Journal: Oncology Reports

Article Title: Expression level of Bcl-XL critically affects sensitivity of hepatocellular carcinoma cells to LIGHT-enhanced and interferon-γ-induced apoptosis

doi: 10.3892/or.17.5.1067

Figure Lengend Snippet: Figure 3. Expression levels of caspase-3, caspase-8, caspase-9, Bcl-XL, Bak and Bid in Hep3B cells treated with IFN-Á/LIGHT. (A) Hep3B cells were treated with 100 ng/ml IFN-Á and various concentrations of LIGHT for 24 h or 72 h as indicated. Cell lysates containing 30 μg of protein were subjected to 12% Tris-glycine gel electrophoresis followed by Western blotting with rabbit polyclonal antibodies against caspase-3, caspase-8, caspase-9, Bcl- XL, Bak, Bid, respectively. Cells treated with 100 ng/ml IFN-Á or 100 ng/ml LIGHT alone and untreated cells were used as the controls. ß-actin was used as an internal control for total protein loading. (B) Hep3B cells (2x105) treated with 100 ng/ml sLIGHT and 100 ng/ml of IFN-Á were also incubated with 100 μM of caspase-3 inhibitor Z-DEVD-fmk, caspase-9 inhibitor Z- IEHD-fmk, caspase-8 inhibitor Z-IEHD-fmk, or normal saline for 96 h. Cell viability was then measured. The viability of untreated (without any cytokine and inhibitor) cells was set at 100%, and the relative viability of other samples was calculated accordingly. The experiments were performed in triplicate, and in at least two separated experiments. The bar indicates the standard error. **p<0.01, versus group treated with normal saline.

Article Snippet: A recombinant LIGHT soluble protein that only contained the extracellular region of human LIGHT and caspase inhibitors Z-DEVD-fmk, Z-IETD-fmk and Z-LEHD-fmk was purchased from R&D System (Minneapolis, MN, USA).

Techniques: Expressing, Nucleic Acid Electrophoresis, Western Blot, Control, Incubation, Saline

Journal: Journal of Virology

Article Title: Hepatitis B Virus X Protein Stimulates Viral Genome Replication via a DDB1-Dependent Pathway Distinct from That Leading to Cell Death

doi: 10.1128/jvi.79.7.4238-4245.2005

Figure Lengend Snippet: FIG. 4. HBx promotes HBV replication and induces cell death through distinct DDB1-dependent pathways. (A) Human hepatoma HepG2 and Huh-7 cells were transfected with wild-type HBV or HBV(X) genomic DNA together with a GFP gene to assess transfection efficiencies by FACS analysis. The amount of core particle-associated HBV DNA replicative intermediates was assessed by Southern blot analysis 3 days after transfection as described in the legend to Fig. 1. Transfection efficiencies were similar for the two genomic constructs in each cell line, but important differences were noticed between the two cell lines (5% in HepG2 cells versus 20% in Huh-7 cells [data not shown]). Hence, the amounts of sample analyzed were corrected accordingly. One of two independent transfection experiments is shown. The single-stranded (ssDNA) and double-stranded (dsDNA) HBV DNA replicative forms are indicated on the right. (B) Western blot analysis. Whole-cell extracts prepared from HepG2 or Huh-7 cells transfected with GFP-HBx or empty vector (vect) (top) or from the indicated untransfected cell lines (bottom) were separated by gel electrophoresis. Immunoblot analyses were performed with antibodies to HBx (top), DDB1 (bottom), and, as a control for loading, -tubulin. In the upper gel, fourfold-larger amounts of HepG2 protein extract were loaded on the gel to correct for transfection efficiencies. (C) Clonogenic cell survival assay. HeLa, Huh-7, and HepG2 cells were either mock transduced (Mock) or transduced with lentivirus vectors expressing the indicated GFP-HBx fusion proteins. Transduction efficiencies were comparable, as assessed by FACS analysis (data not shown). Cells were then seeded at appropriate dilutions in six-well culture dishes. After 16 days of undisturbed growth at 37°C, the surviving cells were fixed and stained with crystal violet. (D) Huh-7 and HepG2 cells were transfected with a GFP-expressing plasmid bearing a hygromycin resistance gene either alone (vect) or together with equal amounts of the indicated HBV genomic DNA. The transfected cells were seeded at appropriate dilutions in a six-well culture dish and cultured in medium containing hygromycin. Drug-resistant colonies were fixed and stained with crystal violet 20 (HepG2) or 15 (Huh-7) days after transfection. Note that the HBV replication assay presented in panel A was performed 3 days after transfection, at which time the HBx-expressing HepG2 and Huh-7 cells do not show any of the obvious changes in morphology that typically precede HBx-mediated cell death (data not shown).

