Journal: Cell reports

Article Title: Limiting Mrs2-dependent mitochondrial Mg 2+ uptake induces metabolic programming in prolonged dietary stress

doi: 10.1016/j.celrep.2023.112155

Figure Lengend Snippet:

Article Snippet: Human: HEK 293 Cell Line , ATCC , Cat# CRL-1573.

Techniques: Plasmid Preparation, Mutagenesis, Recombinant, Western Blot, Stripping, Injection, Bicinchoninic Acid Protein Assay, Citrate Assay, TCA Precipitation, Avidin-Biotin Assay, Blocking Assay, Cholesterol Assay, Knock-Out, Software

Journal: Communications Biology

Article Title: The protease calpain2a limits innate immunity by targeting TRAF6 in teleost fish

doi: 10.1038/s42003-023-04711-7

Figure Lengend Snippet: a List of TRAF6 interactome based on Label-free quantification intensity (section). b , c MKC cells seeded in 6 cm 2 dishes. After 24 h, cell lysates were immunoprecipitated (IP) with anti-TRAF6 or anti-calpain2a affinity gels. Then the immunoprecipitates and cell lysates were analyzed by IB with the anti-TRAF6 and anti-calpain2a Abs, respectively. d , e HEK 293 cells seeded in 6 cm 2 dishes were transfected with the plasmids calpain2a-Flag and TRAF6-Myc (2 μg each). After 24 h, cell lysates were immunoprecipitated (IP) with anti-Myc d or anti-Flag e affinity gels. Then the immunoprecipitates and cell lysates were analyzed by IB with the anti-Myc and anti-Flag Abs, respectively. f The same amino acids among human calpain2a (Hu-calpain2), mouse calpain2a (Mu-calpain2), zebrafish calpain2a (ZF-calpain2a) and miiuy croaker calpain2a (M-calpain2a) are highlighted with black background. g – i EPC cells were transfected with calpain2a-Flag or empty vector together with the NF-κB, IL-1β, and IL-8 luciferase reporters. At 24 h post-transfection, cells were untreated (Mock) or treated with LPS for 6 h. The luciferase activity value was achieved against the Renilla luciferase activity ( n = 3 per group). Western blot analysis was used to measure the expression of transiently transfected calpain2a-Flag. The expression of Tubulin was used as a loading control. Data were analyzed by two-way ANOVA ( g , h , i ). ** p < 0.01. All experiments were performed in at least three independent experiments.

Article Snippet: Human embryonic kidney (HEK) 293 cells lines and Epithelioma papulosum cyprini (EPC) cells lines, purchased from American Type Culture Collection and kept in our lab. Miiuy croaker ( M. miiuy ) kidney cell lines (MKC) and Miiuy croaker intestine cell lines (MIC) were cultured from kidney and intestine tissues of miiuy croaker , .

Techniques: Immunoprecipitation, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Western Blot, Expressing

Journal: bioRxiv

Article Title: The ATM-E6AP-MASTL axis mediates DNA damage checkpoint recovery

doi: 10.1101/2023.02.22.529521

Figure Lengend Snippet: (A) HEK293 cells were treated with 0.5 uM doxorubicin (DOX) as indicated, cell lysates were collected and analyzed by immunoblotting for MASTL, RPA32 and α-tubulin. (B) HEK293 cells were treated with 10 mM hydroxyurea (HU) as indicated, cell lysates were collected and analyzed by immunoblotting for MASTL, RPA32 and α-tubulin. (C) HEK293 cells were treated with 10 nM camptothecin (CPT) as indicated, cell lysates were collected and analyzed by immunoblotting for MASTL, RPA32 and α-tubulin. (D) HeLa cells were treated with 10 mM HU, and incubated as indicated. Cell lysates were collected and analyzed by immunoblotting for MASTL, phospho-H2AX Ser-139, RPA32, phospho-ATM/ATR substrates, and α-tubulin. (E) HeLa cells were treated with or without 0.5 uM DOX, 10 nM CPT, 10 mM HU and 20 Gy Ionizing Radiation (IR) for 4 hours. Cell lysates were collected and analyzed by immunoblotting for MASTL, phospho-H2AX Ser-139, phospho-ATM/ATR substrates, and α-tubulin. (F) HeLa cells were treated with 0.5 μM DOX, and analyzed by immunoblotting for MASTL, α-tubulin, Aurora A, Aurora B, CDK1, Cyclin B1, and phospho-ATM/ATR substrates. (G) SCC38 cells were incubated with or without HU and caffeine, as indicated, and analyzed by immunoblotting for MASTL and β-actin.

Article Snippet: Human cervix carcinoma (HeLa) and human embryonic kidney 293 (HEK293) cell lines were obtained and authenticated by ATCC, and maintained in Dulbecco’s modified Eagle medium (DMEM, Hyclone) with 10% fetal bovine serum (FBS, Hyclone).

