Journal: Antimicrobial Agents and Chemotherapy

Article Title: Cytotoxicological Analysis of a gp120 Binding Aptamer with Cross-Clade Human Immunodeficiency Virus Type 1 Entry Inhibition Properties: Comparison to Conventional Antiretrovirals

doi: 10.1128/aac.01502-08

Figure Lengend Snippet: FIG. 1. Phenotyping of human cord blood stem cell-derived cardiomyocytes. (A) Cardiomyocytes were cultured in chamber slides, permeab- ilized, and treated with rhodamine-labeled secondary antibody. (B) 4,6-Diamidino-2-phenylindole nuclear stain (B). (C) cTnI primary antibody and rhodamine-labeled secondary antibody. (D) A composite image created by superimposing the images in panels B and C.

Article Snippet: The human cord blood stem cell-derived cardiomyocytes were purchased from Celprogen.

Techniques: Derivative Assay, Cell Culture, Labeling, Staining

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Cytotoxicological Analysis of a gp120 Binding Aptamer with Cross-Clade Human Immunodeficiency Virus Type 1 Entry Inhibition Properties: Comparison to Conventional Antiretrovirals

doi: 10.1128/aac.01502-08

Figure Lengend Snippet: FIG. 2. Effect of the B40 aptamer in comparison to the effects of a panel of 17 ARVs on the viability of human cardiomyocytes and PBMCs. Human cord blood stem cell-derived cardiomyocytes or freshly isolated PBMCs were seeded at a density of 20,000 cells/well in 96-well plates and exposed to the respective ARVs at a concentration equivalent to the Cmax shown in Table 1 or the B40 aptamer at 2 M. All experiments were done in triplicate and were independently re- peated twice. ATP levels were measured as a correlate of cell viability after 7 days of exposure by relating the RLU of treated cells to the RLU of untreated cells. The results were normalized and expressed as the percent cell death for the treated cell samples by comparison to the number of untreated cells. The data on the graph show the means, and the error bars represent the standard deviations for three replicates in two sets of independent experiments. ARVs whose toxicity was statis- tically different from that of the untreated controls, as determined by the t test, are indicated as follows: *, P 0.01; **, P 0.001; ***, P 0.0001.

Article Snippet: The human cord blood stem cell-derived cardiomyocytes were purchased from Celprogen.

Techniques: Comparison, Derivative Assay, Isolation, Concentration Assay

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Cytotoxicological Analysis of a gp120 Binding Aptamer with Cross-Clade Human Immunodeficiency Virus Type 1 Entry Inhibition Properties: Comparison to Conventional Antiretrovirals

doi: 10.1128/aac.01502-08

Figure Lengend Snippet: FIG. 3. Caspase 3/7 activity in cardiomyocytes (A) and PBMCs (B) upon exposure to ARVs. Cells seeded at a density of 50,000 cells/well were exposed to EFZ, ABC, TDF, RTV, IND, T20, or the B40 aptamer at the Cmax for 1, 4, and 7 days. No statistically significant increases in caspase 3/7 activity were observed for any ARV, including the B40 aptamer, in cardiomyocytes or PBMCs compared to the ac- tivity in untreated control cells. A statistically significant decrease in caspase 3/7 activity was observed at least at day 4 and 7 in cardiomy- ocytes (A) and PBMCs (B) treated with EFV, ABC, TDF, and RTV compared to the activity in untreated control cells, as determined by the t test. *, P 0.002; **, P 0.0002. The data on the graphs represent the means, and the error bars represent the standard devi- ations for three replicates in two sets of independent experiments.

Article Snippet: The human cord blood stem cell-derived cardiomyocytes were purchased from Celprogen.