Article Snippet: The human hepatoma HepG2 cell line obtained from the American Type Culture Collection and the Huh-7 cell line were grown at 37°C in the presence of 5% CO2 in modified Eagle’s medium (MEM) (Invitrogen) supplemented with 100 U of penicillin/ml, 100 g of streptomycin/ml, 2 mM L-glutamine, 1 mM sodium pyruvate, 1% nonessential amino acids, and 10% (vol/vol) fetal calf serum (Chemie Brunschwig).

Techniques: Transfection, Southern Blot, Construct, Western Blot, Plasmid Preparation, Nucleic Acid Electrophoresis, Control, Clonogenic Cell Survival Assay, Transduction, Expressing, Staining, Cell Culture

Journal: PLoS Pathogens

Article Title: T Cells Contain an RNase-Insensitive Inhibitor of APOBEC3G Deaminase Activity

doi: 10.1371/journal.ppat.0030135

Figure Lengend Snippet: (A) Deaminase activity was measured using an infrared 700 (IR700)–labeled oligo containing the A3G recognition site (CCC) either with or without exogenous recombinant uracil DNA glycosylase (+/- UDG). Oligos were incubated with crude cell lysates containing 10 μg of total cellular protein obtained from H9 cells, H9 cells expressing the HIV genome containing a deletion in Vif (H9-HIV), or from HeLa or 293FT cells transfected with the indicated amounts of A3G plasmid DNA (pA3G). Extent of oligo cleavage (indicating extent of deamination) was determined by gel electrophoresis followed by detection on a LI-COR scanner (top panel), and the percentage of probe cleaved was graphed (second panel). Below, equivalent amounts of cell lysate were analyzed in parallel by western blot (WB) to show A3G protein content. Western blot of calreticulin is shown as a loading control. (B) UDG activity was measured in select lysates from (A) using an IR700-labeled dU-containing oligo in the presence or absence of exogenous UDG (+/- UDG). Results are displayed as in (A) and show that unlike A3G activity shown in (A), UDG activity is similar in all cell lysates analyzed. All assays were performed on RNAse A–treated samples.

Article Snippet: To each well was added 10 μl of cell lysate in NP40 buffer and 70 μl of a master mix containing 10 pmol Taqman probe, 0.4 units uracil DNA glycosylase (NEB, http://www.neb.com/ ), 50 mM Tris (pH 7.4), and 10 mM EDTA.

Techniques: Activity Assay, Labeling, Recombinant, Incubation, Expressing, Transfection, Plasmid Preparation, Nucleic Acid Electrophoresis, Western Blot

Journal: Cancer research

Article Title: Phosphatase 1 nuclear targeting subunit mediates recruitment and function of poly (ADP-ribose) polymerase 1 in DNA repair

doi: 10.1158/0008-5472.CAN-18-1673

Figure Lengend Snippet: (A) HeLa cells were transfected with control, non-targeting, or PNUTS siRNA (#1) for 1 day. The cells were then treated with IR at the indicated doses, incubated for 30 min, harvested and analyzed by immunoblotting for γH2AX, PNUTS, and H2B. (B) HeLa cells were transfected with PNUTS siRNA (#1), and siRNA-resistant (SiR) WT PNUTS, as indicated. The cell lysates were analyzed by immunoblotting for γH2AX, PNUTS, and β-actin. (C, D) HeLa cells were treated with PNUTS siRNA (#1 and #2), as indicated. The comet assay was performed as described in Materials and Methods. Representative images are shown in panel C. The percentage of DNA in the tail section was quantified, the mean values and standard derivations are shown in panel D (N>20). (E) HeLa cells were treated with control or PNUTS siRNA (#1) for 1 day. The cell lysates were analyzed by immunoblotting for γH2AX, phospho-CHK1, phospho-BRCA1, PNUTS, and β-actin. (F) SCC38 cells were transfected with PNUTS siRNA (#1), and siRNA-resistant (SiR) WT or W401A PNUTS, as indicated. The cell lysates were analyzed by immunoblotting for γH2AX, PNUTS, and β-actin. (G) SCC38 cells were treated with control or PNUTS siRNA (#1) at day 0, incubated with doxorubicin (Dox, 2 μg/ml) at day 1, and incubated for 3 days. Cell viability was determined and normalized to that of day 1. The mean value and standard deviation were calculated from 3 independent experiments. (H) SCC38 cells were treated with PNUTS siRNA (#1) and siRNA-resistant (SiR) PNUTS, as in panel G. These cells were then treated with Dox (2 μg/ml), and incubated for 1-4 days. Cell viability was determined and normalized to that of the first day. The mean value and standard deviation were calculated from 3 independent experiments. (I) The colonogenic assay was performed as described in Materials and Methods. The numbers of colonies formed were normalized to the untreated control. The mean value and standard deviation were calculated from 3 independent experiments. Statistical significance was analyzed using an unpaired 2-tailed Student’s t-test. A p-value<0.001 was considered highly significant (***).