Techniques: Western Blot, Incubation

Journal: bioRxiv

Article Title: The ATM-E6AP-MASTL axis mediates DNA damage checkpoint recovery

doi: 10.1101/2023.02.22.529521

Figure Lengend Snippet: HEK293 cells were treated without (A) or with (B) 10 mM HU for 2 hours. These cells were then treated with cycloheximide (CHX, 20 μg/ml) at time 0 to block protein synthesis, and analyzed by immunoblotting for the protein stability of MASTL and α-tubulin.

Article Snippet: Human cervix carcinoma (HeLa) and human embryonic kidney 293 (HEK293) cell lines were obtained and authenticated by ATCC, and maintained in Dulbecco’s modified Eagle medium (DMEM, Hyclone) with 10% fetal bovine serum (FBS, Hyclone).

Techniques: Blocking Assay, Western Blot

Journal: bioRxiv

Article Title: The ATM-E6AP-MASTL axis mediates DNA damage checkpoint recovery

doi: 10.1101/2023.02.22.529521

Figure Lengend Snippet: (A) The sequence alignment of the conserved E6AP Ser-218 motif in human, mouse and Xenopus . (B) A phospho-specific antibody recognizing E6AP Ser-218 was generated, as described in Materials and Methods. WT or E6AP KO HEK293 cells were treated without or with 10 mM HU, and analyzed by immunoblotting for phospho-E6AP Ser-218, E6AP, and α-tubulin. (C) HeLa cells were treated without or with 0.5 μM DOX and 5 μM KU55933 (ATMi), as indicated, and analyzed by immunoblotting for E6AP, phospho-E6AP Ser-218 and α-tubulin. (D) HeLa cells were transfected with HA-tagged WT, S218A or S218D E6AP. Cell lysates were harvest for IP assays. The input, MASTL IP, and a control IP using empty beads products were analyzed by immunoblotting for MASTL and HA. (E) HeLa cells were transfected with HA-tagged WT or S218A E6AP, as in panel D. Cells were treated with or without 0.5 μM DOX for 3 hours, and harvested for IP assays. The input, HA IP, and a control IP using empty beads products were analyzed by immunoblotting for MASTL, phospho-E6AP Ser-218, and HA. (F) E6AP KO HeLa cells were transfected with HA-tagged WT or S218A E6AP, as in panel D. Cells were treated with or without 0.5 μM DOX, incubated as indicated, and harvested for immunoblotting for MASTL and α-tubulin.

Article Snippet: Human cervix carcinoma (HeLa) and human embryonic kidney 293 (HEK293) cell lines were obtained and authenticated by ATCC, and maintained in Dulbecco’s modified Eagle medium (DMEM, Hyclone) with 10% fetal bovine serum (FBS, Hyclone).

Techniques: Sequencing, Generated, Western Blot, Transfection, Incubation

Journal: bioRxiv

Article Title: The ATM-E6AP-MASTL axis mediates DNA damage checkpoint recovery

doi: 10.1101/2023.02.22.529521

Figure Lengend Snippet: (A) WT or E6AP knockout HEK293 cells were treated without or with doxorubicin (DOX, 0.5 μM) for 4 hours. Cells were harvested and analyzed by immunoblotting for phospho-E6AP Ser-218 and α-tubulin. (B) HEK293 cells were treated without or with DOX (0.5 μM), or ATM inhibitor (KU55933, 10 μM), and analyzed by immunoblotting. (C) HeLa cells were treated with hydroxyurea (HU, 10 mM) combined with ATM/ATR inhibitor (caffeine, 4 mM) or ATM inhibitor (KU55933, 10 μM), for 12 hours, as indicated. Cells were harvested and analyzed by immunoblotting.

Article Snippet: Human cervix carcinoma (HeLa) and human embryonic kidney 293 (HEK293) cell lines were obtained and authenticated by ATCC, and maintained in Dulbecco’s modified Eagle medium (DMEM, Hyclone) with 10% fetal bovine serum (FBS, Hyclone).