Techniques: Activity Assay, Control

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Cytotoxicological Analysis of a gp120 Binding Aptamer with Cross-Clade Human Immunodeficiency Virus Type 1 Entry Inhibition Properties: Comparison to Conventional Antiretrovirals

doi: 10.1128/aac.01502-08

Figure Lengend Snippet: FIG. 4. MAO A and B enzyme modulation by the B40 aptamer and T20 in cardiomyocytes (A and C) and PBMCs (B and D). Cells were incubated in triplicate with the serially diluted test molecule at a maximum concentration of 2 M for 2 h. A cell lysate was incubated with MAO A or B substrate for 1 h, followed by the addition of detection reagent, and the resulting luminescence signal was quantified in a luminometer. The no-treatment control was used as the baseline for maximum enzyme activity. No change in enzyme activity was detected for either MAO A or MAO B in the presence of the B40 aptamer and T20 for both cardiomyocytes and PBMCs. However, there was a dose-dependent inhibition of MAO A by clorgyline and MAO B by deprenyl, which are well-documented inhibitors of MAO A and MAO B, respectively, indicating the presence and activity of both enzymes in human cardiomyocytes and PBMCs. Enzyme activity was calculated by relating the RLUs of untreated control cells to that of the treated cells as a percentage. The data on the graphs represent the means, and the error bars represent the standard deviations for three replicates in two sets of independent experiments.

Article Snippet: The human cord blood stem cell-derived cardiomyocytes were purchased from Celprogen.

Techniques: Incubation, Concentration Assay, Control, Activity Assay, Inhibition

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Cytotoxicological Analysis of a gp120 Binding Aptamer with Cross-Clade Human Immunodeficiency Virus Type 1 Entry Inhibition Properties: Comparison to Conventional Antiretrovirals

doi: 10.1128/aac.01502-08

Figure Lengend Snippet: FIG. 5. Effects of the B40 aptamer and T20 on CYP3A4 enzyme activity in cardiomyocytes (A) and PBMCs (B). Cells were seeded at a density of 20,000 cells/well and exposed to the respective test molecule in triplicate for 2 days at serially diluted concentrations. The overlay medium was removed, and fresh medium containing a luciferin luminogenic substrate for the CYP3A4 enzyme was added. After 4 h of incubation, the luciferin detection reagent was added and the resulting luminescence signal was quantified in a luminometer. The no-treatment control was used as the baseline for maximum enzyme activity, and RTV, a known inhibitor of CYP3A4 (36, 56), was used as a positive control. The data on the graphs represent the means, and the error bars represent the standard deviations for three replicates in two sets of independent experiments.

Article Snippet: The human cord blood stem cell-derived cardiomyocytes were purchased from Celprogen.

Techniques: Activity Assay, Incubation, Control, Positive Control

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Cytotoxicological Analysis of a gp120 Binding Aptamer with Cross-Clade Human Immunodeficiency Virus Type 1 Entry Inhibition Properties: Comparison to Conventional Antiretrovirals

doi: 10.1128/aac.01502-08

Figure Lengend Snippet: FIG. 6. Effects of the B40 aptamer and T20 on mitochondrial tox- icity, as measured by determination of the mtDNA/nDNA ratio in cardiomyocytes and PBMCs. Cardiomyocytes and PBMCs were ex- posed to 2 M B40 aptamer, T20, or ddC in triplicate for 7 days. The relative amounts of nDNA and mtDNA were quantified by real-time quantitative PCR amplification of the CCOI nuclear gene and the ASPOLG mitochondrial gene, respectively. The data were extrapo- lated from a standard curve relating the cycle numbers to the relative DNA concentration. The mtDNA/nDNA ratio for the untreated sam- ple was arbitrarily set equal to 1.0. Only the ddC-treated cells showed a statistically significant decrease in the mtDNA/nDNA ratio com- pared to that for the untreated control, as determined by a t test and as indicated as follows: *, P 0.001; **, P 0.0001. The data on the graph represent the means, and the error bars represent the standard deviations for three replicates in two independent experiments.

Article Snippet: The human cord blood stem cell-derived cardiomyocytes were purchased from Celprogen.

Techniques: Real-time Polymerase Chain Reaction, Concentration Assay, Control

Journal: Communications Biology

Article Title: mTORC1 impedes osteoclast differentiation via calcineurin and NFATc1