Article Snippet: Immunoblotting Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were carried out as previously described ( 25 ), using the following antibodies:, γH2AX, PNUTS, PARP1, H2B, BRCA1 phospho-S1524, CHK1 phospho-S317 antibodies from Bethyl Laboratories; β-actin, GFP, Poly (ADP-Ribose) Polymer antibodies from Abcam; α-tubulin, BRCA1 phospho-S1524, active caspase-3 antibodies from Cell Signaling Technology.

Techniques: Transfection, Incubation, Western Blot, Single Cell Gel Electrophoresis, Standard Deviation

Journal: Biochemistry

Article Title: Modeling Suggests TRPC3 Hydrogen Bonding and Not Phosphorylation Contributes to the Ataxia Phenotype of the Moonwalker Mouse

doi: 10.1021/acs.biochem.5b00235

Figure Lengend Snippet: The gain-of-function Mwk mutation in TRPC3 causes increased cell death and calcium signaling in neuronal cells. (A) Equal amounts of TRPC3 are expressed at the cell surface in the mouse cerebellum. Biotinylated cerebellar slice cultures from wild-type (WT) and Mwk mice were lysed and subjected to pull-down experiments using streptavidin beads, followed by immunoblotting for TRPC3 and actin. Abbreviations: I, input; S, supernatant; B, pellet (biotinylated fraction). (B) Overexpression of Mwk (T635A) but not wild-type (WT) TRPC3 significantly induced cell death in mouse Neuro-2a cells (mean ± SEM; n = 3; ANOVA followed by Bonferroni’s post hoc test; p < 0.005). V denotes vector control. (C and D) Overexpression of Mwk (T635A) but not wild-type (WT) TRPC3 causes significant nuclear localization of co-expressed GFP-tagged NFAT (mean ± SEM; n = 3; ANOVA followed by Bonferroni’s post hoc test; p < 0.001). Cells were fixed 24 h after transfection and subjected to indirect immunofluorescence using antibodies against FLAG, GFP, and the DNA dye DAPI. Cells transfected with mutant TRPC3 have a more rounded appearance because of their impending cell death. (E) The in vitro gain-of-function phenotype of Mwk TRPC3 is not likely to be mediated by phosphorylation. Overexpression of the phospho-mimic T635D mutation but also of the control T635N mutation did not induce cell death in mouse Neuro-2a cells (mean ± SEM; n = 3; ANOVA followed by Bonferroni’s post hoc test). (F) Overexpression of the phospho-mimic T635D mutation but also of the control T635N mutation did not cause significant nuclear translocation of GFP-NFAT (mean ± SEM; n = 3; ANOVA followed by Bonferroni’s post hoc test).

Article Snippet: Protein lysates prepared with RIPA buffer (Thermo Scientific) were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) followed by immunoblotting with anti-TRPC3 (1:200, Alomone) and anti-Actin (1:1000, Abcam) antibodies.

Techniques: Mutagenesis, Western Blot, Over Expression, Plasmid Preparation, Transfection, Immunofluorescence, In Vitro, Translocation Assay

Journal: Biochemistry

Article Title: Modeling Suggests TRPC3 Hydrogen Bonding and Not Phosphorylation Contributes to the Ataxia Phenotype of the Moonwalker Mouse

