Journal: Nature Communications

Article Title: Structural basis of the activation of TRPV5 channels by long-chain acyl-Coenzyme-A

doi: 10.1038/s41467-023-41577-z

Figure Lengend Snippet: Whole-cell voltage-clamp recordings in HEK293 cells transiently cotransfected with TRPV6 and ci-VSP were performed as described in Methods. In these measurements, constant holding at −60 mV for 59 s, then a depolarizing step to +100 mV for 1 s was applied to activate ci-VSP and induce PI(4,5)P 2 hydrolysis. This was repeated up to 14 times continuously ( a , bottom). The intracellular pipette solution did not contain ATP. a monovalent TRPV6 currents were measured by application of Mg 2+ - and Ca 2+ -free solution, as described in the methods section. Standard intracellular solution was supplemented with 100 µM diC 8 PI(4,5)P 2 ( b ) or 10 µM palmitoyl-CoA ( c ). d Summary of relative recovered current after +100 mV pulse compared to peak current. e Summary of the ratio between the steady-state current before the +100 mV pulse and inhibited current right after the +100 mV pulse. Line graphs in d and e show mean ± SEM from individual cells.

Article Snippet: Human Embryonic Kidney 293 (HEK293) cells (CRL-1573) and HEK 293T cells (CRL-3216) were purchased from American Type Culture Collection (ATCC).

Techniques: Transferring

Journal: Nature Communications

Article Title: Structural basis of the activation of TRPV5 channels by long-chain acyl-Coenzyme-A

doi: 10.1038/s41467-023-41577-z

Figure Lengend Snippet: a-d Representative traces for whole-cell patch-clamp recordings in HEK293T cells transiently cotransfected with TRPV6 and ci-VSP, performed as described in the Methods section. The membrane potential was clamped at −60 mV for 59 s, then a depolarizing step to +100 mV for 1 s was applied (arrows) to activate ci-VSP, after which the holding potential returned to −160 mV. Monovalent currents were measured in a Ca 2+ - Mg 2+ -free solution, and at the end of the experiment a solution containing 1 mM Mg 2+ was applied to inhibit monovalent currents, see methods for details. The transfected cells were pretreated with ( b ) or without ( a ) 164 µM oleic acid overnight in DMEM containing high glucose (25 mM). For the low glucose condition, transfected cells were pretreated with 164 µM oleic acid ( c ) or 164 µM oleic acid with 100 µM etomoxir ( d ) overnight in the DMEM containing normal glucose (5.5 mM). e Summary of the inhibited currents right after the +100 mV pulse. Statistical significance was calculated with one way analysis of variance, overall ANOVA F = 16.53, p = 3.96 ×10 −6 . f Cartoon explaining the various treatments in these experiments. Long chain fatty acids (oleic acid) are taken up by the cell and converted to LC-CoA, which is transported from the cytoplasm to the mitochondria by CPT-1. High glucose, and etomoxir inhibits CPT-1, leading to increased cytoplasmic LC-CoA levels (created with Biorender). Bar graph shows mean ± SEM and scatter plots. The symbols represent experiments from individual cells.

Article Snippet: Human Embryonic Kidney 293 (HEK293) cells (CRL-1573) and HEK 293T cells (CRL-3216) were purchased from American Type Culture Collection (ATCC).

Techniques: Patch Clamp, Membrane, Transfection

Journal: Journal of the American Chemical Society

Article Title: Selective Recognition of Carbohydrate Antigens by Germline Antibodies Isolated from AID Knockout Mice

doi: 10.1021/jacs.1c12745

Figure Lengend Snippet: Binding properties of representative germline antibodies to Lewis X, sialyl Lewis C, and the Tn antigen. (A) Signals in relative fluorescence units (RFUs) for antibodies on our 738-component array. Data for 11A11, 9A9, and 8C11 are at a concentration approximately 20-fold higher than KD,App for the primary antigen; 2D3.C11 is at 8 μg/mL. Positive and negative controls have been excluded. (B) Binding data for anti-Lewis X antibodies to Lewis X positive MCF7 cell line and Lewis X negative SK-MEL-28 as compared to positive control anti-Lewis X antibody MC-480. (C) Binding data for anti-Tn antibodies to Tn positive MCF7 cell line and Tn negative HEK-293 cell line as compared to positive control anti-Tn antibody SBH-Tn. Error bars (B,C) represent the SD of three experiments. Additional bar graphs can be found in Figure S8. Representative histograms and additional flow cytometry data can be found in Figure S9.