doi: 10.1038/s42003-018-0028-4

Figure Lengend Snippet: Late mTORC1 deletion in myeloid lineage enhances osteoclastogenesis. Analyses of Raptor fl/fl -Lyz-Cre cKO mice and littermate controls. a , b Expression of osteoclast markers ( a ) and osteoclastogenic transcription factors ( b ) in bone marrow osteoclast differentiation cultures ( n = 6). c Representative images of TRAP-stained osteoclast differentiation cultures. d Osteoclast resorptive activity measured by calcium release from bone plates ( n = 16). e Osteoclast precursor proliferation by BrdU incorporation ( n = 10). f , g mTORC1 signaling in bone marrow osteoclast differentiation cultures from Raptor fl/fl -Lyz mice or littermate controls 50 h after RANKL treatment. f Phosphorylation of mTOR; g Phosphorylation of S6K1. p/T, ratio of phosphorylated/total protein. h – t Bone phenotype in 3-month-old male mice. h Serum CTX-1 bone resorption marker ( n = 8). i Serum P1NP bone formation marker ( n = 8). j Representative μCT images of the trabecular bone of the tibial metaphysis (top) and the entire proximal tibia (bottom). k – p Quantification of trabecular bone volume and architecture in proximal tibiae by μCT ( n = 8–10). k BV/TV bone volume/tissue volume ratio; l BS bone surface; m Tb.N trabecular number; n Conn. D. connectivity density; o Tb.Sp trabecular separation; p SMI structure model index. q – t Bone histomorphometry of distal femurs ( n = 6). Oc.S osteoclast surface, B.S bone surface, Oc.N osteoclast number, Ob.S osteoblast surface, Ob.N osteoblast number. Error bars, SEM; *, p < 0.05; **, p < 0.01; ***, p < 0.005; ****, p < 0.001; n.s. non-significant. R raptor. Full-size scans of immunoblots are shown in Supplementary Fig.

Article Snippet: Osteoblast precursors were expanded for 4 days in Mesenchymal Stem Cell (MSC) media using a Mouse MesenCult Proliferation Kit (StemCell Technologies) before the addition of osteoblast differentiation cocktail (StemCell Technologies).

Techniques: Expressing, Staining, Activity Assay, BrdU Incorporation Assay, Phospho-proteomics, Marker, Western Blot

Journal: Communications Biology

Article Title: mTORC1 impedes osteoclast differentiation via calcineurin and NFATc1

doi: 10.1038/s42003-018-0028-4

Figure Lengend Snippet: Constitutive mTORC1 activation in myeloid lineage impedes osteoclast differentiation. Analyses of Tsc1 fl/fl -Lyz-Cre cKO mice and littermate controls. a , b Expression of osteoclast markers ( a ) and osteoclastogenic transcription factors ( b ) in bone marrow osteoclast differentiation cultures ( n = 6). c Representative images of TRAP-stained osteoclast differentiation cultures. d Osteoclast resorptive activity measured by calcium release from bone plates ( n = 16). e Osteoclast precursor proliferation by BrdU incorporation ( n = 10). f , g mTORC1 signaling in bone marrow osteoclast differentiation cultures 60 h after RANKL treatment. f Phosphorylation of mTOR; g Phosphorylation of S6K1. p/T ratio of phosphorylated/total protein. h – r Bone phenotype in 2-month-old male mice. h Serum CTX-1 bone resorption marker ( n = 8). i Serum P1NP bone formation marker ( n = 8). j Bone histomorphometry of distal femurs ( n = 6). Oc.S osteoclast surface, B.S bone surface, Oc.N osteoclast number. k Bone histomorphometry of distal femurs ( n = 6). Ob.S osteoblast surface, B.S bone surface, Ob.N osteoblast number. l Representative μCT images of the trabecular bone of the tibial metaphysis (top) and the entire proximal tibia (bottom). m – r Quantification of trabecular bone volume and architecture in proximal tibiae by μCT ( n = 8). m BV/TV bone volume/tissue volume ratio, n BS bone surface, o Tb.N trabecular number, p Tb.Sp trabecular separation, q Conn. D. connectivity density, r SMI structure model index. Error bars, SEM; * p < 0.05; ** p < 0.01; *** p < 0.005; **** p < 0.001; n.s. non-significant. T Tsc1. Full-size scans of immunoblots are shown in Supplementary Fig.

Article Snippet: Osteoblast precursors were expanded for 4 days in Mesenchymal Stem Cell (MSC) media using a Mouse MesenCult Proliferation Kit (StemCell Technologies) before the addition of osteoblast differentiation cocktail (StemCell Technologies).

Techniques: Activation Assay, Expressing, Staining, Activity Assay, BrdU Incorporation Assay, Phospho-proteomics, Marker, Western Blot