippet: Polyclonal rabbit anti-phospho-Cdc2 (Tyr-15) and anti-phospho-Cdc2 (Thr-161) antibodies were purchased from Cell Signaling Technology, and polyclonal rabbit anti-PSTAIR antibody and polyclonal sheep antihuman PP2C were purchased from Upstate Biotechnology.

Techniques: Recombinant, Residue, Incubation, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Thr-161 Phosphorylation of Monomeric Cdc2

doi: 10.1074/jbc.m202742200

Figure Lengend Snippet: FIG. 7. Monomeric Cdc2 in P60 and F9 is phosphorylated on Thr-161. A, P40 and P60 were prepared in the absence (Mg2) or in the presence of Mg2 (Mg2). They were analyzed by Western blotting with the anti-cyclin B2 antibody (upper panel), the anti-Cdc2 antibody (middle panel), and the antibody recognizing specifically the phospho- Thr-161 residue of Cdc2 (lower panel). B, F9 was prepared in the absence (Mg2) or in the presence of Mg2 (Mg2). As indicated, 25 mM Mg2 with or without Cak1 was added back or not in F9 initially prepared in the absence of Mg2, and the fraction was further incubated for 30 min at 30 °C before Western blotting with the anti-Cdc2 antibody (upper panel) and the antibody recognizing specifically the phospho- Thr-161 residue of Cdc2 (lower panel). C, F9 was prepared in the presence of Mg2 and then incubated in the presence or in the absence of Cak1 for 30 min at 30 °C. PP2C (0.9 mg/ml in the final assay) was added or not, and then incubation was extended for 30 min. Samples were analyzed by Western blotting with the anti-Cdc2 antibody (upper panel) and the antibody recognizing specifically the phospho-Thr-161 residue of Cdc2 (lower panel).

Article Snippet: Polyclonal rabbit anti-phospho-Cdc2 (Tyr-15) and anti-phospho-Cdc2 (Thr-161) antibodies were purchased from Cell Signaling Technology, and polyclonal rabbit anti-PSTAIR antibody and polyclonal sheep antihuman PP2C were purchased from Upstate Biotechnology.

Techniques: Western Blot, Residue, Incubation

Journal: Journal of Biological Chemistry

Article Title: Thr-161 Phosphorylation of Monomeric Cdc2

doi: 10.1074/jbc.m202742200

Figure Lengend Snippet: FIG. 6. Xenopus recombinant PP2C dephosphorylates the Thr- 160 residue of Cdk2. A, recombinant Cdk2 and / casein were first phosphorylated in the presence of [-32P]ATP by Cak1 and PKA, re- spectively. Phosphorylated Cdk2 or casein were then incubated for 20 min in the presence of 2 g Xenopus recombinant PP2C protein, in the presence or in the absence of Mg2 and 106 M okadaic acid, and the percentage of dephosphorylated substrate was measured. B, recombi- nant Cdk2 (5 M) was first phosphorylated by Cak1 in the presence of cold ATP and then incubated for 30 min at 30 °C with increasing concentrations (1.2, 0.24, 0.12, 0.024, and 0.012 mg/ml) of Xenopus recombinant PP2C. Proteins were Western-blotted with an antibody recognizing specifically the phospho-Thr-160 residue of Cdk2 (lower panel) and an anti-PSTAIR antibody recognizing all forms of Cdk2 (upper panel). C, recombinant Cdk2 was first phosphorylated by Cak1 in the presence of [-32P]ATP. Phosphorylated Cdk2 was incubated for 30 min in the presence of increasing concentrations of recombinant cyclin A. Xenopus recombinant PP2C (2 g) was then added in the presence of Mg2, and the release of 32P from Cdk2 was measured.

Article Snippet: Polyclonal rabbit anti-phospho-Cdc2 (Tyr-15) and anti-phospho-Cdc2 (Thr-161) antibodies were purchased from Cell Signaling Technology, and polyclonal rabbit anti-PSTAIR antibody and polyclonal sheep antihuman PP2C were purchased from Upstate Biotechnology.

Techniques: Recombinant, Residue, Incubation, Western Blot