Article Snippet: The human embryonic kidney HEK-293 cell line was purchased from the American Type Culture Collection (ATCC CRL-1573; Manassas, VA).

Techniques: Binding Assay, Fluorescence, Concentration Assay, Positive Control, Flow Cytometry

Journal: eLife

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

doi: 10.7554/eLife.86976

Figure Lengend Snippet: ( A ) HEK293 cells were treated with 0.5 µM 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 µM DOX, 10 nM CPT, 10 mM HU, and 20 Gy ionizing radiation (IR) for 4 hr. 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: eLife

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

doi: 10.7554/eLife.86976

Figure Lengend Snippet: ( A–C ) HeLa cells expressing CFP-MASTL were treated without or with ionizing radiation (IR). Cells were analyzed by direct fluorescence for CFP-MASTL expression, and by immunofluorescence for the phosphorylation of ATM/ATR substrates. Quantifications are shown in panels B and C (>10 cells/time point). ( D, E ) HEK293 cells were treated without ( D ) or with ( E ) 10 mM hydroxyurea (HU) for 2 hr. 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: Expressing, Fluorescence, Immunofluorescence, Blocking Assay, Western Blot

Journal: eLife

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

doi: 10.7554/eLife.86976

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 knockout (KO) HEK293 cells were treated without or with 10 mM hydroxyurea (HU) for 4 hr, and analyzed by immunoblotting for phospho-E6AP Ser-218, E6AP, and α-tubulin. ( C ) HeLa cells were treated without or with 0.5 μM doxorubicin (DOX) and 5 μM KU55933 (ATMi), as indicated, for 1 hr, and analyzed by immunoblotting for phospho-ATM/ATR substrates, phospho-E6AP Ser-218, and α-tubulin. ( D ) HeLa cells were transfected with HA-tagged WT, S218A, or S218D E6AP. Cell lysates were harvested for immunoprecipitation (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 hr, 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, Knock-Out, Western Blot, Transfection, Immunoprecipitation, Incubation

Journal: eLife

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

doi: 10.7554/eLife.86976

Figure Lengend Snippet: ( A ) WT or E6AP knockout HEK293 cells were treated without or with doxorubicin (DOX, 0.5 μM) for 4 hr. 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), for 4 hr, 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 hr, 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: eLife

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

doi: 10.7554/eLife.86976

Figure Lengend Snippet: ( A ) E6AP knockout (KO) HeLa cells were transfected with HA-tagged WT or S218A E6AP, as in . Cells were treated with 0.1 μM etoposide (ETO) for 18 hr, and released in fresh medium for recovery. Cells were then harvested at the indicated time points (after the removal of ETO) for immunofluorescence (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 manually and shown. The mean values and standard deviations were calculated from three experiments. An unpaired two-tailed Student’s t test was used to determine the statistical significance (**p<0.01, n>500 cell numbers/measurement). ( B ) E6AP KO HeLa cells expressing HA-tagged WT or S218A E6AP, as in panel A, were treated with 2 mM hydroxyurea (HU) for 18 hr. Cells were then released in fresh medium, and incubated as indicated, for recovery. Cell cycle progression was analyzed by fluorescence-activated cell sorting (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 hr, released in fresh medium for recovery, and incubated as indicated. Cells were analyzed by immunoblotting for phospho-cyclin-dependent kinase (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 min, 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: Knock-Out, Transfection, Immunofluorescence, Activation Assay, Two Tailed Test, Expressing, Incubation, Fluorescence, FACS, Western Blot