Techniques: Knock-Out, Western Blot

Journal: bioRxiv

Article Title: The ATM-E6AP-MASTL axis mediates DNA damage checkpoint recovery

doi: 10.1101/2023.02.22.529521

Figure Lengend Snippet: (A) E6AP KO HeLa cells were transfected with HA-tagged WT or S218A E6AP, as in . Cells were treated with 0.1 μM ETO for 18 hours, and released in fresh medium for recovery. Cells were then harvested at the indicated time points (after the removal of ETO) for IF using an anti-phospho-Aurora A/B/C antibody. The activation of Aurora phosphorylation (shown in red) and chromosome condensation (in blue) indicated mitosis. The percentages of cells in mitosis were quantified and shown. The mean values and standard deviations were calculated from three experiments. An unpaired 2-tailed Student’s t test was used to determine the statistical significance (** p<0.01). (B) E6AP KO HeLa cells expressing HA-tagged WT or S218A E6AP, as in panel A, were treated with 2 mM HU for 18 hours. Cells were then released in fresh medium, and incubated as indicated, for recovery. Cell cycle progression was analyzed by FACS. (C) WT or S218A E6AP was expressed in E6AP KO HEK293 cells. Cells were treated without or with 0.1 μM ETO for 18 hours, released in fresh medium for recovery, and incubated as indicated. Cells were analyzed by immunoblotting for phospho-CDK substrates and histone H3. (D) WT or S218A E6AP was expressed in E6AP KO HEK293 cells, as in panel C. Cells were treated without or with 1 μM CPT for 90 minutes, and analyzed by immunoblotting for phospho-ATM/ATR substrates, phospho-SMC1 Ser-957, and α-tubulin.

Article Snippet: Human cervix carcinoma (HeLa) and human embryonic kidney 293 (HEK293) cell lines were obtained and authenticated by ATCC, and maintained in Dulbecco’s modified Eagle medium (DMEM, Hyclone) with 10% fetal bovine serum (FBS, Hyclone).

Techniques: Transfection, Activation Assay, Expressing, Incubation, Western Blot

Journal: Gut Pathogens

Article Title: CMTM3 protects the gastric epithelial cells from apoptosis and promotes IL-8 by stabilizing NEMO during Helicobacter pylori infection

doi: 10.1186/s13099-023-00533-4

Figure Lengend Snippet: Proteomics analysis indicates that NEMO is involved in H. pylori -CMTM3-inflammation. a Differentially expressed proteins (P.adj < 0.05). b Kyoto Encyclopedia of Genes and Genomes. enrichment analysis. The TOP regulated signaling pathways were shown of CMTM3 KO vs control (LentiV2) in GES-1 cells (padj < 0.05). c Heat map of the expression levels of the differentially expressed proteins enriched in epithelial cell signaling in H. pylori infection signaling pathway. Red indicates high and blue indicates low protein expression. d NEMO expression was detected in CMTM3 KO GES-1 and HEK 293 T cells by western blotting. β-Actin was used as a control. Data are representative of three independent experiments. e NEMO expression was detected in CMTM3 KO GES-1 cells by qRT-PCR. Data is representative of three independent experiments. f Representative immunohistochemistry images of NEMO by human gastric tissues from the same patient before and after H. pylori infection (n = 11). g Summary of immunohistochemistry results for NEMO. Graph showing the average quantification of NEMO positive cells in at least 6 random fields of one patient. Data is expressed as means ± SEM. *p < 0.05. ns, no statistical difference

Article Snippet: Human cell lines HEK 293 T, and AGS were maintained from ATCC (Manassas, Virginia, USA).

Techniques: Expressing, Infection, Western Blot, Quantitative RT-PCR, Immunohistochemistry

Journal: Gut Pathogens

Article Title: CMTM3 protects the gastric epithelial cells from apoptosis and promotes IL-8 by stabilizing NEMO during Helicobacter pylori infection

doi: 10.1186/s13099-023-00533-4

Figure Lengend Snippet: CMTM3 reduces NEMO degradation. a The immunoprecipitation analysis of GES-1 cells. Cells expressing pCMV-NEMO-3Flag were immunoprecipitated by an anti-Flag antibody and immunoblotted by anti-NEMO and anti-CMTM3 antibodies. b Co-localization of CMTM3 and NEMO in GES-1 cells was investigated by immunofluorescence co-localization analysis after H. pylori infection for 24 h. Scale bar, 10 μm. Hochest was used for nuclear staining. c, d CMTM3 knockout GES-1 cells were treated with cycloheximide (25 µg/mL) and proteins were subjected to immunoblotting (c) . Data is representative of three independent experiments. The remaining NEMO was quantified (d) . e The ubiquitylation of NEMO. HEK 293 T cells were transfected with the indicated vectors. After 48 h transfection, cells were treated with MG132 (10 μM) for 6 h. Then cells were lysed and immunoprecipitated with anti-Flag antibody and analyzed by immunoblotting with the indicated antibodies. f CMTM3 KO GES-1 cells were treated with MG132 (10 μM) and collected at indicated time points. Whole-cell lysates were subjected to immunoblotting with indicated antibodies. g The expression the phosphorylated IKBα (p-IKBα) and phosphorylated p65 (p-p65) were analyzed in CMTM3 KO GES-1 cells.*p < 0.05

Article Snippet: Human cell lines HEK 293 T, and AGS were maintained from ATCC (Manassas, Virginia, USA).

Techniques: Immunoprecipitation, Expressing, Immunofluorescence, Infection, Staining, Knock-Out, Western Blot, Transfection