doi: 10.1021/acs.biochem.5b00235

Figure Lengend Snippet: Structural overview and sequence alignments of TRPC3 and the S4–S5 linker region. (A) Schematic of the six-transmembrane TRPC3 channel protein, in which the six transmembrane helices, S1–S6 (numbered cylinders), are preceded by a coiled coil domain (gray box) and an ankyrin repeat domain (black diamonds) and followed by the TRP domain (checkered box), another predicted coiled coil domain (gray box), and a predicted CIRB domain (light gray box). The Mwk mutation (T635A) resides within the S4–S5 linker region and is indicated by a yellow circle. The sequence alignment of the wild-type and mutant mouse TRPC3 S4–S5 linker region is shown below. Figure adapted from ref . (B) Sequence alignment of the S4–S5 linker regions of several TRP channels and Kv1.2. The position of the Mwk mutation is highlighted in yellow. Other residues found to have significant effects on channel function when mutated are colored red (see the text for details). (C) Structure of apo TRPV1 (PDB entry 3J5P) with mutants in the S4–S5 linker region found to have significant effects on TRP channel function highlighted with red dots. (D) Sequence conservation diagram of the S4 transmembrane helix and the S4–S5 linker region of Kv and TRP channels showing that the threonine mutated in the Mwk mouse is surprisingly more conserved in Kv channels than in TRP channels. The analysis includes Kv’s 1. x –10. x for a total of 40 sequences analyzed for Kv channels, and all representatives of the TRPC, TRPM, TRPV, TRPA, TRPP, and TRPML subfamilies for a total of 28 sequences analyzed for TRP channels. Figures made using WebLogo and MAFFT sequence alignments of all Kv and TRP channels identified in ref .

Article Snippet: Protein lysates prepared with RIPA buffer (Thermo Scientific) were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) followed by immunoblotting with anti-TRPC3 (1:200, Alomone) and anti-Actin (1:1000, Abcam) antibodies.

Techniques: Sequencing, Mutagenesis

Journal: Biochemistry

Article Title: Modeling Suggests TRPC3 Hydrogen Bonding and Not Phosphorylation Contributes to the Ataxia Phenotype of the Moonwalker Mouse

doi: 10.1021/acs.biochem.5b00235

Figure Lengend Snippet: Homology modeling reveals potential for hydrogen bonding. (A) Sequence alignment of mouse TRPC3 to rat TRPV1 and to the S4–S5 linker of the Kv 1.2/2.1 chimera used to create the homology models. A cut of 21 amino acids (indicated by double lines) was made at the end of S3 to facilitate modeling. Transmembrane helices are indicated by black boxes with the corresponding label for each S1–S6 transmembrane helix above. Coloring is according to the Clustal coloring scheme. The TRPC3 Mwk mutation in the S4–S5 linker region is indicated by a double-lined box. Arrows indicate residues highlighted in other panels of the figure. Image made with JalView. (B) Top-ranked (according to MODELLER score) mouse TRPC3 homology model on the TRPV1 apo structure (PDB entry 3J5P) with the Kv 1.2/2.1 channel chimera structure for the S4–S5 linker shown from a top view and a side view. Coloring indicates separate chains of the tetrameric channel. The S1–S4 helices of the purple chain have been removed for the sake of clarity in the side view figure. (C) Homology models of TRPC3 indicate that the threonine 635 (in green) mutated in the Mwk mouse has the potential for hydrogen bonding with the end of helix S6. The first quadrant shows an overlap of four models and the resulting T635 orientation. The remaining quadrants show three possible hydrogen bonding partners: S735 (cyan), Y736 (magenta), and the adjacent R634 (yellow). (D) Overexpression of the TRPC3 mutants Mwk (T635A) and T635V significantly induced cell death in mouse Neuro-2a cells (mean ± SEM; n = 3; ANOVA followed by the Newman–Keuls post hoc test; p < 0.001 and p < 0.05). (E) Overexpression of the TRPC3 mutants Mwk (T635A) and T635V caused significant nuclear translocation of GFP-NFAT (mean ± SEM; n = 3; ANOVA followed by the Newman–Keuls post hoc test; p < 0.0001). (F) Overexpression of the TRPC3 mutants S735A and Y736F did not induce cell death upon overexpression in Neuro-2a cells (mean ± SEM; n = 3; ANOVA followed by the Newman–Keuls post hoc test). See also Figure S2 of the . (G) Other possible hydrogen bonding partners on the S6 helix (indicated as wheat mesh) were not seen as being viable for experimental validation as they pointed away from the site in question. Building homology models using the capsaicin-bound open state TRPV1 structure, however, indicated a further possible hydrogen bonding interaction with N726 (red) on helix S6. This interaction is with an adjacent subunit, and not the same subunit as with residues predicted with models based on the apo TRPV1. N726 is 10 residues toward the center of the membrane along S6 compared to other residues suspected to interact with T635.

Article Snippet: Protein lysates prepared with RIPA buffer (Thermo Scientific) were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) followed by immunoblotting with anti-TRPC3 (1:200, Alomone) and anti-Actin (1:1000, Abcam) antibodies.

Techniques: Sequencing, Mutagenesis, Over Expression, Translocation Assay